U.S. patent application number 09/894967 was filed with the patent office on 2002-10-24 for binding sites for phosphotyrosine binding domains.
Invention is credited to Kavanaugh, William Michael, Williams, Lewis T..
Application Number | 20020156236 09/894967 |
Document ID | / |
Family ID | 23679669 |
Filed Date | 2002-10-24 |
United States Patent
Application |
20020156236 |
Kind Code |
A1 |
Kavanaugh, William Michael ;
et al. |
October 24, 2002 |
Binding sites for phosphotyrosine binding domains
Abstract
The present invention generally provides peptides that comprise
a recognition sequence motif for phosphotyrosine binding proteins.
In particular, the present invention provides peptides which
comprise a core sequence of amino acids, and analogs thereof, which
are recognized and bound by the PTB (phosphotyrosine binding)
domain. Also provided are methods of using the peptides of the
invention in diagnostic, screening and therapeutic
applications.
Inventors: |
Kavanaugh, William Michael;
(Mill Valley, CA) ; Williams, Lewis T.; (Tiburon,
CA) |
Correspondence
Address: |
TOWNSEND AND TOWNSEND AND CREW, LLP
TWO EMBARCADERO CENTER
EIGHTH FLOOR
SAN FRANCISCO
CA
94111-3834
US
|
Family ID: |
23679669 |
Appl. No.: |
09/894967 |
Filed: |
June 27, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09894967 |
Jun 27, 2001 |
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08423646 |
Apr 14, 1995 |
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6280964 |
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Current U.S.
Class: |
530/324 ;
530/325; 530/326; 530/327; 530/328 |
Current CPC
Class: |
C07K 2319/00 20130101;
A61K 38/00 20130101; Y10S 530/81 20130101; G01N 33/68 20130101;
G01N 2333/71 20130101; C07K 14/71 20130101; G01N 33/6842
20130101 |
Class at
Publication: |
530/324 ;
530/325; 530/326; 530/327; 530/328 |
International
Class: |
C07K 014/435; C07K
007/08 |
Goverment Interests
[0001] This invention was made with Government support under Grant
Nos. K11 HL02410 and R01-HL32898, awarded by the National
Institutes of Health. The Government has certain rights in this
invention.
Claims
What is claimed is:
1. A substantially pure peptide which is capable of binding a PTB
domain, wherein the peptide is from 5 to 100 amino acids in length,
and comprises a core sequence of amino acids
NX.sub.3X.sub.1X.sub.2X.sub.4; wherein X.sub.1 is selected from the
group consisting of Y, pY or an analog thereof, E, T, D, Q, A and
F; X.sub.2 is selected from pY or an analog thereof, and Y,
provided that at least one of X.sub.1 and X.sub.2 is pY, or an
analog thereof; X.sub.3 is selected from the group consisting of L
and A; and X.sub.4 is selected from the group consisting of W, L,
S, F and Q.
2. The peptide as recited in claim 1, wherein the peptide is from 6
to 100 amino acids in length, and comprises a core sequence of
amino acids X.sub.5NX.sub.3X.sub.1X.sub.2X.sub.4, wherein X.sub.5
is selected from the group consisting of D, S, E and A.
3. The peptide as recited in claim 2, wherein X.sub.2 is pY.
4. The peptide as recited in claim 3, wherein the peptide is from 6
to 100 amino acids in length, and comprises a core sequence of
amino acids selected from the group consisting of
DNX.sub.3X.sub.1pYX.sub.4 and ENX.sub.3X.sub.1pYX.sub.4, where
X.sub.4 is selected from the group consisting of W and F.
5. The peptide as recited in claim 2, wherein the peptide is from
12 to 100 amino acids in length, and comprises a core sequence of
amino acids selected from the group consisting of AFDNLY(pY)WDQNS,
AFDNL(pY)YWDQNS and AFDNL(pY)(pY)WDQNS.
6. The peptide as recited in claim 2, wherein the peptide is from
21 to 100 amino acids in length, and comprises a core sequence of
amino acids selected from the group consisting of:
PAFSPAFDNLY(pY)WDQNSSEQG; PAFSPAFDNL(pY)YWDQNSSEQG;
PAFSPAFDNL(pY)(pY)WDQNSSEQG; PAFSPAADNLY(pY)WDQNSSEQG;
PAFSPAADNL(pY)YWDQNSSEQG; PAFSPAADNL(pY)(pY)WDQNSSEQG;
PAFSPAFANLY(pY)WDQNSSEQG; PAFSPAFANL(pY)YWDQNSSEQG;
PAFSPAFANL(pY)(pY)WDQNSSEQG; PAFSPAFSNLY(pY)WDQNSSEQG;
PAFSPAFSNL(pY)YWDQNSSEQG; PAFSPAFSNL(pY)(pY)WDQNSSEQG;
PAFSPAFDNAY(pY)WDQNSSEQG; PAFSPAFDNA(pY)YWDQNSSEQG;
PAFSPAFDNA(pY)(pY)WDQNSSEQG; PAFSPAFDNLA(pY)WDQNSSEQG;
PAFSPAFDNLF(pY)WDQNSSEQG; PAFSPAFDNLY(pY)FDQNSSEQG;
PAFSPAFDNL(pY)YFDQNSSEQG; PAFSPAFDNL(pY)(pY)FDQNSSEQG;
PAFSPAFDNLY(pY)WAQNSSEQG; PAFSPAFDNL(pY)YWAQNSSEQG;
PAFSPAFDNL(pY)(pY)WAQNSSEQG; PAFSPAFDNLY(pY)WDANSSEQG;
PAFSPAFDNL(pY)YWDANSSEQG; PAFSPAFDNL(pY)(pY)WDANSSEQG;
PAFSPAFDNLY(pY)WDNNSSEQG; PAFSPAFDNL(pY)YWDNNSSEQG;
PAFSPAFDNL(pY)(pY)WDNNSSEQG; PAFSPAFDNLY (pY)WDDNSSEQG;
PAFSPAFDNL(pY)YWDDNSSEQG; PAFSPAFDNL(pY)(pY)WDDNSSEQG;
PAFSPAFDNLY(pY)WDQASSEQG; PAFSPAFDNL(pY)YWDQASSEQG;
PAFSPAFDNL(pY)(pY)WDQASSEQG; PAFSPAFDNLY(pY)WDQNASEQG; PAFSPAFDNL
(pY)YWDQNASEQG; and PAFSPAFDNL(pY)(pY)WDQNASEQG.
7. The peptide as recited in claim 1, wherein at least one of
X.sub.1 and X.sub.2 is an analog of phosphotyrosine, and said
analog is (phosphonomethyl)phenylalanine.
8. A substantially pure peptide which is capable of binding a PTB
domain, wherein the peptide is from 21 to about 100 amino acids in
length and which comprises a core sequence of amino acids selected
from the group consisting of AFGGAVENPE(pY)LAPRAGTASQ and
EGTPTAENPE(pY)LGLDVPV.
9. A composition comprising a peptide as recited in claim 1, and a
pharmaceutically acceptable carrier.
10. A method of determining whether a protein comprises a PTB
domain, comprising the steps of: contacting the protein with a
peptide, which peptide is from 5 to 100 amino acids in length and
comprises a core sequence of amino acids
NX.sub.3X.sub.1X.sub.2X.sub.4, wherein X.sub.1 is selected from the
group consisting of Y, pY, E, T, D, Q, A and F; X.sub.2 is selected
from pY and Y, provided that at least one of X.sub.1 and X.sub.2 is
pY; X.sub.3 is selected from the group consisting of L and A; and
X.sub.4 is selected from the group consisting of W, L, S, F and Q;
and determining whether the peptide binds to the protein during
said contacting step, where the binding of the peptide to the
protein is indicative that the protein comprises a PTB domain.
11. The method as recited in claim 10, wherein prior to said
contacting step, the protein is attached to a solid support; the
peptide used in said contacting step further comprises a detectable
group fused to the peptide; and said determining step comprises
assaying for the presence of the detectable group.
12. The method as recited in claim 10, wherein prior to said
contacting step, the peptide is attached to a solid support.
13. A method of determining whether a test compound is an agonist
or antagonist of a PTB/phosphorylated ligand interaction,
comprising the steps of: incubating the test compound with a
protein comprising a PTB domain and a peptide, which peptide is
from 5 to 100 amino acids in length and which comprises a core
amino acid sequence NX.sub.3X.sub.1X.sub.2X.sub.4, wherein X.sub.1
is selected from the group consisting of Y, pY, E, T, D, Q, A and
F; X.sub.2 is selected from pY and Y, provided that at least one of
X.sub.1 and X.sub.2 is pY; X.sub.3 is selected from the group
consisting of L and A; and X.sub.4 is selected from the group
consisting of W, L, S, F and Q; and determining the amount of
protein bound to the peptide during said incubating step; and
comparing the amount of protein bound to the peptide during said
incubating step to an amount of protein bound to the peptide in the
absence of the test compound, the increase or decrease in the
amount of protein bound to the peptide in the presence of the test
compound being indicative that the test compound is an agonist or
antagonist of PTB domain/phosphorylated ligand interaction,
respectively.
14. A method of inhibiting the binding of a PTB domain-containing
protein to a tyrosine phosphorylated target, comprising contacting
the PTB domain-containing protein with an effective amount of the
peptide of claim 1.
15. The method as recited in claim 14, wherein the tyrosine
phosphorylated target is c-erbB2.
16. The method as recited claim 15, wherein the PTB
domain-containing protein is SHC.
17. A method of obtaining substantially pure PTB-domain-containing
protein from a mixture of different proteins, comprising the steps
of: providing a peptide which is from 5 to 100 amino acids in
length, and which comprises a core amino acid sequence
NX.sub.3X.sub.1X.sub.2X.sub.4, wherein X.sub.1 is selected from the
group consisting of Y, pY, E, T, D, Q, A and F; X.sub.2 is selected
from pY and Y, provided that at least one of X.sub.1 and X.sub.2 is
pY; X.sub.3 is selected from the group consisting of L and A; and
X.sub.4 is selected from the group consisting of W, L, S, F and Q;
bound to a solid support; contacting the mixture of different
proteins with the peptide bound to the solid support whereby the
PTB domain-containing protein is bound to the peptide; washing the
solid support to remove unbound proteins; and eluting substantially
pure PTB-domain-containing protein from the solid support.
