U.S. patent application number 11/022308 was filed with the patent office on 2005-12-22 for methods and compositions for regulating protein-protein interactions.
Invention is credited to Lu, Kun Ping, Zhou, Xiao Zhen.
Application Number | 20050282740 11/022308 |
Document ID | / |
Family ID | 22955874 |
Filed Date | 2005-12-22 |
United States Patent
Application |
20050282740 |
Kind Code |
A1 |
Lu, Kun Ping ; et
al. |
December 22, 2005 |
Methods and compositions for regulating protein-protein
interactions
Abstract
The invention relates to methods and compositions of WW-domains
as phosphoserine and phosphothreonine binding modules. The
WW-domain containing polypeptides of the invention can be used, for
example, to regulate cell growth; to treat neurodegenerative
diseases; to screen for substances that modulated interactions
between WW-domain containing polypeptides and phosphorylated
ligands; as drug targeting vehicles; to direct protein degradation;
and in the treatment of certain diseases or conditions
characterized by aberrant WW-domain containing polypeptides or
their ligands.
Inventors: |
Lu, Kun Ping; (Newton,
MA) ; Zhou, Xiao Zhen; (Newton, MA) |
Correspondence
Address: |
LAHIVE & COCKFIELD, LLP.
28 STATE STREET
BOSTON
MA
02109
US
|
Family ID: |
22955874 |
Appl. No.: |
11/022308 |
Filed: |
December 21, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11022308 |
Dec 21, 2004 |
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10256865 |
Sep 26, 2002 |
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10256865 |
Sep 26, 2002 |
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09252404 |
Feb 18, 1999 |
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6495376 |
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Current U.S.
Class: |
435/6.16 ;
514/17.7; 514/19.8 |
Current CPC
Class: |
A61K 38/1709 20130101;
A61K 47/62 20170801; A61K 38/17 20130101; C07K 14/47 20130101; A61P
25/28 20180101; C12N 9/90 20130101; A61P 35/00 20180101; G01N
2500/02 20130101; A61K 38/08 20130101; A61K 38/52 20130101; G01N
33/68 20130101; G01N 33/6872 20130101; A61K 38/10 20130101 |
Class at
Publication: |
514/007 |
International
Class: |
A61K 038/17 |
Goverment Interests
[0001] The invention was supported, in whole or in part, by grant
R01 GM56230 from the National Institutes of Health. The Government
has certain rights in the invention.
Claims
1. A method of enhancing the binding of a WW-domain containing
polypeptide with a phosphorylated ligand, the method comprising
contacting the WW-domain containing polylpeptide with a substance
in the presence of the phosphorylated ligand such that binding of
the WW-domain containing polylpeptide to the phosphorylated ligand
is enhanced.
2. The method of claim 1 wherein the WW-domain containing
polypeptide is selected from the group consisting of Pin1, NEDD4,
YAP, FE65, forming binding protein, dystrophin, utropin,
Ess1p/Ptf1p, Rsp5, Pub1, Dodo, Msb1, ORF1, YKB2, DP71, C38D4.5,
P9659.21, Yo61, Yfid, ZK1248.15, KO15c11, CD45AP, FBP11, FBP21,
FBP23, FBP28 and FBP30.
3-5. (canceled)
6. The method of claim 1 wherein the substance is selected from the
group consisting of a phosphoserine peptide, phosphothreonine
peptide, peptide mimetic or small organic molecule.
7-9. (canceled)
10. The method of claim 1 wherein enhancement comprises
phosphorylating the ligand thereby, enhancing binding of the
WW-domain containing polypeptide to the phosphorylated ligand.
11. The method of claim 1 wherein the enhancement comprises
dephosphorylating the WW-domain containing polypeptide or
inhibiting the phosphorylation of the WW-domain containing
polypeptide, thereby enhancing the binding of the WW-domain
containing polypeptide to the ligand.
12-21. (canceled)
22. A method of regulating protein degradation comprising mediating
the binding of the NEDD4 WW-domain to a phosphorylated ligand.
