U.S. patent application number 09/801348 was filed with the patent office on 2004-08-26 for f-box proteins and genes.
Invention is credited to Elledge, Stephen J., Harper, Jeffrey Wade, Winston, Jeffrey T..
Application Number | 20040166530 09/801348 |
Document ID | / |
Family ID | 25491933 |
Filed Date | 2004-08-26 |
United States Patent
Application |
20040166530 |
Kind Code |
A1 |
Harper, Jeffrey Wade ; et
al. |
August 26, 2004 |
F-box proteins and genes
Abstract
The present invention provides compositions and methods for gene
identification, as well as drug discovery and assessment. In
particular, the present invention provides components of an E3
complex involved in ubiquitination of cell cycle regulators and
other proteins, as well as members of a class of proteins that
directly function in recognition of ubiquitination targets. The
present invention also provides sequences of multiple F-box
proteins.
Inventors: |
Harper, Jeffrey Wade;
(Sugarland, TX) ; Elledge, Stephen J.; (Houston,
TX) ; Winston, Jeffrey T.; (Thousand Oaks,
CA) |
Correspondence
Address: |
Tim Headley
GARDERE WYNNE SEWELL LLP
1000 Louisiana, Suite 3400
Houston
TX
77002-5007
US
|
Family ID: |
25491933 |
Appl. No.: |
09/801348 |
Filed: |
March 7, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09801348 |
Mar 7, 2001 |
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09172841 |
Oct 15, 1998 |
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6232081 |
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09801348 |
Mar 7, 2001 |
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08951621 |
Oct 16, 1997 |
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6573094 |
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Current U.S.
Class: |
435/7.1 |
Current CPC
Class: |
G01N 33/6842 20130101;
A61K 38/00 20130101; C07K 16/18 20130101; G01N 33/68 20130101; C07K
14/4702 20130101 |
Class at
Publication: |
435/007.1 |
International
Class: |
G01N 033/53 |
Goverment Interests
[0002] This invention was made with government support under
National Institutes of Health Grant No. R01AG11085. The Government
has certain rights in the invention.
Claims
What is claimed is:
1. A method for the detection of one or more NF-.kappa.B regulatory
factors comprising the steps of: a) providing a slimb protein, and
a sample suspected of containing one or more NF-.kappa.B regulatory
factors; and b) exposing said slimb protein to said sample under
conditions such that said slimb protein binds to said one or more
NF-.kappa.B regulatory factors to form a slimb/regulatory factor
complex.
2. The method of claim 1, further comprising the step of detecting
said slimb/regulatory factor complex.
3. The method of claim 1, further comprising the step of observing
said slimb/regulatory factor complex for degradation of said one or
more NF-.kappa.B regulatory factors.
4. The method of claim 1, further comprising the step of exposing
said slimb protein and one or more NF-.kappa.B regulatory factors
to an F-box protein antagonist.
5. The method of claim 4, wherein said F-box protein antagonist
prevents the formation of said slimb/regulatory factor complex.
6. A method for the detection of a slimb protein complex,
comprising the steps of: a) providing a slimb protein and a sample
suspected of containing one or more proteins capable of forming a
complex with said slimb protein; and b) exposing said slimb protein
to said one or more proteins capable of forming a complex with said
slimb protein under conditions such that said slimb protein binds
to said one or more proteins capable of forming a complex with said
slimb protein to form a slimb protein complex.
7. The method of claim 6, further comprising the step of detecting
said slimb protein complex.
8. The method of claim 6, wherein step b) further comprises
exposing said slimb protein and said one or more proteins capable
of forming a complex with said slimb protein to an F-box protein
antagonist.
9. The method of claim 8, wherein said F-box protein antagonist
prevents the formation of said slimb protein complex.
10. An isolated nucleotide sequence comprising nucleotide sequence
encoding at least one functionally active fragement of an F-box
protein, wherein said sequence consists of a least a portion of the
sequence set forth in SEQ ID NOS: 54 and 56.
Description
[0001] This application is a Continuation-in-Part application of
U.S. patent application Ser. No. 08/951,621, filed Oct. 16, 1997,
pending, which is hereby incorporated herein by reference in its
entirety.
FIELD OF THE INVENTION
[0003] The present invention provides compositions and methods for
identification of F-box proteins, as well as for drug discovery and
assessment. In particular, the present invention provides
components of an E3 complex involved in ubiquitination of cell
cycle regulators and other proteins, as well as members of a class
of proteins that directly function in recognition of ubiquitination
targets.
BACKGROUND OF THE INVENTION
[0004] The proper development and maintenance of a multicellular
organism is a complex process that requires precise spatial and
temporal control of cell proliferation. Cell proliferation is
controlled via an intricate network of extracellular and
intracellular signaling pathways that process growth regulatory
signals. This signaling network is superimposed upon the basic cell
cycle regulatory machinery that controls particular cell cycle
transitions. In eukaryotes, the cell cycle is comprised of an
ordered series of discrete events. In contrast to the periodicity
of eukaryotic DNA replication and mitosis, cellular growth requires
that most metabolic reactions occur continuously. The cell cycle
regulatory machinery coordinates the events that occur during the
cell cycle, as well as cell growth. Protein degradation is an
important aspect of the development and maintenance of
multicellular organisms, as it provides direction, order, and the
appropriate timing for the key events that occur during the cell
cycle.
[0005] The problem of how cell division is controlled has long been
a topic of intense research. Early models suggested the existence
of an initiator that would accumulate during the cell cycle, and
induce DNA replication or mitosis when it reached a critical
concentration. The mitotic process would then inactivate the
initiator, thereby "resetting" the cell cycle. Subsequent research
showed that mitotic cyclins accumulate during interphase to drive
entry of cells into mitosis. These cyclins are then degraded at the
end of mitosis, in order to reset the cycle. Protein degradation
has been shown to have a pervasive role in the regulation of cell
cycle progression. For example, proteolysis is required for
multiple mitotic processes, and for initiating DNA replication
(See, King et al., Science 274:1652-1659 [1996]). Nonetheless, much
remains unknown regarding the proteins and the interactions that
are involved in the proteolytic regulation of the cell cycle and
other processes. Indeed, many proteins are likely to be involved in
proteolysis and cellular maintenance (as well as other processes).
Such information is needed for the development of compounds to
regulate the cell cycle and prevent or treat diseases associated
with abnormal cell proliferation.
SUMMARY OF THE INVENTION
[0006] The present invention provides compositions and methods for
gene identification (e.g., F-box genes), as well as drug discovery
and assessment. The present invention provides components of an E3
complex involved in ubiquitination of cell cycle regulators and
other proteins, as well as members of a class of proteins that
directly function in recognition of ubiquitination targets.
[0007] Thus, the present invention provides the function of a class
of proteins referred to as F-box proteins in targeted
ubiquitination. The present invention finds utility in methods for
developing compounds that affect ubiquitination. The present
invention also provides numerous novel F-box containing mammalian
genes whose encoded proteins are contemplated to function in
processes including, but not limited, to targeted ubiquitination of
cellular proteins.
[0008] The present invention also provides amino acid and DNA
sequence information for eighteen novel F-box-containing human or
mouse genes. As with Cdc4, Grr1, Skp2, and cyclin F, these novel
F-box proteins have the capacity to associate with Skp1 and to
simultaneously interact with other proteins through other
protein-protein interaction motifs encoded by regions of their
genes other than the F-box. Thus, the present invention provides
compositions and methods for determining the interaction of these
proteins with other proteins.
[0009] In one embodiment, the present invention provides an
isolated polypeptide comprising at least one functionally active
fragment of an F-box protein. In a preferred alternative
embodiment, the F-box protein is mammalian, while in a particularly
preferred embodiment, the F-box protein is human or murine.
[0010] In another embodiment, the functionally active fragment
comprises the amino acid sequence selected from the amino acid
sequences set forth in SEQ ID NOS:1, 3, 5, 9, 13, 17, 19, 25, 27,
41, 45, 47, 51, 53, 55, and 57, while in alternative embodiment,
the functionally active fragment comprises the amino acid sequence
selected from the amino acid sequences set forth in SEQ ID NOS:7,
11, 15, 21, 23, 29, 31, 33, 35, 37, 39, 43, and 49.
[0011] The present invention also provides a purified antibody
which binds specifically to the isolated polypeptide encoding an
F-box protein. In one embodiment, the antibody is monoclonal, while
in another embodiment, the antibody is polyclonal. In another
embodiment, the present invention provides a purified antibody
which specifically binds to a complex comprised of an F-box protein
and an F-box protein target. In yet another embodiment, the present
invention provides an antibody which specifically binds to a
complex comprised of an F-box protein and Skp1; it is contemplated
that the Skp1 in the complex may be bound to another protein, but
such binding is not required.
[0012] The present invention also provides an isolated nucleotide
sequence encoding at least one functionally active fragment of an
F-box protein, wherein the nucleotide sequence encodes at least a
portion of an F-box protein. In a preferred embodiment, the F-box
protein is mammalian, while in particularly preferred embodiments,
the F-box protein is human or murine. In one embodiment, the
isolated nucleotide sequence comprises at least a portion of the
sequence set forth in SEQ ID NOS:2, 4, 6, 10, 14, 18, 20, 26, 28,
42, 48, 52, 54, 56, and 58. In another embodiment, the isolated
nucleotide sequence comprises at least a portion of the sequence
set forth in SEQ ID NO:8, 12, 16, 22, 24, 30, 32, 34, 36, 38, 40,
44, and 50.
[0013] The present invention also provides a vector comprising a
nucleotide sequence, wherein the nucleotide sequence comprises the
nucleotide sequence encoding at least one functionally active
fragment of an F-box protein, wherein the nucleotide sequence
encodes at least a portion of an F-box protein. In one preferred
embodiment, the isolated nucleotide sequence comprises at least a
portion of the sequence set forth in SEQ ID NOS:2, 4, 6, 10, 14,
18, 20, 26, 28, 42, 48, 52, 54, 56, and 58, while in another
preferred embodiment, the isolated nucleotide sequence comprises at
least a portion of the sequence set forth in SEQ ID NO:8, 12, 16,
22, 24, 30, 32, 34, 36, 38, 40, 44, and 50.
[0014] The present invention also provides a host cell transformed
with at least one vector comprising a nucleotide sequence, wherein
the nucleotide sequence comprises the nucleotide sequence encoding
at least one functionally active fragment of an F-box protein,
wherein the nucleotide sequence encodes at least a portion of an
F-box protein. In one preferred embodiment, the isolated nucleotide
sequence comprises at least a portion of the sequence set forth in
SEQ ID NOS:2, 4, 6, 10, 14, 18, 20, 26, 28, 42, 48, 52, 54, 56, and
58, while in another preferred embodiment, the isolated nucleotide
sequence comprises at least a portion of the sequence set forth in
SEQ ID NO:8, 12, 16, 22, 24, 30, 32, 34, 36, 38, 40, 44, and
50.
[0015] The present invention also provides an isolated nucleotide
sequence encoding the amino acid sequence selected from group
consisting of SEQ ID NOS:1, 3, 5, 7, 9, 11, 13, 15 ,17, 19, 21, 23,
25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, and
57. In one embodiment, the present invention provides a vector
comprising an isolated nucleotide sequence encoding the amino acid
sequence selected from group consisting of SEQ ID NOS:1, 3, 5, 7,
9, 11, 13,15 ,17, 19, 21, 23, 25, 27, 29,31, 33, 35, 37, 39, 41,
43, 45, 47, 49, 51, 53, 55, and 57. In another embodiment, the
present invention provides a host cell transformed with this
vector.
[0016] The present invention further provides a polynucleotide
sequence comprising at least fifteen nucleotides, which hybridizes
under stringent conditions to at least a portion of a
polynucleotide sequence, wherein the polynucleotide sequence is
selected from the polynucleotide sequences set forth in SEQ ID
NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34,
36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, and 58.
[0017] The present invention also provides methods for detection of
polynucleotides encoding F-box protein in a biological sample
comprising the steps of: hybridizing at least a portion of the
polynucleotide encoding an F-box protein, to nucleic acid material
of a biological sample, thereby forming a hybridization complex;
and detecting the hybridization complex, wherein the presence of
the complex correlates with the presence of a polynucleotide
encoding F-box protein in the biological sample. In one embodiment
of the method, prior to hybridization, the nucleic acid material of
the biological sample is amplified by the polymerase chain
reaction.
[0018] The present invention also provides methods for the
detection of F-box protein targets comprising the steps of:
providing an F-box protein, and a sample suspected of containing an
F-box protein target; exposing the F-box protein to the sample
under conditions such that the F-box protein binds to the F-box
protein target to form an F-box protein and target complex; and
detecting the F-box protein and target complex. In one embodiment
of the method, the box protein target is selected from the group
consisting of cyclins, cyclin-dependent kinases, and I.kappa.B. An
alternative embodiment further comprises the step of analyzing said
F-box protein and target complex, wherein the analyzing comprises
observing the F-box protein and target complex for degradation of
the F-box protein target. In another embodiment, the method further
comprises the step of exposing the F-box protein and F-box protein
target to an F-box protein antagonist. In yet another embodiment of
the method, the F-box protein antagonist prevents the formation of
the F-box protein and the target complex.
[0019] The present invention also provides methods for the
detection of an F-box protein and Skp1 complex, comprising the
steps of: providing an F-box protein, and Skp1; exposing the F-box
protein to Skp1 under conditions such that the F-box protein binds
to Skp1 to form an F-box protein and Skp1 complex; and detecting
the F-box protein and Skp1 complex. One embodiment of the method
further comprises the step of exposing the F-box protein and Skp1
to an F-box protein antagonist. In yet another embodiment of the
method, the F-box protein antagonist prevents the formation of the
F-box protein and Skp1 complex.
[0020] The present invention also provides methods and compositions
useful to determine the complexity and diversity of mammalian F-box
proteins, as well as the identity of F-box proteins from various
species, the protein-protein interaction domains involved, the
proteolytic pathways, and regulatory pathways. Indeed, the present
invention provides methods and compositions to identify the
functions and ubiquitination targets of these and other F-box
containing proteins.
[0021] However, the present invention is not limited to F-box
proteins involved in ubiquitination. Thus, the function of F-box
proteins is not necessarily limited to ubiquitination, and the
present invention provides the methods and compositions to make
this determination. It is contemplated that additional F-box
containing genes will be discovered through the use of two-hybrid
screens with Skp1 or ubiquitination targets as the two-hybrid
"bait" (e.g., as described in the Example 6). It is also
contemplated that additional F-box genes will be discovered through
sequencing of the mammalian genome and sequence analysis, to
determine the homology with existing F-box proteins, such as those
identified in the present invention.
[0022] The present invention also provide compositions and methods
for development of drugs that disrupt at least one pathway in which
F-box proteins function, and are required for biological and/or
biochemical processes.
[0023] The present invention also provides methods and compositions
to identify and/or investigate cell cycle regulators, transcription
regulators, proteins involved in DNA replication, and other
cellular regulatory proteins. It is further contemplated that the
present invention finds use in elucidating inflammatory response
and infectious disease processes involving protein degradation, as
well as development of compounds that control (i.e., either enhance
or retard) protein degradation, as appropriate to ameliorate the
effects of the inflammatory response or disease process.
[0024] The present invention also provides methods and compositions
for identifying and investigating the function of protein targets
whose abundance is altered in disease, as well as for detection,
identification, and characterization of mutations in F-box genes
through various methods, including, but not limited sequence
analysis, Southern blot analysis of DNA, etc. Furthermore, the
present invention also finds use in assessing alterations in
cellular protein abundance due to overexpression of particular
F-box proteins. It is contemplated that such alterations are
associated with particular diseases. The present invention also
finds use in determination of overexpression caused by gene
amplification in DNA samples from diseased tissue or individuals
through such methods as Southern analysis using a particular F-box
gene as probe.
[0025] It is also contemplated that targets of novel human F-box
proteins will be determined by those experienced in the art by
approaches including, but not limited to two-hybrid library
screens, immunoprecipitation analysis followed by immunoblotting
with antibodies against candidate targets, peptide mapping, mass
spectral analysis, peptide sequencing, and/or by screening lambda
based expression libraries with F-box protein probes.
[0026] In addition, the present invention finds use in engineering
F-box proteins to artificially recruit particular proteins into an
E3 complex for ubiquitination. Thus, it is clear that the present
invention provides methods and compositions for detailed
investigation of F-box proteins, as well as proteins that associate
with F-box proteins. Furthermore, the present invention thereby
provides methods and compositions for the detection and analysis of
abnormalities in proteolytic functions, as well as methods and
compositions for the development of compounds suitable for use in
ameliorating such abnormalities.
[0027] The present invention further provides a method for the
detection of one or more NF-.kappa.B regulatory factors comprising
the steps of: providing a slimb protein, and a sample suspected of
containing one or more NF-.kappa.B regulatory factors, and exposing
the slimb protein to the sample under conditions such that the
slimb protein binds to the one or more NF-.kappa.B regulatory
factors to form a slimb/regulatory factor complex. In some
preferred embodiments, the method further comprises the step of
detecting the slimb/regulatory factor complex. In other embodiment,
the method further comprises the step of observing the
slimb/regulatory factor complex for degradation of the one or more
NF-.kappa.B regulatory factors. In yet other embodiments, the
method further comprises the step of exposing the slimb protein and
one or more NF-.kappa.B regulatory factors to an F-box protein
antagonist. In particular embodiments, the F-box protein antagonist
prevents the formation of the slimb/regulatory factor complex.
[0028] The present invention also provides a method for the
detection of a slimb protein complex, comprising the steps of:
providing a slimb protein and a sample suspected of containing one
or more proteins capable of forming a complex with the slimb
protein; and exposing the slimb protein to the one or more proteins
capable of forming a complex with the slimb protein under
conditions such that the slimb protein binds to the one or more
proteins capable of forming a complex with the slimb protein to
form a slimb protein complex. In preferred embodiments, the method
further comprises the step of detecting the slimb protein complex.
In some embodiment, step b) of the method further comprises
exposing the slimb protein and the one or more proteins capable of
forming a complex with the slimb protein to an F-box protein
antagonist. In particularly preferred embodiments, the F-box
protein antagonist prevents the formation of the slimb protein
complex.
DESCRIPTION OF THE FIGURES
[0029] Unless otherwise indicated, a "P" enclosed within a circle
indicates that the protein associated with the symbol is
phosphorylated.
[0030] FIG. 1 shows the assembly of a multiprotein complex
containing Cdc34, Cdc53, Skp1, and Cdc4, with the three panels
showing the enhancement of the formation of a Cdc53/Cdc4 complex by
Skp1. Panel A shows the results of immunoprecipitation with Myc tag
on Cdc53 (Cdc53.sup.M) using anti-Myc antibodies. Panel B shows the
immunoprecipitation results with a Flag tag on Cdc4 (Cdc4.sup.F).
Panel C shows that Skp1 and Cdc34 can associate with Cdc53
simultaneously.
[0031] FIG. 2A shows an SDS-PAGE analysis of purified Cln1
HA/Gst-Cdc28HA/Cks1.
[0032] FIG. 2B is an autoradiograph showing the phosphorylation of
Sic1 by Cln1/Cdc28 complexes in vitro.
[0033] FIG. 2C shows immunoblot results indicating that
phosphorylation of Sic1 is required for its association with
Cdc34/Cdc53/Skp1/Cdc4 complexes.
[0034] FIG. 2D shows immunoblot results indicating that association
of phosphorylated Sic1 with Cdc4 is enhanced by Skp1.
[0035] FIG. 2E shows immunoblot results indicating that association
of phosphorylated Sic1 with Skp1 requires the WD-40 repeats of
Cdc4.
[0036] FIG. 3A shows the immunoblot results demonstrating that
phosphorylated Sic1 is ubiquitinated in vivo and in vitro with
purified Cdc34 E2 and Cdc53/Skp1/Cdc4 complexes.
[0037] FIG. 3B shows immunoblot results for anti-Cdc53.sup.M immune
complexes tested against Cdc53.sup.M/Skp1, Cdc53.sup.M/Skp1/Cdc4,
and supplemented with ATP, ubiquitin, human E1, Cdc34 purified from
E. coli, and either unphosphorylated or phosphorylated Sic1
complexes.
[0038] FIG. 3C shows immunoblot results for anti-Skp1.sup.F immune
complexes tested with SkP1.sup.F/Cdc53.sup.M/Cdc4, Skp1.sup.F/Cdc4,
and Skp1.sup.F/Cdc53.sup.M.
[0039] FIG. 3D shows immunoblot results that indicate
ubiquitination of Sic1 does not require that Cln/Cdc28 be present
in the ubiquitination reaction nor that Sic1 be associated with
Clb5/Cdc28.
[0040] FIG. 3E shows immunoblot results that
Clb5/Cdc28-phosphorylated Sic1 is a substrate for ubiquitination by
Cdc34.
[0041] FIG. 4A shows immunoblot results indicating that Grr1 can
associate with Skp1 and Cdc53.
[0042] FIG. 4B shows an autoradiograph indicating that
phosphorylated Sic1 associates with Cdc4 but not Grr1-containing
complexes.
[0043] FIG. 4C shows an immunoblot indicating that Cdc4, but not
Grr1, supports ubiquitination of Sic1 in vitro.
[0044] FIG. 4D shows an immunoblot used to verify the presence of
reaction components derived from immunoprecipitation (the blot used
for ubiquitination assays was reprobed to detect Grr1G10,
Cdc53.sup.M, and Cdc4).
[0045] FIG. 5A is an autoradiograph showing differential
recognition of Sic1 and Cln proteins by Grr1 and Cdc4.
[0046] FIG. 5B shows an immunoblot verifying the presence of Cdc4,
Grr1G10, Cdc53.sup.M, and Skp1.sup.F.
[0047] FIG. 5C is an immunoblot indicating that phosphorylation of
Cln is required for the association of Cln1/Cdc28 complexes with
Grr1.
[0048] FIG. 5D is an autoradiograph showing that purified
Skp1/Cdc53/Grr1 complexes are not sufficient for Cln1
ubiquitination by Cdc34 in vitro.
[0049] FIG. 5E is an immunoblot showing that phosphorylated Cln1 is
ubiquitinated in a fractionated yeast extract system.
[0050] FIG. 6A is a schematic showing that phosphorylation of
substrates through protein kinase signalling pathways is required
for recognition by F-box receptor proteins.
[0051] FIG. 6B is a schematic showing that distinct F-box complexes
may regulate different biological processes through selective
recruitment of substrates. Hypothetical FEC configurations are
shown together with the signals that are being sensed, the
corresponding substrates and the physiological consequences of
complex function.
[0052] FIG. 6C is a schematic showing the interplay between protein
kinase and the SCF pathway in the G1 to S-phase transition in S.
cerevisiae. In this Figure, perpendicular bars indicate inhibitory
events.
[0053] FIG. 7 shows the alignment of various F-box proteins
provided in the present invention.
[0054] FIG. 8 shows immunoblotting results demonstrating that Skp1
associates with phosphorylated I.kappa.B but not unphosphorylated
I.kappa.B.
[0055] FIG. 9 shows the interaction between various F-box proteins
and phosphorylated and unphosphorylated I.kappa.B. Schematic
representations of the F-box proteins are provided with "F"
representing F-box sequences.
[0056] FIGS. 10A and 10B show lysates from the indicated
transfections that were subjected to immunoblotting using the
indicated antibodies.
[0057] FIGS. 11A and 11B show additional
immunoprecipitation/western blotting experiments using the
indicated transfections and antibodies.
[0058] FIG. 12 shows immunoblotting results with the indicated
antibodies for phosphorylation-specific interaction of SCF slimb
complexes with I.kappa.B peptide sequences.
DEFINITIONS
[0059] To facilitate understanding of the invention, a number of
terms are defined below.
[0060] As used herein, the term "F-box proteins" refers to the
amino acid sequences of substantially purified proteins involved in
proteolysis, including but not limited to proteins involved in the
ubiquitin-ligase complex obtained from any species, including
bovine, ovine, porcine, murine, equine, and human, from any source
whether natural, synthetic, semi-synthetic, or recombinant. The
F-box is a sequence of 35-45 amino acids and allows the F-box
proteins to enter into complexes with Skp1. Thus, the F-box
proteins bind Skp1, and contain a motif that displays sequence
similarity to Grr1 and Cdc4. This conserved structural motif is
included in the sequence alignments shown in FIG. 7 (i.e., the
amino acid residues that are shared by the F-box proteins shown).
However, it is not intended that the term be limited to the exact
sequences set forth in FIG. 7. In some embodiments, the F-box
proteins further comprise additional motifs, in particular motifs
involved in protein-protein interaction. These additional motifs
included, but are not limited to leucine-rich repeats, and WD-40.
In preferred embodiments, the F-box protein is mammalian, while in
particularly preferred embodiments, the F-box protein is human or
murine.
[0061] As used herein, the term "F-box target" refers to any moiety
that is recognized by at least one F-box containing protein. It is
intended that the term encompass such proteins as the cyclins
(e.g., A, D, and E), as well as cyclin kinase inhibitors (e.g.,
p27), and I.kappa.B, as well as other proteins. It is not intended
that the term be limited to any particular protein or compound.
[0062] As used herein, the term "multiprotein complex" refers to
complexes comprising more than one protein. It is intended that the
term encompass complexes with any number of proteins. In preferred
embodiments, the proteins comprising a multiprotein complex
function cooperatively. For example, in particularly preferred
embodiments of the present invention, Cdc34, Cdc53, Skp1, and Cdc4
comprise a multiprotein complex. It is also intended that the term
encompass complexes comprising Skp1, any of the amino acid
sequences set forth in Table 2 or Table 4, and a Cdc53 homolog. In
preferred embodiments, the Cdc53 homolog in such multiprotein
complexes comprises human Cul proteins (e.g., Cul 1 through 5), as
well as murine Cul proteins. It is also intended that this term
encompass complexes comprised of an F-box protein and its target
protein (i.e., an F-box target protein).
[0063] The term "modulate," as used herein, refers to a change or
an alteration in the biological activity of an F-box protein (e.g.,
mammalian F-box proteins). Modulation may be an increase or a
decrease in protein activity, a change in binding characteristics,
or any other change in the biological, functional, or immunological
properties of an F-box protein.
[0064] The term "mimetic," as used herein, refers to a molecule,
the structure of which is developed from knowledge of the structure
of an F-box protein, or portions thereof and, as such, is able to
effect some or all of the actions of F-box proteins and/or F-box
protein-like molecules.
[0065] The term "antagonist" refers to molecules or compounds which
inhibit the action of a composition (e.g., an F-box protein).
Antagonists may or may not be homologous to the targets of these
compositions in respect to conformation, charge or other
characteristics. In particularly preferred embodiments, antagonists
prevent the functioning of F-box proteins. It is contemplated that
antagonists may prevent binding of an F-box protein and its
target(s). It is also contemplated that antagonists prevent or
alter the binding of an F-box protein and Skp1. However, it is not
intended that the term be limited to a particular site of
function.
[0066] The term "derivative," as used herein, refers to the
chemical modification of a nucleic acid encoding an F-box protein
(in particular, mammalian F-box proteins), or the encoded F-box
protein. Illustrative of such modifications would be replacement of
hydrogen by an alkyl, acyl, or amino group. A nucleic acid
derivative would encode a polypeptide which retains essential
biological characteristics of the natural molecule.
[0067] A "variant" of an F-box protein, as used herein, refers to
an amino acid sequence that is altered by one or more amino acids.
The variant may have "conservative" changes, wherein a substituted
amino acid has similar structural or chemical properties (e.g.,
replacement of leucine with isoleucine). More rarely, a variant may
have "nonconservative" changes (e.g., replacement of a glycine with
a tryptophan). Similar minor variations may also include amino acid
deletions or insertions, or both. Guidance in determining which
amino acid residues may be substituted, inserted, or deleted
without abolishing biological or immunological activity may be
found using computer programs well known in the art, for example,
DNASTAR software.
[0068] "Alterations" in the polynucleotide of for example, SEQ ID
NO:4, as used herein, comprise any alteration in the sequence of
polynucleotides encoding human F1 Alpha F-box protein, including
deletions, insertions, and point mutations that may be detected
using hybridization assays. Included within this definition is the
detection of alterations to the genomic DNA sequence which encodes
an F-box protein (e.g., by alterations in the pattern of
restriction fragment length polymorphisms) capable of hybridizing
to a particular sequence, the inability of a selected fragment to
hybridize to a sample of genomic DNA (e.g., using allele-specific
oligonucleotide probes), and improper or unexpected hybridization,
such as hybridization to a locus other than the normal chromosomal
locus for the polynucleotide sequence encoding an F-box protein
(e.g., using fluorescent in situ hybridization [FISH] to metaphase
chromosomes spreads).
[0069] A "consensus gene sequence" refers to a gene sequence which
is derived by comparison of two or more gene sequences and which
describes the nucleotides most often present in a given segment of
the genes; the consensus sequence is the canonical sequence. In
some embodiments, "consensus," refers to a nucleic acid sequence
which has been resequenced to resolve uncalled bases, or which has
been extended using any suitable method known in the art, in the 5'
and/or the 3' direction and resequenced, or which has been
assembled from the overlapping sequences of more than one clone
using any suitable method known in the art, or which has been both
extended and assembled.
[0070] The term "sample," as used herein, is used in its broadest
sense. The term encompasses biological sample(s) suspected of
containing nucleic acid encoding F-box proteins or fragments
thereof, and may comprise a cell, chromosomes isolated from a cell
(e.g., a spread of metaphase chromosomes), genomic DNA (in solution
or bound to a solid support such as for Southern analysis), RNA (in
solution or bound to a solid support such as for northern
analysis), cDNA (in solution or bound to a solid support), an
extract from cells or a tissue, and the like.
[0071] As used herein the terms "protein" and "polypeptide" refer
to compounds comprising amino acids joined via peptide bonds and
are used interchangeably.
[0072] The terms "gene sequences" or "native gene sequences" are
used to indicate DNA sequences encoding a particular gene which
contain the same DNA sequences as found in the gene as isolated
from nature. In contrast, "synthetic gene sequences" are DNA
sequences which are used to replace the naturally occurring DNA
sequences when the naturally occurring sequences cause expression
problems in a given host cell. For example, naturally-occurring DNA
sequences encoding codons which are rarely used in a host cell may
be replaced (e.g., by site-directed mutagenesis) such that the
synthetic DNA sequence represents a more frequently used codon. The
native DNA sequence and the synthetic DNA sequence will preferably
encode the same amino acid sequence.
[0073] As used herein, the term "gene" means the
deoxyribonucleotide sequences comprising the coding region of a
structural gene and the including sequences located adjacent to the
coding region on both the 5, and 3, ends for a distance of about 1
kb on either end such that the gene corresponds to the length of
the full-length mRNA. The sequences which are located 5' of the
coding region and which are present on the mRNA are referred to as
5' non-translated sequences. The sequences which are located 3' or
downstream of the coding region and which are present on the mRNA
are referred to as 3' non-translated sequences; these sequences.
The term "gene" encompasses both cDNA and genomic forms of a gene.
A genomic form or clone of a gene contains the coding region
interrupted with non-coding sequences termed "introns" or
"intervening regions" or "intervening sequences." Introns are
segments of a gene which are transcribed into nuclear RNA (hnRNA);
introns may contain regulatory elements such as enhancers. Introns
are removed or "spliced out" from the nuclear or primary
transcript; introns therefore are absent in the messenger RNA
(mRNA) transcript. The mRNA functions during translation to specify
the sequence or order of amino acids in a nascent polypeptide.
[0074] In addition to containing introns, genomic forms of a gene
may also include sequences located on both the 5' and 3' end of the
sequences which are present on the RNA transcript. These sequences
are referred to as "flanking" sequences or regions (these flanking
sequences are located 5' or 3' to the non-translated sequences
present on the mRNA transcript). The 5' flanking region may contain
regulatory sequences such as promoters and enhancers which control
or influence the transcription of the gene. The 3' flanking region
may contain sequences which direct the termination of
transcription, post-transcriptional cleavage and
polyadenylation.
[0075] As used herein, the term "structural gene" refers to a DNA
sequence coding for RNA or a protein. In contrast, "regulatory
genes" are structural genes which encode products which control the
expression of other genes (e.g., transcription factors).
[0076] As used herein the term "coding region" when used in
reference to structural gene refers to the nucleotide sequences
which encode the amino acids found in the nascent polypeptide as a
result of translation of a mRNA molecule. The coding region is
bounded, in eukaryotes, on the 5' side by the nucleotide triplet
"ATG" which encodes the initiator methionine and on the 3' side by
one of the three triplets which specify stop codons (i.e., TAA,
TAG, TGA).
[0077] The term "portion," as used herein, with regard to a protein
(as in "a portion of a given protein") refers to fragments of that
protein. The fragments may range in size from four amino acid
residues to the entire amino acid sequence minus one amino acid.
Thus, a protein "comprising at least a portion of the amino acid
sequence of SEQ ID NO:3" encompasses the full-length human F1
protein, and fragments thereof.
[0078] "Nucleic acid sequence" as used herein refers to an
oligonucleotide, nucleotide, or polynucleotide, and fragments or
portions thereof, and to DNA or RNA of genomic or synthetic origin
which may be single- or double-stranded, and represent the sense or
antisense strand. Similarly, "amino acid sequence" as used herein
refers to an oligopeptide, peptide, polypeptide, or protein
sequence, and fragments or portions thereof, and to naturally
occurring or synthetic molecules.
[0079] A "composition comprising a given polynucleotide sequence"
as used herein refers broadly to any composition containing the
given polynucleotide sequence. The composition may comprise an
aqueous solution. Compositions comprising polynucleotide sequences
encoding F-box proteins or fragments thereof, may be employed as
hybridization probes. In this case, the F-box-encoding
polynucleotide sequences are typically employed in an aqueous
solution containing salts (e.g., NaCl), detergents (e.g., SDS) and
other components (e.g., Denhardt's solution, dry milk, salmon sperm
DNA, etc.).
[0080] Where "amino acid sequence" is recited herein to refer to an
amino acid sequence of a naturally occurring protein molecule,
"amino acid sequence" and like terms, such as "polypeptide" or
"protein" are not meant to limit the amino acid sequence to the
complete, native amino acid sequence associated with the recited
protein molecule.
[0081] A "deletion," as used herein, refers to a change in either
amino acid or nucleotide sequence in which one or more amino acid
or nucleotide residues, respectively, are absent.
[0082] An "insertion" or "addition," as used herein, refers to a
change in an amino acid or nucleotide sequence resulting in the
addition of one or more amino acid or nucleotide residues,
respectively, as compared to the naturally occurring molecule.
[0083] A "substitution," as used herein, refers to the replacement
of one or more amino acids or nucleotides by different amino acids
or nucleotides, respectively.
[0084] The term "biologically active," as used herein, refers to a
protein having structural, regulatory, or biochemical functions of
a naturally occurring molecule. Likewise, "immunologically active"
refers to the capability of the natural, recombinant, or synthetic
F-box proteins, or any oligopeptide thereof, to induce a specific
immune response in appropriate animals or cells and to bind with
specific antibodies.