18. A method of treating a patient suffering from a proliferative
cell disorder, comprising administering to the patient an effective
amount of the peptide recited in claim 1.
19. The method as recited in claim 18, wherein the proliferative
cell disorder is selected from the group consisting of
atherosclerosis, inflammatory joint disease, psoriasis, restinosis
and cancer.
20. The method as recited in claim 19, wherein the proliferative
cell disorder is cancer.
21. The method as recited in claim 20, wherein the cancer is breast
cancer.
Description
BACKGROUND OF THE INVENTION
[0002] The present invention generally provides peptides that
comprise a recognition sequence motif for phosphotyrosine binding
proteins. In particular, the present invention provides peptides
which comprise a core sequence of amino acids, and analogs thereof,
which are recognized and bound by the PTB phosphotyrosine binding
domain. Also provided are methods of using the peptides of the
invention in diagnostic, screening and therapeutic
applications.
[0003] Receptor signaling pathways are the subject of widespread
research efforts. A better understanding of these signaling
pathways will lead to the design of new and more effective drugs in
the treatment of many diseases. Of particular interest are the
growth factor and related receptor signaling pathways and their
role in cell growth and differentiation. Binding of a particular
growth factor to its receptor on the cell plasma membrane can
stimulate a wide variety of biochemical responses, including
changes in ion fluxes, activation of various kinases, alteration of
cell shape, transcription of various genes and modulation of
enzymatic activities in cellular metabolism.
[0004] In particular, upon binding an external ligand, a receptor
may undergo auto-phosphorylation of specific tyrosine residues,
and/or may phosphorylate other proteins. This tyrosine
phosphorylation creates binding sites for cytoplasmic signaling
proteins which have specific domains that recognize the
phosphorylated tyrosine and adjacent residues. Once bound, these
signaling proteins may in turn be activated. The activated
signaling proteins then may effect downstream processes. Pawson and
Gish, Cell 71:359-362 (1992).
[0005] Src Homologous, or "SH2" domains are amino acid sequences
that are similar to a 100-residue, non-catalytic region of the Src
tyrosine kinase and are present in various signaling molecules.
Sadowski et al., Mol. Cell. Biol. 6, 4396 (1986). SH2 domains are
functional protein motifs that bind tyrosine-phosphorylated targets
by recognizing phosphotyrosine and specific adjacent residues. J.
A. Escobedo et al., Mol. Cell. Biol. 11, 1125 (1991); L. C. Cantley
et al. Cell 64, 281 (1991); T. Pawson and G. D. Gish Cell 71, 359
(1992); S. Zhou et al. Cell 72, 767 (1993); G. Waksman, S. E.
Shoelson, N. Pant, D. Cowburn, J. Kuriyan Cell 72, 779 (1993).
Activation of tyrosine kinases by growth factors, cytokines, and
oncogenic agents therefore serves as a switch for assembling SH2
domain-containing proteins with their tyrosine-phosphorylated
targets in signaling complexes, in which downstream effectors are
activated.
[0006] The use of phosphotyrosine binding domains, including SH2
domains, has been discussed in methods for identifying targets of
tyrosine kinases in cells, and thus identifying intermediates in
cell signaling pathways. See, PCT Patent Application No. WO
92/13001, to Schlessinger et al.
[0007] The specific use of SH2 domains and subdomains in affecting
the SH2/phosphorylated ligand regulatory scheme, or screening for
compounds which affect SH2 binding in this regulatory scheme, has
been previously described. See, U.S. Pat. No. 5,352,660 to A. J.
Pawson. The use of these domains in assaying for the presence of
SH2 binding phosphoproteins has also been described.
[0008] Specific SH2 containing proteins include the products of the
SHC gene. The SHC (which stands for SH2, Collagen) gene encodes a
transforming protein, expressed as 46- and 52-kD proteins that are
tyrosine phosphorylated in response to a number of growth factors,
e.g., PDGF, EGF and FGF, and have been implicated as mediators of
signaling from growth factor receptor and non-receptor tyrosine
kinases to Ras. G. Pelicci et al. Cell 70, 93-104 (1992); M.
Rozakis-Adcock et al. Nature, 360:689 (1992).
[0009] Thus, a great deal of attention has been directed toward
studying these SH2 domains and their role in cell signaling
pathways. However, SH2 domains, and the proteins which comprise
them, are not the only phosphotyrosine binding mediators of such
pathways.
[0010] A new phosphotyrosine binding ("PTBI") domain has been
identified within the sequence of the SHC protein. See, Kavanaugh
and Williams, Science (1994) 266:1862-1865. This PTB domain was
reported to specifically bind the tyrosine phosphorylated version
of a target protein, which target protein was phosphorylated upon
cell activation/stimulation, e.g., anti-IgM stimulated B cells,
IL-6 stimulated HepG2 hepatoma cells, LIF stimulated CCE embryonic
stem cells. The amino acid sequence of this domain is unlike that
of any member of the known SH2 domain family. Therefore, although
the nature of phosphotyrosine binding by the PTB domain is similar
to that of the SH2 domain, functionally, and mechanistically, the
two are very different.
[0011] The study of these cell signaling pathways, and the ability
to control them requires identification and characterization of
proteins which contain phosphotyrosine binding domains and the
protein sequences to which they bind. The present invention meets
these and other needs.
SUMMARY OF THE INVENTION
[0012] The present invention generally provides substantially pure
peptides which are capable of binding a PTB domain, wherein the
peptide is from 5 to 100 amino acids in length, and comprises a
core sequence of amino acids NX.sub.3X.sub.1X.sub.2X.sub.4; where
X.sub.1 is selected from the group consisting of Y, pY or an analog
thereof, E, T, D, Q, A and F; X.sub.2 is selected from pY or an
analog thereof, and Y, provided that at least one of X.sub.1 and
X.sub.2 is pY, or an analog thereof; X.sub.3 is selected from the
group consisting of L and A; and X.sub.4 is selected from the group
consisting of W, L, S, F and Q. In a preferred embodiment, at least
one of X.sub.1 and X.sub.2 will be an analog of phosphotyrosine,
and said analog is (phosphonomethyl)-phenylalanine. In preferred
aspects, the peptides are from 6 to 100 amino acids in length, and
comprise a core sequence of amino acids
X.sub.5NX.sub.3X.sub.1X.sub.2X.sub.4, wherein X.sub.5 is selected
from the group consisting of D, S, E and A. In still more preferred
peptides, X.sub.2 will be pY. In particularly preferred
embodiments, the peptides will be from 6 to 100 amino acids in
length, and comprise a core sequence of amino acids selected from
the group consisting of DNX.sub.3X.sub.1pYX.sub.4 and
ENX.sub.3X.sub.1pYX.sub.4, where X.sub.4 is selected from the group
consisting of W and F.
[0013] Especially preferred peptides will be from 12 to 100 amino
acids in length, and which comprise a core sequence of amino acids
selected from the group consisting of: AFDNLY(pY)WDQNS;
AFDNL(pY)YWDQNS; and AFDNL(pY)(pY)WDQNS. As preferred, are peptides
which are from 21 to 100 amino acids in length and which comprise a
core sequence of amino acids selected from the group consisting
of:
[0014] PAFSPAFDNLY(pY)WDQNSSEQG; PAFSPAFDNL(pY)YWDQNSSEQG;
PAFSPAFDNL(pY)(pY)WDQNSSEQG; PAFSPAADNLY(pY)WDQNSSEQG;
PAFSPAADNL(pY) YWDQNSSEQG; PAFSPAADNL(pY)(pY)WDQNSSEQG; PAFSPAFANLY
(pY) WDQNSSEQG; PAFSPAFANL (pY) YWDQNSSEQG;
PAFSPAFANL(pY)(pY)WDQNSSEQG; PAFSPAFSNLY (pY)WDQSSEQG;
PAFSPAFSNL(pY)YWDQNSSEQG; PAFSPAFSNL(pY)(pY)WDQNSSEQG;
PAFSPAFDNAY(pY)WDQNSSEQG; PAFSPAFDNA(pY)YWDQNSSEQG;
PAFSPAFDNA(pY)(pY)WDQNSSEQG; PAFSPAFDNLA(pY)WDQNSSEQG; PAFSPAFDNLF
(pY)WDQNSSEQG; PAFSPAFDNLY(pY)FDQNSSEQG; PAFSPAFDNL(pY)YFDQNSSEQG;
PAFSPAFDNL(pY)(pY)FDQNSSEQG; PAFSPAFDNLY(pY)WAQNSSEQG;
PAFSPAFDNL(pY)YWAQNSSEQG; PAFSPAFDNL(pY)(pY)WAQNSSEQG;
PAFSPAFDNLY(pY)WDANSSEQG; PAFSPAFDNL (pY)YWDANSSEQG; PAFSPAFDNL
(pY)(pY)WDANSSEQG; PAFSPAFDNLY(pY) WDNNSSEQG; PAFSPAFDNL(pY)
YWDNNSSEQG; PAFSPAFDNL(pY)(pY)WDNNSSEQG; PAFSPAFDNLY(pY)WDDNSSEQG;
PAFSPAFDNL(pY) YWDDNSSEQG; PAFSPAFDNL(pY)(pY)WDDNSSEQG;
PAFSPAFDNLY(pY) WDQASSEQG; PAFSPAFDNL(pY) YWDQASSEQG;
PAFSPAFDNL(pY)(pY)WDQASSEQG; PAFSPAFDNLY(pY)WDQNASEQG;
PAFSPAFDNL(pY)YWDQNASEQG; and PAFSPAFDNL(pY)(pY)WDQNASEQG.