23. (canceled)
24. A method of regulating the interaction of a WW-domain of
dystrophin to a phosphorylated ligand.
25-26. (canceled)
27. A method of regulating the function of a neuronal cell
comprising mediating the binding of the Pin1 WW-domain to a
phosphorylated ligand selected from the group consisting of tau and
amyloid precursor protein.
28. The method of claim 27 comprising enhancing the binding of the
Pin1 WW-domain to phosphorylated threonine-231 tau, whereby
phosphorylated tau binds to microtubules, thereby resulting in
microtubule assembly.
29. A method of identifying a substance that modulates the
interaction of a WW-domain containing polypeptide and a
phosphorylated ligand comprising the steps of: a) contacting the
WW-domain containing polypeptide with one, or more, test
substances, b) maintaining the test substances and the WW-domain
containing polypeptide under conditions suitable for interaction;
and c) determining the interaction between the test substance and
WW-domain containing polypeptide, wherein the interaction indicates
that the test substance modulates the interaction between the
WW-domain-containing polypeptide and the ligand.
30. The method of claim 29, wherein the WW-domain containing
polypeptide is selected from the group consisting of Pin1, NEDD4,
YAP, FE65, forming binding protein, dystrophin, utropin,
Ess1p/Ptf1p-, Rsp5, Pub1, Dodo, Msb1, ORF1, YKB2, DP71, C38D4.5,
P9659.21, Yo61, Yfe1, ZK1248.15, KO15c11, CD45AP, FBP11, FBP21,
FBP23, FBP28 and FBP30.
31. The method of claim 29, wherein the ligand is selected from the
group consisting of tau protein, Cdc25c, Cdc27c, Plk1, NIMA, Myt1,
Rab4, amyloid precursor protein, Weel, Mos, Sox3, Xbr-1b, MP75
(E-MAP-115), MP110 (Cdc5),-MP68, and MP30.
32. The method of claim 29, wherein the substance enhances the
interaction between the WW-domain and the ligand.
33. The method of claim 29, wherein the substance inhibits the
interaction between the WW-domain and the ligand.
34. (canceled)
35. A method of identifying a test substance that modulates the
interaction between a WW-domain containing polypeptide and a ligand
wherein the ligand is phosphorylated comprising the steps of: a)
combining one, or more, test substances with the ligand and
WW-domain containing polypeptide thereby producing a combination;
b) maintaining the combination of step a) under conditions suitable
for interaction between the ligand and the WW-domain containing
polypeptide; c) determining the amount of interaction in the
combination in step b); and d) comparing the amount of interaction
in step c) with the amount of interation in the absence of the test
substance, wherein the difference in the interaction indicates that
the test substance modulates the interaction between the ligand and
the WW-domain containing polypeptide.
36. The method of claim 35, wherein the WW-domain containing
polypeptide comprises the WW-domain of a polypeptide selected from
the group consisting of Pin1, NEDD4, YAP, FE65, formin binding
protein, dystropin, utropin, Ess1p/Ptf1p, Rsp5, Pub1, Dodo, Msb1,
ORF1, YKB2, DP71, C38D4.5, P9659.21, Yo61, Yfid, ZK1248.15,
K015c11, CD45AP, FBP11, FBP21, FBP23, FBP28 and FBP30.
37. The method of claim 35, wherein the ligand is selected from the
group consisting of tau protein, Cdc25c, Cdc27c, Plk1, NIMA, Myt1,
Rab4, amyloid precursor protein, Weel, Mos, Sox3, Xbr-1b, MP75
(E-MAP-115), MP110 (Cdc5), MP68, and MP30.
38. (canceled)
39. A polypeptide comprising a mutant Pin1 WW-domain, wherein the
mutation mutant Pin1 WW-domain comprises a modification of an amino
acid selected from the group consisting of tyrosine at position 23,
tryptophan at position 34, arginine at position 14, serine at
position 16, serine at position 18 of the Pin1 WW-domain.
40. The polypeptide of claim 39, wherein the modified amino acid is
replaced with an amino acid selected from the group consisting of
alanine, glutamic acid or phenylalanine.