[0085] As used herein, the term "purified" or "to purify" refers to
the removal of contaminants from a sample. For example, proteins of
interest are purified by removal of contaminating proteins; they
are also purified by the removal of substantially all proteins that
are not of interest. The removal of non-immunoglobulin proteins
and/or the removal of immunoglobulins that do not bind protein
results in an increase in the percent of protein of
interest-reactive immunoglobulins in the sample. In another
example, recombinant polypeptides are expressed in bacterial host
cells and the polypeptides are purified by the removal of host cell
proteins; the percent of recombinant polypeptides is thereby
increased in the sample.
[0086] The term "substantially purified," as used herein, refers to
nucleic or amino acid sequences that are removed from their natural
environment, isolated or separated, and are at least 60% free,
preferably 75% free, and most preferably 90% free from other
components with which they are naturally associated.
[0087] The term "recombinant DNA molecule" as used herein refers to
a DNA molecule which is comprised of segments of DNA joined
together by means of molecular biological techniques.
[0088] The term "recombinant protein" or "recombinant polypeptide"
as used herein refers to a protein molecule which is expressed from
a recombinant DNA molecule. The term "native protein" as used
herein refers to a protein which is isolated from a natural source
as opposed to the production of a protein by recombinant means.
[0089] As used herein, the term "overproducing" is used in
reference to the production of polypeptides in a host cell, and
indicates that the host cell is producing more of the polypeptide
by virtue of the introduction of nucleic acid sequences encoding
the polypeptide than would be expressed by the host cell absent the
introduction of these nucleic acid sequences. To allow ease of
purification of polypeptides produced in a host cell it is
preferred that the host cell express or overproduce the polypeptide
at a level greater than 1 mg/liter of host cell culture.
[0090] "A host cell capable of expressing a recombinant protein as
a soluble protein at a level greater than or equal to X milligrams
per 1 OD of cells per liter" is a host cell that produces X
milligrams of recombinant protein per liter of culture medium
containing a density of host cells equal to 1 OD.sub.600. The
amount of recombinant protein present per OD per liter is
determined by quantitating the amount of recombinant protein
recovered following affinity purification.
[0091] "A host cell capable of secreting a recombinant protein into
the culture supernatant at a level greater than or equal to 10 mg
recombinant protein per 1 OD of cells per liter" refers to a host
cell that secretes a recombinant protein into the culture
supernatant (i.e., the medium, such as LB broth, used to grow the
host cell) at a level greater than or equal to 10 mg recombinant
protein per liter of medium containing a concentration (i.e.,
density) of host cells equal to 1 OD.sub.600. The host cells may be
grown in shaker flasks (approximately 1 liter culture medium) or in
fermentation tank (approximately 10 liters culture medium) and the
amount of recombinant protein secreted into the culture supernatant
may be determined using a quantitative ELISA assay.
[0092] As used herein, the term "fusion protein" refers to a
chimeric protein containing the protein of interest (i.e., a
ubiquitination complex and/or fragments thereof) joined to an
exogenous protein fragment (the fusion partner which consists of a
non-ubiquitination complex protein). The fusion partner may enhance
solubility of the protein as expressed in a host cell, may provide
an "affinity tag" to allow purification of the recombinant fusion
protein from the host cell or culture supernatant, or both. If
desired, the fusion protein may be removed from the protein of
interest prior to immunization by a variety of enzymatic or
chemical means known to the art.
[0093] As used herein, the term "affinity tag" refers to such
structures as a "poly-histidine tract" or "poly-histidine tag," or
any other structure or compound which facilitates the purification
of a recombinant fusion protein from a host cell, host cell culture
supernatant, or both. As used herein, the term "flag tag" refers to
short polypeptide marker sequence useful for recombinant protein
identification and purification.
[0094] As used herein, the terms "poly-histidine tract" and
"poly-histidine tag," when used in reference to a fusion protein
refers to the presence of two to ten histidine (or more) residues
at either the amino- or carboxy-terminus of a protein of interest.
A poly-histidine tract of six to ten residues is preferred. The
poly-histidine tract is also defined functionally as being a number
of consecutive histidine residues added to the protein of interest
which allows the affinity purification of the resulting fusion
protein on a nickel-chelate or IDA column.
[0095] As used herein, the term "chimeric protein" refers to two or
more coding sequences obtained from different genes, that have been
cloned together and that, after translation, act as a single
polypeptide sequence. Chimeric proteins are also referred to as
"hybrid proteins." As used herein, the term "chimeric protein"
refers to coding sequences that are obtained from different species
of organisms, as well as coding sequences that are obtained from
the same species of organisms.
[0096] As used herein, the term "protein of interest" refers to the
protein whose expression is desired within the fusion protein. In a
fusion protein, the protein of interest will be joined or fused
with another protein or protein domain, the fusion partner, to
allow for enhanced stability of the protein of interest and/or ease
of purification of the fusion protein.
[0097] As used herein "soluble" when in reference to a protein
produced by recombinant DNA technology in a host cell, is a protein
which exists in solution in the cytoplasm of the host cell; if the
protein contains a signal sequence, the soluble protein is secreted
into the culture medium of eukaryotic cells capable of secretion or
by bacterial hosts possessing the appropriate genes. In contrast,
an insoluble protein is one which exists in denatured form inside
cytoplasmic granules (i.e., inclusion bodies) in the host cell.
High level expression (i.e., greater than 1 mg recombinant
protein/liter of culture) of recombinant proteins often results in
the expressed protein being found in inclusion bodies in the host
cells. A soluble protein is a protein which is not found in an
inclusion body inside the host cell or is found both in the
cytoplasm and in inclusion bodies and in this case the protein may
be present at high or low levels in the cytoplasm.
[0098] "Peptide nucleic acid" as used herein, refers to a molecule
which comprises an oligomer to which an amino acid residue, such as
lysine, and an amino group have been added. These small molecules,
also designated anti-gene agents, stop transcript elongation by
binding to their complementary strand of nucleic acid (Nielsen, P.
E. et. al., Anticancer Drug Des., 8:53-63 [1993]).
[0099] The term "hybridization" as used herein, refers to any
process by which a strand of nucleic acid binds with a
complementary strand through base pairing. Hybridization and the
strength of hybridization (i.e., the strength of the association
between the nucleic acids) is impacted by such factors as the
degree of complementary between the nucleic acids, stringency of
the conditions involved, the T.sub.m of the formed hybrid, and the
G:C ratio within the nucleic acids.
[0100] As used herein, the term "T.sub.m" is used in reference to
the "melting temperature." The melting temperature is the
temperature at which a population of double-stranded nucleic acid
molecules becomes half dissociated into single strands. The
equation for calculating the T.sub.m of nucleic acids is well known
in the art. As indicated by standard references, a simple estimate
of the T.sub.m value may be calculated by the equation;
T.sub.m=81.5+0.41(% G+C), when a nucleic acid is in aqueous
solution at 1 M NaCl (See e.g., Anderson and Young, Quantitative
Filter Hybridization, in Nucleic Acid Hybridization [1985]). Other
references include more sophisticated computations which take
structural as well as sequence characteristics into account for the
calculation of T.sub.m.
[0101] The term "hybridization complex," as used herein, refers to
a complex formed between two nucleic acid sequences by virtue of
the formation of hydrogen binds between complementary G and C bases
and between complementary A and T bases; these hydrogen bonds may
be further stabilized by base stacking interactions. The two
complementary nucleic acid sequences hydrogen bond in an
antiparallel configuration. A hybridization complex may be formed
in solution (e.g., C.sub.0t or R.sub.0t analysis) or between one
nucleic acid sequence present in solution and another nucleic acid
sequence immobilized on a solid support (e.g., membranes, filters,
chips, pins or glass slides to which cells have been fixed for in
situ hybridization).
[0102] The terms "complementary" or "complementarity" as used
herein, refer to the natural binding of polynucleotides under
permissive salt and temperature conditions by base-pairing. For
example, for the sequence "A-G-T" binds to the complementary
sequence "T-C-A". Complementarity between two single-stranded
molecules may be "partial", in which only some of the nucleic acids
bind, or it may be complete when total complementarity exists
between the single stranded molecules. The degree of
complementarity between nucleic acid strands has significant
effects on the efficiency and strength of hybridization between
nucleic acid strands. This is of particular importance in
amplification reactions, which depend upon binding between nucleic
acids strands.
[0103] The term "homology," as used herein, refers to a degree of
complementarity. There may be partial homology or complete homology
(i.e., identity). A partially complementary sequence is one that at
least partially inhibits an identical sequence from hybridizing to
a target nucleic acid; it is referred to using the functional term
"substantially homologous." The inhibition of hybridization of the
completely complementary sequence to the target sequence may be
examined using a hybridization assay (Southern or Northern blot,
solution hybridization and the like) under conditions of low
stringency. A substantially homologous sequence or probe will
compete for and inhibit the binding (i.e., the hybridization) of a
completely homologous sequence or probe to the target sequence
under conditions of low stringency. This is not to say that
conditions of low stringency are such that non-specific binding is
permitted; low stringency conditions require that the binding of
two sequences to one another be a specific (i.e., selective)
interaction. The absence of non-specific binding may be tested by
the use of a second target sequence which lacks even a partial
degree of complementarity (e.g., less than about 30% identity); in
the absence of non-specific binding, the probe will not hybridize
to the second non-complementary target sequence. When used in
reference to a single-stranded nucleic acid sequence, the term
"substantially homologous" refers to any probe which can hybridize
(i.e., it is the complement of) the single-stranded nucleic acid
sequence under conditions of low stringency as described.
[0104] As known in the art, numerous equivalent conditions may be
employed to comprise either low or high stringency conditions.
Factors such as the length and nature (DNA, RNA, base composition)
of the sequence, nature of the target (DNA, RNA, base composition,
presence in solution or immobilization, etc.), and the
concentration of the salts and other components (e.g., the presence
or absence of formamide, dextran sulfate and/or polyethylene
glycol) are considered and the hybridization solution may be varied
to generate conditions of either low or high stringency different
from, but equivalent to, the above listed conditions.
[0105] As used herein the term "stringency" is used in reference to
the conditions of temperature, ionic strength, and the presence of
other compounds such as organic solvents, under which nucleic acid
hybridizations are conducted. With "high stringency" conditions,
nucleic acid base pairing will occur only between nucleic acid
fragments that have a high frequency of complementary base
sequences. Thus, conditions of "weak" or "low" stringency are often
required with nucleic acids that are derived from organisms that
are genetically diverse, as the frequency of complementary
sequences is usually less.
[0106] Low stringency conditions comprise conditions equivalent to
binding or hybridization at 42.degree. C. in a solution consisting
of 5.times.SSPE (43.8 g/l NaCl, 6.9 g/l NaH.sub.2PO.sub.4.H.sub.2O
and 1.85 g/l EDTA, pH adjusted to 7.4 with NaOH), 0.1% SDS,
5.times. Denhardt's reagent (50.times. Denhardt's contains per 500
ml: 5 g Ficoll (Type 400, Pharmacia), 5 g BSA [Fraction V; Sigma])
and 100 .mu.g/ml denatured salmon sperm DNA followed by washing in
a solution comprising 5.times.SSPE, 01% SDS at 42.degree. C. when a
probe of about 500 nucleotides in length is employed.
[0107] The art knows well that numerous equivalent conditions may
be employed to comprise low stringency conditions; factors such as
the length and nature (DNA, RNA, base composition) of the probe and
nature of the target (DNA, RNA, base composition, present in
solution or immobilized, etc.) and the concentration of the salts
and other components (e.g., the presence or absence of formamide,
dextran sulfate, polyethylene glycol) are considered and the
hybridization solution may be varied to generate conditions of low
stringency hybridization different from, but equivalent to, the
above listed conditions. In addition, the art knows conditions
which promote hybridization under conditions of high stringency
(e.g., increasing the temperature of the hybridization and/or wash
steps, the use of formamide in the hybridization solution,
etc.).
[0108] The term "antisense," as used herein, refers to nucleotide
sequences which are complementary to a specific DNA or RNA
sequence. The term "antisense strand" is used in reference to a
nucleic acid strand that is complementary to the "sense" strand.
Antisense molecules may be produced by any method, including
synthesis by ligating the gene(s) of interest in a reverse
orientation to a viral promoter which permits the synthesis of a
complementary strand. Once introduced into a cell, this transcribed
strand combines with natural sequences produced by the cell to form
duplexes. These duplexes then block either the further
transcription or translation. In this manner, mutant phenotypes may
be generated. The designation "negative" is sometimes used in
reference to the antisense strand, and "positive" is sometimes used
in reference to the sense strand.
[0109] The term also is used in reference to RNA sequences which
are complementary to a specific RNA sequence (e.g., mRNA). Included
within this definition are antisense RNA ("asRNA") molecules
involved in gene regulation by bacteria. Antisense RNA may be
produced by any method, including synthesis by splicing the gene(s)
of interest in a reverse orientation to a viral promoter which
permits the synthesis of a coding strand. Once introduced into an
embryo, this transcribed strand combines with natural mRNA produced
by the embryo to form duplexes. These duplexes then block either
the further transcription of the mRNA or its translation. In this
manner, mutant phenotypes may be generated. The term "antisense
strand" is used in reference to a nucleic acid strand that is
complementary to the "sense" strand. The designation. (-) (i.e.,
"negative") is sometimes used in reference to the antisense strand
with the designation (+) sometimes used in reference to the sense
(i.e., "positive") strand.
[0110] A gene may produce multiple RNA species which are generated
by differential splicing of the primary RNA transcript. cDNAs that
are splice variants of the same gene will contain regions of
sequence identity or complete homology (representing the presence
of the same exon or portion of the same exon on both cDNAs) and
regions of complete non-identity (for example, representing the
presence of exon "A" on cDNA 1 wherein cDNA 2 contains exon "B"
instead). Because the two cDNAs contain regions of sequence
identity they will both hybridize to a probe derived from the
entire gene or portions of the gene containing sequences found on
both cDNAs; the two splice variants are therefore substantially
homologous to such a probe and to each other.
[0111] "Transformation," as defined herein, describes a process by
which exogenous DNA enters and changes a recipient cell. It may
occur under natural or artificial conditions using various methods
well known in the art. Transformation may rely on any known method
for the insertion of foreign nucleic acid sequences into a
prokaryotic or eukaryotic host cell. The method is selected based
on the host cell being transformed and may include, but is not
limited to, viral infection, electroporation, lipofection, and
particle bombardment. Such "transformed" cells include stably
transformed cells in which the inserted DNA is capable of
replication either as an autonomously replicating plasmid or as
part of the host chromosome. The term "transfection" as used herein
refers to the introduction of foreign DNA into eukaryotic cells.
Transfection may be accomplished by a variety of means known to the
art including calcium phosphate-DNA co-precipitation,
DEAE-dextran-mediated transfection, polybrene-mediated
transfection, electroporation, microinjection, liposome fusion,
lipofection, protoplast fusion, retroviral infection, and
biolistics. Thus, the term "stable transfection" or "stably
transfected" refers to the introduction and integration of foreign
DNA into the genome of the transfected cell. The term "stable
transfectant"in insect cells. For example, these vectors find use
in expression systems for recombinant proteins that require
eukaryotic processing systems. It is intended that the present
invention encompass baculovirus-derived vectors, as well as vectors
derived from other viruses capable of infecting invertebrate cells.
In preferred embodiments, the vectors are used to infect insect
cells.
[0112] As used herein, the term "selectable marker" refers to the
use of a gene which encodes an enzymatic activity that confers the
ability to grow in medium lacking what would otherwise be an
essential nutrient (e.g., the HIS3 gene in yeast cells); in
addition, a selectable marker may confer resistance to an
antibiotic or drug upon the cell in which the selectable marker is
expressed. Selectable markers may be "dominant"; a dominant
selectable marker encodes an enzymatic activity which can be
detected in any eukaryotic cell line. Examples of dominant
selectable markers include the bacterial aminoglycoside 3'
phosphotransferase gene (also referred to as the neo gene) which
confers resistance to the drug G418 in mammalian cells, the
bacterial hygromycin G phosphotransferase (hyg) gene which confers
resistance to the antibiotic hygromycin and the bacterial
xanthine-guanine phosphoribosyl transferase gene (also referred to
as the gpt gene) which confers the ability to grow in the presence
of mycophenolic acid. Other selectable markers are not dominant in
that there use must be in conjunction with a cell line that lacks
the relevant enzyme activity. Examples of non-dominant selectable
markers include the thymidine kinase (tk) gene which is used in
conjunction with tk.sup.- cell lines, the CAD gene which is used in
conjunction with CAD-deficient cells and the mammalian
hypoxanthine-guanine phosphoribosyl transferase (hprt) gene which
is used in conjunction with hprt.sup.- cell lines. A review of the
use of selectable markers in mammalian cell lines is provided in
Sambrook, J. et. al., Molecular Cloning: A Laboratory Manual, 2nd
ed., Cold Spring Harbor Laboratory Press, New York (1989)
pp.16.9-16.15.
[0113] As used herein, the term "vector" is used in reference to
nucleic acid molecules that transfer DNA segment(s) from one cell
to another. The term "vehicle" is sometimes used interchangeably
with "vector."
[0114] The term "expression vector" as used herein refers to a
recombinant DNA molecule containing a desired coding sequence and
appropriate nucleic acid sequences necessary for the expression of
the operably linked coding sequence in a particular host organism.
Nucleic acid sequences necessary for expression in prokaryotes
usually include a promoter, an operator (optional), and a ribosome
binding site, often along with other sequences. Eukaryotic cells
are known to utilize promoters, enhancers, and termination and
polyadenylation signals.
[0115] The terms "in operable combination," "in operable order,"
and "operably linked" as used herein refer to the linkage of
nucleic acid sequences in such a manner that a nucleic acid
molecule capable of directing the transcription of a given gene
and/or the synthesis of a desired protein molecule is produced. The
term also refers to the linkage of amino acid sequences in such a
manner so that a functional protein is produced.
[0116] As used herein, the term "amplifiable nucleic acid" is used
in reference to nucleic acids which may be amplified by any
amplification method. It is contemplated that "amplifiable nucleic
acid" will usually comprise "sample template."
[0117] As used herein, the term "sample template" refers to nucleic
acid originating from a sample which is analyzed for the presence
of "target" (defined below). In contrast, "background template" is
used in reference to nucleic acid other than sample template which
may or may not be present in a sample. Background template is most
often inadvertent. It may be the result of carryover, or it may be
due to the presence of nucleic acid contaminants sought to be
purified away from the sample. For example, nucleic acids from
organisms other than those to be detected may be present as
background in a test sample.
[0118] As used herein, the term "primer" refers to an
oligonucleotide, whether occurring naturally as in a purified
restriction digest or produced synthetically, which is capable of
acting as a point of initiation of synthesis when placed under
conditions in which synthesis of a primer extension product which
is complementary to a nucleic acid strand is induced, (i.e., in the
presence of nucleotides and an inducing agent such as DNA
polymerase and at a suitable temperature and pH). The primer is
preferably single stranded for maximum efficiency in amplification
but may alternatively be double stranded. If double stranded, the
primer is first treated to separate its strands before being used
to prepare extension products. Preferably, the primer is an
oligodeoxyribonucleotide. The primer must be sufficiently long to
prime the synthesis of extension products in the presence of the
inducing agent. The exact lengths of the primers will depend on
many factors, including temperature, source of primer and the use
of the method.
[0119] As used herein, the term "probe" refers to an
oligonucleotide (i.e., a sequence of nucleotides), whether
occurring naturally as in a purified restriction digest or produced
synthetically, recombinantly or by PCR amplification, which is
capable of hybridizing to another oligonucleotide of interest. A
probe may be single-stranded or double-stranded. Probes are useful
in the detection, identification and isolation of particular gene
sequences. It is contemplated that any probe used in the present
invention will be labelled with any "reporter molecule," so that is
detectable in any detection system, including, but not limited to
enzyme (e.g. ELISA, as well as enzyme-based histochemical assays),
fluorescent, radioactive, and luminescent systems. It is not
intended that the present invention be limited to any particular
detection system or label.
[0120] As used herein, the term "target" when used in reference to
the polymerase chain reaction, refers to the region of nucleic acid
bounded by the primers used for polymerase chain reaction. Thus,
the "target" is sought to be sorted oat from other nucleic acid
sequences. A "segment" is defined as a region of nucleic acid
within the target sequence.
[0121] As used herein, the term "polymerase chain reaction" ("PCR")
refers to the method of K. B. Mullis U.S. Pat. Nos. 4,683,195,
4,683,202, and 4,965.188, hereby incorporated by reference, which
describe a method for increasing the concentration of a segment of
a target sequence in a mixture of genomic DNA without cloning or
purification. This process for amplifying the target sequence
consists of introducing a large excess of two oligonucleotide
primers to the DNA mixture containing the desired target sequence,
followed by a precise sequence of thermal cycling in the presence
of a DNA polymerase. The two primers are complementary to their
respective strands of the double stranded target sequence. To
effect amplification, the mixture is denatured and the primers then
annealed to their complementary sequences within the target
molecule. Following annealing, the primers are extended with a
polymerase so as to form a new pair of complementary strands. The
steps of denaturation, primer annealing and polymerase extension
can be repeated many times (i.e., denaturation, annealing and
extension constitute one "cycle"; there can be numerous "cycles")
to obtain a high concentration of an amplified segment of the
desired target sequence. The length of the amplified segment of the
desired target sequence is determined by the relative positions of
the primers with respect to each other, and therefore, this length
is a controllable parameter. By virtue of the repeating aspect of
the process, the method is referred to as the "polymerase chain
reaction" (hereinafter "PCR"). Because the desired amplified
segments of the target sequence become the predominant sequences
(in terms of concentration) in the mixture, they are said to be
"PCR amplified".
[0122] With PCR, it is possible to amplify a single copy of a
specific target sequence in genomic DNA to a level detectable by
several different methodologies (eg., hybridization with a labeled
probe; incorporation of biotinylated primers followed by
avidin-enzyme conjugate detection; incorporation of
.sup.32P-labeled deoxynucleotide triphosphates, such as dCTP or
dATP, into the amplified segment). In addition to genomic DNA, any
oligonucleotide sequence can be amplified with the appropriate set
of primer molecules. In particular the amplified segments created
by the PCR process itself are, themselves, efficient templates for
subsequent PCR amplifications.
[0123] "Amplification" is a special case of nucleic acid
replication involving template specificity. It is to be contrasted
with non-specific template replication (i.e., replication that is
template-dependent but not dependent on a specific template).
Template specificity is here distinguished from fidelity of
replication (i.e., synthesis of the proper polynucleotide sequence)
and nucleotide (ribo- or deoxyribo-) specificity. Template
specificity is frequently described in terms of "target"
specificity. Target sequences are "targets" in the sense that they
are sought to be sorted out from other
[0124] As used herein, the terms "restriction endonucleases" and
"restriction enzymes" refer to bacterial enzymes, each of which cut
double-stranded DNA at or near a specific nucleotide sequence.
[0125] As used herein, the term "recombinant DNA molecule" as used
herein refers to a DNA molecule which is comprised of segments of
DNA joined together by means of molecular biological
techniques.
[0126] DNA molecules are said to have "5' ends" and "3' ends"
because mononucleotides are reacted to make oligonucleotides in a
manner such that the 5' phosphate of one mononucleotide pentose
ring is attached to the 3' oxygen of its neighbor in one direction
via a phosphodiester linkage. Therefore, an end of an
oligonucleotides referred to as the "5' end" if its 5' phosphate is
not linked to the 3' oxygen of a mononucleotide pentose ring and as
the "3' end" if its 3' oxygen is not linked to a 5' phosphate of a
subsequent mononucleotide pentose ring. As used herein, a nucleic
acid sequence, even if internal to a larger oligonucleotide, also
may be said to have 5' and 3' ends. In either a linear or circular
DNA molecule, discrete elements are referred to as being "upstream"
or 5' of the "downstream" or 3' elements. This terminology reflects
the fact that transcription proceeds in a 5' to 3' fashion along
the DNA strand. The promoter and enhancer elements which direct
transcription of a linked gene are generally located 5' or upstream
of the coding region. However, enhancer elements can exert their
effect even when located 3' of the promoter element and the coding
region. Transcription termination and polyadenylation signals are
located 3' or downstream of the coding region.
[0127] As used herein, the term "an oligonucleotide having a
nucleotide sequence encoding a gene" means a nucleic acid sequence
comprising the coding region of a gene or in other words the
nucleic acid sequence which encodes a gene product. The coding
region may be present in either a cDNA, genomic DNA or RNA form.
When present in a DNA form, the oligonucleotide may be
single-stranded (i.e., the sense strand) or double-stranded.
Suitable control elements such as enhancers/promoters, splice
junctions, polyadenylation signals, etc. may be placed in close
proximity to the coding region of the gene if needed to permit
proper initiation of transcription and/or
[0128] As used herein, the term "promoter/enhancer" denotes a
segment of DNA which contains sequences capable of providing both
promoter and enhancer functions (i.e., the functions provided by a
promoter element and an enhancer element, as discussed above). For
example, the long terminal repeats of retroviruses contain both
promoter and enhancer functions. The enhancer/promoter may be
"endogenous," "exogenous," or "heterologous." An "endogenous"
enhancer/promoter is one which is naturally linked with a given
gene in the genome. An "exogenous" or "heterologous"
enhancer/promoter is one which is placed in juxtaposition to a gene
by means of genetic manipulation (i.e., molecular biological
techniques), such that transcription of that gene is directed by
the linked enhancer/promoter.
[0129] The presence of "splicing signals" on an expression vector
often results in higher levels of expression of the recombinant
transcript. Splicing signals mediate the removal of introns from
the primary RNA transcript and consist of a splice donor and
acceptor site (See e.g., J. Sambrook et at, Molecular Cloning: A
Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press,
New York [1989], pp. 16.7-16.8). A commonly used splice donor and
acceptor site is the splice junction from the 16S RNA of SV40.
[0130] Efficient expression of recombinant DNA sequences in
eukaryotic cells requires expression of signals directing the
efficient termination and polyadenylation of the resulting
transcript. Transcription termination signals are generally found
downstream of the polyadenylation signal and are a few hundred
nucleotides in length. The term "poly A site" or "poly A sequence,"
as used herein, denotes a DNA sequence that directs both the
termination and polyadenylation of the nascent RNA transcript.
Efficient polyadenylation of the recombinant transcript is
desirable, as transcripts lacking a poly A tail are unstable and
are rapidly degraded. The poly A signal utilized in an expression
vector may be "heterologous" or "endogenous." An endogenous poly A
signal is one that is found naturally at the 3' end of the coding
region of a given gene in the genome. An heterologous poly A signal
is one which is isolated from one gene and placed 3' to another
gene. A commonly used heterologous poly A signal is the SV40 poly A
signal. The SV40 poly A signal is contained on a 237 bp BamHI/BclI
restriction fragment, and directs both termination and
polyadenylation (S. Sambrook, supra, at 16.6-16.7).
[0131] Eukaryotic expression vectors may also contain "viral
replicons," or "viral origins of replication." Viral replicons are
viral DNA sequences which allow for the extrachromosomal
replication of a vector in a host cell expressing the appropriate
replication factors.
[0132] As used herein, the terms "nucleic acid molecule encoding,"
"DNA sequence encoding," and "DNA encoding" refer to the order or
sequence of deoxyribonucleotides along a strand of deoxyribonucleic
acid. The order of these deoxyribonucleotides determines the order
of amino acids along the polypeptide (protein) chain. The DNA
sequence thus codes for the amino acid sequence.
[0133] The term "Southern blot" refers to the analysis of DNA on
agarose or acrylamide gels to fractionate the DNA according to size
followed by transfer of the DNA from the gel to a solid support,
such as nitrocellulose or a nylon membrane. The immobilized DNA is
then probed with a labeled probe to detect DNA species
complementary to the probe used. The DNA may be cleaved with
restriction enzymes prior to electrophoresis. Following
electrophoresis, the DNA may be partially depurinated and denatured
prior to or during transfer to the solid support. Southern blots
are a standard tool of molecular biologists (See e.g., J. Sambrook
et at, supra at pp 9.31-9-58).
[0134] The term "Northern blot" as used herein refers to the
analysis of RNA by electrophoresis of RNA on agarose gels to
fractionate the RNA according to size followed by transfer of the
RNA from the gel to a solid support, such as nitrocellulose or a
nylon membrane. The immobilized RNA is then probed with a labeled
probe to detect RNA species complementary to the probe used.
Northern blots are a standard tool of molecular biologists (See
e.g., Sambrook et al., supra at pp. 7.39-7.52).
[0135] The term "isolated" when used in relation to a nucleic acid,
as in "an isolated oligonucleotide" refers to a nucleic acid
sequence that is identified and separated from at least one
contaminant nucleic acid with which it is ordinarily associated in
its natural source. Isolated nucleic acid is such present in a form
or setting that is different from that in which it is found in
nature (e.g., in an expression vector). In contrast, non-isolated
nucleic acids are nucleic acids such as DNA and RNA found in the
state they exist in nature. For example, a given DNA sequence
(e.g., a gene) is found on the host cell chromosome in proximity to
neighboring genes; RNA sequences, such as a specific mRNA sequence
encoding a specific protein, are found in the cell as a mixture
with numerous other mRNAs which encode a multitude of proteins.
However, isolated nucleic acid encoding a mammalian F-box protein
includes, by way of example, such nucleic acid in cells ordinarily
expressing an F-box protein where the nucleic acid is in a
chromosomal location different from that of natural cells, or is
otherwise flanked by a different nucleic acid sequence than that
found in nature. The isolated nucleic acid or oligonucleotide may
be present in single-stranded or double-stranded form. When an
isolated nucleic acid or oligonucleotide is to be utilized to
express a protein, the oligonucleotide will contain at a minimum
the sense or coding strand (i.e., the oligonucleotide may
single-stranded), but may contain both the sense and anti-sense
strands (i.e., the oligonucleotide may be double-stranded).
[0136] As used herein, the term "immunogen" refers to a substance,
compound, molecule, or other moiety which stimulates the production
of an immune response. The term "antigen" refers to a substance,
compound, molecule, or other moiety that is capable of reacting
with products of the immune response. For example, F-box proteins
may be used as immunogens to elicit an immune response in an animal
to produce antibodies directed against the subunit used as an
immunogen. The subunit may then be used as an antigen in an assay
to detect the presence of anti-F-box protein antibodies in the
serum of the immunized animal. It is not intended that the present
invention be limited to antigens or immunogens consisting solely of
one protein (i.e., it is intended that the present invention
encompass complexes). Nor is it intended that the present invention
be limited to any particular antigens or immunogens.
[0137] The term "antigenic determinant," as used herein, refers to
that portion of a molecule (i.e., an antigen) that makes contact
with a particular antibody (i.e., an vitro (Cole et al., in
Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, pp. 77-96
[1985]).
[0138] According to the invention, techniques described for the
production of single chain antibodies (U.S. Pat. No. 4,946,778;
herein incorporated by reference) can be adapted to produce F-box
protein-specific single chain antibodies. An additional embodiment
of the invention utilizes the techniques described for the
construction of Fab expression libraries (Huse et al., Science
246:1275-1281 [1989]) to allow rapid and easy identification of
monoclonal Fab fragments with the desired specificity for F-box
proteins
[0139] Antibody fragments which contain the idiotype (antigen
binding region) of the antibody molecule can be generated by known
techniques. For example, such fragments include but are not limited
to: the F(ab')2 fragment which can be produced by pepsin digestion
of the antibody molecule; the Fab' fragments which can be generated
by reducing the disulfide bridges of the F(ab')2 fragment, and the
Fab fragments which can be generated by treating the antibody
molecule with papain and a reducing agent.
[0140] In the production of antibodies, screening for the desired
antibody can be accomplished by techniques known in the art (e.g.,
radioimmunoassay, ELISA [enzyme-linked immunosorbant assay],
"sandwich" immunoassays, immunoradiometric assays, gel diffusion
precipitin reactions, immunodiffusion assays, in situ immunoassays
[using colloidal gold, enzyme or radioisotope labels, for example],
Western Blots, precipitation reactions, agglutination assays (e.g.,
gel agglutination assays, hemagglutination assays, etc.),
complement fixation assays, immunofluorescence assays, protein A
assays, and immunoelectrophoresis assays, etc.
[0141] As used herein the term "immunogenically-effective amount"
refers to that amount of an immunogen required to invoke the
production of protective levels of antibodies in a host upon
vaccination.
[0142] As used herein, the term "reporter reagent" or "reporter
molecule" is used in reference to compounds which are capable of
detecting the presence of antibody bound to antigen. For example, a
reporter reagent may be a colorimetric substance which is attached
to an enzymatic substrate. Upon binding of antibody and antigen,
the enzyme acts on its substrate and causes the production of a
color. Other reporter reagents include, but are not limited to
fluorogenic and radioactive compounds or molecules.
[0143] As used herein the term "signal" is used in reference to the
production of a sign that a reaction has occurred, for example,
binding of antibody to antigen. It is contemplated that signals in
the form of radioactivity, fluorogenic reactions, and enzymatic
reactions will be used with the present invention. The signal may
be assessed quantitatively as well as qualitatively.
[0144] As used herein the term "NF-.kappa.B regulatory factors"
refers to any factors (e.g., proteins, enzymes, peptides, small
molecules, and nucleic acids) involved in the regulation of
NF-.kappa.B signalling pathways. For example, such factors include,
but are not limited to, F-box proteins, I.kappa.BS, IKKs, and
agonists, antagonists, and cofactors that interact with these
factors. It is contemplated that the NF-.kappa.B regulatory factors
can either directly or indirectly (e.g., through other factors)
bind to a target of interest (e.g., a slimb protein).
GENERAL DESCRIPTION OF THE INVENTION
[0145] The present invention provides compositions and methods for
gene identification, as well as drug discovery and assessment. The
present invention provides components of an E3 complex involved in
ubiquitination of cell cycle regulators and other proteins, as well
as members of a class of proteins that directly function in
recognition of ubiquitination targets. These compositions are
involved in protein degradation pathways associated with the
eukaryotic cell cycle, among others.
[0146] Protein degradation is a commonly employed mechanism for the
control of protein abundance. It is also a particularly effective
method for promoting unidirectional cell cycle transitions because
of its rapidity and irreversibility. Three major transitions (i.e.,
entry into S phase, separation of sister chromatids, and exit from
mitosis), require the degradation of specific proteins via the
ubiquitin-26S proteosome pathway (reviewed in King et al., Science
274:1652-1659 [1996]). Ubiquitin is a relatively small protein
(approximately 76 amino acid residues) found in all cells of higher
organisms. Ubiquitin plays major roles in intracellular protein
degradation and histone modification.
[0147] Thus, ubiquitination is an important mechanism used to
regulate protein abundance. However, until the development of the
present invention, the specificity of target selection for
ubiquitin dependent proteolysis was largely unknown. Central to
this process are the E3s which confer substrate specificity on the
ubiquitination reaction and are therefore likely points for
regulation. The present invention provides methods for producing
ubiquitinated Sic1 in vivo and in vitro using recombinant proteins.
The present invention also provides compositions and methods for
the development of drugs and other compounds effective in
correcting abnormalities in protein degradation, based on the
demonstration that 1) Cdc53, Skp1, and Cdc4 form a functional E3
ubiquitin ligase complex that works together with the E2 Cdc34 to
ubiquitinate Sic1; 2) Cdc4 acts as a receptor for phosphorylated
Sic1 recognition; and 3) the sole function Cln/Cdc28 kinases in
this process is to phosphorylate Sic1, allowing recognition by
Cdc4. Importantly, it was found that distinct F-box proteins can
selectively recognize different ubiquitination substrates in a
phosphorylation-dependent manner.