[0015] In an alternate embodiment, the present invention provides
substantially pure peptides which are capable of binding a PTB
domain, wherein the peptides are from 21 to about 100 amino acids
in length and which comprise a core sequence of amino acids
selected from the group consisting of AFGGAVENPE(pY)LAPRAGTASQ and
EGTPTAENPE(pY)LGLDVPV.
[0016] In a further embodiment, the present invention provides
compositions which comprise the peptides of the present invention
and pharmaceutically acceptable carriers.
[0017] In another embodiment, the present invention provides a
method of determining whether a protein comprises a PTB domain. The
method comprises the steps of contacting the protein with a peptide
of the present invention, and determining whether the peptide binds
to the protein. The binding of the peptide to the protein is
indicative that the protein comprises a PTB domain. In preferred
aspects, the protein is attached to a solid support prior to
contacting the protein with the peptide of the present invention,
and the peptide used in the contacting step further comprises a
detectable group fused to the peptide. The determining step then
comprises assaying for the presence of the detectable group.
Alternatively, the peptide of the invention will be attached to a
solid support prior to contacting the protein with the peptide of
the invention.
[0018] In an additional embodiment, the present invention provides
a method of determining whether a test compound is an agonist or
antagonist of a PTB domain/phosphorylated ligand interaction. The
method comprises the steps of incubating the test compound with a
protein comprising a PTB domain, and a peptide of the invention,
determining the amount of protein bound to the peptide during the
incubating step, and comparing the amount of protein bound to the
peptide during the incubating step to an amount of protein bound to
the peptide in the absence of the test compound. The increase or
decrease in the amount of protein bound to the peptide in the
presence of the test compound will be indicative that the test
compound is an agonist or antagonist of PTB domain/phosphorylated
ligand interaction, respectively.
[0019] In yet another embodiment of the present invention is
provided a method of inhibiting the binding of a PTB
domain-containing protein to a tyrosine phosphorylated target,
comprising contacting the PTB domain-containing protein with an
effective amount of the peptide of the invention. In a preferred
aspect, the tyrosine phosphorylated target is c-erbB2. In another
preferred aspect, the PTB domain-containing protein is SHC.
[0020] Also provided by the present invention, is a method of
obtaining substantially pure PTB domain-containing protein from a
mixture of different proteins. The method comprises the steps of
providing a peptide of the present invention bound to a solid
support. The mixture of different proteins is contacted with the
peptide bound to the solid support whereby the PTB
domain-containing protein is bound to the peptide. The solid
support is washed to remove unbound proteins, and substantially
pure PTB domain-containing protein is then eluted from the solid
support.
[0021] In an additional embodiment, the present invention provides
a method of treating a patient suffering from a proliferative cell
disorder. The method comprises administering to the patient an
effective amount of the peptide of the present invention.
Typically, the proliferative cell disorder is selected from the
group consisting of atherosclerosis, inflammatory joint disease,
psoriasis, restinosis and cancer. Preferably, the proliferative
cell disorder is cancer, and more preferably, breast cancer.
BRIEF DESCRIPTION OF THE DRAWINGS
[0022] FIG. 1 shows proteins expressed from a .lambda. gt11 cDNA
library, immobilized on filters, phosphorylated in vitro using
recombinant PDGF receptor kinase, followed by hybridization with
.sup.32P-labeled PTB domain. Shown is a positive (clone 39.1) and
representative negative plaque purified by successive rounds of
screening, then transferred to a filter. Quadrants of the filter
were treated as indicated prior to hybridization with
.sup.32P-labeled PTB domain.
[0023] FIG. 2 shows the association of c-erbB2 with the PTB domain.
Panel A shows GST-PTB and GST-(1-45) (residues 1-45 of SHC,
containing no PTB domain) fusion proteins, tagged with the
influenza hemagglutinin (IHA) epitope, and incubated with lysate of
SKBR3 cells (containing c-erbB2) or with buffer. Anti-IHA
immunoprecipitates of each were separately blotted with
anti-c-erbB2 ("erbB-2 blot") and anti-IHA 12CA5 antibodies ("IHA
blot"). Also shown are blots of immunoprecipitates using preimmune
serum and anti-SHC serum. Panel B shows a blot of IHA tagged
GST-PTB, incubated with SKBR3 lysate in the presence or absence of
the indicated peptides derived from c-erbB2 (upper blot), and with
varied concentrations of the peptide PAFSPAFDNL(pY)(pY)WDQNSSEQG
("pY1221/pY1222") (lower blot). Panel C shows a blot of IHA tagged
GST-PTB, incubated with SKBR3 lysate in the presence of 500 nM of
the indicated pY substituted peptides.
[0024] FIG. 3 is a bar graph showing the effects of various
conditions upon PTB domain/phosphopeptide binding. IHA tagged
GST-PTB domain fusion protein was incubated in the presence of the
following biotinylated peptides: PAFSPAFDNLYYWDQNSSEQG
("b-unphos."); PAFSPAFDNL(pY)(pY)WDQNSSEQ- G ("b-phos."), alone and
in the presence of 100.times.non-biotinylated, unphosphorylated and
phosphorylated peptide ("100.times.unphos." and "100.times.phos.",
respectively); PAFSPAFDQL(pY)(pY)WDQNSSEQG ("b-N1219Q"); and
PAFSPAFDDL(pY)(pY)WDQNSSEQG ("b-N1219D"). Specific binding was
detected using streptavidin-coupled alkaline phosphatase. Also
shown is the level of binding by b-phos. and b-unphos. to an SH2
phosphotyrosine binding domain.
DESCRIPTION OF THE PREFERRED EMBODIMENT
[0025] The present invention generally provides peptides which
comprise a sequence motif which is recognized and bound by
phosphotyrosine binding proteins. More particularly, the peptides
of the present invention are recognized and bound by proteins which
comprise a PTB domain.
[0026] These peptides, or their analogs, may generally be used in
blocking or inhibiting PTB domain/phosphorylated ligand
interactions, both in vitro and in vivo. As a result, the peptides
of the present invention can be useful as antagonists of PTB
domain/phosphorylated ligand interaction, for controlling or
inhibiting cell signalling pathways which rely on these PTB
domain/phosphorylated ligand interactions, i.e.,.growth factor
dependent activation or stimulation of cells, and growth factor
initiated mitogenesis. The peptides of the present invention can
also be useful as affinity ligands or probes, in the
identification, purification, and/or characterization of PTB domain
containing proteins, or, alternatively, as target peptides in
screening for agonists or antagonists of PTB domain/phosphorylated
ligand interaction.
[0027] I. Definitions
[0028] As used herein, the twenty conventional, or natural amino
acids and their abbreviations follow conventional usage
(Immunology--A Synthesis, 2nd Edition, E. S. Golub and D. R. Gren,
Eds., Sinauer Associates, Sunderland, Mass. (1991)). Specifically,
abbreviations for the amino acid residues as used herein are: A,
Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K,
Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T,
Thr; V, Val; W, Trp; Y, Tyr. Phosphotyrosine is denoted by pY, and
(phosphonomethyl)phenylalanine is denoted by Pmp.
[0029] Stereoisomers (e.g., D-amino acids) of the twenty
conventional amino acids, unnatural amino acids such as
.alpha.,.alpha.-disubstituted amino acids, N-alkyl amino acids,
lactic acid, and other unconventional amino acids may also be
suitable components for polypeptides of the present invention.
Examples of unconventional or unnatural amino acids include amino
acids well known in the art, but which are not included in the
twenty conventional amino acids, such as: 4-hydroxyproline,
.gamma.-carboxyglutamate, .epsilon.-N,N,N-trimethyllysine,
.epsilon.-N-acetyllysine, O-phosphoserine, N-acetylserine,
N-formylmethionine, 3-methylhistidine, 5-hydroxylysine,
.omega.-N-methylarginine, and other similar amino acids and imino
acids (e.g., 4-hydroxyproline). In the polypeptide notation used
herein, the lefthand direction is the amino terminal direction and
the righthand direction is the carboxy-terminal direction, in
accordance with standard usage and convention.
[0030] The term "analog" as used herein refers to compounds which
are generally structurally similar to the compound of which they
are an analog, or "parent" compound. Generally analogs will retain
certain characteristics of the parent compound, e.g., biological or
pharmacological activity, while lacking other, less desirable
characteristics, e.g., antigenicity, proteolytic instability,
toxicity, and the like. As applied to polypeptides, the term
"analog" generally refers to polypeptides which are comprised of a
segment of about at least 3 amino acids that has substantial
identity to at least a portion of a PTB domain-binding peptide, and
which has at least one of the following properties: (1)
specifically binds to the PTB domain, and (2) affects or blocks a
PTB domain-containing protein mediated phenotype. Typically, analog
peptides comprise a conservative amino acid substitution (or
addition or deletion) with respect to the naturally-occurring
sequence. Analogs typically are at least 5 amino acids long,
preferably at least 20 amino acids long or longer, most usually
being as long as a minimal length binding/recognition sequence
identified by methods for identifying PTB domain-binding peptides.
Some analogs may lack substantial biological activity but may still
be employed for various uses, such as for raising antibodies to
predetermined epitopes, as an immunological reagent to detect
and/or purify reactive antibodies by affinity chromatography, or as
a competitive or noncompetitive agonist, antagonist, or partial
agonist of PTB domain function.
[0031] As used herein, the term "peptide" and "polypeptide" refer
to macromolecules which comprise a multiplicity of amino or imino
acids (or their equivalents) in peptide linkage, wherein said
peptides may comprise or lack post-translational modifications
(e..g., glycosylation, cleavage, phosphorylation, side-chain
derivation, and the like).