41. (canceled)
42. A method of modulating the interaction between a
WW-domain-containing polypeptide and a phosphorylated ligand in an
individual comprising the steps of: a) providing a drug that
interacts with the WW-domain containing polypeptide; b)
administering the drug to the individual, wherein the drug binds to
the WW-domain containing polypeptide, thereby modulating the
interaction between the WW-domain containing polypeptide and the
phosphorylated ligand.
Description
BACKGROUND OF THE INVENTION
[0002] Homeostasis of the organism depends upon interactions
between protein-interacting modules and ligands to activate and
deactivate cell signaling pathways for biological processes such as
cell proliferation, cell death and protein degradation.
Protein-interacting modules are conserved regions of amino acids
that bind specific sequences in target proteins or position enzymes
in close proximity to their substrates. For example, src homology
domain 2 (SH2) binds phosphotyrosine residues on target cells to
mediate receptor activation and receptor-ligand binding (Pawson,
T., et al., Science 278:2075 (1997)). An example are WW-domains
which are highly conserved regions of approximately 40 amino acids
residues with two invariant tryptophans (W) in a triple stranded
.beta. sheet (Sudol; M. Prog. Biophys. Mol. Biol. 65:113 (1996);
Rotin, D. Curr. Topics Microbiol. Immunol. 228:115 (1998)).
Although the WW-domains of certain polypeptides have been
implicated in protein-protein interactions by binding to proline
rich sequences, many of their ligands do not contain proline rich
sequences. (Sudol, M. Prog. Biophys. Mol. Biol. 65:113 (1996);
Staub, O. et al., Structure 4:495 (1996), Rotin, D., Curr. Top.
Microbiol. Immunol. 228:115 (1998)). Therefore, the role of
WW-domain-containing proteins in mediating cell signaling events in
biological processes is not known. However, due to their potential
importance in cellular processes, it is important to elucidate a
clearer understanding of the role of WW-domains in protein-protein
interactions and cell signaling.
SUMMARY OF THE INVENTION
[0003] The present invention is based upon the discovery that
WW-domains are phosphoserine or phosphothreonine binding modules.
As further described herein, the present invention is also based
upon the discovery that the WW-domain itself is phosphorylated, and
that phosphorylation/dephosphorylation of the WW-domain polypeptide
regulates the interaction of the WW-domain polypeptide with its
phosphorylated ligand. As a result of this discovery, methods and
compositions are available to modulate protein-protein
interactions, e.g., the interaction between a signaling or
regulatory polypeptide and its phosphorylated ligands.
[0004] The invention relates to methods of modulating
protein-protein interactions comprising modulating the binding of
WW-domain polypeptides with phosphorylated ligands. In one
embodiment the binding interaction between the WW-domain containing
polypeptide and phosphorylated ligand is inhibited. In another
embodiment the binding interaction of the WW-domain containing
polypeptide and phosphorylated ligand is enhanced. As used herein,
a phosphorylated ligand is a molecule (e.g., protein, peptide,
peptide mimetic or small organic molecule) containing a
phosphoserine or phosphothreonine that binds to a WW-domain
containing polypeptide. For example, ligands specifically
encompassed by the present invention include tau protein, amyloid
precursor protein, Cdc25C, Cdc27, Plk1, NIMA, Myt1, Rab4, Wee1,
Mos, Sox3, Xbr-1b, MP75 (E-MAP-115), MP110 (Cdc5), MP68, and MP30.
WW-domain containing polypeptides specifically encompassed by the
present invention include Pin1, NEDD4, YAP, FE65, formin binding
protein, dystrophin, utropin, Ess1p/Ptf1p, Rsp5, Pub1, Dodo, Msb1,
ORF1, YKB2, DP71, C38D4.5, P9659.21, Yo61, Yfx1, ZK1248.15,
KO15c11, CD45AP, FBP11, FBP21, FBP23, FBP28 and FBP30.