[0148] The formation of ubiquitin-protein conjugates in protein
degradation pathways involves three components that participate in
a cascade of ubiquitin transfer reactions: a ubiquitin activating
enzyme (E1), a ubiquitin conjugating enzyme (E2), and a specificity
factor (E3) (Hershko et al., J. Biol. Chem., 267:8807-8812 [1983]).
Ubiquitin is activated as a thiol-ester on E1 in an ATP-dependent
reaction, transferred to an E2 as a thiol ester and ultimately
conjugated to the target protein in conjunction with an E3, which
functions in substrate recognition and in some instances may serve
as a thiol-ubiquitin carrier (Scheffner et al., Cell 75:495-505
[1993]; and Scheffner et al., Nature 373:81-83 [1995]). Together,
these enzymes polyubiquitinate lysine residues in target proteins
through formation of isopeptide bonds with ubiquitin, leading to
recognition by the 26S proteosome. This association eventually
results in the degradation of the target protein.
[0149] While E1 and E2 proteins can be identified through sequence
similarity, this is not yet generally true for E3 proteins. Thus,
the present invention provides previously unreported methods and
compositions. This is significant as the identity of E3 components
are a central issue in cell cycle control, among other processes,
because they are potential regulators of both the timing of
ubiquitination and the selection of substrates. Prior to the
development of the present invention, much of the prior knowledge
of E3s was provided by analysis of the HECT domain protein E6-AP
which functions as a ubiquitin-ligase for p53, (Huibregtse et al.,
Proc. Natl. Acad. Sci. USA 92:2563-2567 [1995]; and Scheffner et
al., Nature 373:81-83 [1995]); and the anaphase promoting complex
(APC), which functions in the destruction of mitotic cyclins and
proteins involved in sister chromatid cohesion (reviewed in King et
al., [1996] supra). These APC substrates contain a destruction box
motif, although precisely how the timing and selection of
substrates by the APC is achieved is unknown. In contrast, timing
of ubiquitination of a variety of non-APC substrates is thought to
be regulated in part by the phosphorylation of the substrate
itself. PEST sequences (i.e., sequences that are rich in proline,
glutamic acid, serine and threonine) are frequently found in
unstable proteins such as cyclins and contain sites of
phosphorylation (Rogers et al., Science 234:364-368 [1986]).
Phosphorylation of specific residues has been implicated in the
destruction of G1 cyclins in yeast and mammalian cells (Tyers et
al., EMBO J., 11:1773-84 [1992]; Lanker et al., Science
271:1597-1601 [1996]; Clurman et al., Genes Dev., 10:1979-1990
[1996]; Diehl et al., Genes Dev., 11:957-972 [1997]; and Won and
Reed, EMBO J., 15:4182-4193 [1996]), and the cyclin-kinase
inhibitor (CKI) p27 (Sheaff et al., Genes Dev., 11:1464-1478
[1997]).
[0150] In S. cerevisiae, entry into S-phase requires activation of
the Cdc28 kinase by G1 cyclins (Cln1, Cln2, and Cln3) and S-phase
cyclins (Clb5 and Clb6) (Nasmyth, Curr. Opin. Cell Biol., 5:166-179
[1993]). Although both Cln/Cdc28 and Clb/Cdc28 complexes assemble
during G1, Clb/Cdc28 is sequestered in an inactive form through
association with the CK1 p40Sic1 (Mendenhall, Science 259:216-219
[1993]; and Schwob et. al., Cell 79: 233-244 [1994]). Sic1 levels
vary in the cell cycle, sharply decreasing at the G1/S transition,
and this correlates with activation of Clb5/Cdc28. The decrease in
Sic1 levels depends on the E2 Cdc34, suggesting that ubiquitination
triggers Sic1 destruction (Schwob et al., Cell 79:233-244 [1994]).
Sic1 destruction also requires CLN and CDC28 function; elimination
of Sic1 defines the threshold requirement for Cln/Cdc28 activity in
S-phase entry (Schneider et al., Science 272: 560-562 [1996];
Schwob et al., [1994], supra; and Tyers, Proc. Natl. Acad. Sci.
U.S.A. 93:7772-7776 [1996]). Although Sic1 is a phosphoprotein
(Schneider et al., Science 272:560-562 [1996]), it is not known
whether Cln/Cdc28 complexes directly phosphorylate Sic1 or whether
phosphorylation plays another, perhaps indirect, role in Sic1
destruction. The development of the present invention provides
methods and compositions to resolve these questions.
[0151] Three other genes, SKP1, CDC53, and CDC4, are also required
for S-phase entry (Schwob et al., 1994, supra; and Bai et al., Cell
86:263-274 [1996]). These genes, together with CDC34, show a
pattern of suppression and enhancement consistent with roles in a
common process; conditional alleles of these genes cause arrest
with unreplicated DNA and multiple buds (Yochem and Byers, J. Mol.
Biol., 195:233-245 [1987]; Goebl et al., J. Mol. Biol., 195:233-245
[1988]; Bai et al., [1996] supra; and Mathias et al., Mol. Cell.
Biol., 16:6634-6643 (1996]). Sic1 accumulates in cdc34-1, cdc4-1,
or skp 1-11 mutants, and deletion of SIC1 allows such mutants to
undergo DNA synthesis (Schwob et al., [1996], supra; and Bai et
al., [1996], supra). Components of the Cdc34 pathway have also been
implicated in the destruction of a number of other important
regulatory proteins, including Cln2 (Deshaies et al., EMBO J.,
14:303-312 [1995]; Bai et al., [1996] supra; and Willems et al.,
Cell 86:453-463 [1996]), Cdc6 (Piatti et al., Genes Dev.,
10:1516-1531 [1996]), the CKIs Rum1 and Far1 (McKinney et al.,
Genes Dev., 7:833-843 [1993]; and Kominami and Toda, Genes Dev.,
11:1548-1560 [1997]), and the transcription factor Gcn4 (Kornitzer
et al., EMBO J., 33:6021-6030 [1994]). Thus, it is contemplated
that Cdc34, Cdc53, Skp1, and Cdc4 are utilized for the destruction
of diverse regulatory proteins. A requirement for Cdc34 for Cln2
ubiquitination has been demonstrated in crude yeast lysates
(Deshaies et al., [1995], supra), but this requirement has been
suggested to be indirect (Blondel and Mann, Nature 384:279-282
[1996]). Interestingly, SKP1 is also required for the G2/M
transition (Bai et al., [1996], supra; and Connelly and Heiter,
Cell 86:275-285 [1996]), and has been found to be a component of
the kinetochore complex CBF3 (Connelly and Fleiter, Cell 86:275-285
[1996], supra; and Stemmann and Lechner, EMBO J., 15:3611-3620
[1996]).
[0152] Skp1 binds to Cdc4, and this interaction involves a motif in
Cdc4 referred to as the F-box (Bai et al., [1996], supra). The
F-box motif is found in a large number of proteins including cyclin
F (Bai et al., EMBO J., 15:3611-3620 [1994]) and the cyclin
A/Cdk2-associated protein Skp2 (Zhang et al., Cell 82:915-925
[1995]), both of which bind Skp1. The two largest classes of F-box
proteins either contain WD-40 repeats (e.g., Cdc4) or leucine-rich
repeats (LRR) (e.g., Skp2 and Grr1) (Bai et al., [1996], supra).
GRR1 was initially identified as a gene required for glucose
repression (Flick and Johnston, Mol. Cell. Biol., 11:5101-12
[1991]) but was later also found to be involved in Cln destruction
(Barral et al., Genes Dev., 9:399-409 [1995]). The discovery that
Skp1 is required for the destruction of both Sic1 and Clin2, while
Cdc4 and Grr1 were only implicated in the destruction of one of
these, led to development of one embodiment of the present
invention (i.e., one model) in which F-box proteins function to
recognize targets for ubiquitination, and Skp1 links these
F-box/target complexes to the ubiquitination machinery.
[0153] The present invention was developed in a stepwise fashion,
with an important aspect being the elucidation of the role of Skp1
and F-box proteins in ubiquitination through in vitro
reconstruction of the Sic1 ubiquitination pathway. Sic1
ubiquitination was found to depend upon each of the proteins
implicated in Sic1 destruction in vivo. For example, Skp1 recruits
Cdc4 into a Cdc53/Cdc34 complex, and enhances recognition of Sic1
by Cdc4, with the latter interaction requiring Sic1
phosphorylation. In contrast, Grr1 does not interact with Sic1, but
does recruit phosphorylated Cln1 and Cln2 into Skp1/Cdc53
complexes. Thus, the present invention provides F-box proteins that
function as receptors, which recruit substrates into a
Skp1/Cdc53/Cdc34 complex for ubiquitination by Cdc34.
[0154] Thus, the present invention provides the function of a class
of proteins referred to as F-box proteins in targeted
ubiquitination. The present invention finds utility in methods for
developing compounds that affect ubiquitination. The present
invention also provides numerous novel F-box containing mammalian
genes whose encoded proteins are contemplated to function in
processes including, but not limited, to targeted ubiquitination of
cellular proteins. Specifically, F-box proteins function as
receptors for proteins to be ubiquitinated.
[0155] As described in the Examples, through a series of
experiments using a set of defined proteins found in S. cerevisiae,
it was demonstrated that three proteins (i.e., Cdc53, Skp1, and the
F-box protein Cdc4) form a complex referred to as an "E3" which
functions together with an E1 ubiquitin activating enzyme, and the
E2 ubiquitin conjugating enzyme Cdc34, to ubiquitinate the Cdk
inhibitor Sic1. Recognition of Sic1 by this E3 complex requires
that Sic1 be specifically phosphorylated and phosphorylation may be
a general mechanism used to regulate the timing of ubiquitination
of target proteins. Thus, it is contemplated that compounds that
alter this phosphorylation will in turn, alter the timing of
ubiquitination of target proteins. Such compounds are contemplated
as possible drugs that disrupt at least one pathway in which F-box
proteins function, and are required for biological and/or
biochemical processes.
[0156] Cdc53 was found to function as an adapter and link Skp1 to
the E2, while Skp1 was found also to function as an adapter and
links Cdc53 to the F-box protein Cdc4. Cdc4 was found to function
as an adapter to link ubiquitination targets (e.g., Sic1) to the
Skp1/Cdc53/Cdc34 complex. E1 is not a stable component of the
ubiquitination complex, but is required for ubiquitination of the
target protein. The F-box protein contains minimally two
protein-protein interaction domains. The F-box is a sequence of
35-45 amino acids and allows the F-box proteins to enter into
complexes with Skp1. F-box proteins also contain additional
domains, typically, but not necessarily C-terminal to the F-box
sequence, which based on the results with Cdc4 function as
recognition components for ubiquitination substrates. Cdc4 contains
C-terminal WD-40 repeats. Another F-box protein (Grr1) contains
leucine rich repeats which are protein-protein interaction domains.
Because Skp1 simultaneously forms complexes with Cdc53 proteins and
an F-box protein, these interactions give rise to formation of an
E3 complex. Any particular F-box protein may interact
simultaneously with both Skp1 and at least one ubiquitination
target. F-box proteins may have a single ubiquitination target but
it is contemplated that they (i.e., at least some F-box proteins)
also have multiple in vivo ubiquitination targets. For example, the
data obtained for Cdc4 indicate that it is involved in the
destruction of at least two proteins, Sic1 and Cdc6. Thus, the
present invention provides the necessary components and methods to
alter ubiquitination of target proteins through the use of new
drugs or other compounds.
[0157] Based on the sequence of the yeast genome, it was determined
that S. cerevisiae contains nine F-box proteins. CDC4 is required
for the destruction of Sic1 and Cdc6, while Grr1 is required for
the destruction of the G1 (Cln) cyclins, and MET30 is required for
proper control of methionine biosynthetic pathways and is predicted
to control the abundance of Met4.
[0158] The present invention also provides methods and compositions
useful to determine the complexity and diversity of mammalian F-box
proteins, as well as the identity of F-box proteins from various
species, the protein-protein interaction domains involved, the
proteolytic pathways, and regulatory pathways. For example, the
mammalian proteins (cyclin F, Skp2) contain an F-box and associate
with Skp1, but their functions and ubiquitination targets have not
been demonstrated. Cyclin F contains a cyclin box motif C-terminal
to the F-box. Skp2 contains a leucine rich motif C-terminal to its
F-box. Mouse MD6, an additional mammalian F-box containing protein;
X54352) is in Genbank but its function is unknown. The present
invention provides human MD6, with the following sequences:
1 (SEQ ID NO:57) LPLELSFYLLKWLDPQTLLTCCLVSKQWNKVISACTEVW; and (SEQ
ID NO:58; AA252600)
CTTCCCCTGGAGCTCAGTTTTTATTTGTTAAAATGGCTCGATCCTCAGAC
TTTACTCACATGCTGCCTCGTCTCTAAACAGTGGAATAAGGTGATAAGTG
CCTGTACAGAGGTGTGG.
[0159] Furthermore, the closest homolog of MD6 is MET30; it is
contemplated that MD6 plays a homologous role in methionine
biosynthesis in eukaryotes. The present invention provides methods
and compositions to identify the functions and ubiquitination
targets of these and other F-box containing proteins.
[0160] The present invention also provides amino acid and DNA
sequence information for eighteen novel F-box-containing human or
mouse genes. As with Cdc4, Grr1, Skp2, and cyclin F, these novel
F-box proteins have the capacity to associate with Skp1 and to
simultaneously interact with other proteins through other
protein-protein interaction motifs encoded by regions of their
genes other than the F-box. Thus, the present invention provides
compositions and methods for determining the interaction of these
proteins with other proteins.
[0161] Mammalian Skp1, by analogy with budding yeast, functions as
an adapter linking Skp1 to an E2. It is contemplated that cellular
proteins brought into complexes containing Cdc53 and Skp1 by any
one of these novel F-box proteins has the potential to be
ubiquitinated by an E2 (e.g., Cdc34) in combination with an E1. It
is further contemplated that interaction with an F-box protein may
also produce an alternative regulatory function (e.g., altering
subcellular localization of the associated protein). Thus, the
function of F-box proteins is not necessarily limited to
ubiquitination, and the present invention provides the methods and
compositions to make this determination. It is contemplated that
additional F-box containing genes will be discovered through the
use of two-hybrid screens with Skp1 or ubiquitination targets as
the two-hybrid "bait" (e.g., as described in the Example 6). It is
also contemplated that additional F-box genes will be discovered
through sequencing of the mammalian genome and sequence analysis,
to determine the homology with existing F-box proteins, such as
those identified in the present invention.
[0162] For example, it is contemplated that cell cycle regulators
such as cyclins and cyclin-kinase inhibitors, transcription
regulators, proteins involved in DNA replication, and other
cellular regulatory proteins will be identified and/or investigated
using the methods and compositions provided by the present
invention. It is further contemplated that the present invention
will find use in elucidating inflammatory response and infectious
disease processes involving protein degradation, as well as
development of compounds that control (i.e., either enhance or
retard) protein degradation as appropriate, to ameliorate the
effects of the inflammatory response or disease process.
[0163] Thus, it is also contemplated that F-box proteins are
involved in regulatory pathways important for cellular homostasis
and/or growth control. In this context, F-box proteins may be
involved in the elimination or modification of proteins which
positively or negatively regulate the cell cycle, which positively
or negatively regulate transcription, or which positively or
negatively regulate the abundance of a protein involved in a
signaling pathway. Elimination of proteins could be mediated by the
26S proteosome after targeted ubiquitination by a E3 complex
containing an F-box protein. Ubiquitination without proteolytic
destruction may alter the activity of the target protein either
positively or negatively. Thus, it is contemplated that molecules
that alter the activities or target specificities of F-box
proteins, or the ability of F-box proteins to enter into
macromolecular complexes such as E3 complexes composed of and F-box
protein, a Cdc53 homolog and Skp1, will find utility as
pharmaceutical agents for a variety of diseases. The present
invention provides the compositions and methods for the
identification of molecules (including but not limited to proteins,
peptides, naturally occuring alkaloids, and synthetic alkaloids)
which alter the activities, levels, or targets of F-box
proteins.
[0164] For example, disruption of the F-box protein/Skp1 complex is
achieved using synthetic molecules, proteins, or peptides which
mimic the F-box sequence or its three dimensional structure and
block association of any F-box protein with Skp1. It is
contemplated that blockage of this interaction renders the F-box
protein non-functional with respect to ubiquitination of its target
proteins. Similarly, disruption of such complexes is also achieved
with synthetic molecules, proteins, or peptides which specifically
bind the F-box of a particular F-box protein. This approach
provides specificity for a particular pathway involving a specific
F-box. These classes of molecules can be identified using various
methods, including, but not limited to, peptide phage display
libraries to identify peptide sequences that bind either an F-box
sequence of a specific domain in Skp1 involved in interaction with
the F-box. In this method, F-box sequences or Skp1 sequences are
immobilized on solid supports such as a magnetic bead through the
use of biotinylated F-box or Skp1 sequences and streptavidin coated
magnetic beads. Phage display libraries are then bound to the
coated magnetic beads and phage binding the beads are isolated and
analyzed for binding sequences.
[0165] A similar method involves the use of two-hybrid screens to
identify proteins or fragments of proteins that bind Skp1 or the
F-box sequence. Such molecules find use in blocking assembly of
Skp1/F-box protein complexes in vivo and are useful (either
directly or as precursors) in the generation of pharmacological
agents.
[0166] In another embodiment, disruption of F-box/target
interactions are also contemplated. In addition to the F-box, F-box
containing proteins may also contain an additional interaction
domain including but not limited to WD-40 or leucine rich repeats.
For example, F1 Alpha and F2 Beta contain leucine rich repeats.
Embodiments of the present invention provide methods to identify
targets of F-box proteins which include, but are not limited to
cyclins, cell cycle regulators, cyclin-kinase inhibitors,
.beta.-catenin, I.kappa.B, and transcriptional regulators. It is
contemplated that molecules which either block, enhance, or
otherwise facilitate association of any target with any F-box
protein are useful as pharmaceutical agents in the treatment of
human diseases. The approaches described herein provide examples of
approaches that would yield peptides, proteins, and naturally
occuring or synthetic molecules which can bind target recognition
motifs in F-box proteins or motifs in the target protein
responsible for recognizing the F-box protein. It is also
contemplated that molecules which bind these domains block complex
formation and thereby block, accelerate, or alter the normal
function of the F-box protein, which may include (depending upon
the particular F-box protein), but is not limited to
ubiquitination.
[0167] The present invention also provides experimental strategies
to determine whether molecules identified in these ways can block
complex assembly. It is contemplated that binding assays based on
immobilized Skp1 and soluble F-box protein (or vice versa), or
immobilized F-box protein and soluble target (or vice versa), will
be developed in a manner similar to the development of embodiments
of the present invention (i.e., with Skp1, Cdc4 [an F-box protein]
and Sic1 [the target of Cdc4]). Molecules to be tested for their
ability to alter either Skp1/F-box protein interaction or F-box
protein/target interaction may be added to binding reactions and
the effects of the added agent examined by determining the fraction
of soluble protein bound relative to that bound in the absence of
the agent. It is also contemplated that such an assay be adapted to
high throughput screening strategies through the use of
radiolabeled or otherwise tagged soluble binding protein.
[0168] The present invention also provides evidence for
phosphorylation specific recognition of target proteins and methods
for determining whether recognition of the target requires that the
target be phosphorylated. It is contemplated that agents that block
or enhance specific phosphorylation of target proteins to allow
recognition by F-box proteins will be identified through approaches
disclosed herein. It is contemplated that such agents will find use
as pharmaceutical agents that increase or decrease the rate of
ubiquitination of target proteins.
[0169] In addition, the present invention finds use in the
identification and development of compounds effective against viral
infection and disease. For example, two viral proteins (adenovirus
E3-12.9K and baculovirus ORF11), appear to essentially encode only
an F-box, and a SKP1-related gene is present in Chorella virus. As
viruses subvert the cell cycle in order to replicate, it is
contemplated that disruption of the ubiquitin-mediated proteolysis
pathway would also disrupt viral replication. It is possible that
F-box containing viruses can inhibit degradation of specific
protein subsets (e.g., cyclins) to enhance their replication, or
promote the degradation of specific inhibitory proteins. It is also
possible that these proteins may target the destruction of proteins
that inhibit or kill the virus. The present invention finds use in
development of compositions and methods to inhibit viral
replication by interfering with the ubiquitin-mediated proteolysis
pathway utilized by the virus, as well as by upregulating the
cellular machinery to enhance proteolysis of viral components. In
particular, the present invention finds use in identification and
development of compounds effective against immunodeficiency viruses
(e.g., human immunodeficiency virus, as well as other viruses such
as feline immunodeficiency virus, bovine immunodeficiency virus,
and simian immunodeficiency virus).
[0170] It is further contemplated that targets of novel human F-box
proteins will be determined by those experienced in the art by
approaches including, but not limited to two-hybrid library
screens, immunoprecipitation analysis followed by immunoblotting
with antibodies against candidate targets, peptide mapping, mass
spectral analysis, peptide sequencing, and/or by screening lambda
based expression libraries with F-box protein probes.
[0171] For example, the present invention provides an example
whereby a novel E3 ubiquitin ligase complex has been identified
using the methods and compositions described herein. In particular,
the F-box protein slimb (TRCP), was found to associate with
I.kappa.B, providing the potential to screen for factors that
regulate the NF-.kappa.B pathway. This has important implications
in the regulation and control of cancer and the immune system,
among other important physiological effects.
[0172] The present invention also finds use in investigating the
function and methods of altering protein targets whose abundance is
altered in disease. For example, cyclins are frequently
overexpressed in cancer cells. Thus, mutations in F-box proteins
involved in cyclin destruction will lead to cyclin accumulation;
such cyclin accumulation may promote inappropriate cell division
characteristic of cancer. The present invention also finds utility
in the identification of mutations in F-box genes through various
methods, including, but not limited sequence analysis, Southern
blot analysis of DNA, etc. Furthermore, the present invention also
finds use in assessing alterations in cellular protein abundance
due to overexpression of particular F-box proteins. It is
contemplated that such alterations are associated with particular
diseases. The present invention also finds use in determination of
overexpression caused by gene amplification in DNA samples from
diseased tissue or individuals through such methods as Southern
analysis using a particular F-box gene as probe.
[0173] Furthermore, the present invention thereby provides methods
and compositions for the detection and analysis of abnormalities in
proteolytic functions, as well as methods and compositions for the
development of compounds suitable for use in ameliorating such
abnormalities.
DETAILED DESCRIPTION OF THE INVENTION
[0174] As discussed above, the present invention provides
compositions and methods for gene identification and
characterization, as well as drug discovery and assessment. In
particular, the present invention provides components of an E3
complex involved in ubiquitination of cell cycle regulators and
other proteins, as well as members of a class of proteins that
directly function in recognition of ubiquitination targets (i.e.,
F-box proteins). These compositions are involved in protein
degradation pathways associated with the eukaryotic cell cycle.
[0175] Assembly of a Complex Containing Cdc53/Skp1/Cdc4 and the E2
Cdc34
[0176] Strong genetic evidence implicated Cdc34, Cdc53, Skp1, and
Cdc4 as molecules involved in the control of S-phase entry through
destruction of Sic1. In preliminary work, SKP1 and CDC4 were found
to show reciprocal overproduction suppression of their respective
temperature sensitive mutants and that Cdc4 physically associated
with Skp1. A further search for suppressors using a GAL-driven cDNA
library revealed that CDC53 overexpression suppresses skp1-11.
These observations, coupled with genetic and physical evidence of a
Cdc53/Cdc34 interaction resulted in the development of embodiments
of the present invention.
[0177] The first step in assembling the complexes of interest
involved co-infection of insect cells with various baculovirus
expression vectors. Insect cells were co-infected with various
combinations of baculoviruses expressing Myc-tagged Cdc53
(Cdc53.sup.M), Cdc34, Cdc4, and Skp1. Anti-Myc immune complexes
from lysates of these infected cells were immunoblotted to identify
associated proteins (See, FIG. 1A). As shown in FIG. 1A, in the
presence of all four proteins, anti-Cdc53.sup.M complexes contained
Cdc4, Cdc34, and Skp1 (FIG. 1A, lane 5). However, in the absence of
Skp1, only low levels of Cdc4 were found to bind with Cdc53.sup.M,
regardless of the presence of Cdc34 (See, FIG. 1A, lanes 7 and 8).
To confirm this result, the association of Cdc53.sup.M with
anti-Cdc4.sup.F immune complexes was analyzed. These results
indicated the association of Cdc53.sup.M with anti-Cdc4.sup.F
immune complexes was also greatly enhanced in the presence of Skp1
(See, FIG. 1B). Thus, one function of Skp1 is to facilitate
association of Cdc53 with Cdc4. In contrast to Cdc4, both Skp1 and
Cdc34 were shown to associate with Cdc53.sup.M in the absence of
other yeast proteins (See, FIG. 1A). Furthermore, it appeared that
Cdc53 can simultaneously associate with both Cdc34 and Skp1, as the
association of Gst-Skp1 with Cdc34 is enhanced in the presence of
Cdc53.sup.M (See, FIG. 1C). These data indicated that Cdc34, Cdc53,
Skp1, and Cdc4 form a multiprotein complex.
[0178] Phosphorylation of Sic1 by Cln/Cdc28 is Required for its
Recognition by a Cdc4/Skp1/Cdc53 Complex
[0179] While previous studies implicated involvement of
Cln/Cdc28-dependent phosphorylation in Sic1 degradation (Schwob et
al., Cell 79:233-244 [1994]; Schneider et al., Science 272:560-562
[1996]; and Tyers, Proc. Natl. Acad. Sci. U.S.A. 93:7772-7776
[1996]), until the development of the present invention, it was not
clear whether Sic1 was directly phosphorylated by Cln/Cdc28, or
whether this phosphorylation was correlative or causative for
subsequent Sic1 degradation (and if causative, whether this
modification played a role in Sic1 recognition by the
ubiquitination machinery). Nor was it known whether Cln/Cdc28 might
also directly regulate the activity of the ubiquitination
machinery. Once the methods to generate and purify Cln1/Gst-Cdc28
and Sic1/Clb5/Gst-Cdc28 complexes from insect cells were
established in vitro during the development of the present
invention, the determination was made as to whether any of these
components might function in Sic1 recognition, and if Sic1
phosphorylation plays a role in this process. This aspect of the
present invention finds use in providing methods for the
development of drugs or other compounds suitable for prevention
and/or treatment of cancers (i.e., uncontrolled cellular growth),
as well as treatment of other diseases associated with
abnormalities in cell cycle control.
[0180] In order to accomplish this, Sic1 was purified to near
homogeneity from insect cells by virtue of its association with
Clb5/Gst-Cdc28 complexes (See, FIG. 2B). Initially, it was believed
that such a complex would represent the primary form of Sic1
ubiquitinated in vivo. However, it was found that uninhibited
Clb5/Cdc28 in these preparations phosphorylated Sic1, making it
impossible to directly assess the role of specific phosphorylation
by Clns. Therefore, a kinase-impaired Gst-Cdc28(K-) containing a
mutation in a critical catalytic residue (D145N) was used to
assemble Sic1 complexes. In such complexes, Sic1 remains
essentially unphosphorylated, however the Sic1 is readily
phosphorylated by Cln1/Cdc28 (See, FIG. 2A). In vitro
phosphorylation of Sic resulted in a reduction in its
electrophoretic mobility (See, FIG. 2B), reminiscent of that
observed with Sic1 in vivo.
[0181] In the absence of Cln1 kinase, the extent of Sic1
phosphorylation was found to be less than 2% of that of
phosphorylated Sic1, but this modification did not result in
alterations in electrophoretic mobility. For simplicity, this
weakly phosphorylated form of Sic1 is herein referred to as
"unphosphorylated Sic1."
[0182] Cdc4 is the Specificity Factor for Recognition of
Phosphorylated Sic1
[0183] Phosphorylated and unphosphorylated Sic1 were used in
binding reactions with anti-Cdc53.sup.M immune complexes assembled
and purified from insect cells (See, FIG. 2C). Phosphorylated Sic1
was found to efficiently associate with Cdc53/Skp1/Cdc4 complexes;
this association was dependent upon the presence of Skp1 (See, FIG.
2C, lanes 6 and 8). Typically 10-20% of the input phosphorylated
Sic1 was bound at about 20 nM Sic1. In contrast, the extent of
binding of unphosphorylated Sic1 (See, FIG. 2C, lane 7) was
comparable to that observed in control immune complexes generated
from uninfected cells (See, FIG. 2C, lane 3), and was less than 1%
of the input Sic1. It was also observed that, consistent with the
results in FIG. 1, the level of Cdc4 found in immune complexes
lacking Skp1 were more than 10-fold lower than that found in the
presence of Skp1. Thus, Cdc4 and/or Skp1 function as binding
factors for Sic1, and association of Sic1 with this complex
requires phosphorylation by Cln1/Cdc28.
[0184] In addition, to directly examine the roles of Skp1 and Cdc4
in Sic1 recognition, binding experiments were performed using
series of complexes assembled in vivo that contained constant high
levels of Flag-tagged Skp1 (Skp1.sup.F), and increasing quantities
of Cdc4. These experiments, as described in the Examples, showed
that association of phosphorylated Sic1 with anti-Skp1.sup.F immune
complexes was absolutely dependent upon the presence of Cdc4 (e.g.,
compare lanes 3 and 9 of FIG. 2E). Moreover, deleting the last
three WD-40 repeats from the C-terminus of Cdc4 (Cdc4.DELTA.AWD)
abolished its ability to associate with phosphorylated Sic1 (See,
FIG. 2E, lanes 10-16). Therefore, these experiments indicated that
Cdc4 functions as the specificity factor for binding of
phosphorylated Sic1, and the Cdc4-Sic1 interaction requires an
intact WD-40 repeat domain in Cdc4. While Skp1 alone does not
interact with Sic1, it stimulates association of Sic1 with with
Flag-Cdc4 (Cdc4.sup.F) by about 5-fold (See, FIG. 2D). The weak
association of Sic1 with Cdc4 alone (See, FIG. 2D, lane 3) may
reflect the participation of an insect cell Skp1 homolog. Although
it is not clear if Skp1 physically contacts Sic1 or stabilizes a
form of Cdc4 compatible with Sic1 binding, and such an
understanding is not necessary in order to use the present
invention, these results clearly demonstrated that there is a
positive contribution of Skp1 in the Cdc4/Sic1 interaction.
[0185] Sic1 is Ubiquitinated In Vivo
[0186] While the finding that Cdc4, Skp1, and Cdc53 form a complex
that binds both phosphorylated Sic1 and the E2 Cdc34 was consistent
with a role for ubiquitination in the regulation of Sic1 abundance,
prior to the development of the present invention, Sic1 had not
been demonstrated to be ubiquitinated in vivo. In order to directly
accomplish this, insect cell lysates were generated from either
wild type cells or sic1 deletion mutants expressing
His.sub.6-Ub.sup.RA or Ub.sup.RA as a negative control, and
ubiquitinated proteins purified using Ni.sup.+2 beads (Willems et
al., Cell 86:453-463 [1996]) prior to immunoblotting with anti-Sic1
antibodies (See, FIG. 3A).
[0187] The K48R mutation in Ub.sup.RA blocks polyubiquitination and
therefore recognition by the proteolytic machinery (i.e.,
proteosome recognition) (Chau et al., Science 243:1576-1583
[1989]), while the G76A mutation reduces the rate at which
hydrolases remove ubiquitin conjugates (Hodgins et al., J. Biol.
Chem., 267:8807-8812 [1992]). A ladder of bands recognizable by
anti-Sic1 antibodies was detected in the Ni.sup.+2-bead bound
proteins from wild type lysates expressing His.sub.6-Ub.sup.RA
(See, FIG. 3A, lane 8) but not in conjugates derived from
Ub.sup.RA-expressing cells or a sic1 deletion strain (See, FIG. 3A,
lanes 5 and 6). This result demonstrates that Sic1 is ubiquitinated
in vivo. Thus, the present invention also provides an important
therapeutic target for development of drugs and other compounds for
disease prevention and/or treatment.
[0188] Reconstitution of the Sic1 Ubiquitination Pathway Using
Purified Proteins
[0189] Once a strategy to generate Cdc4/Skp1/Cdc53 complexes that
recognized phosphorylated Sic1 was developed, the next step was to
determine whether these complexes can catalyze ubiquitination of
Sic1 in vitro when supplemented with Cdc34, E1, ATP, and ubiquitin.
It was observed that in the presence of all reaction components,
phosphorylated Sic1 in complexes with Clb5/Cdc28 was efficiently
convened to higher molecular weight conjugates detectable with
anti-Sic1 antibodies (See, FIG. 5B, lane 6; and FIG. 5C, lane 5).
In contrast, unphosphorylated Sic1 was not detectably
ubiquitinated. Sic1 ubiquitination absolutely required Cdc34, Cdc4,
Cdc53, Skp1, E1 and ubiquitin (See e.g, FIG. 5B and FIG. 5C), as
well as yeast Skp1. The pattern of high molecular weight Sic1
conjugates obtained in reactions with ubiquitin was different from
that observed when Gst-Ub.sup.RA was used as the ubiquitin source,
(See, FIG. 5C, compare lanes 5 and 11) confirming that the high
molecular weight forms observed were products of ubiquitination.
With Gst-Ub.sup.RA, the Sic1 reaction products were integrated into
a ladder of bands differing by approximately 35 kDa, the size of
Gst-Ub.sup.RA (See, FIG. 3C, lane 11). Since Gst-Ub.sup.RA had a
reduced ability to form polyubiquitin chains, the number of bands
observed is likely to reflect the number of individual lysines
ubiquitinated on a single Sic1 molecule. The ubiquitination
reaction was time-dependent and the reaction efficiency ranged from
10-40% of the input Sic1 protein (See e.g., FIGS. 3B and 3C). When
the reaction was performed with pre-bound Sic1, the efficiency was
greater than 50%. In addition, it was found that greater than 50%
of the Sic1 ubiquitin conjugates formed after 60 minutes had
dissociated from the Cdc4/Skp1/Cdc53 complex. Neither Gst-Cdc28,
Clb5, Cdc53, Skp1, or Cdc4 formed ubiquitin conjugates under the
reaction conditions employed, although Cdc34 was ubiquitinated as
previously reported.
[0190] To test whether Sic1 ubiquitination requires association
with Clb5/Cdc28 complexes, ubiquitination reactions using Sic1
produced in bacteria were performed both with and without
phosphorylation with Cln2/Cdc28 (See, FIG. 3D). As in the case of
Sic1 assembled in insect cells with Clb5/Cdc28, phosphorylated Sic1
from bacteria was efficiently ubiquitinated with greater than 90%
of the Sic1 forming ubiquitin conjugates (See, FIG. 3D, lane 8),
and ubiquitination absolutely required Sic1 phosphorylation (i.e.,
unphosphorylated Sic1 was not ubiquitinated; See e.g., FIG. 3D,
lane 4). Thus, phosphorylation of Sic1 was shown to be required for
its recognition by Cdc4 and Skp1.