[0032] As used herein, the terms "label" or "labeled" refer to
incorporation of a detectable marker, e.g., by incorporation of a
radiolabeled amino acid or attachment of biotinyl moieties to a
polypeptide, wherein the attached biotinyl moieties can be detected
by marked avidin (e.g., streptavidin containing a fluorescent
marker or enzymatic activity that can be detected by optical or
colorimetric methods). Various methods of labeling polypeptides and
glycoproteins are known in the art and may be used. Examples of
labels for polypeptides include, but are not limited to, the
following: radioisotopes (e.g., .sup.3H, .sup.14C, .sup.35S,
.sup.125I, .sup.131I), fluorescent labels (e.g., FITC, rhodamine,
lanthanide phosphors), enzymatic labels (e.g., horseradish
peroxidase, .beta.-galactosidase, luciferase, alkaline
phosphatase), biotinyl groups, predetermined polypeptide epitopes
recognized by a secondary reporter (e.g., leucine zipper pair
sequences, binding sites for secondary antibodies, metal binding
domains, epitope tags). In some embodiments, labels are attached by
spacer arms of various lengths to reduce potential steric
hindrance.
[0033] As used herein, "substantially pure" means that the
particular peptide is the predominant species present (i.e., on a
weight/volume percentage, it is the most abundant single species
within the composition), and preferably a substantially purified
fraction is a composition wherein the peptide comprises at least
about 50 percent (w/v) of all macromolecular species present.
Generally, a substantially pure composition will comprise more than
about 80 to 90 percent of all protein present in the composition.
Most preferably, the peptide is purified to essential homogeneity
(contaminant proteins cannot be detected in the composition by
conventional detection methods) wherein the composition consists
essentially of a single protein species.
[0034] II. Identification of Peptides
[0035] The PTB domain was originally identified as a 186-residue
segment of the signaling protein SHC which binds specifically to
the tyrosine-phosphorylated form of an unidentified 145 kDa protein
in response to many growth factors, but which is structurally
dissimilar to members of the SH2 domain family. See, Kavanaugh and
Williams, Science (1994) 266:1862-1865.
[0036] To determine the targets to which PTB domains bind, a method
of screening a library of tyrosine phosphorylated proteins was
developed. Standard expression cloning systems are generally
unsuitable, because they do not permit screening for
phosphorylation-dependent protein-protein interactions. An
expression cloning approach which allowed identification of
proteins which bound PTB domain only when tyrosine-phosphorylated,
was developed. Standard methods were used to express proteins from
a .lambda. gt11 cDNA library and immobilize them on filters. See,
e.g., Sambrook, et al., Molecular Cloning: A Laboratory Manual
(Cold Spring Harbor Laboratory Press, 2nd ed. 1989). The proteins
on filters were then phosphorylated in vitro with recombinant
tyrosine kinases, washed, incubated with .sup.32P-labeled PTB
domain protein derived from SHC as a probe, and autoradiography was
performed. A clone was identified which bound the PTB domain probe
only when subjected to phosphorylation conditions prior to
hybridization (FIG. 1).
[0037] The positive clone was identified as corresponding to amino
acids 1086 to 1255 of c-erbB2/c-neu/HER2 protein, a tyrosine kinase
receptor proto-oncogene (See FIG. 1). This region of c-erbB2
contains seven tyrosines, five of which have been shown to be
autophosphorylation sites. Hazan, et al., Cell Growth Differ (1990)
1:3-7, Segatto, et al., New Biol. (1990) 2:187-195, Akiyama, et
al., Mol. Cell. Biol. (1991) 11:833-842.
[0038] To verify that the PTB domain binds to c-erbB2 which had
been autophosphorylated in vivo, PTB domain was incubated with
lysate from SKBR3 human breast carcinoma cells, which contain
overexpressed and autophosphorylated c-erbB2. C-erbB2 from these
cells specifically associated with GST-PTB domain fusion protein,
but not with GST fusion protein containing SHC residues 1-45, which
lie outside of the PTB domain (FIG. 2A, left panel, see also,
Kavanaugh and Williams, Science (1994) 266:1862-1865). Further,
dephosphorylation of the c-erbB2 from SKBR3 cells with
tyrosine-specific phosphatases completely eliminated binding to the
PTB domain. Taken together, these data demonstrate that the PTB
domain specifically associates with the tyrosine-phosphorylated
form of c-erbB2. C-erbB2 associates with SHC in vivo (FIG. 2A,
right panel) through a mechanism which requires c-erbB2
autophosphorylation at these sites. Segatto, et al., Oncogene
(1993) 8:2105-2112. Therefore, c-erbB2 is also an apparent target
of the PTB domain in vivo.
[0039] Peptides derived from the c-erbB2 sequence were synthesized,
substituting phosphotyrosine for each of the seven tyrosines in the
c-erbB2 sequence. These peptides were tested for their ability to
compete with c-erbB2 from SKBR3 lysate for binding to PTB domain.
The peptides tested and their respective IC.sub.50 values, are
listed in Table 1. The IC.sub.50 is the concentration of peptide
required to inhibit 50% of normal binding of PTB to c-erbB2.
1 TABLE 1 Apparent Inhibition Peptide Sequence (IC.sub.50)
PAFSPAFDNL (pY) (pY) WDQNSSEQG 50 nM ("Y1221/pY1222") AFDNLY (pY)
WDQNS ("Y1221/pY1222") 30 nM AFGGAVENPE (pY) LAPRAGTASQ (("pY1196")
1 .mu.M EGTPTAENPE (pY) LGLDVPV ("pY1248") 1 .mu.M APLACSPQPE (pY)
VNQPEVRPQS ("pY1139") >100 .mu.M SPHDLSPLQR (pY) SEDPTLPL
("pY1112") >100 .mu.M TLPLPPETDG (pY) VAPLACSPQ (pY1127")
>100 .mu.M
[0040] The peptides PAFSPAFDNL(pY)(pY)WDQNSSEQG, AFDNLY(pY)WDQNS,
AFGGAVENPE(pY)LAPRAGTASQ and EGTPTAENPE(pY)LGLDVPV showed
relatively strong inhibition of PTB domain/c-erbB2 binding with
approximate IC.sub.50s of 50 nM, 30 nM, 1 .mu.M and 1 .mu.M,
respectively. The phosphopeptides SPHDLSPLQR(pY)SEDPTLP,
APLACSPQPE(pY)VNQPEVRPQS and TLPLPPETDG(pY)VAPLACSPQ, on the other
hand appeared to be ineffective.
[0041] Comparison of the sequences of the c-erbB2 derived peptides
which were able to bind PTB indicated a common sequence motif of
NXX(pY). Furthermore, a similar sequence motif is also found in a
number of other signalling proteins associated with cell
proliferation, including polyomavirus middle T antigen, the
principal transforming protein of the polyomavirus (Campbell, et
al., Proc. Nat'l Acad. Sci. U.S.A. (1994) 91:6344-6348); Trk
tyrosine kinase, associated with signal transduction from nerve
growth factors (Obermeier, et al., J. Biol. Chem. (1993)
268(31):22963-22966); the EGF receptor (Okabayashi, et al., J.
Biol. Chem. (1994) 269(28):18674-18678); erbB3, a member of the
Type-I (EGF receptor related) family of growth factor receptors
(Prigent and Gullick, EMBO J. (1994) 13(12):2831-2841); mouse CD3
epsilon chain, integrins and the insulin and IGF receptors. A
number of these proteins have been reported to associate with the
SHC protein, and the specific sequence motifs are shown in Table 2,
below.
2 TABLE 2 Protein Peptide Sequence Middle T Ag. LLSNPT (pY) SVMR
erbB3 AFDNPD (pY) WHSRLF Trk IENPQ (pY) FSDA EGF Receptor SLDNPD
(pY) QQDFF
[0042] From the above data, a common PTB recognition sequence,
NXXpY is indicated, and more particularly, the motifs NPXpY and
NLXpY. These sequence motifs appear to be conserved in a variety of
signalling proteins, and are present in the peptides which show the
greatest affinity for the PTB domain.
[0043] To further characterize the nature of PTB domain binding,
peptides were prepared based upon the lead peptide derived from the
c-erbB2 protein, PAFSPAFDNL(pY) (pY)WDQNSSEQG ("pY1221/pY1222").
These peptides were then tested for their ability to block PTB
domain/c-erbB2 binding. The peptides and binding results are shown
in Table 3, below.
3TABLE 3 Affinity Peptide (IC.sub.50) PAFSPAFDNLYYWDQNSSEQG
("unphos") >30 .mu.N PAFSPAFDNL (pS) (pS) WDQNSSEQG ("ser phos")
>30 .mu.M PAFSPAFDNLEEWDQNSSEQG ("glu-glu") >30 .mu.M
PAFSPAFDNLFFWDQNSSEQG ("phe-phe") >30 .mu.M AFDNL (pY) (pY)
WDQNS ("pY1221/pY1222 short") 30 nM AFDNL (pY) YWDQNS
("pY1221/Y1222") 1 .mu.M AFDNLY (pY) WDQNS ("Y1221/pY1222") 30 nM
DSWDQNQLFS (pY) (pY) SFAPEGPAN (scrambled 1) >30 .mu.M DSW (pY)
SQNQLFDSFAPEG (pY) PAN (scrambled 2) >30 .mu.M
[0044] Peptides in which phosphotyrosine was substituted with
phosphoserine or glutamic acid did not compete with c-erbB2 for PTB
domain binding (See, also FIG. 2, Panel C). Phosphorylated peptide
or "phosphopeptide", PAFSPAFDNL(pY)(pY)WDQNSSEQG, which had been
dephosphorylated with tyrosine-specific phosphatases, also was
unable to block the PTB domain/c-erbB2 interaction. This data
demonstrates that the PTB domain specifically recognizes the
phosphotyrosine residue.