[0005] Also encompassed by the present invention are molecules
which mimic a WW-domain, referred to herein as WW-domain mimic
molecules or pseudo-WW-domain molecules. Such molecules possess
structural similarity with the WW-domains described herein or
contain the consensus sequence LxxGWtx.sub.6Gtx(Y/F)(Y/F)h(N/D)
Hx(T/S)tT(T/S).sub.tWxtPt (where x=any amino acid, t=turn like or
polar residue, and h=hydrophobic amino acid as described by Rotin,
D., Curr. Top. Microbiol. Immunol. 228:115-133 (1998) the teachings
of which are incorporated herein by reference in their entirety).
For example, a WW-domain can contained the consensus sequence
LP.sub.XGWE.sub.XXXXXXXG.sub.XXYY.sub.XNH.sub.XT.sub.XXT.sub.XW.sub.XXP,
where x=any amino acid. The WW-domain mimic molecules are amino
acid sequences, peptides, peptide mimetics, or polypeptides. The
WW-domain mimic molecules are capable of interacting with, or
binding to, phosphoserine/phosphothreonine ligands, thus modulating
the activity of the phosphorylated ligand.
[0006] Also encompassed by the present invention are phosphorylated
ligand sequences, referred to herein as phosphorylated ligand
mimics, or phosphorylated pseudo-ligands. Phosphorylated ligand
mimics are amino acid sequences, peptides, peptide mimetics, or
polypeptides that contain a phosphoserine or phosphothreonine
residue(s) and are of sufficient length and share sufficient amino
acid identity with the ligand that the ligand mimics and interacts
with, or binds to, the WW-domain containing polypeptide and thus
modulates the activity of the WW-domain containing polypeptide.
[0007] A method of modulating the activity of a phosphorylated
ligand or ligand mimic for a WW-domain, or a WW-domain containing
polypeptide, comprises providing a WW-domain or WW-domain mimic
which interacts with the ligand, wherein the activity of the
phosphorylated ligand, ligand mimic, WW-domain polypeptide or
WW-domain mimic is modulated (e.g., inhibited or enhanced). The
activity can be binding activity between the ligand and WW-domain;
enzymatic/regulatory activity of the WW-domain polypeptide; or
both. For example, the prolyl-peptidyl cis-trans isomerase activity
of Pin1 or ubiquitin ligase activity of Nedd4 can increase
following binding of the WW-domain to a phosphorylated ligand.
[0008] Another aspect of the invention relates to regulating cell
growth comprising mediating the binding of the WW-domain of Pin1 to
a mitotic regulatory protein. The WW-domain can bind to a
phosphorylated ligand (e.g., NIMA) resulting in cell proliferation.
Cell proliferation can be regulated by regulating the
phosphorylation state of the WW-domain. Dephosphorylation of the
WW-domain of Pin1 leads to binding of the WW-domain to a
phosphorylated ligand resulting in cell proliferation. Likewise,
phosphorylation of the WW-domain inhibits binding to phosphorylated
ligands resulting in cell death.
[0009] The invention also encompasses methods of regulating
neurodegenerative diseases by modulating the interaction of a
WW-domain and a ligand in cells (e.g., neurons, glial cells,
Schwann cells) of the central (e.g., brain and spinal cord) and
peripheral nervous system and any cells associated with the central
or peripheral nervous systems (e.g., skeletal muscle). The
interaction between the WW-domain and a neural cellular target can
inhibit, halt, prevent or reverse neural degeneration by, for
example, interfering with neuronal cell death (e.g., apoptosis,
necrosis) or restoring neuronal function.
[0010] A further aspect of the invention encompasses a method of
regulating the function of phosphorylated ligands of WW-domain
containing polypeptides comprising mediating the binding of the
ligand to the WW-domain. Specifically encompassed by the invention
is a method of regulating the activity of hyperphosphorylated tau
protein in Alzheimer's disease comprising enhancing the binding of
the WW-domain of Pin1 to the phosphorylated threonine 231 of tau
whereby the binding of the WW-domain to tau results in binding of
tau to microtubules leading to microtubule assembly. Another method
of the invention relates to a method of regulating the interaction
between the WW-domain of dystrophin and phosphorylated ligands.