[0191] Next, it was determined whether Cln/Cdc28, present in small
amounts in the ubiquitination reaction, is also required for
additional steps in the ubiquitination process (e.g, to
phosphorylate the ubiquitination machinery). This was accomplished
by treating bacterial Sic1 with Cln2/Gst-Cdc28 complexes
immobilized on GSH-Sepharose beads, removing the complexes from the
beads prior to use in ubiquitination reactions, and determining
whether the complexes were free of soluble kinase by immunoblotting
with anti-HA antibodies (See, FIG. 3D, lane 3). These results
indicated that Sic1 phosphorylated in this manner was also
efficiently ubiquitinated (See, FIG. 3D, lane 9). Thus, these data
indicated that Sic1 phosphorylation constitutes the primary
requirement of Cln/Cdc28 kinases in Sic1 ubiquitination in the in
vitro reaction.
[0192] Although Sic1 was found to be an inhibitor of Cdc28/Clb5
complexes, when the kinase complex contained an excess of Sic1, it
was incapable of phosphorylating Sic1 and converting it into a
substrate for ubiquitination (FIG. 3E shows the reduced
electrophoretic mobility) and .sup.32P incorporation. This
Clb5/Cdc28-phosphorylated Sic1 was also a substrate for
ubiquitination (See, FIG. 3E). Although it is not necessary to
understand the mechanisms involved in order to use the present
invention, overexpression of CLB5 can drive S-phase entry in cln-
cells and suggests that active Clb5/Cdc28 formed during Sic1
destruction may collaborate with Cln/Cdc28 to complete the Sic1
ubiquitination process.
[0193] F-box Proteins are Receptors for Ubiquitination
Substrates
[0194] The determination that Cdc4 functions in the recognition and
ubiquitination of phosphorylated Sic1 is consistent with a function
of F-box proteins being recognition of ubiquitination targets.
During the development of the present invention, investigations
into whether specific F-box proteins could have broad specificity
and interact with multiple targets, or could be relatively
restricted in their target specificity, perhaps associating with
only a single target, were conducted.
[0195] To elucidate the selectivity of F-box proteins, experiments
were conducted to determine whether substitution of Cdc4 by another
F-box protein (Grr1) could support Sic1 binding and ubiquitination.
Grr1 has an F-box near its N-terminus and can interact
simultaneously with Skp1 and Cdc53 when co-expressed in insect
cells. Gene 10-tagged Grr1 (Grr1.sup.10) was also found to interact
simultaneously with Skp1 and Cdc53, when co-expressed in insect
cells (See, FIG. 4A). It was found that Grr1 and Cdc4 interact with
Skp1/Cdc53 in a mutually exclusive manner. In contrast with Cdc4,
however, the Grr1/Cdc53 interaction in insect cells was not
enhanced by co-expression of Skp1, although Skp1 assembled with
these complexes.
[0196] Importantly, Grr1 assembled with Cdc53/Skp1 (i.e.,
Cdc53/Skp1/Grr1 complex) was unable to associate with
phosphorylated Sic1 and did not support ubiquitination of
phosphorylated Sic1 complexes in the in vitro system with purified
proteins under conditions where Cdc4 readily facilitates Sic1
binding and ubiquitination (See, FIGS. 4B and C). Therefore, the
F-box proteins of some embodiments of the present invention display
selectivity toward particular targets.
[0197] Recognition of Phosphorylated Cln1 and Cln2 by Grr1
[0198] Previous studies have shown that mutations of potential
Cdc28 phosphorylation sites in the C-terminal PEST domain in Cln2
increase its stability in vivo (Lanker et al., Science
273:1597-1601 [1996]), and that only the phosphorylated form of
Cln2 is associated with Cdc53 in vivo (Willems et al., [1996],
supra), implicating this interaction in the Cln destruction
pathway. Cdc28 is required for Cln phosphorylation although it has
not been determined that the requisite phosphorylation reflects
autophosphorylation or phosphorylation by a distinct protein
kinase. The finding that Sic1 is recognized by the F-box protein
Cdc4, together with a genetic requirement for the F-box protein
Grr1 in Cln destruction, led to the next step in the development of
the present invention, namely the examination of whether Grr1
functions in recognition of phosphorylated Clns.
[0199] To generate Cln proteins for binding reactions,
Cln/Gst-Cdc28 complexes were isolated from insect cells. In the
presence of ATP, both Cln1 and Cln2 were found to be
autophosphorylated, a modification that reduces their
electrophoretic mobility (see below). To examine whether Grr1 can
associate with phosphorylated Clns and to compare the extent of
selectivity of Grr1 and Cdc4 toward Cln binding, anti-Skp1.sup.F
immune complexes from cells co-expressing Grr1 or Cdc4 in the
presence or absence of Cdc53 were used in binding reactions with
.sup.32P-labeled Cln1 or Cln2 kinase complexes. .sup.32P-labeled
Sic1 was used as a control for Cdc4 binding. Both Cln1 and Cln2
complexes were found to associate with Grr1/Skp1.sup.F/Cdc53
complexes (See, FIG. 5A) with an efficiency of about 40% of the
input Cln1 or Cln2 (See, FIG. 5A, lanes 8 and 12) and this
association did not require Cdc53 (lane 16). In contrast, about 6%
of the input Cln proteins associated with Cdc4/Skp1.sup.F complexes
independent of the presence of Cdc53 (lanes 7, 11, and 15),
compared with 1% association in the absence of an F-box protein
(lanes 6, 10, 14). The extent of selectivity of these F-box
proteins for Cln and Sic1 was further reflected by the observation
that Cln1 protein present in the phosphorylated Sic1 preparation
was selectively enriched in Grr1 complexes (FIG. 5A, lane 4). The
presence of all proteins in the binding reaction was confirmed by
immunoblotting (FIG. 5B) and the quantities of Cdc4 and Grr1 were
comparable, based on Coomassie staining of SDS gels of immune
complexes. Thus, Grr1 and Cdc4 display specificity toward
physiological substrates.
[0200] Cln1 Phosphorylation is Required for Recognition by Grr1
[0201] If Cln phosphorylation is required for ubiquitination as
suggested by genetic studies (Lanker et al., [1996], supra; and
Willem et al., [1996], supra), and if Grr1 is the receptor for
Clns, then the Grr1/Cln interaction would be expected to be
phosphorylation dependent. Thus, the next step in the development
of the present invention was to examine Grr1 alone and in complexes
with Skp1 or Skp1/Cdc53. Thus, Grr1 alone, or in complexes with
Skp1 or Skp1/Cdc53 was immunoprecipitated from insect cell lysates
and used in binding assays with phosphorylated or unphosphorylated
Cln1 complexes (FIG. 5C).
[0202] Unphosphorylated Cln1 was produced in insect cells as a
complex with kinase deficient Gst-Cdc28(K-), which minimized Cln1
autophosphorylation during expression and allowed the role of
phosphorylation to be tested. As isolated, this Cln1 protein
migrated as a homogeneous species of approximately 66 kDa (FIG. 5C,
lane 1). In contrast, phosphorylated Cln1 (lane 2) undergoes a
dramatic mobility shift to approximately 80 kDa, consistent with
the results observed in vivo. Phosphorylated Cln1 (and its
associated Cdc28 protein) efficiently associated with all Grr1
complexes (FIG. 5C, lanes 6, 8, 10), but was absent from control
binding reactions lacking Grr1 (FIG. 5C, lane 4). In contrast, the
levels of unphosphorylated Cln1 associated with Grr1 complexes were
compared to that found in binding reactions lacking Grr1 (FIG. 5C,
lanes 3, 5, 7, 9). Thus, association of both Cln1 with Grr1 and
Sic1 with Cdc4 is greatly enhanced by phosphorylation. Although the
Grr1/Skp1/Cdc53 complex is capable of binding efficiently to
phosphorylated Cln1, it was not competent for Cln1 ubiquitination
when supplemented with Cdc34 and E1 (FIG. 5D). Moreover, Cdc4
complexes that functioned in Sic1 ubiquitination also failed to
catalyze ubiquitination of Cln1 (FIG. 5D), despite the fact that
Cln1 can associate, albeit weakly, with Cdc4 (FIG. 5A). In
contrast, identical preparations of phosphorylated Cln1 protein
were efficiently ubiquitinated in partially purified yeast lysates
in a Cdc34-dependent manner (See e.g, FIG. 5E), indicating that
this preparation of Cln1 is competent for ubiquitination. Although
an understanding of the mechanism is not necessary in order to use
the present invention, the absence of Cln1 ubiquitination in the
purified system may reflect the requirement of additional factors
or modifications.
[0203] F-box Proteins as Receptors for Ubiquitination Targets
[0204] The present invention contemplates that a large number of
proteins contain the F-box, and are thereby implicated in the
ubiquitin pathway. The development of the present invention has
revealed that F-box proteins directly contact ubiquitination
substrates and can display selectivity in recognition of potential
targets for ubiquitination, as would be expected of E3 proteins.
For example, both Grr1 and Cdc4 assemble into mutually exclusive
complexes with Cdc53 and Skp1 (FIG. 4). However, Grr1 does not
associate with Sic1, nor does it support Sic1 ubiquitination. In
contrast, it was found that Cln proteins efficiently associate with
Grr1/Skp1.sup.F complexes and with Cdc4/Skp1.sup.F (although less
efficiently) (See e.g., FIG. 5). Although Cdc53 was originally
isolated as a Cln2-interacting protein (Willems et al., [1996],
supra), the present invention provides evidence that this original
interaction was bridged by Grr1 and possibly Cdc4. The Grr1/Cln
interaction is of interest in view of the fact that GRR1, CDC53,
and SKP1 are required for destruction of Cln proteins, and suggests
that Grr1 functions as a component of an E3 for Cln ubiquitination.
The absence of Cln ubiquitination by purified Grr1 complexes is
likely to indicate the absence of an essential factor(s) or
modifications that are not required for Sic1 ubiquitination in
vitro, and provides evidence that Cln ubiquitination may be more
complex than is Sic1 ubiquitination. Nonetheless, the present
invention provides methods, compositions, and models for the
development of compounds that interact with the ubiquitination
process, and thereby affect protein degradation through any number
of routes.
[0205] Despite the observation that F-box proteins may show
selectivity towards potential substrates, it is unlikely that F-box
proteins will be monospecific. For example, in S. pombe, recent
genetic data have linked the CDC4 homolog pop + with the
ubiquitination of both the CK1 Rum1 and Cdc18, a regulator of DNA
replication (Kominami and Toda, Genes Dev., 11:1548-1560 [1997]).
In budding yeast, CDC4 has also been implicated in destruction of
the Cdc18 homolog Cdc6 (Piatti et al., Genes Dev., 10:1516-1531
[1996]), indicating that it too has multiple targets. It was also
determined that Cdc4 can associate with Clns, albeit less
efficiently than with Grr1 (FIG. 5). Of importance is the fact that
all of the targets of F-box protein mediated destruction identified
to date are central regulators of key events in the cell, including
DNA replication, cell cycle progression, and nutritional
sensing.
[0206] A Cdc53/Cdc4/Skp1 E3 Complex is Required for Sic1
Ubiquitination by Cdc34
[0207] Sic1 destruction is genetically dependent upon Cdc34, Cdc4,
Cdc53, and Skp1. During the development of the present invention,
it was determined that these proteins are directly involved in the
ubiquitination process. As Cdc53 can simultaneously bind the E2
Cdc34 and Skp1, it frictions as an adapter linking the Skp1/F-box
protein complex to E2s (FIG. 1). In turn, Skp1 has the ability to
link Cdc4 to Cdc53. Cdc4 binds both Skp1 and the ubiquitination
substrate Sic1. The interaction of Cdc4 with Skp1 was shown to
involve the F-box located in the N-terminus of Cdc4, while the
interaction with Sic1 involves Cdc4's C-terminal WD-40 repeats
(FIG. 2). Skp1 was also shown to be involved in substrate
recognition because it enhances the association of Cdc4 with
phosphorylated Sic1. Cdc4 was shown to act as a receptor that, in
conjunction with Skp1, recruits substrates to the ubiquitination
complex. It is contemplated that any of these proteins could also
have carrier roles in the transfer of ubiquitin like E6AP (See
e.g., Scheffner et al., Cell 75:495-505 [1995]). However, it was
determined that mutation of the only conserved cysteine in Skp1 or
all 6 cysteines in Cdc53 did not impair complementation of skp1 or
cdc53 null mutations, respectively, indicating that these two
proteins are unlikely to transfer ubiquitin by a thio-ester
intermediate.
[0208] Phosphorylation Directly Regulates Association of Sic1 and
Cln Proteins with E3s
[0209] A central feature in the recognition of Sic1 and Cln by
F-box proteins is the phosphorylation dependent nature of the
interaction. Association of Sic1 with Cdc4-containing complexes and
subsequent ubiquitination requires Sic1 phosphorylation, as shown
in FIGS. 2 and 3. It was also shown that Sic1 phosphorylated by
excess Clb5/Cdc28 kinase can be ubiquitinated in vitro (See, FIG.
3E). It is contemplated that the initial generation of Clb5/Cdc28
activity at the G1/S transition could potentially accelerate Sic1
destruction facilitating the sharp and unidirectional change of
state characteristic of cell cycle transitions.
[0210] Similarly, association of Grr1 with Cln proteins is greatly
enhanced by phosphorylation, as indicated in FIG. 5.
Phosphorylation of specific residues in the C-terminal PEST domain
of Cln1 is required for Cln2 instability (Lanker et al., [1996],
supra), and phosphorylated Cln2 is found in complexes with Cdc53 in
vivo (Willems et al. [1996], supra). The present invention shows
that Cln/Cdc28 can provide a system that functions in vitro. The
present invention also provides methods, compositions, and models
for the determination of whether Cln ubiquitination is activated by
autophosphorylation in trans, as the accumulation of active
Cln/Cdc28 complexes may be required to achieve sufficient Cln
phosphorylation to promote its destruction.
[0211] While regulating the association of F-box proteins through
substrate phosphorylation is an effective method controlling the
timing of ubiquitination, it is not necessarily the case that all
F-box proteins will recognize their substrates in a phosphorylation
dependent manner. Observations made during the development of the
present invention indicate that WD-40 and LRR containing F-box
proteins can interact with phosphorylated substrates, but
approximately half of the known F-box proteins do not have obvious
protein interaction motifs. Nonetheless, the present invention
provides methods, compositions, and models to determine whether the
interaction of these proteins with their targets is regulated by
phosphorylation or even involves ubiquitination. The timing of
ubiquitination could be controlled by mechanisms unrelated to
substrate phosphorylation, such as controlled accessibility of
substrates or regulated expression, localization, or modification
of the F-box protein, thus providing methods for development of
compounds that affect proteolysis.
[0212] While the abundance of Cdc4 is not cell cycle regulated, the
F-box protein Skp2 displays cell cycle-regulated mRNA abundance
which peaks in S-phase, consistent with its association with cyclin
A during that phase of the cycle (Zhang et al., [1995], supra). In
vivo, association of Grr1 and Skp1 is enhanced in the presence of
glucose in a post-translational mechanism.
[0213] A large number of proteins contain PEST sequences and in a
subset of these proteins, these sequences have been shown to be
phosphorylated and to mediate instability. The development of one
embodiment of the present invention focused on the role of Skp1 and
F-box proteins in assembly of a ubiquitination complexes that
recognizes specific phosphorylated proteins. While the particular
complex defined by this embodiment of the present invention is
unlikely to be responsible for recognition of all PEST-dependent
proteolysis substrates, this complex is likely to be the prototype
for a diverse set of complexes in higher eukaryotes. Five CDC53
homologs have been identified in mammals (Cul1-5; Kipreos et al.,
Cell 85:829-839 [1996]). approximately 15 E2-related genes exist in
S. cerevisiae alone, several dozen F-box containing proteins have
been identified in several species, and several SKP1 related genes
exist in C elegans and are likely to exist in mammals as well. It
is clear that the present invention provides methods, compositions,
and models to identify PEST-dependent proteolysis substrates in
these and other organisms, as well as providing the flexibility to
differentially regulate the ubiquitination of a very large number
of substrates.
[0214] Other Applications
[0215] In addition, various embodiments of the present invention
find use in other settings. For example, the methods, compositions,
and models of the present invention provide the tools to determine
the function of such proteins as elongin C, a Skp1-related protein
is part of a complex containing the Cdc53-related protein Cul2, the
von Hippel-Lindau (VHL) tumor suppressor protein, elongin B, and
elongin A, a protein that is also found in association with elongin
C, and contains an F-box. Thus, the present invention provides the
means to develop compounds that affect systems other than
ubiquitination-mediated proteolysis.
[0216] Indeed, the F-box-directed ES complex (FEC) embodiment
described in detail herein, represents one example of a pathway
through which protein kinases control the stability of target
proteins. In view of the large number of protein kinases and
possible FECs, this pathway may be second only to transcriptional
regulation in the control of protein abundance. While the specific
examples described herein focus on the concern the cell cycle, the
present invention provides methods, compositions and models
applicable to other, diverse regulatory systems.
[0217] Although an understanding of the mechanism is not necessary
in order to use the present invention, FIG. 6A provides a model in
which a protein kinase phosphorylates target proteins, thus
activating them for association with their receptors, the F-box
proteins. Although some F-box proteins may already be associated
with a Skp1/Cdc53 complex prior to association with substrates, as
shown in FIG. 6A, it is also possible that F-box proteins exist in
a unbound form, and that association of the F-box protein with the
substrate drives association with Skp1/Cdc53. Since Skp1 enhances
the association of Cdc4 with Sic1, depending on the relative Kd
values for individual interactions and concentrations of the
constituents, association of the target with an F-box protein may
enhance association with Skp1. Once the ubiquitination complex is
formed and polyubiquitination takes place with the assistance of E1
and E2 proteins, the substrate is then released and recognized to
the 26S proteosome where it is proteolyzed.
[0218] As indicated in FIG. 6B, it is contemplated that other
combinations of FEC (or "SCF") complexes exist in cells. For
example, the F-box protein Met30 is closely related to Cdc4, and is
required for repression of genes in the methionine biosynthetic
pathway in the presence of S-adenosylmethionine (AdoMet) (See,
Thomas et al., Mol Cell. Biol., 15:6526-6534 [1995]). Met30 forms a
complex with Met4, a transcription factor required for methionine
biosynthetic gene expression. The present invention provides the
means to determine whether Met4 is ubiquitinated in response to
adomethionine. Furthermore, although the primary embodiment of the
present invention has focused on Cdc34, the present invention
provides means to determine whether other E2s are capable of
functioning in the context of FECs.
[0219] Also, as shown in FIG. 6C, SCF complexes (i.e., Skp1, Cdc53,
and Cdc4 present in a multiprotein complex), work together with
protein kinase signalling pathways to control protein abundance.
FIG. 6C illustrates one such pathway, in which SCF pathways
function multiple times in the transition from G1 to S phase in S.
cerevisiae.
[0220] Like protein synthesis, protein destruction is a fundamental
mechanism used by organisms to manipulate their function. In one
embodiment, the present invention provides the composition of an E3
complex, FEC, involved in selection of ubiquitination substrates.
Because the constituents of this complex are members of protein
families, the present invention provides the prototype for a large
class of E3s formed by combinatorial interactions of related family
members as indicated in FIG. 6B. The identification of F-box
proteins as the receptor components of this ubiquitin ligase
provides the means for identification of the key regulatory
molecules controlled by ubiquitin-mediated proteolysis. Thus, the
present invention provides means for the elucidation of the
biochemistry of this general ubiquitination pathway is likely to
have important ramifications for many aspects of biology including
cell proliferation, development, and differentiation.
[0221] The Present Invention in Action
[0222] The following example is provided to illustrate one specific
application of the present invention. In this example, the methods
and compositions of the present invention are used to identify a
novel E3 ubiquitin ligase complex that finds use in such
applications as the ubiquitination of I.kappa.B, which has direct
impact on the regulation of NF-.kappa.B activity and associated
cellular pathways. The findings of these studies provide new
therapeutic targets for the NF-.kappa.B pathway that can diversify
the existing programs for drug development.
[0223] The NF-.kappa.B pathway has many important physiological
roles and has become the focus of intense interest as a target for
drug development. For example, the NF-.kappa.B pathway has been
implicated in regulation of apoptosis. Hallmarks of transformed
cells include the ability to proliferate with reduced growth factor
levels and defects in the ability to undergo apoptosis. Many cell
types contain signaling systems that recognize inappropriate
proliferation and respond by activating an intrinsic apoptotic
pathway leading to cell loss. For example, it has been shown that
loss of the Cdk inhibitor p57 in the lens leads to both
inappropriate proliferation and increased apoptosis (Zhang et al.,
Nature 387:151 [1997]). As such, transformation pathways frequently
include some process that either inactivates a component of the
apoptotic machinery, activates a survival pathway, or both.
TNF-.alpha., a pro-inflanmmatory cytokine, functions in part to
activate NF-.kappa.B, a transcription factor composed of p50 and
p65/Rel subunits (Baeuerle and Baltimore, Cell 87:13 [1996]; Beg et
al., Mol. Cell. Biol. 13:3301 [1993]; DiDonato et al., Mol. Cell.
Biol. 15:1302 [1995]; and Tewari and Dixit, Genes & Devel. 6:39
[1996]). NF-.kappa.B also activates the expression of a large
number of genes, including growth factors, chemokines, and adhesion
molecules which mediate inflammatory responses.
[0224] TNF-.alpha. has also been shown to induce particular cell
types to undergo apoptosis, although the cytotoxic effects are
revealed most frequently only if protein/RNA synthesis is blocked
(Tewari and Dixit, supra). Recent studies have revealed that the
inability of cells to undergo apoptosis in response to TNF-.alpha.
reflects activation of a survival pathway, which is programmed by
NF-.kappa.B action (Beg and Baltimore, Science 274:782 [1996]; Liu
et al., Cell 87:565 [1996]; Van Antwerp et al., Science 274:787
[1996]; and Wang et al., Science 274:784 [1996]). Cells lacking
RelA or blocked for NF-.kappa.B nuclear translocation are sensitive
to TNF-mediated killing (Beg and Baltimore [1996], supra; and Wang
et al., supra). Moreover, induction of NF-.kappa.B activity
protects cells against TNF-mediated cell death (Van Antwerp et al.,
supra). TNF-.alpha. may induce cell death through one pathway and
simultaneously induce a protective mechanism through NF-.kappa.B
(Beg and Baltimore [1996], supra). These studies indicated for the
first time an important role for NF-.kappa.B in cell survival
pathways and suggested inhibition of NF-.kappa.B function might be
used to predispose cancer cells to killing by TNF-.alpha. or
chemotherapeutic compounds.
[0225] In principle, agents that block NF-.kappa.B function could
inactivate the cell survival pathway set in motion by NF-.kappa.B,
rendering cells capable of undergoing apoptosis. In addition to its
survival functions, there is evidence that NF-.kappa.B may play
growth promoting roles by activating transcription of myc, which
may drive the cell cycle forward (reviewed by Sovak et al., J.
Clin. Invest. 100:2952 [1997]). There is accumulating evidence that
NF-.kappa.B is used to set up a survival pathway in transformed
mammary cells. Activated nuclear NF-.kappa.B is prominent in
mammary tumor lines (Nakshatri et al., Mol. Cell. Biol. 17:3629
[1997]), but rare in normal mammary epithelial cells, and recent
studies indicate blocking NF-.kappa.B in this setting can induce
apoptosis (Sovak et al., supra). Other cell types such as B-cells
also undergo apoptosis when NF-.kappa.B is inhibited (Wu et al.,
EMBO J. 15:4682 [1996]). It is possible that NF-.kappa.B is
normally used to protect particular mammary cells from apoptosis,
which is occurring as part of the normal biology of the system, and
that transformation takes advantage of this property. In addition,
NF-.kappa.B activation in mammary tumor cells correlates with
ER-independent proliferation (Nakshatri et al., supra), suggesting
a possible link between estrogen responsiveness and apoptosis.
[0226] For the last several years, there has been interest in the
drugs that block NF-.kappa.B activation for use in
anti-inflammatory diseases (See e.g., Vogel, Science 281:1943
[1998]), an interest that has been strengthened by the finding that
aspirin functions to block the NF-.kappa.B pathway (Grilli et al.,
Science 274:1383 [1996]). The finding that NF-.kappa.B also
functions in cell survival has led to the realization that drugs
that affect this pathway may also be useful in cancer treatment.
The insensitivity of some tumor cells to chemotherapeutics may
reflect an inability to undergo apoptosis and interestingly,
inhibitors of NF-.kappa.B can correct the radiation sensitivity of
cells mutant in the AT gene (Jung et al., Science 268:1619 [1995]).
Thus, NF-.kappa.B inhibitors may find use as an adjunct to
chemotherapy/radiotherapy. Thus, it is contemplated that a more
complete understanding of the NF-.kappa.B activation pathway will
lead to the identification of new therapeutic targets.
[0227] NF-.kappa.B activation involves a multi-step signal
transduction pathway (Baeuerle and Baltimore, supra) involving
receptor activation, activation of kinases (IKK.alpha. and
IKK.beta.) that phosphorylate I.kappa.B (the endogenous inhibitor
of NF-.kappa.B), ubiquitination of I.kappa.B, proteolysis of
I.kappa.B, and translocation of NF-.kappa.B to the nucleus. Recent
advances include identification of IKKs (DiDonato et al., Nature
388:548 [1997]; Mercurio et al., Science 278:860 [1997]; Regnier et
al., Cell 90:373 [1997]; Woronicz et al., Science 278:866 [1997];
and Zandi et al., Nature 387:151 [1997]) and the components of the
TNF receptor complex (reviewed by Tewari and Dixit, supra). In
contrast, prior to the present invention, virtually nothing was
known about the molecules that function in the ubiquitination
step.
[0228] As discussed above, the present invention provides a novel
E3 ubiquitin ligase complex that provides means to identify
therapeutic targets for regulating NF-.kappa.B activity, to
identify the molecular determinants that confer the ability of this
ligase to recognize phosphorylated I.kappa.B, and to identify
molecules that can disrupt this interaction.
[0229] A. Background Regulation of NF-.kappa.B Function
[0230] NF-.kappa.B activity is regulated primarily through its
sub-cellular localization (Baeuerle and Baltimore, supra). In the
absence of signal, NF-.kappa.B is sequestered in the cytoplasm by
interaction with a member of the I.kappa.B (inhibitor of .kappa.B)
family of proteins (Baeuerle and Baltimore, Science 242:540
[1988]). I.kappa.B binds to p50/p65 heterodimers and simultaneously
blocks both the nuclear localization signal and the ability of
NF-.kappa.B to bind DNA (Beg et al., Genes & Devel. 6:1899
[1992]; Luque and Gelinas, Mol. Cell. Biol. 18:1213 [1998]; and
Thompson et al., Cell 80:573 [1995]). In response to stimuli
intended to activate NF-.kappa.B, I.kappa.B is rapidly
phosphorylated (Beg et al., Mol. Cell. Biol. 13:3301 [1993]; Brown
et al., Science 267:1485 [1995]; Chen et al., Genes & Devel.
9:1586 [1995]; DiDonato et al., [1995], supra; Finco et al., Proc.
Natl. Acad. Sci. 91:11884 [1994]; Lin et al., Proc. Natl. Acad.
Sci. 92:552 [1995]; and Liu et al., Cell 87:565 [1996]). This
signals I.kappa.B to be destroyed by ubiquitin mediated
proteolysis, allowing NF-.kappa.B to translocate to the nucleus to
activate target genes (Alkalay et al., Proc. Natl. Acad. Sci.
92:10599; Henkel et al., Nature 365:182 [1993]; and Scherer et al.,
Proc. Natl. Acad. Sci. 92:11259 [1995]). The identity and
regulation of the ubiquitin ligase that functions in NF-.kappa.B
ubiquitination was unknown in the art.
[0231] A key component of this signaling pathway involves
activation of kinases responsible for I.kappa.B phosphorylation,
since this step (i.e., I.kappa.B phosphorylation) is thought to be
the rate-limiting step in NF-.kappa.B activation. Signaling
molecules such as TNF, which promoter NF-.kappa.B activation in
particular cell types, bind to TNF receptors that link to the death
domain protein TRADD, and TRAF1/2 which contain a TRAF domain
(Tewari and Dixit, supra). These proteins function in the transient
activation of two kinases, IKK.alpha. and IKK.beta., which are part
of a large (700 kd) complex whose other components are not yet
fully defined (DiDonato et al., [1997], supra; Mercurio et al.,
supra; Regnier et al., Cell 90:373 [1997]; Woronicz et al., Science
278:866 [1997]; and Zandi et al., supra). In vitro, each of these
kinases specifically phosphorylate I.kappa.B on two serine residues
(Ser-32 and Ser-36) and this combination of phospho-serine residues
is thought to be the targeting signal for I.kappa.B ubiquitination,
although how this signal is recognized and utilized is not known in
the art Importantly, overexpression of non-phosphorylatable
I.kappa.B has been shown to be effective in blocking NF-.kappa.B
activation (Woronicz et al., supra).
[0232] B. Phosphorylation Specific Association of I.kappa.B with
Skp1
[0233] The role of SCF complexes in phosphorylation-dependent
ubiquitination led to the examination of whether I.kappa.B might
associate with Skp1. HeLa cell lysates were incubated with agarose
beads (Affigel beads) containing unphosphorylated and
phosphorylated I.kappa.B sequences overlapping the ubiquitination
targeting signal previously identified in I.kappa.B (Yaron et al.,
EMBO J. 16:6486 [1997]; containing Ser-32 and Ser-36) and the
presence of Skp1 in I.kappa.B-associated proteins examined by
immunoblotting, as shown in FIG. 8. The results demonstrated that
Skp1 specifically associated with phosphorylated I.kappa.B but not
unphosphorylated I.kappa.B. Skp1 is a highly abundant protein and
is thought to be distributed among multiple F-box proteins and
possibly kinetochore complexes. It is estimated that .about.1% of
the Skp1 in these extracts can associate with I.kappa.B in
vitro.
[0234] C. Slimb F-Box Protein Associates with Phosphorylated
I.kappa.B
[0235] Having found that Skp1 can associate with phosphorylated
I.kappa.B, a number of F-box proteins were surveyed for association
with phosphorylated I.kappa.B. Various F-box proteins were produced
by in vitro translation and tested for binding to phospho-I.kappa.B
and I.kappa.B. In particular, a variety of in vitro translated
F-box proteins containing LRRs (Skp2, F alpha), WD40 repeats (MD6,
Met30), a cyclin box (cyclin F), and no obvious additional domains
(F gamma) failed to interact with phosphorylated I.kappa.B, as
shown in FIG. 9. In contrast, the slimb protein (also referred to
herein as "TRCP protein") specifically associated with
phosphorylated I.kappa.B, suggesting that slimb F-box protein plays
a role in I.kappa.B/NF-.kappa.B regulation.
[0236] To determine cell types where slimb might function,
asystematic in situ hybridization analysis was initiated to
determine patterns of slimb expression in adult mouse tissues and
during development. A section through a E12.5 day mouse was
subjected to in situ hybridization .sup.35S-labeled mouse slimb
antisense RNA using established procedures (Zhang et al., supra).
Analysis demonstrated that slimb is expressed at maximal levels in
the ventricles of the forebrain and hindbrain, lung, and liver.
Weaker expression was observed throughout most of the embryo.
[0237] D. slimb/Skp1 Associates with Phosphorylated I.kappa.B
[0238] The finding that Skp1 and slimb can both form complexes with
phosphorylated I.kappa.B beads, together with the fact that slimb
contains an F-box, led to the examination of whether slimb can
associate with Skp1 and Cul1 in vivo. Although every F-box protein
tested to date interacts with Skp1, there are 6 Cul homologs and it
is not clear at present whether they all bind to Skp1 or only a
subset bind to Skp1. To examine these interactions, 293 T-cells
were transfected with vectors expressing various tagged versions of
Skp1, slimb, and cul1 as shown in FIGS. 10A and 10B. To assemble
the SCF/slimb complex, plasmids expressing cul1HA, Skp1HA3, and
slimbMYC9 were transfected in the indicated combinations (FIG. 10A)
into 293 cells using lipofection. After 48 hours, cells were
disrupted in lysis buffer (10 mM Tris-HCl, 0.5% nonidet P-40, 150
mM NaCl, 10 mM beta-glycerolphosphate) and insoluble material
removed by centrifugation. Lysates (1 mg of protein) were subjected
to immunoprecipitation using anti-myc antibodies. Immune complexes
were washed three times in lysis buffer and were separated by
SDS-PAGE and transferred to nitrocellulose. Blots were developed
using anti-HA, anti-Skp1, and anti-myc antibodies. FIG. 10B shows
lysates from the indicated transfections that were subjected to
immunoblotting using the indicated antibodies.
[0239] Additionally immunoprecipitation/Western blotting
experiments were performed as shown in FIGS. 11A and 11B. In FIG.
11A, the indicated plasmids were transfected into 293T cells and
after 48 hours, lysates were made and subjected to
immunoprecipitation using anti-HA antibodies to precipitate Cul1.
The presence of slimb and Skp1 were determined using anti-myc and
anti-Skp1 antibodies. The myc9-tagged Skp1 migrates at
approximately 30 kilodaltons compared to 19 kd for untagged
Skp1.
[0240] The data in FIGS. 10 and 11 demonstrate that Cul1
immunocomplexes contain Skp1, as expected, but also contain slimb.
Likewise, although the data are not included herein, it was shown
that slimb immunocomplexes contain Skp1 and Cul1. To examine
whether slimb/Skp1 complexes are capable of associating with
phosphorylated I.kappa.B, 293 T-cells were transfected with CMV-HA
slimb and CMV-HA Skp1 and lysates subsequently incubated with
I.kappa.B or phosphorylated I.kappa.B beads prior to SDS-PAGE and
Western analysis with anti-HA antibodies as shown in FIG. 11B. In
this Figure, lysates from the indicated transfected cells were
subjected to binding reactions using immobilized I.kappa.B or
phospho-I.kappa.B. After washing, bound proteins were subjected to
immunoblotting with anti-HA to visualize slimb and Skp1 proteins.
As shown, Skp1 and slimb associate specifically with
phospho-I.kappa.B (i.e., transfected slimb and Skp1 assemble into
complexes that are recognized by phospho-I.kappa.B).
[0241] HA slimb was found to associate with phosphorylated but not
unphosphorylated I.kappa.B beads with or without transfection of
Skp1. HA Skp1 also associated with I.kappa.B in a
phosphorylation-specific manner. Upon longer exposure of this blot,
HA Skp1 was detectable in complexes with phosphorylated (but not
unphosphorylated) I.kappa.B in lysates from cells transfected with
HA Skp1 alone, suggesting that HA Skp1 can assemble with the
endogenous slimb protein. Previous studies indicated that a peptide
containing the sequence KKERLLDDRHDSGLDSMKDEE (residues 21-41 from
I.kappa.B; SEQ ID NO:60) will not inhibit I.kappa.B ubiquitination
when added in vitro to a crude cell lysate which supports I.kappa.B
ubiquitination in a manner that is dependent upon the
phosphorylation of Ser-32 and Ser-36 in I.kappa.B. In contrast, the
same peptide that has been phosphorylated on Ser-32 and Ser-36 will
block the ubiquitination of I.kappa.B. Similarly, phosphorylated
I.kappa.B peptide will block nuclear translocation of NF-.kappa.B
in intact cells in response to stimuli while the unphosphorylated
peptide will not. It is known that I.kappa.B needs to be
phosphorylated on these two serines by IKK for ubiquitination to
occur and this phosphorylation serves as the signal. These
phosphopeptides derived from I.kappa.B are thought to block
I.kappa.B ubiquitination by competing with the full-length
I.kappa.B substrate for the recognition factor of the ubiquitin
ligase that is normally functioning in I.kappa.B ubiquitination.