[0045] The above data indicate that the mere presence of
phosphotyrosine alone may not be the only determinant of effective
PTB domain binding and competition. The truncated peptide
AFDNLY(pY)WDQNS, which contained a single phosphotyrosine in the
second tyrosine position, had an IC.sub.50 approximately equal to
that of the double-phosphorylated peptide AFDNL(pY)(pY)WDQNS (See,
FIG. 2, Panel C). However, the peptide AFDNL(pY)YWDQNS,
phosphorylated at only the first tyrosine residue, was 30-fold less
effective in competition. While this latter peptide still shows
strong inhibition of PTB domain/c-erbB2 interaction, it appears
that the PTB domain binds preferentially to phosphotyrosine in the
second position. Further, scrambled peptides, which contained the
phosphotyrosine residues but a rearranged primary sequence, failed
to compete for binding. These data demonstrate that PTB not only
binds phosphotyrosine, but also recognizes a range of specific
adjacent amino acids.
[0046] Accordingly, to determine which residues in the peptide
PAFSPAFDNLY(pY)WDQNSSEQG were important for binding to the PTB
domain, a series of peptides containing point mutations in the
sequence were prepared and tested for inhibition of PTB
domain/c-erbB2 binding. The results are shown in Table 4, below.
The substituted residues are underlined. Relative inhibition scales
denote IC.sub.50 values of 50-500 nM ("+++"), 500 nM to 5 .mu.M
("++") 5 to 50 .mu.M ("+") and >50 .mu.M ("-").
4 TABLE 4 Peptide Inhibition PAFSPAADNLY (pY) WDQNSSEQG ++
PAFSPAFANLY (pY) WDQNSSEQG + PAFSPAFSNLY (pY) WDQNSSEQG +
PAFSPAFDALY (pY) WDQNSSEQG - PAFSPAFDQLY (pY) WDQNSSEQG -
PAFSPAFDDLY (pY) WDQNSSEQG - PAFSPAFDNAY (pY) WDQNSSEQG ++
PAFSPAFDNLAY (pY) WDQNSSEQG ++ PAFSPAFDNLFY (pY) WDQNSSEQG ++
PAFSPAFDNLY (pY) ADQNSSEQG - PAFSPAFDNLY (pY) FDQNSSEQG ++
PAFSPAFDNLY (pY) WAQNSSEQG +++ PAFSPAFDNLY (pY) WDANSSEQG ++
PAFSPAFDNLY (pY) WDNNSSEQG ++ PAFSPAFDNLY (pY) WDDNSSEQG ++
PAFSPAFDNLY (pY) WDQASSEQG ++ PAFSPAFDNLY (pY) WDQNASEQG ++
[0047] From the above data, it can be seen that substitution of the
asparagine in the 9th position can have a negative effect on PTB
binding. Replacement of aspartic acid in the 8th position also
impaired the peptides blocking ability, however this specific
residue was not required for competition. Replacement of tryptophan
in the 13th position with phenylalanine generally resulted in
little loss of affinity, although substitution of this tryptophan
with alanine resulted in reduced affinity. This suggests that large
hydrophobic or aromatic residues at this position may confer higher
affinity. Mutations outside of the central motif DNLY(pY)W
generally resulted in only moderate losses in the affinity of the
peptide.
[0048] To demonstrate directly that the phosphopeptides bind to the
PTB domain, biotinylated peptides were incubated with PTB
domain-containing protein ("PTB domain"). The PTB domain was
immunoprecipitated and the washed pellet assayed for the presence
of bound peptide with streptavidin-coupled alkaline phosphatase.
PTB domain was able to bind directly to phosphorylated peptide
PAFSPAFDNL(pY)(pY)WDQNSSEQG ("pY1221/pY1222"), but did not bind to
unphosphorylated peptide (See FIG. 3). Further, PTB domain did not
bind to phosphorylated peptides containing conservative point
mutations at the asparagine in the ninth position. The specificity
of this sequence for PTB domain was shown by the inability of the
SH2 domain of SHC to bind phosphorylated peptide
PAFSPAFDNL(pY)(pY)WDQNSSEQG. Additionally, this peptide also blocks
association of the SHC PTB domain in vitro with pp145, a previously
identified target of the SHC protein, derived from activated B
cells. See, Kavanaugh and Williams, supra.
[0049] III. Peptides of the Invention
[0050] The peptides of the present invention generally comprise a
core sequence which corresponds to a PTB recognition sequence
motif. This general PTB recognition sequence motif can be readily
identified from the above described data. Typically, the peptides
will comprise the sequence motif NX.sub.3X.sub.1X.sub.2X.sub.4,
where X.sub.1 is Y, pY or an analog thereof, E, T, D, A, F or Q;
X.sub.2 is pY or an analog thereof, or Y, provided that at least
one of X.sub.1 and X.sub.2 are pY, or an analog thereof; X.sub.3
can be any natural or unnatural amino acid, but is preferably L or
A; X.sub.4 is W, F, L, S or Q. Generally, this sequence motif may
be present as its own peptide, or may be a core of a longer
sequence. Generally, the peptides of the present invention will
comprise the above motif as a portion or a whole of a peptide of
from 5 to about 100 amino acids in length. Typically, the peptides
will be from about 6 to about 100 amino acids in length, preferably
the peptides will be from about 12 to about 100 amino acids in
length, more preferably from about 12 to about 50 amino acids in
length, and most preferably, from about 21 to about 50 amino acids
in length.
[0051] In particularly preferred aspects of the present invention,
the peptides are characterized by the core sequence of amino acids
X.sub.5NX.sub.3X.sub.1X.sub.2X.sub.4, where X.sub.1, X.sub.2,
X.sub.3 and X.sub.4 are as described above, and X.sub.5 can be any
natural or unnatural amino acid, but is preferably D, E, S or A.
Still more preferred are peptides which comprise the core sequence
of amino acids DNX.sub.3X.sub.1pYX.sub.4 and
ENX.sub.3X.sub.1pYX.sub.4. The most preferred peptides will
generally comprise one of the following core sequences of amino
acids:
[0052] PAFSPAFDNLY(pY)WDQNSSEQG; PAFSPAFDNL(pY)YWDQNSSEQG;
PAFSPAFDNL (pY) (pY) WDQNSSEQG; AFDNLY (pY) WDQNS; AFDNL (pY)
YWDQNS; AFDNL (pY) (pY) WDQNS; PAFSPAADNLY (pY) WDQNSSEQG;
PAFSPAADNL(pY)YWDQNSSEQG; PAFSPAADNL(pY)(pY)WDQNSSEQG;
PAFSPAFANLY(pY)WDQNSSEQG; PAFSPAFANL(pY)YWDQNSSEQG;
PAFSPAFANL(pY)(pY)WDQNSSEQG; PAFSPAFSNLY(pY)WDQNSSEQG;
PAFSPAFSNL(pY)YWDQNSSEQG; PAFSPAFSNL(pY)(pY)WDQNSSEQG;
PAFSPAFDNAY(pY)WDQNSSEQG; PAFSPAFDNA(pY)YWDQNSSEQG;
PAFSPAFDNA(pY)(pY)WDQNSSEQG; PAFSPAFDNLA(pY)WDQNSSEQG;
PAFSPAFDNLF(pY)WDQNSSEQG; PAFSPAFDNLY(pY)FDQNSSEQG;
PAFSPAFDNL(pY)YFDQNSSEQG; PAFSPAFDNL(pY)(pY)FDQNSSEQG;
PAFSPAFDNLY(pY)WAQNSSEQG; PAFSPAFDNL(pY)YWAQNSSEQG;
PAFSPAFDNL(pY)(pY)WAQNSSEQG; PAFSPAFDNLY(pY)WDANSSEQG;
PAFSPAFDNL(pY)YWDANSSEQG; PAFSPAFDNL(pY)(pY)WDANSSEQG;
PAFSPAFDNLY(pY)WDNNSSEQG; PAFSPAFDNL(pY)YWDNNSSEQG;
PAFSPAFDNL(pY)(pY)WDNNSSEQG; PAFSPAFDNLY(pY)WDDNSSEQG;
PAFSPAFDNL(pY)YWDDNSSEQG; PAFSPAFDNL(pY)(pY)WDDNSSEQG;
PAFSPAFDNLY(pY)WDQASSEQG; PAFSPAFDNL(pY)YWDQASSEQG;
PAFSPAFDNL(pY)(pY)WDQASSEQG; PAFSPAFDNLY(pY)WDQNASEQG;
PAFSPAFDNL(pY)YWDQNASEQG; PAFSPAFDNL(pY)(pY)WDQNASEQG;
AFGGAVENPE(pY)LAPRAGTASQ and EGTPTAENPE(pY)LGLDVPV.
[0053] Also included within the present invention are truncated
versions of the above described peptides, as well as peptides which
are modified at the carboxy and/or amino terminals, e.g., amidated
or acetylated, respectively.
[0054] The polypeptides of the present invention may be used as
isolated polypeptides, or may exist as fusion proteins. A "fusion
protein" generally refers to a composite protein made up of two or
more separate, proteins which are normally not fused together as a
single protein. Thus, a fusion protein may comprise a fusion of two
or more similar and homologous sequences, provided these sequences
are not normally fused together. Fusion proteins will generally be
made by either recombinant nucleic acid methods, i.e., as a result
of transcription and translation of a gene fusion comprising a
segment encoding a peptide of the invention and a segment which
encodes one or more heterologous proteins, or by chemical synthesis
methods well known in the art.
[0055] Additionally, the polypeptides may be free in solution or
may be covalently attached to a solid support. Support bound
polypeptides may be particularly useful in, e.g., screening and
purification applications. Suitable solid supports include those
generally well known in the art, e.g., cellulose, agarose,
polystyrene, divinylbenzene and the like. Many suitable solid
supports are commercially available from, e.g., Sigma Chemical Co.,
St Louis, Mo., or Pharmacia, Uppsala, Sweden, and come prepared for
immediate coupling of affinity ligands.
[0056] These fusion proteins may be prepared to exhibit a
combination of properties or activities of the derivative proteins.