[0011] The present invention further relates to a method of
identifying a substance that modulates the interaction of a
WW-domain containing polypeptide and a ligand, wherein the ligand
is a phosphoserine or phosphothreonine ligand comprising contacting
the WW-domain containing polypeptide with one, or more, test
substances; maintaining the test substances and the WW-domain
containing polypeptide under conditions suitable for interaction;
and determining the interaction between the test substance and
WW-domain containing polypeptide, wherein the interaction indicates
that the test substance modulates the interaction between the
WW-domain-containing polypeptide and the ligand. In one embodiment
the interaction between the WW-domain and ligand that is modulated
by the test substance is binding interaction. In another embodiment
the interaction is enzymatic activity, in particular
prolyl-peptidyl cis-trans isomerase activity of Pin1 or the
ubiquitin ligase activity of Nedd4. The binding interaction or
enzymatic activity between the WW-domain and ligand can be
increased or decreased in the presence of the test substance. Thus,
the test substance can be an antagonist or agonist of the
interaction between the WW-domain and the ligand.
[0012] The present invention also provides mutants of WW-domain
containing polypeptides comprising at least one mutation in the
WW-domain. The ability of the mutant WW-domain containing
polypeptides to bind a ligand is altered. In one embodiment the
binding ability is enhanced. In another embodiment the binding
ability is reduced. The mutant WW-domain containing polypeptides
can also have altered enzymatic, catalytic or regulatory activity.
In one embodiment the enzymatic activity of the WW-domain
containing polypeptide is enhanced. In another embodiment the
enzymatic activity is reduced. The mutant can have a mutation
comprising a modification of an amino acid wherein the amino acid
is selected from the group consisting of tyrosine at position 23,
tryptophan at position 34, arginine at position 14, serine at
position 16, serine at position 18 in Pin1, or equivalent positions
in other WW-domain-containing proteins. The modified amino acid is
replaced with an amino acid residue selected from the group
consisting of alanine, glutamic acid or phenylalanine.
[0013] The invention also relates to a method of regulating protein
degradation comprising regulating the phosphorylation of a serine
residue of a WW-domain polypeptide. In particular, the WW-domain
containing polypeptide is the ubiquitin ligase Nedd4. In one
embodiment phosphorylation of the serine residue leads to binding
of the WW-domain containing polypeptide and ligand to initiate
polypeptide degradation. In another embodiment dephosphorylation of
the serine residue of the WW-domain containing polypeptide prevents
binding of the WW-domain containing polypeptide and ligand thereby
preventing polypeptide degradation. The regulation of protein
degradation by the methods of the invention can result in
regulation of cell growth. In yet another embodiment modulations in
the protein degradation lead to regulation of cell growth. In
particular, inhibition of Cdc25 degradation by the ubiquitin
pathway results in cell death.
[0014] In yet another aspect of the invention relates to a method
of treating a WW-domain containing polypeptide-mediated condition
in a mammal, wherein the condition results from an alteration in a
ligand for the WW-domain containing polypeptide, wherein the ligand
is a phosphoserine or phosphothreonine ligand comprising
introducing into the mammal an amount of a WW-domain containing
polypeptide effective to regulate the ligand, thereby alleviating
the condition.
[0015] In another embodiment the present invention relates to a
method of treating a WW-domain containing polypeptide-mediated
condition in a mammal, wherein the condition results from an
alteration in the WW-domain containing polypeptide wherein a ligand
for the WW-domain contains a phosphoserine or phosphothreonine,
comprising introducing into the mammal an amount of a WW-domain
containing polypeptide effective to alleviate the condition.
[0016] The invention further relates to a method of delivering a
drug to treat a condition in a mammal, wherein the condition
results from an alteration in a phosphorylated ligand for a
WW-domain containing polypeptide, comprising combining the drug and
the WW-domain containing polypeptide or a fragment under conditions
suitable to form a complex; and administering the complex to the
mammal, wherein the complex and phosphorylated ligand interact
thereby alleviating the condition.