Thus, the finding of the present invention that this same
phosphorylated I.kappa.B peptide, but not the unphosphorylated
peptide, will specifically interact with the SCF slimb complex
suggests that this slimb complex is the ubiquitin ligase for
I.kappa.B. Thus, the present invention provides a novel E3
ubiquitin ligase complex, thereby providing means to identify
therapeutic targets for regulating NF-.kappa.B activity, to
identify the molecular determinants that confer the ability of this
ligase to recognize phosphorylated I.kappa.B, and to identify
molecules that can disrupt this interaction.
[0242] These studies have revealed that slimb recognizes the
phosphorylated targeting signal in I.kappa.B. It is contemplated
that other cellular or viral proteins contain these sequences and
will be therefore targeted to the slimb ubiquitin ligase. Although
this sequence is recognized by slimb, it is further contemplated
that other unrelated sequences may also interact with slimb
possibly through independent domains. It is also contemplated that
other F-box proteins containing analogous mutations will find use
to demonstrate the specificity of the dominant negative effect.
[0243] E. Further Characterization
[0244] Using the methods and compositions of the present invention,
there are several approaches available to further characterize the
relationship between the SCF slimb complex and I.kappa.B. These
include both in vivo and in vitro approaches.
[0245] In vivo: In one embodiment of the present invention,
NF-.kappa.B activation or I.kappa.B destruction is blocked using a
dominant negative form of slimb. A dominant negative form of slimb
is one that will still bind to I.kappa.B but will not assemble with
the cul1/Skp1 complex. Therefore, the dominant negative slimb
protein, when expressed at sufficient levels in transfected cells,
would bind phosphorylated I.kappa.B, thereby blocking access of the
endogenous slimb protein to I.kappa.B. Since this dominant negative
I.kappa.B is not assembled with cul1/Skp1 complexes, appropriate E2
conjugating enzymes would not be physically coupled to I.kappa.B
and would therefore not carry out the ubiquitination reaction. Many
forms of slimb find use as dominant negative proteins and are made
using methods standard in the art. For example, in preferred
embodiments, versions of slimb that either lack the F-box domain or
contain one or more point mutations in the F-box domain are used.
This domain is required for interaction with Skp1, and mutation of
the F-box in the appropriate residues blocks association with Skp1.
The preferred residues to be useful in this regard include those
that are highly conserved in other F-boxes. Association with Skp1
in vitro could be used to demonstrate that the mutant slimb protein
no longer interacts with Skp1. The function of the slimb dominant
negative protein is assessed, for example, by monitoring
NF-.kappa.B activity on a reporter construct, the translocation of
NF-.kappa.B to the nucleus in response to TNF treatment, or
stabilization of I.kappa.B protein levels.
[0246] In vitro: In one embodiment of the present invention, the
rate of I.kappa.B ubiquitination in cells is directly altered by
blocking or activating slimb function. For example, in one series
of experiments, the ubiquitination of I.kappa.B is blocked using
slimb mutants (i.e., dominant negative F-box mutants) that bind
I.kappa.B but not Skp1, thereby uncoupling I.kappa.B's ability to
associate with endogenous SCF slimb when the mutant is
overexpression. A set of conserved residues in the F-box whose
mutation abolishes interaction of the F-box protein Cdc4 with Skp1
has previously been identified (Bai et al., Cell 86:263 [1996]). In
one set of experiments, two sets of conserved F-box residues (LP
and IL) in slimb are mutated to AA and act to verify binding to
phospho-I.kappa.B but not Skp1 in vitro. Appropriate mutants are
transfected into HeLa cells and the effects on TNF-induced
activation of NF-.kappa.B is assessed using three primary assays:
1) pulse chase analysis of I.kappa.B (when a high level of
transfection is achieved), 2) NF-.kappa.B activated reporter (e.g.,
luciferase) activity, and/or 3) entry of Rel into the nucleus by
immunofluorescence. Other F-box proteins (including the WD40
containing MD6), mutant in the F-box, are used as controls.
[0247] The results of experiments conducted during the development
of the present invention indicate that slimb levels are low
compared to the levels of transfected slimb. Thus, in preferred
embodiments of the present invention, the dominant approach is
used. For confirmation or as alternative embodiments, other
approaches such as antisense are used. For example, the antisense
approach has been used to successfully block IKK activity (DiDonato
et al., [1997], supra).
[0248] In yet other embodiments of the present invention, the role
of slimb in the ubiquitination of I.kappa.B is characterized. In
one embodiment, overexpression of SCF slimb components is used to
enhance the unstimulated rate of I.kappa.B ubiquitination.
[0249] In other embodiments, the activity of the SCF slimb complex
toward I.kappa.B is demonstrated in vitro. For example, experiments
conducted during the development of the present invention have
demonstrated that cells can be transfected with slimb, cul1, and
Skp1 to generate complexes. In the in vitro embodiments,
experiments are conducted to examine whether slimb alone or in
combination with Skp1 and Cul1 accelerates ubiquitination of
endogenous or co-transfected I.kappa.B, using pulse chase analysis
or direct ubiquitination assays. Overexpression of I.kappa.B
increases its levels such that the endogenous slimb complex does
not efficiently ubiquitinate it, thereby providing a window for
acceleration by exogenous slimb. Direct ubiquitination analysis is
achieved by co-transfection of a tagged ubiquitin plasmid followed
by immunoprecipitation of I.kappa.B and immunoblotting for the
tagged ubiquitin.
[0250] In yet other embodiments of the present invention, methods
to determine whether slimb transfection can force NF-.kappa.B
activation in the absence of stimulation as a result of residual
IKK activity, or with reduced levels of stimuli are conducted.
Controls include slimb mutants that cannot bind I.kappa.B, and
F-box proteins that do not associate with I.kappa.B (as described
above).
[0251] As described above, the present invention provides
approaches for reconstruction the SCFCdc4 ubiquitin ligase pathway
for the Cdk inhibitor Sic1 (See also, Skowyra et al., Cell 91:209
[1997]). This approach also finds use with slimb. First, in some
embodiments, experiments are conducted to determine whether slimb
immune complexes from transfected cells contain I.kappa.B ubiquitin
ligase activity using phosphorylated I.kappa.B or I.kappa.B point
mutants in phosphorylation sites as substrates. Cdc34 is the most
likely candidate for the E2, however other E2 are also tested
(e.g., Ubc4, 5, and 10). The development of this system provides a
screening assay to examine whether particular molecules function to
block I.kappa.B ubiquitination.
[0252] In yet other embodiments of the present invention, methods
are provided to determine whether interference with slimb inhibits
NF-.kappa.B function and induces apoptosis. There is clear evidence
that blocking NF-.kappa.B action in mammary tumor cells (578T)
(Sovak et al., supra) and in other cell types (Van Antwerp et al.,
supra; Wang et al., Science 274:784 [1996]; and Wu et al., supra)
can lead to apoptosis. In one set of experiments, synthetic
peptides overlapping the I.kappa.B recognition sequence (in either
the phosphorylated or unphosphorylated forms) are generated and
microinjected (as described by Connell-Crowley et al., Curr. Biol.
8:65 [1997]) into 578T human mammary tumor cells. Apoptosis, as
well as the fate of NF-.kappa.B/I.kappa.B, is assessed by TUNEL and
immunofluorescence, respectively, using standard methods. In other
embodiments methods are provided to test whether dominant negative
slimb or appropriate controls will induce apoptosis.
[0253] F. Slimb/I.kappa.B Interaction Surfaces as a Target for Drug
Design
[0254] Due to the widespread interest in the generation of
anti-NF-.kappa.B therapeutics, many steps in the NF-.kappa.B
pathway are being targeted. Because the nature of the ubiquitin
ligase for I.kappa.B was unknown in the art, prior to the present
invention, this step had not yet been explored. The slimb complex
of the present invention provides a novel target and provide means
to identify anti-NF-.kappa.B therapeutics For example, one major
advantage of slimb is that it recognizes a small phosphopeptide
sequence. It is contemplated that molecules that mimic this
phosphopeptide and block NF-.kappa.B activation will be identified
using the method of the present invention.
[0255] In some embodiments, the first steps in generating
slimb/I.kappa.B interaction surfaces involve identification of the
molecular interaction surfaces (interacting motifs) between slimb
and I.kappa.B, and identification of peptides or proteins that, by
virtue of binding to slimb, block binding to I.kappa.B. These steps
identify and provide motifs and assays that find use in screening
combinatorial libraries for small molecule inhibitors of the
interaction. As there are many alternative approaches that could be
taken to identify molecular interaction surfaces, it is not
intended that the present invention be limited to any specific
approach. Preferred approaches are illustrated below, although the
present invention is not limited to these particular
approaches.
[0256] In a first embodiment of the present invention, a modified
version of the reverse two-hybrid approach (See e.g., Vidal et al.,
Proc. Natl. Acad. Sci. 93:10315 [1996]) is applied to identify
point mutants in slimb that abolish I.kappa.B binding. This
approach uses the power of genetics to screen a large library of
point mutants in slimb (e.g., generated by either chemical
mutagenesis or PCR using standard methods) to identify those that
have lost the ability to bind to a target. Slimb mutants that fail
to interact with I.kappa.B will be counter-screened for interaction
with Skp1 and for expression of full-length slimb mutant protein
using methods similar to those illustrated in FIG. 12. In this
Figure, phosphorylation-specific interaction of SCF slimb complexes
with I.kappa.B peptide sequences were analyzed. Lysates (1 mg)
prepared as described above for FIG. 10 were incubated with 10
microliters of affigel beads containing either the I.kappa.B
peptide or the same peptide containing phosphoserine at both serine
residues. Beads were washed three times with lysis buffer and bound
proteins separated by SDS-PAGE. Proteins were transferred to
nitrocellulose and used for immunoblotting with the indicated
antibodies. A subset of slimb mutants (determined by sequencing)
that pass the secondary tests likely reside in I.kappa.B contact
sequences.
[0257] Preferred interaction surfaces for use in screening assays
are those that have clustered mutations (e.g., those that are
localized nearby on the same surface). To ensure that mutations
reflect an interaction site as opposed to structural alterations,
the samples are assayed for second-site revertants in I.kappa.B
that regenerate interaction with a mutant slimb protein. These
studies, together with conventional deletion analysis provide
information about the necessary and sufficient sequences in
slimb.
[0258] In other embodiments of the present invention, consensus
sequences are determined for interaction with the phosphopeptide
binding site(s) in slimb. The small size and simplicity of the
I.kappa.B sequence makes it an attractive candidate for determining
a consensus binding sequence. In one embodiment of the present
invention, a peptide library approach is used to identify consensus
sequences for phosphopeptide recognition. The technique (See e.g.,
Songyang et al., Cell 72:767 [1993]; Songyang et al., Mol. Cell.
Biol. 14:2777 [1994]; and Songyang et al., J. Biol. Chem. 270:14863
[19951]) involves applying a highly complex mixture of peptide
sequences that contain phosphoserines three residues apart (as in
I.kappa.B), but are otherwise degenerate, to immobilized slimb or
the minimal interaction domain identified above. After the column
is washed, peptides are eluted and sequenced to determine consensus
sequences. Individual sequences are then tested for binding. The
goal here is to define how selective the interaction site is. It is
known for instance that the spacing between the phosphoserines is
required for I.kappa.B to be destroyed (Yaron et al., supra). The
elucidation of such a consensus provides a theoretical "sequence
space" and a starting point for drug discovery. It is also
contemplated that this motif will find use to search databases for
other potential slimb substrates and/or regulators. In alternate
embodiments, a particular peptide sequence in the context of two
glutamic acids (which can mimic phosphoserine) may be able to
associate with slimb. In yet Rother embodiments, peptide library
experiments are performed with fixed glutamic acids to determine if
any sequences exist that compete in a phosphorylation-independent
manner. This provides a starting point for non-phosphorylatable
slimb inhibitors. To determine whether the peptides identified
inactivate slimb in cells, peptides are microinjected into tissue
culture cells and NF-.kappa.B function, as well as apoptosis, in
578T cells is determined (as described above).
[0259] In other embodiments of the present invention, an
alternative approach to the directed search for competitive binding
components is used, which combines the complex nature of the human
genome or peptide aptomer libraries coupled with the power of the
reverse two hybrid approach. In this embodiment, cDNA or peptide
aptomer libraries are be transformed into yeast strains expressing
GAL4-I.kappa.KB and ACT-slimb and cells are selected for the loss
of the I.kappa.B/slimb interaction. Library plasmids are rescued
and sequenced to identify binding components, with further analysis
revealing whether these proteins/peptides disrupt the interaction
by binding to one or both of the proteins. Peptide aptomers are
then assessed as described above for synthetic peptides.
[0260] From these illustrative examples, it is clear that the
present invention provides means to develop anti-NF-.kappa.B
therapies based on the blocking of I.kappa.B ubiquitination. More
generally, the identification and characterization of slimb as a
member of an SCF complex illustrates that the methods and
compositions of the present invention are capable of identifying
and isolating F-box proteins and detecting F-box protein targets
and F-box protein complexes.
EXPERIMENTAL
[0261] The following examples are provided in order to demonstrate
and further illustrate certain preferred embodiments and aspects of
the present invention, and are not to be construed as limiting the
scope thereof.
[0262] In the experimental disclosure which follows, the following
abbreviations apply: h (human); Sc (Saccharomyces cerevisiae); m
(mouse); Ub (ubiquitin); E1 (Ub activating enzyme); E2 (Ub carrier
protein); E3 (Ub-protein ligase); .degree. C. (degrees Centigrade);
rpm (revolutions per minute); BSA (bovine serum albumin); CFA
(complete Freund's adjuvant); IFA (incomplete Freund's adjuvant);
IgG (immunoglobulin G); IM (intramuscular); IP (intraperitoneal);
IV (intravenous or intravascular); Sc (subcutaneous); H.sub.20
(water); HCl (hydrochloric acid); aa (amino acid); bp (base pair);
kb (kilobase pair); kd (kilodaltons); gm (grams); .mu.g
(micrograms); mg (milligrams); ng (nanograms); .mu.l (microliters);
ml (milliliters); mm (millimeters); nm (nanometers); .mu.m
(micrometers); M (molar); mM (millimolar); MW (molecular weight);
sec(s) (second/seconds); min(s) (minute/minutes); hr(s)
(hour/hours); MgCl.sub.2 (magnesium chloride); NaCl (sodium
chloride); DTT (dithiothreitol); OD.sub.280 (optical density at 280
nm); OD.sub.600 (optical density at 600 nm); PAGE (polyacrylamide
gel electrophoresis); PBS (phosphate buffered saline [150 mM NaCl,
10 mM sodium phosphate buffer, pH 7.2]); PEG (polyethylene glycol);
PMSF (phenylmethylsulfonyl fluoride); SDS (sodium dodecyl sulfate);
SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel
electrophoresis); LMA (low melting temperature agarose gel; Tris
(tris(hydroxymethyl)aminomethane); NETN (20 mM Tris-HCl, pH 8, 100
mM NaCl, 1 mM EDTA, 0.5% NP-40, 5 mM NaF, 30 mM
p-nitrophenylphosphate, 1 .mu.g/ml each leupeptin and antipain, and
1 mM PMSF); TBST (20 mM Tris (pH 8), 100 mM NaCl, 0.5% Tween-20);
IPTG (isopropyl-.beta.-D-thiogalacto- pyranoside); LB
(Luria-Bertani medium; per liter: 10 g tryptone, 5 g yeast extract,
10 g NaCl, pH 7; sterilized by autoclaving for 20 minutes at 15
lbs/in.sup.2); vol (volume); w/v (weight to volume); V/V (volume to
volume); Amersham (Amersham Life Science, Inc., Arlington Heights,
Ill.); ICN (ICN Pharmaceuticals, Inc., Costa Mesa, Calif.); Amicon
(Amicon, Inc., Beverly, Mass.); ATCC (American Type Culture
Collection, Rockville, Md.); Becton Dickinson (Becton Dickinson
Labware, Lincoln Park, N.J.); BioRad (BioRad, Richmond, Calif.);
Clontech (CLONTECH Laboratories, Palo Alto, Calif.); Difco (Difco
Laboratories, Detroit, Mich.); GIBCO BRL or Gibco BRL (Life
Technologies, Inc., Gaithersburg, Md.); Babco (Berkeley Antibody
Company, Richmond, Calif.); Invitrogen (Invitrogen Corp., San
Diego, Calif.); Kodak (Eastman Kodak Co., New Haven, Conn.); New
England Biolabs (New England Biolabs, Inc., Beverly, Mass.);
Novagen (Novagen, Inc., Madison, Wis.); Qiagen (Chatsworth,
Calif.); Pharmacia (Pharmacia, Inc., Piscataway, N.J.); Sigma
(Sigma Chemical Co., St. Louis, Mo.); Sorvall (Sorvall Instruments,
a subsidiary of DuPont Co., Biotechnology Systems, Wilmington,
Del.); Stratagene (Stratagene Cloning Systems, La Jolla, Calif.);
Whatman (Whatman LabSales, Hillsboro, Oreg.); Bethyl Laboratories
(Bethyl Laboratories, Montgomery, Tex.); and Zeiss (Carl Zeiss,
Inc., Thornwood, N.Y.).
[0263] Unless otherwise indicated, all restriction enzymes were
obtained from New England BioLabs and were used according to the
manufacturer's instructions; all oligonucleotide primers, adapter
and linkers were synthesized using standard methodologies on an ABI
DNA synthesizer. All chemicals were obtained from Sigma unless
otherwise indicated.
EXAMPLE 1
Preparation of Antibodies
[0264] In this Example, anti-Skp1 and anti-Sic1 antibodies were
prepared. Using standard methods as known in the art, anti-Skp1 and
anti-Sic1 polyclonal antibodies were generated in rabbits, with
bacterial Gst fusion protein described below, used as the
antigen.
[0265] A. Antigen Preparation
[0266] Expression plasmids for GST-SKP1 and GST-SIC1 were generated
by ligating open reading frames for the encoded proteins into
pGEX2TK (Pharmacia), using established procedures known in the art
(See e.g., J. Sambrook et al, supra). The Genbank accession numbers
for SKP1 and SIC1 are U61764 and X78309, respectively.
[0267] Plasmids were transformed into E. coli strain BL21
(DE3)(Novagen). For expression, 1 L of E. coli cells were grown in
LB medium containing 0.1 mg/ml ampicillin at 37.degree. C., until
the OD.sub.600 reached 0.8. Expression was induced with 400 mM IPTG
for three hours. Cells were harvested by centrifugation
(2,000.times.g for 10 minutes), and then lysed in 70 ml NETN buffer
for 30 minutes, on ice. The insoluble material was then removed by
centrifugation (14,000.times.g, for 20 minutes). The lysate was
then incubated with 0.5 ml glutathione Sepharose (Pharmacia) for 1
hour at 4.degree. C. The Sepharose beads were washed three times
with 10 ml NETN buffer, and washed twice with 5 ml of 100 mM NaCl,
and the protein was eluted with buffer containing 0.5 ml 100 mM
Tris (pH 7.5), 100 mM NaCl, 40 mM glutathione. The protein was then
stored at -80.degree. C., prior to its use in the affinity
purification of antibodies.
[0268] B. Antibody Production and Affinity Purification
[0269] Polyclonal rabbit anti-Cdc34 and anti-Cdc4 sera (provided by
M. Goebl), as well as anti-Sic1, were affinity purified using
recombinant antigens immobilized on nitrocellulose. The anti-Skp1
antibodies were not affinity purified.
[0270] To affinity purify the anti-Sic1 antibodies, GST-Sic1
protein (0.1 mg) was subjected to electrophoresis on a 12%
polyacrylamide (SDS-PAGE) gel, the protein was blotted to
nitrocellulose (3 hours, at 350 mA). Nitrocellulose filters
containing GST-Sic1 protein were incubated with 1 ml of anti-Sic1
antibodies for 3 hours, the filters were washed twice with 10 ml of
buffer containing 50 mM Tris (pH 7.5), 50 mM NaCl, 0.5% Tween-20,
and then eluted with 1 ml of 100 mM glycine (pH 2), and stored at
4.degree. C. until use.
[0271] In addition to the anti-Skp1 and anti-Sic1 polyclonal rabbit
antibodies generated in this Example, and the anti-Cdc34 and
anti-Cdc4 polyclonal rabbit antibodies from Dr. Goebl, monoclonal
antibodies were also used in the following Examples. These
commercially available monoclonal antibodies were obtained from
Babco (anti-HA, anti-Myc), Novagen (anti-T7 gene10, [i.e., "G10"]),
and Kodak (anti-Flag, M2).
EXAMPLE 2
Expression, Purification and Phosphorylation of Recombinant
Proteins
[0272] In this Example, recombinant proteins were expressed,
purified and phosphorylated. In these experiments, insect cells and
baculoviruses were used. Baculovirus expression vectors were
generated in this Example using the vectors in combination with
linearized BaculoGold or AcMNPV wild-type DNA (Pharmingen). The
viruses, their tags, and base vectors are listed in Table 1.
[0273] Cdc4.DELTA.WD is a mutant version of Cdc4 that contains a
stop codon at residue 566, which removes the last three WD-40
repeats. Gst-Cdc28HA (D154N), also referred to as
"Gst-Cdc28HA(K-)," is a kinase-impaired form of Cdc28. In complexes
with either Cln1 or Clb5, this kinase was found to exhibit <2%
activity toward histone H1.
[0274] For expression of His.sub.6Cdc34 and His.sub.6-Sic1,
plasmids were transformed into BL21 (DE3) cells (Novagen). One
liter of cells were grown in LB containing 0.1 mg/ml ampicillin, at
37.degree. C., until an OD.sub.600 of 0.8 was reached. Expression
was then induced with 400 mM IPTG for three hours. Cells were
harvested by centrifugation (2,000.times.g, for 10 minutes), lysed
in 70 ml of 20 mM sodium phosphate buffer (pH 7.5) containing 500
mM NaCl, and 0.1 mg/ml lysozyme (Sigma), and incubated for 45
minutes on ice. Insoluble material was removed by centrifugation
(14,000.times.g, for 20 minutes). The lysate was then incubated
with 0.5 ml Ni.sup.+2-NTA (Qiagen) resin as directed by the
manufacturer. The protein was eluted with 20 mM sodium phosphate
(pH 6) containing 500 mM NaCl and 200 mM imidazole, and stored at
-80.degree. C.
2TABLE 1 Baculovirus Expression Vectors Virus Tag Base Vector Cak1
None pVL Cdc4 None pBBIII Cdc4.DELTA.WD None pBBIII Cdc4.sup.F
C-terminal Flag pBBIII Cdc34 None pBBIII Cdc53.sup.M N-terminal Myc
pBBIII Clb5 None pVL Cln1.sup.HA C-terminal HA pBBIII Cln2.sup.HA
C-terminal HA pVL Gst-Cdc28.sup.HA N-terminal Gst pVL C-terminal HA
Gst-Cdc28.sup.HA (D154N) N-terminal Gst pVL C-terminal HA
Grr1.sup.G10 N-terminal His.sub.6-G10 pBBHis His.sup.6-Cks1
N-terminal His.sub.6 pVL Sic1 None pBBIII Skp1 None pVL Skp1.sup.F
N-terminal Flag pBBIII Gst-Skp1 N-terminal Gst pVL
[0275] For recombinant protein expression and assembly of
complexes, 4.times.10.sup.5 insect cells (Hi5, Invitrogen) were
infected with the indicated virus combinations for 40 hours. These
combinations included baculoviruses expressing Myc-tagged Cdc53
(Cdc53.sup.M), Cdc34, Cdc4, and Skp1. Cells were then harvested and
disrupted in lysis buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl,
0.5% Nonidet P40, 10 mM NaF, 10 mM .beta.-glycerol phosphate, 1 mM
PMSF, and 5 .mu.g/ml each leupeptin, antipain, and aprotinin). For
isolation of protein complexes, typically about 3 ml of lysis
buffer was used per 0.5.times.10.sup.8 cells.
[0276] To examine the assembly of recombinant yeast proteins, 0.4
ml lysate were typically derived from 2.times.10.sup.6 cells. In
both cases, cell lysates were centrifuged for 2 minutes at
14,000.times.g, prior to affinity- or immuno-purification.
Immunopurification was performed by incubating the lysates at
4.degree. C. for 2 hours with 4 .mu.g of the anti-Myc or anti-G10
antibody and 8 .mu.l of Protein A-Sepharose, or with 8 .mu.l of
immobilized anti-Flag antibodies (Kodak; See, Example 1). Immune
complexes were washed three times with 1 ml of lysis butter prior
to SDS-PAGE.
[0277] For SDS-PAGE, an equal volume of 2.times. sample buffer (250
mM Tris (pH 6.8), 4% SDS, 20% glycerol, 10% 2-mercaptoethanol) was
added to the samples to be tested, and boiled for 2 minutes.
Samples were then electrophoresed in 12% polyacrylamide gels with
35 mA constant current. Proteins were transferred to nitrocellulose
filters using a BioRad transfer apparatus in 50 mM Tris/glycine
buffer (pH 8), containing 20% methanol, for three hours, at 350 mA.
The nitrocellulose filters were then blocked with 5% non-fat dry
milk solution for 1 hour, followed by incubation overnight with
primary antibody. The antibody dilution used was 1:1000 for
anti-Cdc4, anti-Cdc34, anti-gene10, anti-Sic1, anti-myc, and
anti-HA; the anti-Skp1 antibody was diluted 1:4000. Blots were
washed in TBST (20 mM Tris (pH 8), 100 mM NaCl, 0.5% Tween-20) for
30 minutes, and then incubated with either goat anti-rabbit
conjugated horseradish peroxidase (HRP) or rabbit anti-goat
conjugated HRP (Promega), as appropriate, at a dilution of
1:25,000, for 30 minutes. Immunoblots were then washed with TBST
for 30 minutes, and developed using enhanced chemiluminescence
detection (Amersham) as described by the supplier.
[0278] As shown in FIG. 1A, in the presence of all four proteins
(Cdc53.sup.M, Cdc34, Cdc4, and Skp1), anti-Cdc53.sup.M complexes
contained Cdc4, Cdc34, and Skp1. However, in the absence of Skp1,
only low levels of Cdc4 bound to Cdc53.sup.M, regardless of the
presence of Cdc34 (FIG. 1A, lanes 7 and S). This result was
confirmed through the analysis of Cdc53.sup.M association with
anti-Cdc4 immune complexes (See, FIG. 1B). Thus, Skp1 was shown to
facilitate association of Cdc53 with Cdc4. In contrast, both Skp1
and Cdc34 can simultaneously associate with Cdc53.sup.M in the
absence of other yeast proteins (See; FIGS. 1A and 1C). Together,
these data indicated that Cdc34, Cdc53, Skp1, and Cdc4 form a
multiprotein complex.
[0279] A. Sic1/Clb5/Gst-Cdc28HA(K-) Complexes
[0280] Sic1/Clb5/Gst-Cdc28HA(K-) complexes were purified from
4.times.10.sup.8 cells, as described by Connell-Crowley et al.
(Connell-Crowley et al., Mol. Biol. Cell., 8:287-301 [1997]).
Briefly, eight T-150 flasks of insect cells (Highfive, Invitrogen)
were infected with 1 ml each of baculoviruses expressing either
GST-Cdc28HA, Cln1HA, Cks1, and Cak1, or baculoviruses expressing
Gst-Cdc28HA(K-), Clb5, and Sic1. After 40 hours, the cells were
lysed at 4.degree. C., in 6 ml of NETN (20 mM Tris-HCl, pH 8, 100
mM NaCl, 1 mM EDTA, 0.5% NP-40, 5 mM NaF, 30 mM
p-nitrophenylphosphate, 1 .mu.g/ml each leupeptin and antipain, and
1 mM PMSF). Lysates were cleared by centrifugation at
14,000.times.g for 10 minutes. Supernatants were rotated with 0.2
ml of GSH-Sepharose for 60 minutes at 4.degree. C., and the beads
were washed three times with 2 ml of the lysis buffer, followed by
two washes with 100 mM Tris (pH 8), 100 mM NaCl. Proteins were then
eluted with 0.2 ml of 100 mM Tris (pH 8), 100 mM NaCl, 40 mM
glutathione (Sigma), and 10% glycerol. The proteins were then
stored at -80.degree. C. until use.
[0281] B. Gst-Cdc28HA/ClnHA/Cks1 and Gst-Cdc28HA(K-)/ClnHA/Cks1
Complexes
[0282] Gst-Cdc28HA/ClnHA/Cks1 (i.e., "ClnHA/Gst-Cdc28HA/Cks1" in
the legend for FIG. 2A) and kinase impaired
Gst-Cdc28HA(K-)/Cln1HA/Cks1 complexes were prepared as described
above, as were cells co-infected with viruses expressing
appropriate proteins, and CAK1 expressing virus generated from a
cDNA generously provided by C. Mann (See, Thuret et al., Cell
86:565-576 [1996]). The presence of Cks1 and Cak1 resulted in a
5-fold increase in the yield of active Cln/Cdc28 kinase complexes,
as purified after insect cell co-infection (determined using
histone HI as a substrate). FIG. 2A shows an SDS-PAGE analysis of
purified Cdc28HA/Cks1. In this Figure, the asterisk indicates the
position of endogenous GST protein.
[0283] C. Phosphorylated Sic1 Complexes
[0284] Phosphorylated Sic1 complexes were generated by incubating
2.5 .mu.M Sic1/Clb5/Gst-Cdc28HA(K-) with Gst-Cdc28HA/Cln1HA/Cks1
(50 nM) and 1 mM ATP in kinase buffer (50 mM Tris HCl (pH 7.5), 50
mM NaCl, 10 mM MgCl.sub.2) for 45 minutes at 25.degree. C. Control
unphosphorylated Sic1 complexes were produced in an identical
fashion by omitting Cln1 kinase. Cln/Cdc28 autophosphorylation was
performed by incubating 200 nM Cln/Cdc28 complexes with 1 mM ATP in
kinase buffer at 25.degree. C. for 1 hour. To generate
phosphorylated Sic1 free of Cln/Cdc28 kinase, bacterial Sic1 (0.5
.mu.M) was incubated with 2 mM ATP and Cln2/Gst-Cdc28/Cks1
immobilized on GST-Sepharose (Pharmacia) for 60 minutes at
37.degree. C. Forty ng of phosphorylated Sic1 were removed from the
beads for use in ubiquitination reactions, at a final concentration
of 1 nM. For .sup.32P-labeling of Sic1 and Cln1 proteins, kinase
reactions were performed at 25.degree. C. for 30 minutes, using 50
.mu.M (.gamma.-.sup.32P ATP (0.3 nCi/pmol)) followed by incubation
with 1 mM unlabeled ATP for an additional 30 minutes.
[0285] FIG. 2B shows the gel results of phosphorylation of Sic1 by
Cln1/Cdc28 complexes in vitro. The result for Sic1/Clb5/Gst-Cdc28HA
(K-) incubated with ATP are shown in lane 1, while the result for
Cln1/Cdc28 and ATP is shown in lane 2. Lane 3 shows the reaction
products obtained when Cln1/Cdc28 complexes alone were incubated
with .gamma.-.sup.32P ATP. In lanes 4 and 5, the results from
experiments in which smaller amounts of Sic1 phosphorylation
reactions with 50 nM of Sic1 were performed in the presence of
.gamma.-.sup.32P ATP.
[0286] D. Grr1 Complexes
[0287] The Grr1 complexes were prepared by infecting one T-150
flask of insect cells as described above, with baculoviruses
expressing Grr1G10, Skp1, and Cdc53.sup.M, or variations thereof.
Forty hours after infection, the cells were lysed in 3 ml of NETN,
and the lysates cleared by centrifugation at 14,000.times.g for 10
minutes. Ten percent of each lysate was used for
immunoprecipitation with 5 .mu.g of anti-gene 10 antibodies
(Novagen), and 8 .mu.l of protein A-Sepharose (4.degree. C., for 90
minutes). The immune complexes were washed three times with 1 ml
NETN prior to use in binding experiments or ubiquitination
reactions.
[0288] The complexes were immunoprecipitated with either (A) a Myc
tag on Cdc53 (Cdc53.sup.M) using anti-Myc antibodies or (B) a Flag
tag on Cdc4 (Cdc4.sup.F) as described in Example 3. Immune
complexes were immunoblotted and probed with anti-Myc to detect
Cdc53.sup.M, anti-Cdc4, anti-Cdc34, and anti-Skp1 as described in
Example 3 (See, FIG. 1).
EXAMPLE 3
In Vitro Binding Assays
[0289] Binding reactions were performed at 4.degree. C. for 1 hour,
in 100-250 ml mixtures containing appropriate immunopurified
complexes prepared as described in Example 2, and affinity purified
Sic1 (20 nM) or Cln (2 nM) complexes. Associated proteins were then
washed three times with 1 ml of lysis buffer prior to SDS-PAGE and
immunoblotting, were performed as described above.
[0290] In some experiments, .sup.32 P-labeled Sic1 or Cln complexes
were employed at similar concentrations, and detected by
autoradiography and phosphoimager analysis. Based on protein
staining with Coomassie Blue or silver, the quantities of proteins
in anti-Skp1.sup.F immune complex from Skp1.sup.F/Cdc53.sup.M/Cdc4
expression cells was estimated to be: Skp1.sup.F (1 .mu.g),
Cdc53.sup.M (200 ng), and Cdc4 (200 ng). Likewise, the levels of
proteins in the anti-Grr1G10 complex were: Grr1G10 (100 ng),
Cdc53.sup.M (40 ng), and Skp1 (20 ng).
[0291] In additional experiments, insect cells were co-infected
with constant quantities of baculovirus expressing Skp1.sup.F and
increasing quantities of baculoviruses expressing either Cdc4, or a
C-terminal truncated form of Cdc4 lacking the last three WD-40
repeats (i.e., Cdc4.DELTA.WD; lanes 12-17). Lysates were
immunoprecipitated with anti-Flag antibodies to precipitate
Skp1.sup.F complexes. Binding reactions with phosphorylated Sic1
complexes and detection of bound protein were performed as
described above.
[0292] FIG. 2C indicates that phosphorylation of Sic1 is required
for its association with Cdc34/Cdc53/Skp1/Cdc4 complexes. As shown
in this Figure, phosphorylated Sic1 efficiently associates with
Cdc53/Skp1/Cdc4 complexes, and this association is dependent upon
the presence of Skp1 (See, FIG. 2C, lanes 6 and 8). Typically,
10-20% of the input phosphorylated Sic1 was bound at about 20 nM
Sic1. In contrast, the extent of binding of unphosphorylated Sic1
(lane 7) was comparable to that observed in control immune
complexes generated from uninfected cells (lane 3) and was <1%
of the input Sic1. Consistent with the results in FIG. 1, the level
of Cdc4 found in immune complexes lacking Skp1 were >10-fold
lower than that found in the presence of Skp1. These data suggest
that Cdc4 and/or Skp1 function as binding factors for Sic1 and that
association of Sic1 with this complex requires phosphorylation by
Cln1/Cdc28.