Typical fusion proteins may include a PTB domain-binding peptide
fused to a reporter polypeptide, e.g., a substrate, cofactor,
inhibitor, affinity ligand, antibody binding epitope tag, or an
enzyme which is capable of being assayed. Because of their ability
to recognize and bind PTB domains within a protein, the peptides of
the present invention may act as an affinity ligand to direct the
activity of the fused protein directly to tyrosine phosphorylated
proteins. In the case of a reporter peptide/PTB domain-binding
peptide fusion, this allows the presence and or location of PTB
domain containing proteins to be easily determined. Typical fusion
partners can include bacterial .beta.-galactosidase, trpE, protein
A, .beta.-lactamase, .alpha.-amylase, alcohol dehydrogenase and
yeast a-mating factor. See, e.g., Godowski et al., Science
241:812-816 (1988).
[0057] The peptides of the present invention may be prepared by a
variety of means, e.g., recombinant or synthetic methods. In
general, techniques for recombinant production of proteins are
described, for example, in Sambrook et al., Molecular Cloning: A
Laboratory Manual (2nd ed.) Vols. 1-3, Cold Spring Harbor
Laboratory, (1989). Techniques for the synthesis of polypeptides
are generally described in Merrifield J. Amer. Chem. Soc.
85:2149-2456 (1963), Atherton et al., Solid Phase Peptide
Synthesis: A Practical Approach, IRL Press, Oxford (1989), and
Merrifield, Science 232:341-347 (1986).
[0058] In addition to the above peptides which consist only of
naturally-occurring amino acids, peptidomimetics of the PTB
domain-binding peptides are also provided. Peptide analogs are
commonly used in the pharmaceutical industry as non-peptide drugs
with properties analogous to those of the template peptide. These
types of non-peptide compounds are termed "peptide mimetics" or
"peptidomimetics" (Fauchere, J. (1986) Adv. Drug Res. 15:29; Veber
and Freidinger (1985) TINS p.392; and Evans et al. (1987) J. Med.
Chem. 30: 1229) and are usually developed with the aid of
computerized molecular modeling. Peptide mimetics that are
structurally similar to therapeutically useful peptides may be used
to produce an equivalent therapeutic or prophylactic effect.
Generally, peptidomimetics are structurally similar to a paradigm
peptide (i.e., a peptide that has a biological or pharmacological
activity), such as naturally-occurring PTB domain-binding
polypeptide, but have one or more peptide linkages optionally
replaced by a linkage selected from the group consisting of:
--CH.sub.2NH--, --CH.sub.2S--, --CH.sub.2--CH.sub.2--,
--CH.dbd.CH-- (cis and trans), --COCH.sub.2--, --CH(OH)CH.sub.2--,
and --CH.sub.2SO--, by methods known in the art and further
described in the following references: Spatola, A. F. in "Chemistry
and Biochemistry of Amino Acids, Peptides, and Proteins," B.
Weinstein, eds., Marcel Dekker, New York, p. 267 (1983); Spatola,
A. F., Vega Data (March 1983), Vol. 1, Issue 3, "Peptide Backbone
Modifications" (general review); Morley, J. S., Trends Pharm Sci.
(1980) pp. 463-468 (general review); Hudson, D. et al., Int J Pept
Prot Res. (1979) 14:177-185 (--CH.sub.2NH--, CH.sub.2CH.sub.2--);
Spatola, A. F. et al., Life Sci. (1986) 38:1243-1249
(--CH.sub.2--S); Hann, M. M., J Chem Soc Perkin Trans I (1982)
307-314 (--CH--CH--, cis and trans); Almquist, R. G. et al., J Med
Chem (1980) 23:1392-1398 (--COCH.sub.2--); Jennings-White, C. et
al., Tetrahedron Lett. (1982) 23:2533 (--COCH.sub.2--); Szelke, M.
et al., European Appln. EP 45665 (1982) CA: 97:39405 (1982)
(--CH(OH)CH.sub.2--); Holladay, M. W. et al., Tetrahedron Lett.
(1983) 24:4401-4404 (--C(OH)CH.sub.2--); and Hruby, V. J., Life
Sci. (1982) 31:189-199 (--CH.sub.2--S--).
[0059] Peptide mimetics may have significant advantages over
peptide embodiments, including, for example: more economical
production, greater chemical stability, enhanced pharmacological
properties (half-life, absorption, potency, efficacy, etc.),
altered specificity (e.g., a broad-spectrum of biological
activities), reduced antigenicity, and others. Labeling of
peptidomimetics usually involves covalent attachment of one or more
labels, directly or through a spacer (e.g., an amide group), to
non-interfering position(s) on the peptidomimetic that are
predicted by quantitative structure-activity data and/or molecular
modeling. Such non-interfering positions generally are positions
that do not form direct contacts with the macromolecules(s) (e.g.,
PTB domains) to which the peptidomimetic binds to produce the
therapeutic effect. Derivitization (e.g., labelling) of
peptidomimetics should not substantially interfere with the desired
biological or pharmacological activity of the peptidomimetic.
Generally, peptidomimetics of PTB domain-binding peptides will bind
to the PTB domain with high affinity and possess detectable
biological activity (i.e, are agonistic or antagonistic to one or
more PTB domain-mediated phenotypic changes).
[0060] In a preferred aspect of the present invention, the
phosphotyrosine (pY) group within the above described peptides can
be substituted with an analog of phosphotyrosine which possesses a
phosphate group which is nonhydrolyzable, e.g by tyrosine
phosphatases. Inclusion of a nonhydrolyzable phosphotyrosine analog
allows the peptides of the invention to retain binding and/or
inhibitory activity for longer periods of time, in the presence of
agents which may remove the phosphate group from the
phosphotyrosine, e.g., tyrosine phosphatases, thereby allowing for
more effective inhibition and reduced effective amounts, among
other benefits. Examples of phosphotyrosine analogs having
nonhydrolyzable phosphate groups include, e.g.,
(phosphonomethyl)phenylalanine ("Pmp"). Pmp is a phosphotyrosine
analog in which the >C--O--PO.sub.3H.sub.2 group of pY has been
replaced by >C--CH.sub.2--PO.sub.3H.sub.2. Inclusion of this
analog within sequences recognized by other phosphotyrosine binding
domains yields comparable binding as with their
phosphotyrosine-containing counterparts. See, Domchek, et al.,
Biochem. (1992) 31:9865-9870. Thus, in an aspect of the present
invention, the peptides of the present invention which comprise a
core sequence NX.sub.3X.sub.1X.sub.2X.sub.4, where X.sub.1,
X.sub.2, X.sub.3 and X.sub.4 are as previously described, the
phosphotyrosine residues in X.sub.1 and/or X.sub.2 are substituted
with Pmp.
[0061] Systematic substitution of one or more amino acids of a
consensus sequence with a D-amino acid of the same type (e.g.,
D-lysine in place of L-lysine) may also be used to generate more
stable peptides. D-amino acids are generally denoted by the lower
case abbreviation for the corresponding L-amino acid. In addition,
constrained peptides comprising a consensus sequence or a
substantially identical consensus sequence variation may be
generated by methods known in the art (Rizo and Gierasch (1992)
Ann. Rev. Biochem. 61:387; for example, by adding internal cysteine
residues capable of forming intramolecular disulfide bridges which
cyclize the peptide.
[0062] VI. Methods of Use
[0063] In general, the peptides of the present invention may be
particularly useful as affinity ligands which are capable of
binding proteins that comprise a PTB domain. Further,
phosphotyrosine recognition and binding is a common mediator in
cellular signaling and cellular functioning. Accordingly, the
polypeptides of the present invention may find a variety of uses in
diagnostic, screening and therapeutic applications related to these
areas.
[0064] A. Diagnostics and Screening
[0065] In diagnostic applications, for example, the peptides of the
present invention may generally be useful in methods for
identifying proteins which comprise PTB domains. These methods may
allow for the identification of proteins which are specifically
involved in signaling pathways, such as cell activation following
the binding of a ligand to a cell surface receptor. Specifically,
these methods are useful in identifying downstream signals
following growth factor, hormone, antibody and cytokine activation
of cells. In particular, because of their specificity, the peptides
of the present invention may generally be used as probes for
identifying PTB domain-containing proteins.
[0066] Therefore, in one aspect, the peptides of the present
invention may be used to determine whether a particular protein
comprises a PTB domain. Determination of whether a protein
comprises a PTB domain may be carried out by a variety of means.
For example, in some instances, it may be useful to immobilize the
protein to be tested upon a solid support, e.g., a microtiter well,
or nitrocellulose membrane. After blocking the remaining groups on
the support, the protein to be tested may be exposed to an
appropriate amount of the labelled peptide, as described herein.
Detection of the label bound to the test protein indicates that the
protein contains a PTB domain. As a specific example, following
SDS-PAGE, the gel may be electroblotted onto an appropriate solid
support, e.g., a nitrocellulose or PVDF membrane. Remaining unbound
regions of the membrane may then be blocked with an appropriate
inert protein, e.g., bovine serum albumin, or unphosphorylated
peptide. Following buffer rinses, the blot is then contacted with a
peptide of the invention to which has been coupled a detectable
group, e.g., a radiolabel or enzyme. Radiographs of the blot may be
compared to simultaneously run, stained SDS-PAGE gels, and the
label bound proteins may be identified.
[0067] Additionally, as an affinity ligand, the peptides of the
present invention may also be useful in the purification of
proteins which comprise a PTB domain, from a mixture of different
proteins. Affinity purification of PTB domain-containing proteins
may be carried out using general affinity purification methods well
known in the art. For example, a peptide of the present invention
may be attached to a suitable solid support, as described
above.
[0068] The mixture of proteins may then be contacted with the
peptide bound to the solid support, such that the peptide
selectively binds the PTB domain-containing proteins present within
the mixture of proteins. The bound protein can then be washed to
eliminate unbound proteins. Finally, substantially pure PTB
domain-containing protein may be eluted from the solid support by
generally known elution protocols, e.g., washing with an excess of
phosphotyrosine, which will compete with the binding of PTB to the
target peptide.