[0017] The inventions which are described herein provide
compositions and methods to modulate protein-protein interactions
such as binding interactions between signaling or regulatory
proteins and their phosphorylated ligands. The methods permit
inhibiting or enhancing the interaction between a WW-domain
containing polypeptide and its phosphorylated ligand. The methods
described herein can be used for regulating cell growth; targeting
proteins for cellular degradation; restoring the function of tau to
bind microtubules and promote or restore microtubule assembly in
neurodegenerative diseases such as Alzheimer's disease, Dementia
pugilistica, Down's syndrome, Parkinson's disease, Pick's disease;
identifying a substance which alters the interaction of WW-domain
containing polypeptides and their phosphorylated ligands; and
targeting drugs to ligands of WW-domain containing polypeptides to
treat disease conditions in a mammal. The methods provide a means
to assess the interaction of a phosphoserine/phosphothreonine
binding module (WW-domain containing polypeptide) and its cellular
ligands.
BRIEF DESCRIPTION OF THE FIGURES
[0018] FIG. 1 is a graphic representation of the competition of
Pin1 WW-domain binding to phosphoproteins (pSer) by phosphopeptides
but not by nonphosphorylated (Ser) or proline-rich (Pro)
peptides.
[0019] FIG. 2 is the amino acid sequence alignment of selected
WW-domains. Pin1/human (SEQ ID NO: 1); Ess1/S.c. (SEQ ID NO: 2);
Nedd4/mouse (SEQ ID NO: 3); Dmd/human (SEQ ID NO: 4); Fbp11/mouse
(SEQ ID NO: 5); FE65/rat (SEQ ID NO: 6) and Yap/mouse (SEQ ID NO:
7). The top and bottom lines illustrate the X-ray structural
elements in native Pin 1 and the NMR structural elements in the
isolated YAP WW-domain, respectively. The black boxes with white
letter define the residues in the Pin1 WW-domain, whose mutations
affected the interactions with phosphoproteins. White boxes with
black letters define the residues whose mutations had no detectable
effect. The numbers above the sequences refer to human Pin 1
sequence.
[0020] FIG. 3 depicts the coding sequence of a fully functional
PTF1 genomic fragment replaced with Pin1 or its mutant cDNAs in a
YEP vector. An HA tag was added at the N-terminus to detect protein
expression.
[0021] FIG. 4A is a graphic representation of the binding affinity
of Pin1 to tau peptides detected by an enzyme linked
immunoabsorbant assay using Pin1 antibodies (Pin1 Ab).
[0022] FIG. 4B is a graphic representation of the binding affinity
of Pin1 and phosphorylated (pT231) or nonphosphorylated (T231) tau
peptide.
[0023] FIG. 5A is a graphic representation of the inability of Pin1
to affect tau induced tubulin assembly.
[0024] FIG. 5B is a graphic representation of the ability of
phosphorylated Tau (pTau) to microtubules assembly in the presence
of Pin1, but not the Pin1.sup.Y23A mutant.
[0025] FIG. 6 depicts the amino acid sequence of the WW-domain of
Pin1/human (SEQ ID NO: 33), beginning with the sixth amino acid;
ESS1/9C (SEQ ID NO: 34); Yap/Human (SEQ ID NO: 35); Nedd4/Mouse
(SEQ ID NO: 36); RSPS/9C (SEQ ID NO: 37); Dmd/human (SEQ ID NO: 38)
and FE65/Rat (SEQ ID NO: 39).
DETAILED DESCRIPTION OF THE INVENTION
[0026] The present invention relates to the discovery that
WW-domains bind serine or threonine phosphoproteins, polypeptides,
or peptides with high affinity in a phosphate dependent manner. The
WW-domain-containing serine or threonine phosphorylated binding
polypeptides of the present invention inhibit dephosphorylation of
ligands when bound to the ligand. Binding of the WW-domain
containing polypeptide to a ligand can alter the activity of the
WW-domain containing polypeptide, ligand or both.