[0293] FIG. 2D shows that association of phosphorylated Sic1 with
Cdc4 is enhanced by Skp1. In this Figure, lanes 3-9 contain
anti-Flag immune complexes derived from cells infected with
constant high quantities of a baculovirus expressing Cdc4.sup.F,
while lanes 4-10 contain increasing quantities of a baculovirus
expressing Skp1 in in vitro binding reactions with purified
Cln1/Cdc28-phosphorylated Sic1. While Skp1 alone did not interact
with Sic1, it stimulated association of Sic1 with Cdc4 by about
5-fold (FIG. 2D). The weak association of Sic1 with Cdc4 alone
(FIG. 2D, lane 3) may reflect the participation of an insect cell
Skp1 homolog. The results described herein clearly demonstrate a
positive contribution of Skp1 in the Cdc4/Sic1 interaction.
[0294] FIG. 2E shows that association of phosphorylated Sic1 with
Skp1 requires the WD-40 repeats of Cdc4. In this Figure, lanes 4-9
contain proteins obtained from insect cells co-infected with
constant quantities of baculovirus expressing Skp1.sup.F, and
increasing quantities of baculoviruses expressing Cdc4, while lanes
12-17 contain lysates from cells co-infected with constant
quantities of baculovirus expressing Skp1.sup.F and increasing
quantities of baculoviruses expressing a C-terminal truncated form
of Cdc4 lacking the last three WD-40 repeats (i.e., Cdc4.DELTA.WD).
Association of phosphorylated Sic1 with anti-Skp1.sup.F immune
complexes was absolutely dependent upon the presence of Cdc4 (See,
FIG. 2E, lanes 3 and 9). Moreover, deleting the last three WD-40
repeats from the C-terminus of Cdc4 abolished its ability to
associate with phosphorylated Sic1 (FIG. 2E, lanes 10-16).
Therefore, Cdc4 functions as the specificity factor for binding of
phosphorylated Sic1 and the Cdc4-Sic1 interaction requires an
intact WD-40 repeat domain in Cdc4.
EXAMPLE 4
Ubiquitination Assays
[0295] In this Example, ubiquitination reactions were conducted. In
these experiments, Ni.sup.2+-NTA resin was used to isolate
ubiquitinated proteins from extracts of wild-type cells or sic1
deletion mutants expressing His.sub.6-Ub.sup.RA or Ub.sup.RA
(Willems et al., [1996], supra). In addition, once the strategy to
generate Cdc4/Skp1/Cdc53 complexes that recognized phosphorylated
Sic1 was developed, experiments to determine whether these
complexes can catalyze ubiquitination of Sic1 in vitro when
supplemented with Cdc34, E1, ATP, and ubiquitin were conducted.
[0296] In some experiments, bacterial Sic1 was used and where
indicated, was phosphorylated with soluble or immobilized
Gst-Cdc28HA/Cln2HA prior to use. Bacterial Sic1 ubiquitination
reactions employed 100 nM yeast E1 (a gift from S. Sadis and D.
Firley, Department of Cell Biology, Harvard Medical School).
[0297] A. Ubiquitination of Sic1 In Vivo
[0298] To identify Sic1-ubiquitin conjugates in vivo, 200 ml
(10.sup.7 cells/ml) of wild-type (MT235), or a sic1 deletion
(MT767) cells expressing either pCUP1-UB1.sup.RA (<pUB204>)
or pCUP1-UB1.sup.HIS-MYC-RA (<pUB223>) were prepared, and
lysates were generated in 50 mM Tris-HCl (pH 7.5), 100 mM NaCl,
0.1% NP-40, 1 mM PMSF, 0.6 mM dimethylaminopurine, 1 .mu.g/ml
leupeptin, 1 .mu.g/ml pepstatin, 10 .mu.g/ml Tosylphenyl
chloromethyl ketone and 10 .mu.g/ml soybean trypsin inhibitor as
described by Willems et al. (Willems et al., Cell 86:453-463
[1996]). Briefly, 8 .mu.g of yeast protein was incubated with 12
.mu.l of N+.sup.2NTA beads (Qiagen) for 1 hour at 4.degree. C., as
described by the manufacturer. The beads were then washed 3 times
in lysis buffer, 1 time in high salt buffer (50 mM Tris-HCl, pH
8.0, 0.5 M NaCl), and the proteins were eluted with 10 .mu.l of 100
mM Tris-HCl, pH 6.8, 1% SDS, 100 .mu.M DTT, 100 .mu.M EDTA.
Proteins were separated by SDS-PAGE and immunoblotted with
anti-Sic1 antibodies, as described above.
[0299] FIG. 3 shows that phosphorylated Sic1 is ubiquitinated in
vivo and in vitro with purified Cdc34 E2 and Cdc53/Skp1/Cdc4
complexes. In this Figure, the position of Sic1 and Sic1-ubiquitin
conjugates are indicated (i.e., "Sic1" and "Sic1-Ub,"
respectively).
[0300] B. Reconstitution of the Sic1 Ubiquitination Pathway Using
Recombinant Proteins
[0301] Ubiquitination reactions contained immune complexes prepared
from 2.times.10.sup.6 cells and equilibrated with ubiquitination
buffer (100 mM Tris-HCl (pH 7.5), 5 mM MgCl.sub.2, 0.6 mM DTT), 500
nM bacterial Cdc34, 300 nM human E1 (a gift from M. Rolfe,
Mitotix), 2 mM ATP, and 7 mM yeast ubiquitin (Sigma) or
Gst-UB.sup.RA (purified from bacteria expressing GEX-U6.sup.RA
[provided by M. Tyers, University of Toronto], and the method
described in Example 1 for GST-Skp1), and 80 ng of Sic1 complexes
in a final volume of 14 .mu.l, excluding bead volume. The human E1
purification was described by Rolfe et al. (Rolfe et al., Proc.
Natl. Acad. Sci. USA 92:3264-3268 [1995]). Reactions were allowed
to proceed at 25.degree. C. for 1 hour or as indicated, quenched
with 2.times. sample buffer (250 mM Tris (pH 6.8), 4% SDS, 20%
glycerol, and 10% 2-mercaptoethanol), and analyzed by SDS-PAGE and
immunoblotting with anti-Sic1 antibodies as described above.
[0302] The results indicated that in the presence of all reaction
components, phosphorylated Sic1 was efficiently converted to higher
molecular weight conjugates detectable with anti-Sic1 antibodies
(See, FIG. 3B, lane 6; and FIG. 3C, lane 5). In contrast,
unphosphorylated Sic1 was not detectably ubiquitinated. Sic1
ubiquitination absolutely required Cdc34, Cdc4, Cdc53, Skp1, E1 and
ubiquitin (See e.g, FIG. 3B and FIG. 3C). The pattern of high
molecular weight Sic1 conjugates obtained in reactions with
ubiquitin was different from that observed when Gst-Ub.sup.RA was
used as the ubiquitination source as shown in lanes 5 and 11 of
FIG. 3C. These results confirm that the high molecular weight forms
observed are products of ubiquitination. With Gst-Ub.sup.RA, the
Sic1 reaction products were integrated into a ladder of bands
differing by approximately 35 kDa, the size of Gst-Ub.sup.RA (See,
FIG. 3C, lane 11).
[0303] The ubiquitination reaction was time dependent and the
reaction efficiency ranged from 10-40% of the input Sic1 protein
(FIGS. 3B and 3C). When the reaction was performed with pre-bound
Sic1, the efficiency was greater than 50%. In addition, greater
than 50% of the Sic1 ubiquitin conjugates formed after 60 minutes
were found to have dissociated from the Cdc4/Skp1/Cdc53 complex. In
addition, neither Gst-Cdc28, Clb5, Cdc53, Skp1, or Cdc4 formed
ubiquitin conjugates under the reaction conditions employed,
although Cdc34 was ubiquitinated as previously reported (Haas et
al., J. Biol. Chem., 266:5104-5112 [1991]).
[0304] C. Ubiquitination of Sic1 in Association with Clb5-Cdc28
Complexes
[0305] To test whether Sic1 ubiquitination requires association
with Clb5/Cdc28 complexes, ubiquitination reactions using Sic1
produced in bacteria, with or without phosphorylation with
Cln2/Cdc28 were performed as described above, with yeast E2
replacing human E1. To verify the absence of Cln2HA/Cdc28HA in the
ubiqutination reaction, Sic1 proteins were also immunoblotted with
anti-HA antibodies.
[0306] The results shown in FIG. 3D indicate that ubiquitination of
Sic1 does not require that Cln/Cdc28 be present in the
ubiquitination reaction, nor that Sic1 be associated with
Clb5/Cdc28. In this Figure, lane 1 contains Sic1 purified from
bacteria, while lane 2 contains Sic1 treated with soluble
Cln2/Gst-Cdc28, and lane 3 contains immobilized Cln2/Gst-Cdc28. Use
of phosphorylated Sic1 that was free of Cln2 kinase is indicated by
an asterisk (lanes 3 and 9). As in the case of Sic1 assembled in
insect cells with Clb5/Cdc28, phosphorylated Sic1 from bacteria was
efficiently ubiquitinated, with greater than 90% of the Sic1
forming ubiquitin conjugates (lane 8), and ubiquitination
absolutely required Sic1 phosphorylation (lane 4).
[0307] Although phosphorylation of Sic1 was required for its
recognition by Cdc4 and Skp1, it remained possible that Cln/Cdc28,
present in small amounts in the ubiquitination reaction, is also
required for additional steps in the ubiquitination process, for
instance, to phosphorylate the ubiquitination machinery. To rule
out this caveat, bacterial Sic1 was treated with Cln2/Gst-Cdc28
complexes immobilized on Gst-Sepharose beads, removed from the
beads prior to use in ubiquitination reactions, and determined to
be free of soluble kinase by immunoblotting with anti-HA
antibodies. These results are shown in FIG. 3D, lane 3. Sic1
phosphorylated in this manner was also efficiently ubiquitinated
(See, FIG. 3D, lane 9). These data indicate that Sic1
phosphorylation constitutes the primary requirement of Cln/Cdc28
kinases in Sic1 ubiquitination in the in vitro reaction.
[0308] D. Clb5/Cdc28-phosphorylated Sic1 as a Substrate for
Ubiquitination
[0309] In these experiments, it was found that
Clb5/Cdc28-phosphorylated Sic1 was also a substrate for
ubiquitination. In these experiments, constant amounts of Sic1 were
treated with increasing amounts of Clb5/Cdc28, until the kinase was
in excess as determined by histone kinase assays. Under these
conditions, Sic1 electrophoretic mobility was reduced (FIG. 3E,
lanes 1-6, top), as determined by immunoblotting.
[0310] Aliquots of differentially phosphorylated Sic1 were used in
ubiquitination reactions with immunopurified Cdc53.sup.M/Cdc4/Skp1
complexes supplemented with Cdc34, E1, ubiquitin, and ATP for 30
minutes, as described above (See, FIG. 3E, lanes 1-6). As a
negative control, partially phosphorylated Sic1 corresponding to
the Sic1 protein in lane 5 (top) of FIG. 3E, was reacted in the
absence of Cdc34 (lane 7) or the Cdc53.sup.M/Cdc4/Skp1 complex
(FIG. 3E, lane 8). Sic1 ubiquitination was determined by
immunoblotting with anti-Sic1 antibodies (FIG. 3E, bottom).
[0311] Although Sic1 is an inhibitor of Cdc28/Clb5 complexes, when
the kinase complex was in excess of Sic1, Sic1 was phosphorylated
as determined both by reduced electrophoretic mobility (See, FIG.
3E) and .sup.32P incorporation. This result may explain the fact
that overexpression of CLB5 can drive S-phase entry in cln- cells,
and suggests that active Clb5/Cdc28 formed during Sic1 destruction
may collaborate with Cln/Cdc28 to complete the Sic1 ubiquitination
process.
[0312] E. Sic1 Binding and Ubiquitination with Grr1
[0313] In these experiments, the Cdc4 was substituted with another
F-box protein (Grr1) in order to determine if this protein could
support Sic1 binding and ubiquitination. Grr1 has an F-box near its
N-terminus and can interact simultaneously with Skp1 and Cdc53 when
co-expressed in insect cells (See e.g., Figure A).
[0314] These experiments were conducted as described above, with
the exception being that Grr1 was substituted for Cdc4
(approximately 100 ng). Proteins were separated by SDS-PAGE, and
blotted with anti-Skp1 anti-Myc to detect Grr1G10 and Cdc53.sup.M,
with anti-Skp1 antibodies.
[0315] It was found that that Grr1 and Cdc4 with Skp1/Cdc53 are
mutually exclusive. In contrast with Cdc4, it was not possible to
demonstrate enhancement of the Grr1/Cdc53 interaction in insect
cells by co-expression of Skp1, even though Skp1 assembled with
these complexes. Importantly, Grr1 assembled with Cdc53/Skp1
complexes was unable to associate with phosphorylated Sic1, and was
unable to support ubiquitination of phosphorylated Sic1 complexes
in the in vitro system with purified proteins under conditions
where Cdc4 readily facilitated Sic1 binding and ubiquitination
(See, FIGS. 4B and 4C). Therefore, F-box proteins display
selectivity toward particular targets.
[0316] FIG. 4A shows that Grr1 can associate with Skp1 and Cdc53,
while FIG. 4B shows that phosphorylated Sic1 associates with Cdc4
but not Gir1-containing complexes. In this Figure, lanes 2-5
contain anti-Skp1.sup.F immune complexes derived from insect cells
infected with the indicated baculovirus combinations were used for
binding reactions with .sup.32P-labeled Sic1 complexes. Ten percent
of the input Sic1 complex (lane 1) was included as a control. The
presence of Cdc4, Skp1, Cdc53, and Grr1 was verified by
immunoblotting.
[0317] FIG. 4C shows that Cdc4, but not Grr1, supports
ubiquitination of Sic1 in vitro. The indicated anti-Skp1.sup.F
immune complexes were used in ubiquitination assays as described
for FIG. 3 (above) employing Gst-Ub.sup.RA as the ubiquitin source.
Finally, FIG. 4 shows the results verifying the presence of
reaction components derived from immunoprecipitation (in this
Figure, the blot used for ubiquitination assays was reprobed to
detect Grr1G10, Cdc53.sup.M, and Cdc4).
EXAMPLE 4
Binding of Grr1 to Cln1 and Cln2
[0318] In this Example, the binding of Grr1 to Cln1 and Cln2 was
investigated. In particular, experiments were conducted in order to
determine whether Grr1 binds to Cln1 and/or Cln2 in a
phosphorylation-dependent manner. Indeed, the finding that Sic1 is
recognized by the F-box protein Cdc4, together with a genetic
requirement for the F-box protein Grr1 in Cln destruction, led to
these experiments to examine whether Grr1 functions in recognition
of phosphorylated Clns.
[0319] To generate Cln proteins for binding reactions,
Cln/Gst-Cdc28/Cks complexes were isolated from insect cells as
described in Example 2B. In the presence of ATP, both Cln1 and Cln2
are autophosphorylated, a modification that reduces their
electrophoretic mobility (see below). To examine whether Grr1 can
associate with phosphorylated Clns and to compare the extent of
selectivity of Grr1 and Cdc4 toward Cln binding, anti-Skp1.sup.F
immune complexes from cells co-expressing Grr1 or Cdc4 in the
presence or absence of Cdc53 prepared as described above, were used
in binding reactions with .sup.32P-labeled Cln1 or Cln2 kinase
complexes. .sup.32P-labeled Sic1 was used as a control for Cdc4
binding.
[0320] As shown in FIG. 5A, both Cln1 and Cln2 complexes associated
with Grr1/Skp1.sup.F/Cdc53 complexes with an efficiency of about
40% of the input Cln1 or Cln2 (FIG. 5A, lanes 5 and 12), and this
association did not require Cdc53 (FIG. 5A, lane 16). In contrast,
about 6% of the input Cln proteins associated with Cdc4/Skp1.sup.F
complexes independent of the presence of Cdc53 (FIG. 5A, lanes 7,
11, and 15), compared with 1% association in the absence of an
F-box protein (FIG. 5A, lanes 6, 10, and 14). The extent of
selectivity of these F-box proteins for Cln and Sic1 is further
reflected by the observation that Cln1 protein present in the
phosphorylated Sic1 preparation was selectively enriched in Grr1
complexes (FIG. 5A, lane 4). In this Figure, controls for the
extent of binding (indicated by the asterisk) were 20% of input Cln
and 10% of input Sic1. The presence of all proteins in the binding
reaction was confirmed by immunoblotting (FIG. 5B; in this Figure,
complexes used for binding experiments in FIG. 5A were
immunoblotted with the indicated antibodies to verify the presence
of Cdc4, Grr1G10, Cdc53.sup.M, and Skp1.sup.F), and the quantities
of Cdc4 and Grr1 were found to be comparable, based on Coomassie
staining of SDS gels of immune complexes. Thus, Grr1 and Cdc4
display specificity toward physiological substrates.
[0321] Next, Grr1 alone or in complexes with Skp1 or Skp1/Cdc53
were inmmunoprecipitated from insect cell lysates and used in
binding assays with phosphorylated or unphosphorylated Cln1
complexes prepared as described above. The results are shown in
FIG. 5C. As shown, unphosphorylated Cln1 was produced in insect
cells as a complex with kinase deficient Gst-Cdc28(K-), which
minimized Cln1 autophosphorylation during expression, and allowed
the role of phosphorylation to be tested. FIG. 5C, lane 1 shows
that, as isolated, this Cln1 protein migrates as a homogeneous
species at approximately 66 kDa. In contrast, phosphorylated Cln1
(FIG. 5C, lane 2) undergoes a dramatic mobility shift to
approximately 80 kDa, consistent with in vivo observations. Lanes
4-11 contain anti-Grr1G10 complexes derived from the indicated
insect cell infections used in binding reactions with either
unphosphorylated Cln1HA complexes generated using kinase impaired
Gst-Cdc28(K-)HA (FIG. 5C, lane 1) or phosphorylated
Cln1HA/Gst-Cdc28HA complexes (FIG. 5C, lane 2). Anti-HA antibodies
were used to detect Cln1HA and Gst-Cdc28HA. Twenty percent of the
input Cln1HA complexes were run as controls (FIG. 5C, lanes 1 and
2). Cln1HA isolated from insect cells in complexes with active
Cdc28 migrated as a series of modified forms, reflecting partial
phosphorylation of Cln in vivo in insect cells (See, FIG. 2A).
Incubation of such ClnHA/Cdc28HA complexes with ATP quantitatively
shifts Cln1 HA to a single form migrating as an approximately 84
kDa protein. The blot was reprobed to verify the presence of
Grr1G10, Cdc53.sup.M, and Skp1.sup.F.
[0322] Phosphorylated Cln1 (and its associated Cdc28 protein)
efficiently associated with all Grr1 complexes (FIG. 5C, lanes 6,
8, and 10), but was absent from control binding reactions lacking
Grr1 (FIG. 5C, lane 4). In contrast, the levels of unphosphorylated
Cln1 associated with Grr1 complexes were comparable to that found
in binding reactions lacking Grr1 (FIG. 5C, lanes 3, 5, 7, and
9).
[0323] It was also determined that purified Skp1/Cdc53/Grr1
complexes are not sufficient for Cln1 ubiquitination by Cdc34 in
vitro. Thus, association of both Cln1 with Grr1 and Sic1 with Cdc4
was found to be greatly enhanced by phosphorylation.
Anti-Skp1.sup.F immune complexes were purified from insect cells
infected with the indicated baculoviruses and supplemented with E1,
Cdc34, Gst-Ub.sup.RA, ATP, and either .sup.32P-labeled Sic1 or
Cln1, as described above (e.g., FIG. 3).
[0324] As shown in FIG. 5D, although the Grr1/Skp1/Cdc53 complex is
capable of binding efficiently to phosphorylated Cln1, it was not
competent for Cln1 ubiquitination when supplemented with Cdc34 and
E1. Moreover, FIG. 5D shows that Cdc4 complexes that functioned in
Sic1 ubiquitination also failed to catalyze ubiquitination of Cln1,
despite the fact that Cln1 can associate, albeit weakly, with Cdc4
(See, FIG. 5A). In contrast, identical preparations of
phosphorylated Cln1 protein were efficiently ubiquitinated in
partially purified yeast lysates in a Cdc34 dependent manner (FIG.
5E), indicating that this preparation of Cln1 is competent for
ubiquitination.
EXAMPLE 5
Ubiquitination of Phosphorylated Cln1
[0325] In this Example, preparations of phosphorylated Cln1 (as
described above), were ubiquitinated in partially purified yeast
lysates in a Cdc34-dependent manner.
[0326] In these experiments, 0-100 .mu.g YFII (a 250 mM NaCl eluate
from a DEAE-cellulose column prepared exactly as described in
Deshaies et al. [1995], supra) was supplemented with 500 nM Cdc34,
100 nM human E1, ubiquitin, and an ATP regenerating system (2 mM
ATP, 600 mM creatine phosphate, and 0.15 mg/ml creatine kinase).
The ubiquitination reaction was initiated by addition of 20 ng
Cln1HA/Gst-Cdc28HA/Cks1. After incubation for 60 minutes at
25.degree. C., the reactions were quenched and immunoblotted with
anti-HA antibodies to detect Cln1HA and Gst-Cdc28HA. In FIG. 5E,
the protein indicated by an asterisk is a yeast protein in YFII
that cross-reacts with the anti-HA antibodies used. As indicated in
this Figure, this preparation of Cln1 is competent for
ubiquitination.
EXAMPLE 6
Identification of Human F-Box Proteins
[0327] In this Example, new human F-box proteins were identified,
using a two hybrid system. The SKPI open reading frame (as an
NdeI/BamHI restriction fragment) was subcloned into pAS2 (See,
Harper et al., Cell 75:805-816 [1993]). pAS2-SKP1 was transformed
into yeast strain Y190, and this strain was then used in a two
hybrid screen with a human breast cDNA library generated in
.lambda.ACTII as described by below.
[0328] Yeast strain Y190 was deposited with the ATCC and assigned
number (96400). Y190 was grown in YPD medium (10 g/l yeast extract,
20 g/l peptone and 20 g/l dextrose) containing 10 mg/ml
cycloheximide or on YPD plates (YPD medium containing 20 g/l agar)
containing 10 mg/ml cycloheximide. Y190 contains two chromosomally
located reporter genes whose expression is regulated by Gal4.
[0329] The first reporter gene is the E. coli lacZ gene which is
under the control of the GAL1 promoter. The second reporter gene is
the selectable HIS3 gene which encodes the enzyme imidazole
glycerol phosphate (IGP) dehydrogenase. Yeast cells which express
the HIS3 gene product can be selected by their ability to grow in
medium lacking histidine (i.e., SC-his medium). The .lambda. ACTII
phage cloning vector was deposited with the ATCC and assigned
number 87006. This .lambda. ACTII phage cloning vector was
deposited as a lysogen in JM107 cells which are grown in LB
containing 50 .mu.g/ml ampicillin.
[0330] Yeast cells (strain Y190) containing specific nutritional
markers were grown on SC medium lacking one or more amino acids. SC
medium lacking a particular amino acid is referred to as dropout
media. SC medium is made using the following components:
10.times.YNB (67 g yeast nitrogen base without amino acids in 1
liter water, filter-sterilized and stored in the dark).
[0331] Dropout mixture components:
3 adenine 800 mg arginine 800 mg aspartic acid 4000 mg histidine
800 mg leucine 2400 mg lysine 1200 mg methionine 800 mg
phenylalanine 2000 mg threonine 8000 mg tryptophan 800 mg tyrosine
1200 mg uracil 800 mg
[0332] To make a dropout mixture, the above components are weighed
out, leaving out the amino acids to be selected for, combined, and
ground into a fine powder using a mortar and pestle.
[0333] SC-Trp plates comprise per liter: 870 mg dropout mixture
(minus tryptophan), 20 g dextrose, 1 ml 1N NaOH, 20 g agar, water
to 900 ml. The mixture is then autoclaved. After autoclaving, 100
ml 10.times.YNB is added just prior to pouring the plates.
[0334] The bacterial strain used was E. coli strain BNN132 (ATCC
47059). These cells were grown in LB (10 g/l bacto-tryptone
[DIFCO], 5 g/l bacto-yeast extract [DIFCO], 10 g/l NaCl, pH
adjusted to 7.0 with NaOH). E. coli strain BL21(DE3) (Invitrogen)
was grown in LB.
[0335] As described in more detail below, the pAS2/Skp1/Y190 strain
was transformed with 0.05 mg of plasmid library and 5 mg of carrier
total yeast RNA, and transformants were plated on a minimal media
lacking histidine, leucine, and tryptophan, but containing 25 mM
3-aminotriazole. After 5 days at 30.degree. C., plasmids were
recovered from .beta.-galactosidase positive colonies (See, Harper
et at, Cell 75:805-816 [1993]).
[0336] Also as described in more detail below, sequencing of cDNA
inserts from positive plasmids revealed the presence of one cDNA
containing significant sequence identity to Cdc4 in the F-box
domain of Cdc4. This cDNA is referred to as "F3 gamma." Other F-box
containing, cDNAs were identified by searching the EST (expressed
sequence tag) database, with the F-box region from F3 gamma. As
novel F-box containing proteins were identified these were used to
further search the EST database, in order to identify other novel
F-box proteins. For some of these, both the human and mouse
homologs were identified. It is contemplated that these new F-box
proteins act as components of E3 complexes in mammalian cells
(i.e., analogous to Cdc4 in budding yeast). Table 2 below lists the
protein sequences identified in these experiments, while Table 3
provides the corresponding DNA sequences. FIG. 7 provides the
alignments of these F-box proteins, with gaps indicated by dashes.
Table 4 provides longer (i.e., more complete) cDNA and amino acid
sequences for some of the F-box proteins identified in the
preliminary experiments. The sequences included in Table 4 contain
at least a large portion of the open reading frames (ORFs), and
contain potential target binding domains. Both F1 and F2 contain
leucine rich repeats (e.g., similar to Grr1). Thus, the present
invention provides numerous sequences suitable for detection and
identification of additional F-box proteins, as well as targets for
intervention in the proteolysis pathways (e.g., for drugs and other
compounds suitable for use to either enhance or reduce the
efficiency and/or function of the F-box).
[0337] A. Generation of Human Breast Tissue cDNA Library in
pACTII
[0338] In order to facilitate the isolation of F-box gene sequences
using the yeast two-hybrid system, an human breast tissue cDNA
expression library was constructed in the .lambda. ACTII phage
cloning vector. This cloning vector allows for the construction of
cDNA libraries fused to sequences encoding the Gal4 transcriptional
activation domain. The phage can be converted to a plasmid form
(pACTII) as described below.
[0339] An human breast tissue cDNA library was constructed using
.lambda. ACTII as follows. Total RNA from breast tissue of an adult
female obtained from reductive mammoplasty was provided by Dr. Anne
Bowcock (University of Texas Southwestern Medical Center). PolyA+
mRNA was produced using an mRNA isolation system (GIBCO-BRL). cDNA
synthesis was accomplished using a directional cDNA synthesis kit
from Stratagene as described by the manufacturer.
[0340] After the synthesis of the second strand, the cDNA (in a
volume of 400 .mu.l) was spermine precipitated by the addition of
22 .mu.l of 100 mM spermine. The mixture was incubated on ice for
30 min and then pelleted by centrifugation in a microcentrifuge
(Eppendorf) for 15 min at 4.degree. C. The cDNA pellet was washed
three times for 30 min/wash with 1 ml of spermine wash buffer (70%
ethanol, 10 mM Mg (Ac).sub.2, 0.3 M NaAc at pH 7) and once with 1
ml of 70% ethanol. The cDNA was then dissolved in 50 .mu.l of TE
buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA).
[0341] The ends of the cDNA were made blunt by treatment with T4
DNA polymerase using conditions recommended by the manufacturer
(Stratagene). Following treatment with T4 DNA polymerase, 5 .mu.l
of 0.5 M EDTA was added and the mixture was extracted with
phenol/chloroform and precipitated with ethanol.
[0342] The precipitated cDNA, approximately 4 .mu.g, was
resuspended in 7 .mu.l of TE buffer and then ligated to 2 .mu.g of
a kinased adapter oligonucleotide in a total volume of 10 .mu.l at
4.degree. C. overnight (12-18 hr). The hybridized oligonucleotide
pair contained an EcoRI overhang. For example, the oligonucleotide
CGCGCG hybridized with AATTCGCGCG (SEQ ID NO:59) will create a
suitable EcoRI linker.
[0343] Following the ligation reaction, 170 .mu.l of TE buffer, 20
.mu.l of 1 M KCl and 10 .mu.l of 100 mM spermine were added. The
mixture was incubated on ice for 30 min and precipitated and washed
as described above. The adapted cDNA was resuspended in 20 .mu.l of
TE buffer and digested with XhoI prior to electrophoresis on a 1%
LMA gel. cDNA having a length of 600 bp or longer was excised from
the gel and purified using standard techniques prior to ligation
into .lambda. ACTII arms.
[0344] cDNA (0.1 .mu.g) was ligated with 2 .mu.g of .lambda. ACTII
plasmid DNA prepared as follows. One hundred micograms of .lambda.
ACTII plasmid DNA was digested with XhoI and EcoRI; the digestion
products were then precipitated with ethanol, briefly dried and
then resuspended in 190 .mu.l of TE buffer. Ten microliters of 10
mM spermine were added to the side of the tube and the contents
were mixed by rapid inversion of the tube. An immediate and obvious
precipitate formed and was pelleted by centrifugation for 2 sec in
a tabletop microcentrifuge. The pellet was then washed with
spermine wash buffer followed by a wash with 70% ethanol as
described above. The washed pellet was resuspended in 100 .mu.l of
TE buffer. This preparation of .lambda. ACTII plasmid DNA was then
used for ligation into the cDNA containing adapters (prepared as
described above).
[0345] The ligation of the adapted cDNA and digested .lambda. ACTII
plasmid DNA was performed in a volume of 4 .mu.l at 4.degree. C.
overnight. The ligation mixture was packaged using one Gigapack
Gold packaging extract (Stratagene) according to the manufacturer's
instructions. Approximately 1.times.10.sup.8 total recombinants
were obtained. The phage library was amplified on the LE392 strain
of E. coli (Stratagene).
[0346] Automatic subcloning conversion of the cDNA library in
.lambda. ACTII into plasmid (pACTII) was accomplished by the
incubation of 10.sup.9 phage particles with 2 ml of a fresh
overnight culture of E. coli strain BNN132 (ATCC 47059) in 10 mM
MgCl.sub.2 for 30 min at 30.degree. C. without shaking. Two
milliliters of LB (10 g/l bacto-tryptone, 5 g/l bacto-yeast
extract, 10 g/l NaCl, pH adjusted to 7.0 with NaOH) was then added
and the cells were incubated with shaking for 1 hr at 30.degree. C.
The cells were then plated on ten 150 mm LB plates (15 g/l
bacto-agar in LB) containing 50 .mu.g/ml ampicillin and incubated
overnight at 37.degree. C.
[0347] Ampicillin-resistant cells were collected by scraping the
plates; the cells were then added to 3 liters of terrific broth (12
g/l bacto-tryptone, 24 g/l bacto yeast extract and 100 ml/1 of a
solution comprising 0.17 M KH.sub.2PO.sub.4, 0.72 M
K.sub.2HPO.sub.4) containing 50 .mu.g/ml ampicillin. The culture
was grown to stationary phase and plasmid DNA was isolated using
CsCl density gradients by standard methods (J. Sambrook et al.,
supra, at pp. 1.33-1.48)
[0348] B. Isolation of F-Box Sequences
[0349] In this portion of the Example, an improved version of the
yeast two-hybrid system was employed to identify proteins that bind
to Skp1. The two-hybrid system employs genetic selection to allow
the isolation of interacting proteins. The use of genetic selection
for the detection of interacting proteins allows much larger cDNA
libraries to be screened for associating clones than could be
accomplished using other techniques (i.e., screening expression
libraries, such as .lambda.gt11, with labelled proteins).
[0350] The improved two-hybrid system employs the yeast strain Y190
as the recipient cell line. The yeast strain Y190 contains two
chromosomally located reporter genes whose expression is regulated
by Gal4. The first reporter gene is the E. coli lacZ gene, which is
under the control of the GAL1 promoter. The second reporter gene is
the selectable HIS3 gene. The two-hybrid system is improved by the
use of an additional assay to eliminate false positives. "False
positives" are defined as library clones that activate
transcription in cells expressing fusions unrelated to the target
protein (i.e., Skp1). To isolate interacting proteins, Y190 cells
are first transformed with a first expression plasmid which encodes
a fusion protein comprising a hybrid between the DNA-binding domain
of the yeast transcription factor Gal4 (amino acids 1-147) and a
target protein (i.e., a protein which is used to identify proteins
capable of interacting with this target protein). The transformed
Y190 cells are next analyzed to determine the effect of the
introduction of the first expression plasmid. If the transformation
of Y190 cells with the expression plasmid which expresses the
target protein does not activate either the HIS3 or lacZ reporter
genes, this transformed strain can now be used for screening an
activation domain cDNA library.
[0351] The activation domain library comprises plasmids capable of
expressing the second hybrid molecules of the two-hybrid system.
The second hybrids comprise fusion proteins containing the
sequences encoding the Gal4 activation domiain II (amino acids
768-881) fused to a cDNA library generated from human breast tissue
(described above). When the Y190 cells transformed with the first
expression plasmid are transformed with a second expression vector
(from the expression library) capable of expressing a protein or
portion of a protein which can bind to the Skp1 hybrid,
transcription of the His3 and lacZ genes is activated as the
binding of the second hybrid brings the Gal4 activation domain II
in close proximity to the DNA binding domain of the Gal4 protein
which is bound to the UAS.sub.G upstream of the His3 and lacZ genes
on the chromosome.
[0352] In this two-hybrid system, Y190 cells were transformed with
the expression plasmid pAS2-Skp1 using lithium acetate according to
standard techniques (F. M. Ausubel, et al., Short Protocols in
Molecular Biology, John Wiley & Sons, New York [1992], pp.
13-29-13,30). The expression plasmid, pAS2-Skp1 encodes a fusion
protein comprising a hybrid between the DNA-binding domain of the
yeast transcription factor Gal4 (amino acids 1-147) and the Skp1
molecule. This first hybrid acts as "bait" for the second hybrid
molecule; the Gal4/Skp1 hybrid binds to the upstream activating
sequence from GAL1 (UAS.sub.G) sequences located upstream of the
His3 and lacZ genes in the host cell chromosome. Because the
GAL4-Skp1 hybrid lacks trans-activating sequences, Y190 cells
transformed with pAS1-Skp 1 were His.sup.- and white.