[0069] As a target of PTB domain binding, the peptides of the
present invention may also be used as probes in screening for
compounds which may be agonists or antagonists of that binding, and
more particularly, the cell signaling pathways which lead up to,
and include, the binding of PTB domain to its phosphorylated
ligand, e.g., SHC/c-erbB2 interactions, middle T antigen/SHC
interactions, Trk/SHC interactions, and the like.
[0070] An agonist, antagonist, or test compound may be a chemical
compound, a mixture of chemical compounds, a biological
macromolecule, or an extract made from biological materials such as
bacteria, plants, fungi, or animal cells or tissues. Test compounds
may be evaluated for potential activity as agonists or antagonists
of pathways which lead up to, and include the PTB
domain/phosphorylated ligand interaction. Thus, an agonist or
antagonist may directly affect PTB domain/phosphorylated ligand
interaction, or alternatively, may act upon an upstream event in
the pathway, whereby the level of PTB domain/phosphorylated ligand
interaction is affected.
[0071] Thus, an "agonist" of the pathway will enhance the level of
PTB domain/phosphorylated ligand interaction, while an "antagonist"
will diminish the level of that interaction. The terms "agonist"
and "antagonist", as used herein, do not imply a particular
mechanism of function.
[0072] In screening embodiments, the polypeptides of the present
invention may be used as a model in vitro system for determining
whether a test compound is an agonist or antagonist of the binding
of the PTB domain to its target recognition sequence motif. Such a
system permits the screening of a large number of potential drugs,
or drug candidates, for the ability to enhance or inhibit PTB
domain/phosphorylated ligand interactions, and resulting associated
downstream events.
[0073] The screening methods comprise providing a polypeptide which
contains a PTB domain, and a peptide of the present invention,
whereby the protein and peptide form a complex. The complex may
then be incubated with a test compound. Binding between the PTB
domain and the peptide may then be determined. An increase or
decrease in the level of binding between the PTB domain-containing
protein and the peptide of the invention in response to a
particular compound would indicate that the test compound is an
agonist or antagonist of that binding, respectively. In some cases,
it may be desirable to preincubate the PTB domain-containing
protein, or the peptide of the invention with the test compound,
prior to introduction of the peptide of the invention. The duration
and conditions of preincubation will generally vary depending upon
the compound being tested. Further, other reaction conditions of
the preincubation, e.g., pH and salt concentration, will generally
correspond to the conditions which are most effective for PTB
domain binding to the peptide. Accordingly, these conditions will
likely reflect the conditions normal to the particular cell-line
from which the PTB domain was derived.
[0074] For many of the methods described herein, the peptides of
the invention, or the PTB domain, may be covalently attached or
linked to a detectable group, or label, to facilitate screening and
detection. Useful detectable groups, or labels, are generally well
known in the art. For example, a detectable group may be a
radiolabel, such as, .sup.125I, .sup.32P or .sup.35S, or a
fluorescent or chemiluminescent group. Alternatively, the
detectable group may be a substrate, cofactor, inhibitor, affinity
ligand, antibody binding epitope tag, or an enzyme which is capable
of being assayed. Suitable enzymes include, e.g., horseradish
peroxidase, luciferase, or other readily assayable enzymes. These
enzyme groups may be attached to the peptide by chemical means or
expressed recombinantly, as a fusion protein, by methods well known
in the art.
[0075] It may also be desirable to provide the peptide or PTB
domain-containing protein immobilized upon a solid support, to
facilitate screening of test compounds. Examples of suitable solid
supports include agarose, cellulose, dextran, Sephadex.TM.,
Sepharose.TM., carboxymethyl cellulose, polystyrene, filter paper,
nitrocellulose, ion exchange resins, plastic films, glass beads,
polyaminemethylvinylether maleic acid copolymer, amino acid
copolymer, ethylene-maleic acid copolymer, nylon, silk, etc. The
support may be in the form of, e.g., a test tube, microtiter plate,
resins, beads, test strips, or the like. The coupling of the
peptide or PTB domain-containing protein with the particular solid
support may be carried out by methods well known in the art.
[0076] As a specific example, a PTB domain-containing protein may
be coupled to the wells of a microtiter plate. The test compound
may then be added to the well of the microtiter plate to
preincubate with the PTB domain-containing protein. The peptide of
the invention, to which a detectable group has been attached, may
then be added to the microtiter well. Following sufficient
incubation, the wells may be rinsed, and binding of the peptide to
the PTB domain may be assessed, e.g., by assaying for the presence
of residual detectable groups. Those of skill in the art will
recognize that the screening assay format may be set up in either
direction, i.e., either the peptide or the PTB domain-containing
protein may be bound to the support, while the other is labeled.
The level of binding may then be compared to suitable positive and
negative controls. Alternatively, by providing the polypeptide
containing the PTB domain, and/or the peptide in known
concentrations, one can assay for free, or unbound PTB domain
and/or peptide, and by negative implication, determine the level of
PTB domain/peptide complex which is formed.
[0077] The amount or concentration of agonist/antagonist added
will, when known, vary depending on the compound, but will
generally range from about 10 pM to 100 .mu.M. Typically, a range
of concentrations will be used. In the case of uncharacterized test
compounds it may not be possible, and it is not necessary, to
determine the concentration of agonist/antagonist.
[0078] It will also be desirable to include various experimental
controls in the above assay. Examples of appropriate controls
include negative controls and positive controls. In testing for
agonist activity, negative controls can include incubation of cells
with inert compounds (i.e., compounds known not to have agonist
activity) or in the absence of added compounds. Positive controls
can include incubation with compounds known to have agonist
activity (e.g., the natural ligand). Logically, similar (though
complementary) controls can be included in assays for antagonist
activity, as will be apparent to one of ordinary skill in the art
of biology, as will various additional controls. The description of
controls is meant to be illustrative and in no way limiting.
[0079] In an alternative embodiment, the peptides of the present
invention may be useful in modelling small molecules which
interfere with PTB binding in vivo. In particular, the structure of
the PTB domain recognition sequence motif, as described herein, may
be applied in generating synthetic analogs and mimics of the PTB
domain recognition sequence. Synthetic elements may be pieced
together based upon their analogy to the structural and chemical
aspects of the PTB recognition sequence motif. Such mimics and
analogs may be used in blocking or inhibiting specific aspects of
the cell signaling pathways, e.g., growth factor activation, and
may therefore be useful as therapeutic treatments according to the
methods described herein.
[0080] B. Therapeutic Applications
[0081] In addition to the above described uses, the polypeptides of
the present invention, or analogs thereof, may also be used in
therapeutic applications for the treatment of human or non-human
mammalian patients.
[0082] PTB domain-containing proteins have been shown to bind
proteins which are phosphorylated in response to the activation of
a cell by various growth factors. See Kavanaugh and Williams,
supra. Accordingly, the polypeptides of the present invention may
be used to inhibit or block the interaction of PTB
domain-containing proteins with their phosphorylated ligands by
competing with those ligands.
[0083] In particular, the peptides of the present invention can be
used to block or inhibit growth factor dependent activation or
stimulation of cells, or more specifically, inhibit or block growth
factor initiated mitogenesis. These methods may generally be used
in the treatment of a variety of proliferative cell disorders, or
in screening compounds effective for such treatment. "Proliferative
cell disorder" refers generally to disorders which are
characterized by excessive stimulation or activation of the
mitogenic signaling pathways resulting in excessive or abnormal
cell growth and/or differentiation. Specific disorders include,
e.g., atherosclerosis, inflammatory joint diseases, psoriasis,
restinosis following angioplasty, and cancer. The methods and
compositions of the present invention may be particularly useful in
the case of cancers where there are deregulated tyrosine kinases,
such as thyroid, breast carcinoma, stomach cancer and
neuroblastoma. Alternatively, the methods and compositions may be
useful as a prophylactic treatment, or in screening for compounds
effective in prophylactic treatments. Such prophylactic treatments
will generally be administered to inhibit or block "normal" cell
proliferation, for example, in immunosuppression to prevent graft
rejection, and to alleviate allergic responses involving mast cell
activation.
[0084] In a particularly preferred aspect, the peptides of the
present invention are be used to block or inhibit the interaction
between PTB domain containing proteins and the product of the
c-erbB2 oncogene. More specifically, the peptides can be used to
block or inhibit the interaction between the SHC protein and
c-erbB2.
[0085] Gene amplification of c-erbB2 is known to result in
overexpression of the c-erbB2 product in a variety of
adenocarcinomas, and a number of studies link this overexpression
to the neoplastic process. c-erbB2 amplification has been described
as being associated with human gastric tumor, non-small cell lung,
colon, ovarian and pancreatic adenocarcinomas. Overexpression of
c-erbB2 product has also been found in a significant percentage of
breast carcinomas. For a review of c-erbB2, see Dougall, et al.,
Oncogene (1994) 9:2109-2123.
[0086] Studies have demonstrated the relationship between c-erbB2
overexpression and cellular transformation, using monoclonal
antibodies. Antibodies to the c-erbB2 protein, as well as its
murine homolog, have proven effective in inhibiting tumor
formation, or otherwise shown antiproliferative effects. These
studies indicate that the continued expression of the c-erbB2
product is necessary for the maintenance of the neoplastic
phenotype in c-erbB2 transformed cells, and that expression of the
c-erbB2 product can be functionally linked to cellular
transformation. Dougall, et al. Further, studies indicate that
several critical tyrosine residues within the c-erbB2 protein are
important for conveying the mitogenic signals of the c-erbB2
protein. The peptides of the present invention are particularly
useful in blocking these phosphotyrosine mediated mitogenic
signals.
[0087] The use of the peptides of the invention in methods for
inhibiting or blocking c-erbB2/PTB domain interaction can be useful
in the treatment of disorders which result from the overexpression
of the c-erbB2 gene product, including, e.g., human gastric tumor,
non-small cell lung, colon, ovarian and pancreatic adenocarcinomas,
as well as breast carcinomas. Typically, such treatment will
comprise administering to a patient suffering from one of the above
disorders, an effective amount of a polypeptide of the present
invention, generally in combination with a pharmaceutically
acceptable carrier.