[0353] Y190 cells were transformed with the pAS2-Skp1 plasmid as
follows. Y190 cells were grown in 5 ml of YPD medium (10 g/l yeast
extract, 20 g/l peptone and 20 g/l dextrose) overnight to
saturation at 30.degree. C. The next day, a liter sterile flask
containing 300 ml of YPAD medium (YPD containing 30 mg/l adenine
hemisulfate) was inoculated with the overnight culture and grown
overnight at 30.degree. C. to a density of 1.times.10.sup.7
cells/ml. The cells were then collected by centrifugation at
4000.times.g for 5 min at room temperature. The cell pellet was
then washed by resuspending the cells in 10 ml sterile H.sub.2O
followed by centrifugation at 5000.times.g for 5 min at room
temperature. The washed cells were resuspended in 1.5 ml of LiAc TE
(1 vol 10.times.TE buffer [100 mM Tris-HCl, 10 mM EDTA], pH 7.5),
plus 1 vol of 10.times.LiAc stock solution (1M lithium acetate, pH
7.5) plus 8 vol sterile H.sub.2O. Five micrograms of pAS2-Skp1 DNA
and 200 .mu.g carrier DNA (single-stranded, high molecular weight
carrier DNA was prepared from salmon sperm DNA using standard
protocols; yeast total RNA may also be used as a carrier) were
placed in a sterile 1.5 ml microcentrifuge tube in a total volume
of 20 .mu.l. Two hundred microliters of the yeast suspension was
added to the tube followed by the addition of 1.2 ml of a LiAcPEG
solution (8 vol of 50% (w/v) polyethylene glycol, MW 3350 plus 1
vol of 10.times.TE buffer, pH 7.5 plus 1 vol 10.times.LiAc stock
solution). The cells were then shaken for 30 min at 30.degree. C.,
followed by a heat shock (15 min at 42.degree. C.). Following the
heat shock, the cells were collected by centrifugation for 5 sec at
room temperature in a tabletop microcentrifuge. The cell pellet was
the resuspended in 1 ml of TE buffer and 200 .mu.l of the
suspension were spread onto SC-Trp medium.
[0354] The transformed Y190 cells (Y190/pAS2-Skp1) were then
transformed with a pACTII-human breast tissue cDNA library as
described below. The plasmids contained within this library encode
the second hybrids of the two-hybrid system. The second hybrids
comprised fusion proteins containing the sequences encoding the
Gal4 activation domain II (amino acids 768-881) fused to a cDNA
library generated from human breast tissue. When a Y190/pAS2-Skp1
cell is transformed with a pACTII expression vector capable of
expressing a protein or portion of a protein which can bind to the
Gal4-Skp1 hybrid, transcription of the His3 and LacZ genes is
activated as the binding of the second hybrid brings the Gal4
activation domain II in close proximity to the DNA binding domain
of the Gal4 protein which is bound to the UAS.sub.G upstream of the
His3 and lacZ genes on the chromosome.
[0355] Y190/pAS2-Skp1 cells were transformed with the pACTII-human
breast cDNA library as follows. Briefly, the recipient strain
(Y190/pAS2-Skp1 cells) were grown to mid-log phase
(1.times.10.sup.7 cells/ml) in SC-Trp medium (SC medium lacking
tryptophane). The OD.sub.600 of this culture was determined and 1
liter of YPD medium was inoculated with enough of the culture such
that in 2 generations the cell density became 1.times.10.sup.7
cells/ml. The cells where pelleted by centrifugation and the pellet
was resuspended in LiAcTE (the volume is not critical as this is a
wash step). The cells were pelleted by centrifugation and the cells
were resuspended in 25 ml of LiSORB (100 mM LiAc, 10 mM Tris-HCl
(pH 8.0), 1 mM EDTA, 1 M sorbitol). The cells were then incubated
for 30 min at 30.degree. C. with shaking. The cells were then
pelleted by centrifugation as described above and resuspended in
2.5 ml of LiSORB. After removing 100 .mu.l of cells for a negative
control, 50 .mu.g of pACTII library DNA and 5 mg of yeast total RNA
carrier was added. The mixture was mixed well and then incubated
for 10 min at 30.degree. C. without shaking. The cells were then
transferred to a 250 ml flask and 22.5 ml of LiAcPEG (LiAcTE
containing 40% polyethylene glycol, MW 3350) was added and the
suspension was well mixed. The flask was then placed in a
42.degree. C. water bath for 12 min to heat shock the cells.
Following the heat shock, the transformation mixture was added to
500 ml of SC-Trp, -Leu, -His medium and the culture was allowed to
recover at 30.degree. C. for 4 hours; at this point the cells are
established as transformants. Next, 4.times.10.sup.5 transformants
were obtained by transformation of 1.times.10.sup.10 Y190/pAS2-Skp1
cells with 50 .mu.g of pACTII library DNA.
[0356] Transformants were subjected to selection for histidine
prototrophy by plating 300 .mu.l of the culture on 15 cm petri
dishes containing SC-Trp, -Leu, -His medium containing 50 mM 3
amino-triazole (Sigma), and incubated for 30.degree. C. for 3-5
days.
[0357] The rare surviving colonies were screened for their ability
to produce .beta.-galactosidase using a filter lift assay (L.
Breeden and K. Nasmyth, Cold Spring Harbor Symp. Quant. Biol.,
50:643-650 [1985]). Briefly, colonies were transferred onto
nitrocellulose filters (Scheicher and Schuell, BA85 45 .mu.m
circular filters) by laying the filters onto plates containing the
yeast colonies and allowing the filter to wet completely. The
filters were lifted off the plates and then submerged in liquid
nitrogen for 5-10 sec. The filters were then placed cell side up
into a petri dish containing 3 MM chromatography paper (Whatman)
saturated 0.3 ml/square inch with a solution comprising 100 mM
sodium phosphate, pH 7.0, 10 mM KCl, 0.7 mM magnesium sulfate, 10
mM 2-mercaptoethanol and 1 mg/ml X-gal
(5-bromo-4-chloro-3-indolyl-.beta.-ga- lactoside). The filters were
incubated at 30.degree. C. until blue color developed (30 min to
overnight). Positive (i.e., blue) colonies were then patched onto a
master plate for further analysis.
[0358] Plasmids recovered from 20 His+ blue (i.e., lacZ expressing)
colonies were sequenced from their 5' ends using the chain
termination method in conjunction with the Sequenase.RTM. enzyme
(U.S. Biochemicals); sequencing was performed according the
manufacturer's instructions. The amino acid sequence was deduced
from the nucleotide sequences located within the 20 inserts and
were compared to sequences listed in the GenBank. One of these 20
clones were found to be related to the F-box protein met30 from S.
cerevisiae in the F-box domain. The F-box region of Fgamma was used
to search the EST database of Genbank, and identified two novel
mammalian F-box-containing cDNAs (F13 Omicron and F14 Pi), in
addition to two F-box proteins from C. elegans (C02FS7 and
YK18A11). The Genbank accession numbers for these four cDNAs are
AA422959, AA462249, R12719, and D35163).
[0359] These novel F-box sequences were used to search the EST
sequences listed in Genbank. This search yielded F7 Theta
(AA008567), F1 alpha (F12916), F4 Delta (AA167804), TRCP
(AA478504), F6 Eta (AA027176), F15 Rho (AA538102), F8 Iota
(AA295683), MD6 (AA145853), and Skp2 homologs (U33761). These F-box
protein sequences were then used in additional EST database
searches and yielded sequences for F2 Beta (H58848), F5 Zeta
(R17328), F9 Kappa (AA459120), F10 Lambda (AA501293), F11 Mu
(AA069757), F12 Nu (AA000239), F16 Sigma (H49462), F17 Tau
(AA381895), and F18 Phi (AA309734). cDNA clones in bacterial
plasmids including pBluescript (Stratagene) were retrieved from the
EST cDNA Image Consortium and subjected to sequence analysis using
dideoxy DNA sequencing (See e.g., Sambrook et al., supra) to verify
the F-box sequences. For three cDNAs (F1 alpha, F2 beta, and F4
delta, complete sequences of the available cDNA clones were
obtained by standard primer walking (See e.g., Sambrook et al.,
supra).
[0360] This approach finds use in identification of other novel
F-box containing proteins, either using cDNA libraries from other
tissues or by performing additional database searches. The use of
cDNA libraries from various tissues allows the identification of
F-box proteins that are cell-type specific. Additional alternative
approaches, such as expression screening of .lambda.gt11-based
plaque libraries with Skp1 protein are also contemplated as methods
for yielding novel F-box proteins.
4TABLE 2 F-Box Sequences Identified Name, Source & Genbank #
Amino Acid Sequence SEQ ID NO: TRCP
LPARGLDHIAENILSYLDAKSLCAAELVCKEWYRVTSDGMLW SEQ ID NO:1 (human)
AA478504 F1 (Alpha) LPKELLLRIFSFLDIVTLCRCAQISKAWNIL- ALDGSNW SEQ ID
NO:3 (human) F12916 F2 (Beta)
LPYELIQLILNHLTLPDLCRLAQTCKLLSQHCCDPLQY SEQ ID NO:5 (human) H58848
F2 (Beta) LPYELIQLILNHLSLPDLCRLAQTCRLLHQHCCDPLQY SEQ ID NO:7
(mouse) F3 (Gamma) LPTDPLLLILSFLDYRDLINCCYVSRRLSQLSSHD- PLW SEQ ID
NO:9 (human) AA101399 F3 (Gamma)
LPTDPLLLIVSFVDYRDLINCCYVSRSVSQLSTHDPLW SEQ ID NO:11 (mounse) F4
(Delta) LPPEVMLSIFSYLNPQELCRCSQVSMKWSQLTKTGSLW SEQ ID NO:13 (human)
AA167804 F4 (Delta) LPPEVMLSIFSYLNPQELCRCSQVSTKWSQL- AKTGSLW SEQ ID
NO:15 (mouse) F5 (Zeta) LPLEMLTYILSFLPLSDQKEASLVSWAWYRAAQNALRERLW
SEQ ID NO:17 (human) R17328 F6 (Eta)
LPPELSFTILSYLNATDLCLASCVWQDLANDELLW SEQ ID NO:19 (human) AA027176
F6 (Eta) LPPELSFTILSYLNAIDLCLASCVWQDLANDELLW SEQ ID NO:21 (mouse)
AA213046 F7 (Theta) LPRVLSVYIFSFLDPRSLCRCAQVSWYWKSLAELDQLW SEQ ID
NO:23 (mouse) AA008567 F8 (Iota)
LPIDVQLYILSFLSPHDLCQLGSTNHYWNETVRHPILW SEQ ID NO:25 (human)
AA295683 F9 (Kappa) LPLELWRMILAYLHLPDLGRCSLVCRAWYELILSLDSTRW SEQ ID
NO:27 (human) AA459120 F10 (Lambda)
LPAEITFKIFSQLDIRSLCRASLTCRSWNDFKS SEQ ID NO:29 (mouse) AA501293 F11
(Mu) LPLLQQPLLCSVAHPIASFTMLSYLTGKEAAHLSVELW SEQ ID NO:31 (mouse)
AA069757 F12 (Nu) LPDSLVYQIFLSLGPADVLAAGLVCRQWQAVSRDEFLW SEQ ID
NO:33 (mouse) AA000239 F13 LPEEVLALIFRDLPLRDLAVATRVCRAWAAA SEQ ID
NO:35 (Omicron) (mouse) AA422959 F14 (Pi)
LPSVPMMEILSYLDAYSLLQAAQVNKNWNELASSDVLW SEQ ID NO:37 (mouse)
AA462249 F15 (Rho) MPSEILVKILSYLDAVTLVCIGCVSRRFYHLADDNLIW SEQ ID
NO:39 (mouse) AA538102 F16 (Sigma)
LPMEVLMYIFRWVVSSDLDLRSLEQLSLVCRGFYICARDPEIW SEQ ID NO:41 (human)
AA49462 F16 (Sigma) LSLVCRGFYICARDPEIW SEQ ID NO:43 (mouse)
AA410485 F17 (Tau) LPYELAINIFXYLDRKELGRCAQVSKTWEGD SEQ ID NO:45
(human) AA381895 F18 (Phi) LPLELKLRIFRLLDVRSVLSLSAVCRDLFTASNDPLLW
SEQ ID NO:47 (mouse) AA309734 F18 (Phi)
LPLELKLRIFRLLDVHSVLALSAVCHDLLIASNDPLLW SEQ ID NO:49 (human) W20645
MD6 LPLELSFYLLKWLDPQTLLTCCLVSKQ- WNKVISACTEVW SEQ ID NO:57
(human)
[0361]
5TABLE 3 F-Box Sequences Identified Name and SEQ ID Source DNA
Sequence NO: TRCP CTGCCAGCTCGGGGATTGGATCATATTGC SEQ ID (human)
TGAGAACATTCTGTCATACCTGGATGCCA NO:2 AATCACTATGTGCTGCTGAACTTGTGTGC
AAGGAATGGTACCGAGTGACCTCTGATGG CATGCTGTGG F1 (Alpha)
TTACCCAAAGAACTTCTGTTAAGAATATT SEQ ID (human)
TTCCTTCTTGGATATAGTAACTTTGTGCC NO:4 GATGTGCACAGATTTCCAAGGCTTGGAAC
ATCTTAGCCCTGGATGGAAGCAACTGG F2 (Beta) CTACCTTATGAGCTTATTCAGCTGATTCT
SEQ ID (human) GAATCATCTTACACTACCAGACCTGTGTA NO:6 GATTAGCACAGAC F2
(Beta) CTACCATATGAGCTCATTCAACTGATTCT SEQ ID (mouse)
GAATCATCTTTCACTACCAGACCTGTGTA NO:8 GATTAGCCCAGACTTGCAGGCTTCTCCAC
CAGCATTGCTGTGATCCTCTGCAATAT F3 (Gamma)
CTGCCCACCGATCCCCTGCTCCTCATCTT SEQ ID (human)
ATCCTTTTTGGACTATCGGGATCTAATCA NO:10 ACTGTTGTTATGTCAGTCGAAGACTTAGC
CAGCTATCAAGTCATGATCCGCTGTGG F3 (Gamma)
CTACCCACCGACCCTCTGCTCCTCATAGT SEQ ID (mouse)
ATCCTTCGTGGACTACAGGGACCTAATCA NO:12 ATTGTTGCTATGTTAGTCGAAGCGTTAGC
CAGCTATCAACTCATGATCCACTGTGG F4 (Delta)
CTTCCTCCTGAGGTAATGCTGTCAATTTT SEQ ID (human)
CAGCTATCTTAATCCTCAAGAGTTATTCG NO:14 ATGCAGTCAAGTAAGCATGAAATGGTCTC
AGCTGACAAAAACGGGATCGCTTTGG F4 (Delta) CTTCCTCCTGAGGTAATGCTGTCCATTTT
SEQ ID (mouse) CAGTTACCTTAATCCTCAAGAATTGTGTC NO:16
GGTGTAGTCAAGTCAGTACTAAGTGGTCT CAGCTGGCAAAAACAGGATCTTTGTGG F5 (Zeta)
CTGCCCCTGGAGATGCTCACATATATTCT SEQ ID (human)
GAGCTTCCTGCCTCTGTCAGATCAGAAAG NO:18 AGGCCTCCCTCGTGAGTTGGGCTTGGTAC
CGTGCTGCCCAGAATGCCCTTCGGGAGAG GCTGTGG F6 (Eta)
TTGCCTCCTGAGCTAAGCTTTACCATCTT SEQ ID (human)
GTCCTACCTGAATGCAACTGACCTTTGCT NO:20 TGGCTTCATGTGTTTGGCAGGACCTTGCG
AATGATGAACTTCTCTGG F6 (Eta) CTGCCTCCTGAGCTGAGCCTCACCATCCT SEQ ID
(mouse) ATCCCACCTGGATGCAACTGACCTTTGCC NO:22
TAGCTTCCTGTGGTTGGCAAGAACTCGCT AATGATGAACTTCTCTGG F7 (Theta)
CTTCCAAGGGTGTTATCTGTCTACATCTT SEQ ID (mouse)
TTCCTTCCTGGATCCCCGGAGTCTTTGCC NO:24 GTTGTGCACAGGTGAGCTGGTACTGGAAG
AGCTTGGCTGAGTTGGACCAGCTCTGG F8 (Iota) CTGCCGATTGATGTACAGCTATATATTTT
SEQ ID (human) GTCCTTTCTTTCACCTCATGATCTGTGTC NO:26
AGTTGGGAAGTACAAATCATTATTGGAAT GAAACTGTAAGACATCCAATTCTTTGG F9
(Kappa) CTCCCCTTGGAGCTGTGGCGCATGATCTT SEQ ID (human)
AGCCTACTTGCACCTTCCCGACCTGGGCC NO:28 GCTGCAGCCTGGTATGCAGGGCCTGGTAT
GAACTGATCCTCAGTCTCGACAGCACCCG CTGG F10 (Lambda)
CTGCCTGCAGAAATCACTTTTAAAATTTT SEQ ID (mouse)
CAGTCAGCTGGACATTCGGAGTCTGTGCA NO:30 GGGCTTCATTGACATGCAGGAGCTGGAAT
GAC F11 (Mu) CTGCCATTACTGCAGCAGCCACTTCTGTG SEQ ID (mouse)
TTCTGTGGCTCATCCCATCGCCAGCTTCA NO:32 CCATGCTGTCATACCTCACGGGAAAGGAG
GCCGCTCATCTGTCAGTGGAGTTGTGG F12 (Nu) CTCCCCGACAGCCTTGTCTACCAGATCTT
SEQ ID (mouse) CCTGAGTTTGGGCCCTGCAGATGTGCTGG NO:34
CTGCTGGGCTGGTATGCCGCCAATGGCAG GCTGTGTCCCGGGATGAGTTCTTATGG F13
CTGCCAGAGGAAGTGTTGGCGCTCATCTT SEQ ID (Omicron)
CCGTGACCTGCCTCTCAGGGACCTTGCTG NO:36 (mouse)
TAGCCACCAGAGTCTGCAGGGCCTGGGCG GCGGCT F14 (Pi)
TTACCTAGTGTGCCGATGATGGAAATCCT SEQ ID (mouse)
CTCCTATCTGGATGCCTACAGTTTGCTAC NO:38 AGGCTGCCCAAGTGAACAAGAACTGGAAT
GAACTTGCAAGCAGTGATGTCCTGTGG F15 (Rho) ATGCCATCGGAAATCTTGGTGAAGATACT
SEQ ID (mouse) TTCTTACTTGGATGCGGTGACCTTGGTGT NO:40
GCATTGGATGTGTGAGCAGACGCTTTTAT CATTTGGCTGATGACAATCTTATTTGG F16
(Sigma) CTGCCAATGGAGGTCCTGATGTACATCTT SEQ ID (human)
CCGATGGGTGGTGTCTAGTGACTTGGACC NO:42 TCAGATCATTGGAGCAGTTGTCGCTGGTG
TGCAGAGGGTTCTACATCTGTGCCAGAGA CCCTGAAATATGG F16 (Sigma)
GACTTGGACCTCAGATCGTTAGAGCAGTT SEQ ID (mouse)
GTCACTGGTGTGCAGAGGATTCTATATCT NO:44 GTGCCAGAGACCCTGAAATCTGG F17
(Tau) CTGCCTTACGAATTGGCAATCAATATATT SEQ ID (human)
TNAGTATCTGGACAGGAAAGAACTAGGAA NO:46 GATGTGCACAGGTGAGCAAGACGTGGGAA
GGTGATT F18 (Phi) CTCCCATTGGAACTGAAACTACGGATCTT SEQ ID (human)
CCGACTTCTGGATGTTCGTTCCGTCTTGT NO:48 CTTTGTCTGCGGTTTGTCGTGACCTCTTT
ACTGCTTCAAATGACCCACTCCTGTGG F18 (Phi) CTTCCACTGGAGCTGAAACTACGCATCTT
SEQ ID (mouse) CCGACTTTTGGATGTTCATTCTGTCCTGG NO:50
CCCTGTCTGCAGTCTGTCATGACCTCCTC ATTGCGTCAAATGACCCACTGCTGTGG MD6
CTTCCCCTGGAGCTCAGTTTTTATTTGTT SEQ ID (human)
AAAATGGCTCGATCCTCAGACTTTACTCA NO:58 CATGCTGCCTCGTCTCTAAACAGTGGAAT
AAGGTGATAAGTGCCTGTACAGAGGTGTG G
[0362]
6TABLE 4 Sequences of Some F-Box Proteins Name & SEQ ID Source
Sequence NO: F1 SAMVFSNNDEGLINKKLPKELLLRIFSFLDIVTLCR SEQ ID Alpha
CAQISKAWNILALDGSNWQRIDLFNFQIDVEGRVVE NO:51 (human)
NISKRCGGFLRKLSLRGCIGVGDSSLKTFAQNCRNI EHLNLNGCTKITDSTCYSLSRFCSKLKH-
LDLTSCVS ITNSSLKGISEGCRNLEYLNLSWCDQITKDGIEALV
RGCRGLKALLLRGCTQLEDEALKHIQNYCHELVSLN LQSCSRITDEGVVQICRGCHRLQALCLS-
GCSNLTDA SLTALGLNCPRLQILEAARCSHLTDAGFTLLARNCH
ELEKMDLEECILITDSTLIQLSIHCPKLQALSLSHC ELITDDGILHLSNSTCGHERLRVLELDN-
CLLITDVA LEHLETAEAWSASSCTTASRLPVQASSGCGLSSLMS
KSTPTLLPSPHRQQWQEVDSDCAGAVSFSDSSCLGP RGDEASFPLEDLSLPDRLHHHPIC F1
TTCGGCCATGGTTTTCTCAAACAATGATGAAGGCCT SEQ ID Alpha
TATTAACAAAAAGTTACCCAAAGAACTTCTGTTAAG NO:52 (human)
AATATTTTCCTTCTTGGATATAGTAACTTTGTGCCG ATGTGCACAGATTTCCAAGGCTTGGAAC-
ATCTTAGC CCTGGATGGAAGCAACTGGCAAAGAATAGATCTTTT
TAACTTTCAAATAGATGTAGAGGGTCGAGTGGTGGA AAATATCTCGAAGCGATGCGGTGGATTC-
CTGAGGAA GCTCAGCTTGCGAGGCTGCATTGGTGTTGGGGATTC
CTCCTTGAAGACCTTTGCACAGAACTGCCGAAACAT TGAACATTTGAACCTCAATGGATGCACA-
AAAATCAC TGACAGCACGTGTTATAGCCTTAGCAGATTCTGTTC
CAAGCTGAAACATCTGGATCTGACCTCCTGTGTGTC TATTACAAACAGCTCCTTGAAGGGGATC-
AGTGAGGG CTGCCGAAACCTGGAGTACCTGAACCTCTCTTGGTG
TGATCAGATCACGAAGGATGGCATCGAGGCACTGGT GCGAGGTTGTCGAGGCCTGAAAGCCCTG-
CTCCTGAG GGGCTGCACACAGTTAGAAGATGAAGCTCTGAAACA
CATTCAGAATTACTGCCATGAGCTTGTGAGCCTCAA CTTGCAGTCCTGCTCACGTATCACGGAT-
GAAGGTGT GGTGCAGATATGCAGGGGCTGTCACCGGCTACAGGC
TCTCTGCCTTTCGGGTTGCAGCAACCTCACAGATGC CTCTCTTACAGCCCTGGGTTTGAACTGT-
CCGCGACT GCAAATTTTGGAGGCTGCCCGATGCTCCCATTTGAC
TGACGCAGGTTTTACACTTTTAGCTCGGAATTGCCA CGAATTGGAGAAGATGGATCTTGAAGAA-
TGCATCCT GATAACCGACAGCACACTCATCCAGCTCTCCATTCA
CTGTCCTAAACTGCAAGCCCTGAGCCTGTCCCACTG TGAACTCATCACAGATGATGGGATCCTG-
CACCTGAG CAACAGTACCTGTGGCCATGAGAGGCTGCGGGTACT
GGAGTTGGACAACTGCCTCCTCATCACTGATGTGGC CCTGGAACACCTAGAAACTGCCGAGGCC-
TGGAGCGC CTCGAGCTGTACGACTGCCAGCAGGTTACCCGTGCA
GGCATCAAGCGGATGCGGGCTCAGCTCCCTCATGTC AAAGTCCACGCCTACTTTGCTCCCGTCA-
CCCCACCG ACAGCA F2 RPRFGTSDIEDDAYAEKDGCGMDSLNKKFSS- AVLGE SEQ ID
Beta GPNNGYFDKLPYELIQLILNHLTLPDLCRLAQTCKL NO:53 (human)
LSQHCCDPLQYIHLNLQPYWAKLDDTSLEFLQSRCT
LVQWLNLSWTGNRGFISVAGFSRFLKVCGSELVRLE LSCSHFLNETCLEVISEMCPNLQALNLS-
SCDKLPPQ AFNHIAKLCSLKRLVLYRTKVEQTALLSILNFCSEL
QHLSLGSCVMIEDYDVIASMIGAKCKKLRTLDLWRC KNITENGIAELASGCPLLEELDLGWCPT-
LQSSTGCF TRLAHQLPNLQKLFLTANRSVCDTDIDELACNCTRL
QQLDILGKVTIYKFVLNVCFLDRKANLRLFVRKKKI FGYNKNFILIRWLGLIGNAR F2
AGGCCAAGATTCGGCACGAGTGATATAGAAGATGAT SEQ ID Beta
GCCTATGCAGAAAAGGATGGTTGTGGAATGGACAGT NO:54 (human)
CTTAACAAAAAGTTTAGCAGTGCTGTCCTCGGGGAA GGGCCAAATAATGGGTATTTTGATAAAC-
TACCTTAT GAGCTTATTCAGCTGATTCTGAATCATCTTACACTA
CCAGACCTGTGTAGATTAGCACAGACTTGCAAACTA MISTYCTGAGCCAGCATTGCTGTGATCC-
TCTGCAAT ACATCCACCTCAATCTGCAACCATACTGGGCAAAAC
TAGATGACACTTCTCTGGAATTTCTACAGTCTCGCT GCACTCTTGTCCAGTGGCTTAATTTATC-
TTGGACTG GCAATAGAGGCTTCATCTCTGTTGCAGGATTTAGCA
GGTTTCTGAAGGTTTGTGGATCCGAATTAGTACGCC TTGAATTGTCTTGCAGCCACTTTCTTAA-
TGAAACTT GCTTAGAAGTTATTTCTGAGATGTGTCCAAATCTAC
AGGCCTTAAATCTCTCCTCCTGTGATAAGCTACCAC CTCAAGCTTTCAACCACATTGCCAAGTT-
ATGCAGCC TTAAACGACTTGTTCTCTATCGAACAAAAGTAGAGC
AAACAGCACTGCTCAGCATTTTGAACTTCTGTTCAG AGCTTCAGCACCTCAGTTTAGGCAGTTG-
TGTCATGA TTGAAGACTATGATGTGATAGCTAGCATGATAGGAG
CCAAGTGTAAAAAACTCCGGACCCTGGATCTGTGGA GATGTAAGAATATTACTGAGAATGGAAT-
AGCAGAAC TGGCTTCTGGGTGTCCACTACTGGAGGAGCTTGACC
TTGGCTGGTGCCCAACTCTGCAGAGCAGCACCGGGT GCTTCACCAGACTGGCACACCAGCTCCC-
AAACTTGC AAAAACTCTTTCTTACAGCTAATAGATCTGTGTGTG
ACACAGACATTGATGAATTGGCATGTAATTGTACCA GGTTACAGCAGCTGGACATATTAGGTAA-
GGTTACAA TATATAAATTTGTTTTAAATGTCTGTTTCCTTGACA
GAAAAGCCAATCTCAGACTTTTTGTTAGGAAAAAGA AAATTTTTGGATACAATAAAAATTTTAT-
CCTGATAA GATGGCTTGGTTTGATAGGAAATGCCAGATAGATCA
GTTAATATAGGGAATAATTATATATGTACTTTAATA AAATAGTGAGGACAATAACAATTTTATA-
GTTGAACT GTAAAAAACTATAACCATTAATTCTTGGTCTACTTG
TAAGAGTGAGAATTTACATGAGCTGCGCTCTCTATT TTTATTAAGGAGAGAAGAAATTAATTCA-
TTTGTATA ATGAATTCAAGCTAGTTTTTTTAAGTTTCTTAATTA AGCGGCCGCAAGCTTA F4
WVIMLZERQKFFKYSVDEKSDKEAEVSEHSTGITHL SEQ ID Delta
PPEVMLSIFSYLNPQELCRCSQVSMKWSQLTKTGSL NO:55 (human)
WKHLYPVHWARGDWYSGPATELDTEPDDEWVKNRKD
ESRAFHEWDEDADIDESEESAEESIAISIAQMEKRL LHGLIHNVLPYVGTSVKTLVLAYSSAVS-
SKMVRQIL ELCPNLEHLDLTQTDISDSAFDSWSWLGCCQSLRHL
DLSGCEKITDVALEKISRALGILTSHQSGFLKTSTS KITSTAWKNKDITMQSTKQYACLHDLTN-
KGIGEEID NEHPWTKPVSSENFTSPYVWMLDAEDLADIEDTVEW
RHRNVESLCVMETASNFSCSTSGCFSKDIVGLRTSV CWQQHCASPAFAYCGHSFCCTGTALRTM-
SSLPESSA MCRKAARTRLPRGKDLIYFGSEKSDQETGRVLLFLS
LSGCYQITDHGLRVLTLGGGLPYLEHLNLSGCLTIT GAGLQDLVSACPSLNDEYFYYCDNINGP-
HADTASGC QNLQCGFRACCRSGE F4 ATGGTAATCATGCTGTAAGAGCGACAGAAATTTTTT
SEQ ID Delta AAATATTCCGTGGATGAAAAGTCAGATAAAGAAGCA NO:56 (human)
GAAGTGTCAGAACACTCCACAGGTATAACCCATCTT CCTCCTGAGGTAATGCTGTCAATTTTCA-
GCTATCTT AATCCTCAAGAGTTATGTCGATGCAGTCAAGTAAGC
ATGAAATGGTCTCAGCTGACAAAAACGGGATCGCTT TGGAAACATCTTTACCCTGTTCATTGGG-
CCAGAGGT GACTGGTATAGTGGTCCCGCAACTGAACTTGATACT
GAACCTGATGATGAATGGGTGAAAAATAGGAAAGAT GAAAGTCGTGCTTTTCATGAGTGGGATG-
AAGATGCT GACATTGATGAATCTGAAGAGTCTGCGGAGGAATCA
ATTGCTATCAGCATTGCACAAATGGAAAAACGTTTA CTCCATGGCTTAATTCATAACGTTCTAC-
CATATGTT GGTACTTCTGTAAAAACCTTAGTATTAGCATACAGC
TCTGCAGTTTCCAGCAAAATGGTTAGGCAGATTTTA GAGCTTTGTCCTAACCTGGAGCATCTGG-
ATCTTACC CAGACTGACATTTCAGATTCTGCATTTGACAGTTGG
TCTTGGCTTGGTTGCTGCCAGAGTCTTCGGCATCTT GATCTGTCTGGTTGTGAGAAAATCACAG-
ATGTGGCC CTAGAGAAGATTTCCAGAGCTCTTGGAATTCTGACA
TCTCATCAAAGTGGCTTTTTGAAAACATCTACAAGC AAAATTACTTCAACTGCGTGGAAAAATA-
AAGACATT ACCATGCAGTCCACCAAGCAGTATGCCTGTTTGCAC
GATTTAACTAACAAGGGCATTGGAGAAGAAATAGAT AATGAACACCCCTGGACTAAGCCTGTTT-
CTTCTGAG AATTTCACTTCTCCTTATGTGTGGATGTTAGATGCT
GAAGATTTGGCTGATATTGAAGATACTGTGGAATGG TTGTACAGGAACAGCTTTAAGAACTATG-
TCATCACT CCCAGAATCTTCTGCAATGTGTAGAAAAGCAGCAAG
GACTAGATTGCCTAGGGGAAAAGACTTAATTTACTT TGGGAGTGAAAAATCTGATCAAGAGACT-
GGACGTGT ACTTCTGTTTCTCAGTGGAGGGCTGCCTTATTTGGA
GCACCTTAATCTCTCTGGTTGTCTTACTATAACTGG TGCAGGCCTGCAGGATTTGGTTTCAGCA-
TGTCCTTC TCTGAATGATGAATACTTTTACTACTGTGACAACAT
TAACGGTCCTCATGCTGATACCGCCAGTGGATGCCA GAATTTGCAGTGTGGTTTTCGAGCCTGC-
TGCCGCTC TGGCGAATGACCCTTGACTTCTGATCTTTGTCTACT
TCATTTAGCTGAGCAGGCTTTCTTTCATGCACTTTA CTCATAGCACATTTCTTGTGTTAACCAT-
CCCTTTTT GAGCGTGACTTGTTTTGGCCCCATTTCTTACAACTT
CAGAAATCTTAATTTACCAGTGAATTGTAATGTTGT TTCTCTTGCAAATTATACTTTTGGTTTA-
GAAAGGGA TTAGGTCTTTTCAAAAGGGTGAGAACAGTCTTACAT
TTTTCTTTTAAATGAAATGCTTTAAAGAATGTTGGT AATGCCATGTCATTTAAAGTATTTCATA-
GATAATTT TGAGTTTTAAAGTCCATGGAGGTGATTGGTTCTCTT
TACACATTAACACTGTACCAAGCTTTGCAGATCTTT TCCGACACACATGTCTGAAGACTTATTT-
TCAAAGAC AGCACATTTTTGGAAACTAATCTCTTTTCCGTAATA
TTTCCTTTATTTCAATGATTCTCAGAAGGCCAATTC AAACAAACCCACATTTAAGGTTCTTTAG-
GATTATAG AATAAATTGGCTTCTGAGTGTTAGCTCAGTGAGTAG
GAAAGCACCAATCGATATTTGTTTCCTTTAGGGATA CTTTGTTCTCACCACTGTCCCTATGTCA-
TCAAATTT GGGAGAGATTTTTTAAAATACCACAATCATTTGAAG
AAATGTATAAATAAAATCTACTTTGAGGACTTTACC AAGTAA
[0363] All publications and patents mentioned in the above
specification are herein incorporated by reference. Various
modifications and variation of the described method and system of
the invention will be apparent to those skilled in the art without
departing from the scope and spirit of the invention. Although the
invention has been described in connection with specific preferred
embodiments, it should be understood that the invention as claimed
should not be unduly limited to such specific embodiments. Indeed,
various modifications of the described modes for carrying out the
invention which are obvious to those skilled in molecular biology
or related fields are intended to be within the scope of the
following claims.
Sequence CWU 1
1
60 1 42 PRT Homo sapiens 1 Leu Pro Ala Arg Gly Leu Asp His Ile Ala
Glu Asn Ile Leu Ser Tyr 1 5 10 15 Leu Asp Ala Lys Ser Leu Cys Ala
Ala Glu Leu Val Cys Lys Glu Trp 20 25 30 Tyr Arg Val Thr Ser Asp
Gly Met Leu Trp 35 40 2 126 DNA Homo sapiens 2 ctgccagctc
ggggattgga tcatattgct gagaacattc tgtcatacct ggatgccaaa 60
tcactatgtg ctgctgaact tgtgtgcaag gaatggtacc gagtgacctc tgatggcatg
120 ctgtgg 126 3 38 PRT Homo sapiens 3 Leu Pro Lys Glu Leu Leu Leu
Arg Ile Phe Ser Phe Leu Asp Ile Val 1 5 10 15 Thr Leu Cys Arg Cys
Ala Gln Ile Ser Lys Ala Trp Asn Ile Leu Ala 20 25 30 Leu Asp Gly
Ser Asn Trp 35 4 114 DNA Homo sapiens 4 ttacccaaag aacttctgtt
aagaatattt tccttcttgg atatagtaac tttgtgccga 60 tgtgcacaga
tttccaaggc ttggaacatc ttagccctgg atggaagcaa ctgg 114 5 38 PRT Homo
sapiens 5 Leu Pro Tyr Glu Leu Ile Gln Leu Ile Leu Asn His Leu Thr
Leu Pro 1 5 10 15 Asp Leu Cys Arg Leu Ala Gln Thr Cys Lys Leu Leu
Ser Gln His Cys 20 25 30 Cys Asp Pro Leu Gln Tyr 35 6 71 DNA Homo
sapiens 6 ctaccttatg agcttattca gctgattctg aatcatctta cactaccaga
cctgtgtaga 60 ttagcacaga c 71 7 38 PRT Mus musculus 7 Leu Pro Tyr
Glu Leu Ile Gln Leu Ile Leu Asn His Leu Ser Leu Pro 1 5 10 15 Asp
Leu Cys Arg Leu Ala Gln Thr Cys Arg Leu Leu His Gln His Cys 20 25
30 Cys Asp Pro Leu Gln Tyr 35 8 114 DNA Mus musculus 8 ctaccatatg
agctcattca actgattctg aatcatcttt cactaccaga cctgtgtaga 60
ttagcccaga cttgcaggct tctccaccag cattgctgtg atcctctgca atat 114 9
38 PRT Homo sapiens 9 Leu Pro Thr Asp Pro Leu Leu Leu Ile Leu Ser
Phe Leu Asp Tyr Arg 1 5 10 15 Asp Leu Ile Asn Cys Cys Tyr Val Ser
Arg Arg Leu Ser Gln Leu Ser 20 25 30 Ser His Asp Pro Leu Trp 35 10
114 DNA Homo sapiens 10 ctgcccaccg atcccctgct cctcatctta tcctttttgg
actatcggga tctaatcaac 60 tgttgttatg tcagtcgaag acttagccag
ctatcaagtc atgatccgct gtgg 114 11 38 PRT Mus musculus 11 Leu Pro
Thr Asp Pro Leu Leu Leu Ile Val Ser Phe Val Asp Tyr Arg 1 5 10 15
Asp Leu Ile Asn Cys Cys Tyr Val Ser Arg Ser Val Ser Gln Leu Ser 20
25 30 Thr His Asp Pro Leu Trp 35 12 114 DNA Mus musculus 12
ctacccaccg accctctgct cctcatagta tccttcgtgg actacaggga cctaatcaat
60 tgttgctatg ttagtcgaag cgttagccag ctatcaactc atgatccact gtgg 114
13 38 PRT Homo sapiens 13 Leu Pro Pro Glu Val Met Leu Ser Ile Phe
Ser Tyr Leu Asn Pro Gln 1 5 10 15 Glu Leu Cys Arg Cys Ser Gln Val
Ser Met Lys Trp Ser Gln Leu Thr 20 25 30 Lys Thr Gly Ser Leu Trp 35
14 113 DNA Homo sapiens 14 cttcctcctg aggtaatgct gtcaattttc
agctatctta atcctcaaga gttattcgat 60 gcagtcaagt aagcatgaaa
tggtctcagc tgacaaaaac gggatcgctt tgg 113 15 38 PRT Mus musculus 15
Leu Pro Pro Glu Val Met Leu Ser Ile Phe Ser Tyr Leu Asn Pro Gln 1 5
10 15 Glu Leu Cys Arg Cys Ser Gln Val Ser Thr Lys Trp Ser Gln Leu
Ala 20 25 30 Lys Thr Gly Ser Leu Trp 35 16 114 DNA Mus musculus 16
cttcctcctg aggtaatgct gtccattttc agttacctta atcctcaaga attgtgtcgg
60 tgtagtcaag tcagtactaa gtggtctcag ctggcaaaaa caggatcttt gtgg 114
17 41 PRT Homo sapiens 17 Leu Pro Leu Glu Met Leu Thr Tyr Ile Leu
Ser Phe Leu Pro Leu Ser 1 5 10 15 Asp Gln Lys Glu Ala Ser Leu Val
Ser Trp Ala Trp Tyr Arg Ala Ala 20 25 30 Gln Asn Ala Leu Arg Glu
Arg Leu Trp 35 40 18 123 DNA Homo sapiens 18 ctgcccctgg agatgctcac
atatattctg agcttcctgc ctctgtcaga tcagaaagag 60 gcctccctcg
tgagttgggc ttggtaccgt gctgcccaga atgcccttcg ggagaggctg 120 tgg 123
19 35 PRT Homo sapiens 19 Leu Pro Pro Glu Leu Ser Phe Thr Ile Leu
Ser Tyr Leu Asn Ala Thr 1 5 10 15 Asp Leu Cys Leu Ala Ser Cys Val
Trp Gln Asp Leu Ala Asn Asp Glu 20 25 30 Leu Leu Trp 35 20 105 DNA
Homo sapiens 20 ttgcctcctg agctaagctt taccatcttg tcctacctga
atgcaactga cctttgcttg 60 gcttcatgtg tttggcagga ccttgcgaat
gatgaacttc tctgg 105 21 35 PRT Mus musculus 21 Leu Pro Pro Glu Leu
Ser Phe Thr Ile Leu Ser Tyr Leu Asn Ala Ile 1 5 10 15 Asp Leu Cys
Leu Ala Ser Cys Val Trp Gln Asp Leu Ala Asn Asp Glu 20 25 30 Leu
Leu Trp 35 22 105 DNA Mus musculus 22 ctgcctcctg agctgagcct
caccatccta tcccacctgg atgcaactga cctttgccta 60 gcttcctgtg
gttggcaaga actcgctaat gatgaacttc tctgg 105 23 38 PRT Mus musculus
23 Leu Pro Arg Val Leu Ser Val Tyr Ile Phe Ser Phe Leu Asp Pro Arg
1 5 10 15 Ser Leu Cys Arg Cys Ala Gln Val Ser Trp Tyr Trp Lys Ser
Leu Ala 20 25 30 Glu Leu Asp Gln Leu Trp 35 24 114 DNA Mus musculus
24 cttccaaggg tgttatctgt ctacatcttt tccttcctgg atccccggag
tctttgccgt 60 tgtgcacagg tgagctggta ctggaagagc ttggctgagt
tggaccagct ctgg 114 25 38 PRT Homo sapiens 25 Leu Pro Ile Asp Val
Gln Leu Tyr Ile Leu Ser Phe Leu Ser Pro His 1 5 10 15 Asp Leu Cys
Gln Leu Gly Ser Thr Asn His Tyr Trp Asn Glu Thr Val 20 25 30 Arg
His Pro Ile Leu Trp 35 26 114 DNA Homo sapiens 26 ctgccgattg
atgtacagct atatattttg tcctttcttt cacctcatga tctgtgtcag 60
ttgggaagta caaatcatta ttggaatgaa actgtaagac atccaattct ttgg 114 27
40 PRT Homo sapiens 27 Leu Pro Leu Glu Leu Trp Arg Met Ile Leu Ala
Tyr Leu His Leu Pro 1 5 10 15 Asp Leu Gly Arg Cys Ser Leu Val Cys
Arg Ala Trp Tyr Glu Leu Ile 20 25 30 Leu Ser Leu Asp Ser Thr Arg
Trp 35 40 28 120 DNA Homo sapiens 28 ctccccttgg agctgtggcg
catgatctta gcctacttgc accttcccga cctgggccgc 60 tgcagcctgg
tatgcagggc ctggtatgaa ctgatcctca gtctcgacag cacccgctgg 120 29 33
PRT Mus musculus 29 Leu Pro Ala Glu Ile Thr Phe Lys Ile Phe Ser Gln
Leu Asp Ile Arg 1 5 10 15 Ser Leu Cys Arg Ala Ser Leu Thr Cys Arg
Ser Trp Asn Asp Phe Lys 20 25 30 Ser 30 90 DNA Mus musculus 30
ctgcctgcag aaatcacttt taaaattttc agtcagctgg acattcggag tctgtgcagg
60 gcttcattga catgcaggag ctggaatgac 90 31 38 PRT Mus musculus 31
Leu Pro Leu Leu Gln Gln Pro Leu Leu Cys Ser Val Ala His Pro Ile 1 5
10 15 Ala Ser Phe Thr Met Leu Ser Tyr Leu Thr Gly Lys Glu Ala Ala
His 20 25 30 Leu Ser Val Glu Leu Trp 35 32 114 DNA Mus musculus 32
ctgccattac tgcagcagcc acttctgtgt tctgtggctc atcccatcgc cagcttcacc
60 atgctgtcat acctcacggg aaaggaggcc gctcatctgt cagtggagtt gtgg 114
33 38 PRT Mus musculus 33 Leu Pro Asp Ser Leu Val Tyr Gln Ile Phe
Leu Ser Leu Gly Pro Ala 1 5 10 15 Asp Val Leu Ala Ala Gly Leu Val
Cys Arg Gln Trp Gln Ala Val Ser 20 25 30 Arg Asp Glu Phe Leu Trp 35
34 114 DNA Mus musculus 34 ctccccgaca gccttgtcta ccagatcttc
ctgagtttgg gccctgcaga tgtgctggct 60 gctgggctgg tatgccgcca
atggcaggct gtgtcccggg atgagttctt atgg 114 35 31 PRT Mus musculus 35
Leu Pro Glu Glu Val Leu Ala Leu Ile Phe Arg Asp Leu Pro Leu Arg 1 5
10 15 Asp Leu Ala Val Ala Thr Arg Val Cys Arg Ala Trp Ala Ala Ala
20 25 30 36 93 DNA Mus musculus 36 ctgccagagg aagtgttggc gctcatcttc
cgtgacctgc ctctcaggga ccttgctgta 60 gccaccagag tctgcagggc
ctgggcggcg gct 93 37 38 PRT Mus musculus 37 Leu Pro Ser Val Pro Met
Met Glu Ile Leu Ser Tyr Leu Asp Ala Tyr 1 5 10 15 Ser Leu Leu Gln
Ala Ala Gln Val Asn Lys Asn Trp Asn Glu Leu Ala 20 25 30 Ser Ser
Asp Val Leu Trp 35 38 114 DNA Mus musculus 38 ttacctagtg tgccgatgat
ggaaatcctc tcctatctgg atgcctacag tttgctacag 60 gctgcccaag
tgaacaagaa ctggaatgaa cttgcaagca gtgatgtcct gtgg 114 39 38 PRT Mus
musculus 39 Met Pro Ser Glu Ile Leu Val Lys Ile Leu Ser Tyr Leu Asp
Ala Val 1 5 10 15 Thr Leu Val Cys Ile Gly Cys Val Ser Arg Arg Phe
Tyr His Leu Ala 20 25 30 Asp Asp Asn Leu Ile Trp 35 40 114 DNA Mus
musculus 40 atgccatcgg aaatcttggt gaagatactt tcttacttgg atgcggtgac
cttggtgtgc 60 attggatgtg tgagcagacg cttttatcat ttggctgatg
acaatcttat ttgg 114 41 43 PRT Homo sapiens 41 Leu Pro Met Glu Val
Leu Met Tyr Ile Phe Arg Trp Val Val Ser Ser 1 5 10 15 Asp Leu Asp
Leu Arg Ser Leu Glu Gln Leu Ser Leu Val Cys Arg Gly 20 25 30 Phe
Tyr Ile Cys Ala Arg Asp Pro Glu Ile Trp 35 40 42 129 DNA Homo
sapiens 42 ctgccaatgg aggtcctgat gtacatcttc cgatgggtgg tgtctagtga
cttggacctc 60 agatcattgg agcagttgtc gctggtgtgc agagggttct
acatctgtgc cagagaccct 120 gaaatatgg 129 43 18 PRT Mus musculus 43
Leu Ser Leu Val Cys Arg Gly Phe Tyr Ile Cys Ala Arg Asp Pro Glu 1 5
10 15 Ile Trp 44 81 DNA Mus musculus 44 gacttggacc tcagatcgtt
agagcagttg tcactggtgt gcagaggatt ctatatctgt 60 gccagagacc
ctgaaatctg g 81 45 31 PRT Homo sapiens 45 Leu Pro Tyr Glu Leu Ala
Ile Asn Ile Phe Xaa Tyr Leu Asp Arg Lys 1 5 10 15 Glu Leu Gly Arg
Cys Ala Gln Val Ser Lys Thr Trp Glu Gly Asp 20 25 30 46 93 DNA Homo
sapiens 46 ctgccttacg aattggcaat caatatattt agtatctgga caggaaagaa
ctaggaagat 60 gtgcacaggt gagcaagacg tgggaaggtg att 93 47 38 PRT
Homo sapiens 47 Leu Pro Leu Glu Leu Lys Leu Arg Ile Phe Arg Leu Leu
Asp Val Arg 1 5 10 15 Ser Val Leu Ser Leu Ser Ala Val Cys Arg Asp
Leu Phe Thr Ala Ser 20 25 30 Asn Asp Pro Leu Leu Trp 35 48 114 DNA
Homo sapiens 48 ctcccattgg aactgaaact acggatcttc cgacttctgg
atgttcgttc cgtcttgtct 60 ttgtctgcgg tttgtcgtga cctctttact
gcttcaaatg acccactcct gtgg 114 49 38 PRT Mus musculus 49 Leu Pro
Leu Glu Leu Lys Leu Arg Ile Phe Arg Leu Leu Asp Val His 1 5 10 15
Ser Val Leu Ala Leu Ser Ala Val Cys His Asp Leu Leu Ile Ala Ser 20
25 30 Asn Asp Pro Leu Leu Trp 35 50 114 DNA Mus musculus 50
cttccactgg agctgaaact acgcatcttc cgacttttgg atgttcattc tgtcctggcc
60 ctgtctgcag tctgtcatga cctcctcatt gcgtcaaatg acccactgct gtgg 114
51 456 PRT Homo sapiens 51 Ser Ala Met Val Phe Ser Asn Asn Asp Glu
Gly Leu Ile Asn Lys Lys 1 5 10 15 Leu Pro Lys Glu Leu Leu Leu Arg
Ile Phe Ser Phe Leu Asp Ile Val 20 25 30 Thr Leu Cys Arg Cys Ala
Gln Ile Ser Lys Ala Trp Asn Ile Leu Ala 35 40 45 Leu Asp Gly Ser
Asn Trp Gln Arg Ile Asp Leu Phe Asn Phe Gln Ile 50 55 60 Asp Val
Glu Gly Arg Val Val Glu Asn Ile Ser Lys Arg Cys Gly Gly 65 70 75 80
Phe Leu Arg Lys Leu Ser Leu Arg Gly Cys Ile Gly Val Gly Asp Ser 85
90 95 Ser Leu Lys Thr Phe Ala Gln Asn Cys Arg Asn Ile Glu His Leu
Asn 100 105 110 Leu Asn Gly Cys Thr Lys Ile Thr Asp Ser Thr Cys Tyr
Ser Leu Ser 115 120 125 Arg Phe Cys Ser Lys Leu Lys His Leu Asp Leu
Thr Ser Cys Val Ser 130 135 140 Ile Thr Asn Ser Ser Leu Lys Gly Ile
Ser Glu Gly Cys Arg Asn Leu 145 150 155 160 Glu Tyr Leu Asn Leu Ser
Trp Cys Asp Gln Ile Thr Lys Asp Gly Ile 165 170 175 Glu Ala Leu Val
Arg Gly Cys Arg Gly Leu Lys Ala Leu Leu Leu Arg 180 185 190 Gly Cys
Thr Gln Leu Glu Asp Glu Ala Leu Lys His Ile Gln Asn Tyr 195 200 205
Cys His Glu Leu Val Ser Leu Asn Leu Gln Ser Cys Ser Arg Ile Thr 210
215 220 Asp Glu Gly Val Val Gln Ile Cys Arg Gly Cys His Arg Leu Gln
Ala 225 230 235 240 Leu Cys Leu Ser Gly Cys Ser Asn Leu Thr Asp Ala
Ser Leu Thr Ala 245 250 255 Leu Gly Leu Asn Cys Pro Arg Leu Gln Ile
Leu Glu Ala Ala Arg Cys 260 265 270 Ser His Leu Thr Asp Ala Gly Phe
Thr Leu Leu Ala Arg Asn Cys His 275 280 285 Glu Leu Glu Lys Met Asp
Leu Glu Glu Cys Ile Leu Ile Thr Asp Ser 290 295 300 Thr Leu Ile Gln
Leu Ser Ile His Cys Pro Lys Leu Gln Ala Leu Ser 305 310 315 320 Leu
Ser His Cys Glu Leu Ile Thr Asp Asp Gly Ile Leu His Leu Ser 325 330
335 Asn Ser Thr Cys Gly His Glu Arg Leu Arg Val Leu Glu Leu Asp Asn
340 345 350 Cys Leu Leu Ile Thr Asp Val Ala Leu Glu His Leu Glu Thr
Ala Glu 355 360 365 Ala Trp Ser Ala Ser Ser Cys Thr Thr Ala Ser Arg
Leu Pro Val Gln 370 375 380 Ala Ser Ser Gly Cys Gly Leu Ser Ser Leu
Met Ser Lys Ser Thr Pro 385 390 395 400 Thr Leu Leu Pro Ser Pro His
Arg Gln Gln Trp Gln Glu Val Asp Ser 405 410 415 Asp Cys Ala Gly Ala
Val Ser Phe Ser Asp Ser Ser Cys Leu Gly Pro 420 425 430 Arg Gly Asp
Glu Ala Ser Phe Pro Leu Glu Asp Leu Ser Leu Pro Asp 435 440 445 Arg
Leu His His His Pro Ile Cys 450 455 52 1230 DNA Homo sapiens 52
ttcggccatg gttttctcaa acaatgatga aggccttatt aacaaaaagt tacccaaaga
60 acttctgtta agaatatttt ccttcttgga tatagtaact ttgtgccgat
gtgcacagat 120 ttccaaggct tggaacatct tagccctgga tggaagcaac
tggcaaagaa tagatctttt 180 taactttcaa atagatgtag agggtcgagt
ggtggaaaat atctcgaagc gatgcggtgg 240 attcctgagg aagctcagct
tgcgaggctg cattggtgtt ggggattcct ccttgaagac 300 ctttgcacag
aactgccgaa acattgaaca tttgaacctc aatggatgca caaaaatcac 360
tgacagcacg tgttatagcc ttagcagatt ctgttccaag ctgaaacatc tggatctgac
420 ctcctgtgtg tctattacaa acagctcctt gaaggggatc agtgagggct
gccgaaacct 480 ggagtacctg aacctctctt ggtgtgatca gatcacgaag
gatggcatcg aggcactggt 540 gcgaggttgt cgaggcctga aagccctgct
cctgaggggc tgcacacagt tagaagatga 600 agctctgaaa cacattcaga
attactgcca tgagcttgtg agcctcaact tgcagtcctg 660 ctcacgtatc
acggatgaag gtgtggtgca gatatgcagg ggctgtcacc ggctacaggc 720
tctctgcctt tcgggttgca gcaacctcac agatgcctct cttacagccc tgggtttgaa
780 ctgtccgcga ctgcaaattt tggaggctgc ccgatgctcc catttgactg
acgcaggttt 840 tacactttta gctcggaatt gccacgaatt ggagaagatg
gatcttgaag aatgcatcct 900 gataaccgac agcacactca tccagctctc
cattcactgt cctaaactgc aagccctgag 960 cctgtcccac tgtgaactca
tcacagatga tgggatcctg cacctgagca acagtacctg 1020 tggccatgag
aggctgcggg tactggagtt ggacaactgc ctcctcatca ctgatgtggc 1080
cctggaacac ctagaaactg ccgaggcctg gagcgcctcg agctgtacga ctgccagcag
1140 gttacccgtg caggcatcaa gcggatgcgg gctcagctcc ctcatgtcaa
agtccacgcc 1200 tactttgctc ccgtcacccc accgacagca 1230 53 380 PRT
Homo sapiens 53 Arg Pro Arg Phe Gly Thr Ser Asp Ile Glu Asp Asp Ala
Tyr Ala Glu 1 5 10 15 Lys Asp Gly Cys Gly Met Asp Ser Leu Asn Lys
Lys Phe Ser Ser Ala 20 25 30 Val Leu Gly Glu Gly Pro Asn Asn Gly
Tyr Phe Asp Lys Leu Pro Tyr 35 40 45 Glu Leu Ile Gln Leu Ile Leu
Asn His Leu Thr Leu Pro Asp Leu Cys 50 55 60 Arg Leu Ala Gln Thr
Cys Lys Leu Leu Ser Gln His Cys Cys Asp Pro 65 70 75 80 Leu Gln Tyr
Ile His Leu Asn Leu Gln Pro Tyr Trp Ala Lys Leu Asp 85 90 95 Asp
Thr Ser Leu Glu Phe Leu Gln Ser Arg Cys Thr Leu Val Gln Trp 100 105
110 Leu Asn Leu Ser Trp Thr Gly Asn Arg Gly Phe Ile Ser Val Ala Gly
115 120 125 Phe Ser Arg Phe Leu Lys Val Cys Gly Ser Glu Leu Val Arg
Leu Glu 130 135 140 Leu Ser
Cys Ser His Phe Leu Asn Glu Thr Cys Leu Glu Val Ile Ser 145 150 155
160 Glu Met Cys Pro Asn Leu Gln Ala Leu Asn Leu Ser Ser Cys Asp Lys
165 170 175 Leu Pro Pro Gln Ala Phe Asn His Ile Ala Lys Leu Cys Ser
Leu Lys 180 185 190 Arg Leu Val Leu Tyr Arg Thr Lys Val Glu Gln Thr
Ala Leu Leu Ser 195 200 205 Ile Leu Asn Phe Cys Ser Glu Leu Gln His
Leu Ser Leu Gly Ser Cys 210 215 220 Val Met Ile Glu Asp Tyr Asp Val
Ile Ala Ser Met Ile Gly Ala Lys 225 230 235 240 Cys Lys Lys Leu Arg
Thr Leu Asp Leu Trp Arg Cys Lys Asn Ile Thr 245 250 255 Glu Asn Gly
Ile Ala Glu Leu Ala Ser Gly Cys Pro Leu Leu Glu Glu 260 265 270 Leu
Asp Leu Gly Trp Cys Pro Thr Leu Gln Ser Ser Thr Gly Cys Phe 275 280
285 Thr Arg Leu Ala His Gln Leu Pro Asn Leu Gln Lys Leu Phe Leu Thr
290 295 300 Ala Asn Arg Ser Val Cys Asp Thr Asp Ile Asp Glu Leu Ala
Cys Asn 305 310 315 320 Cys Thr Arg Leu Gln Gln Leu Asp Ile Leu Gly
Lys Val Thr Ile Tyr 325 330 335 Lys Phe Val Leu Asn Val Cys Phe Leu
Asp Arg Lys Ala Asn Leu Arg 340 345 350 Leu Phe Val Arg Lys Lys Lys
Ile Phe Gly Tyr Asn Lys Asn Phe Ile 355 360 365 Leu Ile Arg Trp Leu
Gly Leu Ile Gly Asn Ala Arg 370 375 380 54 1380 DNA Homo sapiens 54
aggccaagat tcggcacgag tgatatagaa gatgatgcct atgcagaaaa ggatggttgt
60 ggaatggaca gtcttaacaa aaagtttagc agtgctgtcc tcggggaagg
gccaaataat 120 gggtattttg ataaactacc ttatgagctt attcagctga
ttctgaatca tcttacacta 180 ccagacctgt gtagattagc acagacttgc
aaactactga gccagcattg ctgtgatcct 240 ctgcaataca tccacctcaa
tctgcaacca tactgggcaa aactagatga cacttctctg 300 gaatttctac
agtctcgctg cactcttgtc cagtggctta atttatcttg gactggcaat 360
agaggcttca tctctgttgc aggatttagc aggtttctga aggtttgtgg atccgaatta
420 gtacgccttg aattgtcttg cagccacttt cttaatgaaa cttgcttaga
agttatttct 480 gagatgtgtc caaatctaca ggccttaaat ctctcctcct
gtgataagct accacctcaa 540 gctttcaacc acattgccaa gttatgcagc
cttaaacgac ttgttctcta tcgaacaaaa 600 gtagagcaaa cagcactgct
cagcattttg aacttctgtt cagagcttca gcacctcagt 660 ttaggcagtt
gtgtcatgat tgaagactat gatgtgatag ctagcatgat aggagccaag 720
tgtaaaaaac tccggaccct ggatctgtgg agatgtaaga atattactga gaatggaata
780 gcagaactgg cttctgggtg tccactactg gaggagcttg accttggctg
gtgcccaact 840 ctgcagagca gcaccgggtg cttcaccaga ctggcacacc
agctcccaaa cttgcaaaaa 900 ctctttctta cagctaatag atctgtgtgt
gacacagaca ttgatgaatt ggcatgtaat 960 tgtaccaggt tacagcagct
ggacatatta ggtaaggtta caatatataa atttgtttta 1020 aatgtctgtt
tccttgacag aaaagccaat ctcagacttt ttgttaggaa aaagaaaatt 1080
tttggataca ataaaaattt tatcctgata agatggcttg gtttgatagg aaatgccaga
1140 tagatcagtt aatataggga ataattatat atgtacttta ataaaatagt
gaggacaata 1200 acaattttat agttgaactg taaaaaacta taaccattaa
ttcttggtct acttgtaaga 1260 gtgagaattt acatgagctg cgctctctat
ttttattaag gagagaagaa attaattcat 1320 ttgtataatg aattcaagct
agtttttttt aagtttctta attaagcggc cgcaagctta 1380 55 519 PRT Homo
sapiens 55 Met Val Ile Met Leu Glx Glu Arg Gln Lys Phe Phe Lys Tyr
Ser Val 1 5 10 15 Asp Glu Lys Ser Asp Lys Glu Ala Glu Val Ser Glu
His Ser Thr Gly 20 25 30 Ile Thr His Leu Pro Pro Glu Val Met Leu
Ser Ile Phe Ser Tyr Leu 35 40 45 Asn Pro Gln Glu Leu Cys Arg Cys
Ser Gln Val Ser Met Lys Trp Ser 50 55 60 Gln Leu Thr Lys Thr Gly
Ser Leu Trp Lys His Leu Tyr Pro Val His 65 70 75 80 Trp Ala Arg Gly
Asp Trp Tyr Ser Gly Pro Ala Thr Glu Leu Asp Thr 85 90 95 Glu Pro
Asp Asp Glu Trp Val Lys Asn Arg Lys Asp Glu Ser Arg Ala 100 105 110
Phe His Glu Trp Asp Glu Asp Ala Asp Ile Asp Glu Ser Glu Glu Ser 115
120 125 Ala Glu Glu Ser Ile Ala Ile Ser Ile Ala Gln Met Glu Lys Arg
Leu 130 135 140 Leu His Gly Leu Ile His Asn Val Leu Pro Tyr Val Gly
Thr Ser Val 145 150 155 160 Lys Thr Leu Val Leu Ala Tyr Ser Ser Ala
Val Ser Ser Lys Met Val 165 170 175 Arg Gln Ile Leu Glu Leu Cys Pro
Asn Leu Glu His Leu Asp Leu Thr 180 185 190 Gln Thr Asp Ile Ser Asp
Ser Ala Phe Asp Ser Trp Ser Trp Leu Gly 195 200 205 Cys Cys Gln Ser
Leu Arg His Leu Asp Leu Ser Gly Cys Glu Lys Ile 210 215 220 Thr Asp
Val Ala Leu Glu Lys Ile Ser Arg Ala Leu Gly Ile Leu Thr 225 230 235
240 Ser His Gln Ser Gly Phe Leu Lys Thr Ser Thr Ser Lys Ile Thr Ser
245 250 255 Thr Ala Trp Lys Asn Lys Asp Ile Thr Met Gln Ser Thr Lys
Gln Tyr 260 265 270 Ala Cys Leu His Asp Leu Thr Asn Lys Gly Ile Gly
Glu Glu Ile Asp 275 280 285 Asn Glu His Pro Trp Thr Lys Pro Val Ser
Ser Glu Asn Phe Thr Ser 290 295 300 Pro Tyr Val Trp Met Leu Asp Ala
Glu Asp Leu Ala Asp Ile Glu Asp 305 310 315 320 Thr Val Glu Trp Arg
His Arg Asn Val Glu Ser Leu Cys Val Met Glu 325 330 335 Thr Ala Ser
Asn Phe Ser Cys Ser Thr Ser Gly Cys Phe Ser Lys Asp 340 345 350 Ile
Val Gly Leu Arg Thr Ser Val Cys Trp Gln Gln His Cys Ala Ser 355 360
365 Pro Ala Phe Ala Tyr Cys Gly His Ser Phe Cys Cys Thr Gly Thr Ala
370 375 380 Leu Arg Thr Met Ser Ser Leu Pro Glu Ser Ser Ala Met Cys
Arg Lys 385 390 395 400 Ala Ala Arg Thr Arg Leu Pro Arg Gly Lys Asp
Leu Ile Tyr Phe Gly 405 410 415 Ser Glu Lys Ser Asp Gln Glu Thr Gly
Arg Val Leu Leu Phe Leu Ser 420 425 430 Leu Ser Gly Cys Tyr Gln Ile
Thr Asp His Gly Leu Arg Val Leu Thr 435 440 445 Leu Gly Gly Gly Leu
Pro Tyr Leu Glu His Leu Asn Leu Ser Gly Cys 450 455 460 Leu Thr Ile
Thr Gly Ala Gly Leu Gln Asp Leu Val Ser Ala Cys Pro 465 470 475 480
Ser Leu Asn Asp Glu Tyr Phe Tyr Tyr Cys Asp Asn Ile Asn Gly Pro 485
490 495 His Ala Asp Thr Ala Ser Gly Cys Gln Asn Leu Gln Cys Gly Phe
Arg 500 505 510 Ala Cys Cys Arg Ser Gly Glu 515 56 2276 DNA Homo
sapiens 56 atggtaatca tgctgtaaga gcgacagaaa ttttttaaat attccgtgga
tgaaaagtca 60 gataaagaag cagaagtgtc agaacactcc acaggtataa
cccatcttcc tcctgaggta 120 atgctgtcaa ttttcagcta tcttaatcct
caagagttat gtcgatgcag tcaagtaagc 180 atgaaatggt ctcagctgac
aaaaacggga tcgctttgga aacatcttta ccctgttcat 240 tgggccagag
gtgactggta tagtggtccc gcaactgaac ttgatactga acctgatgat 300
gaatgggtga aaaataggaa agatgaaagt cgtgcttttc atgagtggga tgaagatgct
360 gacattgatg aatctgaaga gtctgcggag gaatcaattg ctatcagcat
tgcacaaatg 420 gaaaaacgtt tactccatgg cttaattcat aacgttctac
catatgttgg tacttctgta 480 aaaaccttag tattagcata cagctctgca
gtttccagca aaatggttag gcagatttta 540 gagctttgtc ctaacctgga
gcatctggat cttacccaga ctgacatttc agattctgca 600 tttgacagtt
ggtcttggct tggttgctgc cagagtcttc ggcatcttga tctgtctggt 660
tgtgagaaaa tcacagatgt ggccctagag aagatttcca gagctcttgg aattctgaca
720 tctcatcaaa gtggcttttt gaaaacatct acaagcaaaa ttacttcaac
tgcgtggaaa 780 aataaagaca ttaccatgca gtccaccaag cagtatgcct
gtttgcacga tttaactaac 840 aagggcattg gagaagaaat agataatgaa
cacccctgga ctaagcctgt ttcttctgag 900 aatttcactt ctccttatgt
gtggatgtta gatgctgaag atttggctga tattgaagat 960 actgtggaat
ggagacatag aaatgttgaa agtctttgtg taatggaaac agcatccaac 1020
tttagttgtt ccacctctgg ttgttttagt aaggacattg ttggactaag gactagtgtc
1080 tgttggcagc agcattgtgc ttctccagcc tttgcgtatt gtggtcactc
attttgttgt 1140 acaggaacag ctttaagaac tatgtcatca ctcccagaat
cttctgcaat gtgtagaaaa 1200 gcagcaagga ctagattgcc taggggaaaa
gacttaattt actttgggag tgaaaaatct 1260 gatcaagaga ctggacgtgt
acttctgttt ctcagtttat ctggatgtta tcagatcaca 1320 gaccatggtc
tcagggtttt gactctggga ggagggctgc cttatttgga gcaccttaat 1380
ctctctggtt gtcttactat aactggtgca ggcctgcagg atttggtttc agcatgtcct
1440 tctctgaatg atgaatactt ttactactgt gacaacatta acggtcctca
tgctgatacc 1500 gccagtggat gccagaattt gcagtgtggt tttcgagcct
gctgccgctc tggcgaatga 1560 cccttgactt ctgatctttg tctacttcat
ttagctgagc aggctttctt tcatgcactt 1620 tactcatagc acatttcttg
tgttaaccat ccctttttga gcgtgacttg ttttggcccc 1680 atttcttaca
acttcagaaa tcttaattta ccagtgaatt gtaatgttgt ttctcttgca 1740
aattatactt ttggtttaga aagggattag gtcttttcaa aagggtgaga acagtcttac
1800 atttttcttt taaatgaaat gctttaaaga atgttggtaa tgccatgtca
tttaaagtat 1860 ttcatagata attttgagtt ttaaagtcca tggaggtgat
tggttctctt tacacattaa 1920 cactgtacca agctttgcag atcttttccg
acacacatgt ctgaagactt attttcaaag 1980 acagcacatt tttggaaact
aatctctttt ccgtaatatt tcctttattt caatgattct 2040 cagaaggcca
attcaaacaa acccacattt aaggttcttt aggattatag aataaattgg 2100
cttctgagtg ttagctcagt gagtaggaaa gcaccaatcg atatttgttt cctttaggga
2160 tactttgttc tcaccactgt ccctatgtca tcaaatttgg gagagatttt
ttaaaatacc 2220 acaatcattt gaagaaatgt ataaataaaa tctactttga
ggactttacc aagtaa 2276 57 39 PRT Homo sapiens 57 Leu Pro Leu Glu
Leu Ser Phe Tyr Leu Leu Lys Trp Leu Asp Pro Gln 1 5 10 15 Thr Leu
Leu Thr Cys Cys Leu Val Ser Lys Gln Trp Asn Lys Val Ile 20 25 30
Ser Ala Cys Thr Glu Val Trp 35 58 117 DNA Homo sapiens 58
cttcccctgg agctcagttt ttatttgtta aaatggctcg atcctcagac tttactcaca
60 tgctgcctcg tctctaaaca gtggaataag gtgataagtg cctgtacaga ggtgtgg
117 59 10 DNA Artificial Sequence Description of Artificial
Sequence Synthetic 59 aattcgcgcg 10 60 21 PRT Artificial Sequence
Description of Artificial Sequence Synthetic 60 Lys Lys Glu Arg Leu
Leu Asp Asp Arg His Asp Ser Gly Leu Asp Ser 1 5 10 15 Met Lys Asp
Glu Glu 20
* * * * *