[0088] It will also be appreciated by those of skill in the art,
that peptidomimetics of the present invention may also be effective
in blocking growth factor dependent activation of cells, or PTB
domain/c-erbB2 interaction. Specifically, synthetic analogs to the
PTB domain recognition motif as described herein, may also be
applied in the treatment methods described.
[0089] The quantities of reagents necessary for effective therapy,
also referred to herein as an "effective amount," or
"therapeutically effective amount," will depend upon many different
factors, including means of administration, target site,
physiological state of the patient and other medicants
administered. Thus, treatment doses will need to be titrated to
optimize safety and efficacy. Typically, dosages used in vitro may
provide useful guidance in the amounts useful for in situ
administration of these reagents. Animal testing of effective doses
for treatment of particular disorders will provide further
predictive indication of human dosage. Generally, therapeutically
effective amounts of the peptides of the present invention will be
from about 0.0001 to about 100 mg/kg, and more usually, from about
0.001 to about 0.1 mg/kg of the host's body weight. Various
considerations are described, e.g., in Gilman et al., (Eds.),
Goodman and Gilman's: The Pharmacological Basis of Therapeutics,
(8th ed. 1990), Pergamon Press, and Remington's Pharmaceutical
Sciences (7th ed. 1985) Mack Publishing Co., Easton, Pa. Methods of
administration, also discussed in the above references, include,
e.g., oral, intravenous, intraperitoneal or intramuscular
administration, and local administration, including topical,
transdermal diffusion and aerosol administration, for therapeutic,
and/or prophylactic treatment.
[0090] While it is possible to administer the active ingredient
alone, it is preferable to present it as part of a pharmaceutical
composition or formulation. These formulations comprise the
peptides and/or analogs of the invention in a therapeutically or
pharmaceutically effective dose together with one or more
pharmaceutically or therapeutically acceptable carriers and
optionally other ingredients, e.g., other therapeutic ingredients,
or additional constituents which may be required to approximate
physiological conditions, such as pH adjusting and buffering
agents, tonicity adjusting agents, wetting agents and the like.
Additional constituents of the pharmaceutical compositions may
include those generally known in the art for the various
administration methods used, e.g., oral forms may contain
flavorants, sweeteners and the like. For solid compositions,
conventional nontoxic solid carriers may be used which include,
e.g., pharmaceutical grades of mannitol, lactose, starch, magnesium
stearate, sodium saccharin, talcum, cellulose, glucose, sucrose,
magnesium carbonate and the like. Various considerations are
described, e.g., in Gilman et al. (eds) (1990) GOODMAN AND
GILMAN'S: THE PHARMACOLOGICAL BASES OF THERAPEUTICS, 8th Ed.,
Pergamon Press; NOVEL DRUG DELIVERY SYSTEMS, 2nd Ed., Norris (ed.)
Marcel Dekker Inc. (1989), and REMINGTON'S PHARMACEUTICAL
SCIENCES.
[0091] Methods for administration are also discussed in the above
references, e.g., for oral, intravenous, intraperitoneal, or
intramuscular administration, and others. Pharmaceutically
acceptable carriers will include water, saline, buffers, and other
compounds described, e.g., in the MERCK INDEX, Merck & Co.,
Rahway, N.J. See, also, BIOREVERSIBLE CARRIERS IN DRUG DESIGN,
THEORY AND APPLICATION, Roche (ed.), Pergamon Press, (1987). For
some methods of administration, e.g., oral, it may be desirable to
provide the active ingredient in a liposomal formulation. This is
particularly desirable where the active ingredient may be subject
to degradative environments, for example, proteolytic digestive
enzymes. Liposomal formulations are well known in the art, and are
discussed in, e.g., REMINGTON'S PHARMACEUTICAL SCIENCES, supra.
Administration may also be carried out by way of a controlled
release composition or device, whereby a slow release of the active
ingredient allows continuous administration over a longer period of
time.
[0092] The present invention is further illustrated by the
following examples. These examples are merely to illustrate aspects
of the present invention and are not intended as limitations of
this invention.
EXAMPLES
Example 1
Expression Cloning of Tyrosine-Phosphorylated Targets of a PTB
Domain
[0093] Sf9 cells expressing residues 526 to 1067 of mouse PDGF
receptor cytoplasmic domain (tyrosine kinase) in recombinant
baculovirus were prepared and lysed as described by Kavanaugh and
Williams, Science (1994) 266:1862-1865, and Collawn, et al., (1990)
Cell 63:1061-1072. 1.1.times.10.sup.6 plaques of an oligo-dT primed
Balb/c 3T3 fibroblast cDNA .lambda. gt11 library were plated and
transferred to IPTG-impregnated PVDF filters using standard
techniques. See, Sambrook, et al. Molecular Cloning: A Laboratory
Manual (Cold Spring Harbor Laboratory Press, New York, 2nd ed.,
1989). The filters were blocked in TBSTM, 5% BSA (20 mM Tris-HCl,
pH 7.4, 137 mM NaCl, 10 mM MgCl.sub.2 and 0.1% Triton X-100) and
then incubated in TBSTM containing one fifth volume PDGF receptor
cytoplasmic domain lysate, 250 .mu.M ATP and 1 mM sodium
orthovanadate at room temperature for 30 minutes. The filters were
washed and incubated with .sup.32P-labeled GST-PTB domain fusion
protein as described in Kavanaugh and Williams, supra.
Example 2
Association of c-erbB2 with PTB Domain
[0094] Influenza hemagglutinin (IHA) tagged GST-PTB domain fusion
proteins were expressed from recombinant baculovirus in sf9 cells.
Sf9 cells or confluent SKBR3 cells were lysed in
2.times.hybridization buffer containing protease inhibitors and 1
mM sodium orthovanadate, as described in Kavanaugh and Williams,
Science (1994) 266:1862-1865. Approximately 100 ng of GST-PTB
domain was incubated with 1 .mu.g of total SKBR3 lysate protein in
1.times.hybridization buffer for 30 minutes at 4.degree. C.,
immunoprecipitated with 2 .mu.g of 12CA5 and protein-A sepharose,
and the pellets washed 3 to 5 times prior to immunoblot analysis
with anti c-neu/c-erbB2 antibodies. The results are shown in FIG.
2A. Equal amounts of GST-PTB domain protein were immunoprecipitated
as determined by immunoblotting with 12CA5.
Example 3
Inhibition of PTB/c-erbB2 Interaction
[0095] IHA-tagged GST-PTB fusion protein was incubated with SKBR3
lysate as described above, in the presence and absence of the
peptides pY1112, pY1127, pY1139, pY1196, pY1221/pY1222 and pY1248.
The mixture was immunoprecipitated with 12CA5, and immunoblotted
with anti-c-neu/c-erbB2 antibodies. These results are shown in FIG.
2B. PTB domain was pre-incubated with the indicated concentrations
of peptide for 30 minutes at 4.degree. C. prior to adding SKBR3
cell lysate. This experiment was repeated with varying
concentrations of the peptide pY1221/pY1222 and the results are
shown in FIG. 2B, lower blot. Substantial inhibition is shown at as
low as 50 nM peptide concentration. This experiment was also
repeated using the peptides shown in Table 3, and the results are
shown in FIG. 2C. Of the peptides tested, peptides pY1221/pY1222
and Y1221/pY1222 appear to completely block PTB/c-erbB2
interaction, whereas peptide pY1221/Y1222 showed some inhibition of
this interaction. In the experiments involving
serine-phosphorylated peptides, 1 .mu.M okadaic acid and 1 mM EGTA
were included in the buffers. Peptides were synthesized as
described by Escobedo, et al., Mol. Cell. Biol. (1991)
11:1125-1132, and HPLC purified. In this latter experiment, 300 nM
peptides were used.
Example 4
Binding of Phosphopeptides to PTB Domain
[0096] Peptides were biotinylated during synthesis and HPLC
purified. 100 ng of GST-PTB domain or GST-SH2 domain fusion protein
were incubated in 1.times.hybridization buffer with 500 nM
biotinylated phosphopeptide for 1 hour at 4.degree. C.,
immunoprecipitated as described in Example 2, above, washed once,
and the pellets incubated with 0.25 units of streptavidin-alkaline
phosphatase for 5 minutes at 4.degree. C. The pellets were washed
twice more, incubated for 3 minutes at room temperature with 1
mg/ml p-nitrophenylphosphate in 100 mM glycine, pH 10.1, 1 mM
ZnCl.sub.2 and 1 mM MgCl.sub.2. The absorbance was measured at 405
nm.
[0097] The direct binding of the phosphorylated peptide
PAFSPAFDNL(pY)(pY)WDQNSSEQG ("b-phos.") to the PTB domain is shown
in FIG. 3. This peptide bound the PTB domain both in the presence
and absence of a 100.times.concentration of unphosphorylated,
non-biotinylated peptide. PTB binding was inhibited in the presence
of 100.times.concentration of phosphorylated peptide, which
competed for the PTB domain. Unphosphorylated, biotinylated peptide
did not bind the PTB domain. Neither the phosphorylated nor
unphosphorylated form of this peptide were able to specifically
bind to an SH2 domain.
[0098] The peptides PAFSPAFDQL(pY)(pY)WDQNSSEQG ("b-N1219Q") and
PAFSPAFDDL(pY)(pY)WDQNSSEQG ("b-N1219D") which carried point
mutations in the asparagine residue in the ninth position, also
show substantially reduced binding to the PTB domain in these
assays (FIG. 3).
[0099] While the foregoing invention has been described in some
detail for purposes of clarity and understanding, it will be clear
to one skilled in the art from a reading of this disclosure that
various changes in form and detail can be made without departing
from the true scope of the invention. All publications and patent
documents cited in this application are incorporated by reference
in their entirety for all purposes to the same extent as if each
individual publication or patent document were so individually
denoted.
* * * * *