[0027] The term "WW-domain containing polypeptide" as used herein
refers to a protein (also referred to herein as a polypeptide)
which binds phosphorylated ligands. For example, the
WW-domain-containing polypeptides encompassed by the present
invention include Pin1, Nedd4, YAP, FE65, formin binding protein,
dystrophin, utropin and Ess1p/Ptf1p, Rsp5, Pub1, Dodo, Msb1, ORF1,
YKB2, DP71, C38D4.5, P9659.21, Yo61, Yfx1, ZK1248.15, KO15c11,
CD45AP, FBP11, FBP21, FBP23, FBP28 and FBP30. (Rotin, D. Curr.
Topics Microbiol. Immunol. 228:115 (1998)). Database accession
numbers for the nucleotide and amino acid sequences for these
WW-domain-containing proteins are known. (Rotin, D. Curr. Topics
Microbiol. Immunol. 228:115 (1998)). It is understood that any
additional WW-domain-containing proteins to be discovered are
within the scope of the invention.
[0028] "WW-domain-containing polypeptide", as the term is used
herein, can also include any polypeptide which shows sequence and
structural identity to a WW-domain which contains an amino acid
sequence with identity to any known WW-domain containing
polypeptides such as Pin1, Nedd4, YAP, FE65, formin binding
protein, dystropin, utropin, Ess1p/Ptf1p, Rsp5, Pub1, Dodo, Msb1,
ORF1, YKB2, DP71, C38D4.5, P9659.21, Yo61, Yfx1, ZK1248.15,
KO15c11, CD45AP, FBP11, FBP21, FBP23, FBP28 and FBP30. (FIG. 6)
(See, for example, Hunter, T., et al., WO 97/17986 (1997); Rotin,
D., Curr. Top. Microbiol. Immunol. 228:115-133 (1998), the
teachings of which are incorporated herein in their entirety).
Sequence identity can be determined using database search
strategies well known in the art including, for example, Basic
Local Alignment Search Tool (BLAST) (Altschul, S. F., et al., J.
Mol. Biol. 215:403410 (1990)) and FASTA (Pearson, W. R., et al.,
Proc. Natl. Acad. Sci. U.S.A. 85:2444-2448 (1988)) algorithms. In
one embodiment, the BLAST parameters are set such that they yield a
sequence having at least about 60% sequence identity with the
corresponding known WW-domain sequence, preferably, at least about
70% sequence. In another embodiment, the percent sequence identity
is at least about 85%, and in yet another embodiment, at least
about 95%. Such molecules are also referred to herein as WW-domain
mimic molecules and are characterized by highly conserved regions
of approximately 40 amino acids residues with two invariant
tryptophans (W) in a triple stranded .beta. sheet (Sudol, M. Prog.
Biophys. Mol. Biol. 65:113 (1996); Rotin, D. Curr. Topics
Microbiol. Immunol. 228:115 (1998)). Thus, the WW-domain mimic
molecules possess structural similarity with the WW-domains
described herein or contain the consensus sequence
LxxGWtx.sub.6Gtx(Y/F)(Y/F)h(N/D)Hx(T/S)tT(T/S)tWxtPt (where x=any
amino acid, t=turn like or polar residue, and h=hydrophobic amino
acid as described by Rotin, D., Curr. Top. Microbiol. Immunol.
228:115-133 (1998)). For example, the WW-domain of a WW-domain
mimic molecule can have the consensus sequence
LP.sub.XGWE.sub.XXXXXXXG.sub.XXYY.sub.XNH.sub- .XT.sub.XXT.sub.XXP,
where x=any amino acid. (FIG. 6). The WW-domain mimic molecules can
be about 38-40 amino acids in length, or they can be shorter or
longer than 38-40 amino acids. The WW-domain mimic molecules are
capable of interacting with, or binding to,
phosphoserine/phosphothre- onine ligands, thus modulating the
activity of the phosphorylated ligand.
[0029] It is also envisioned that any WW-domain or WW-domain
containing polypeptide functionally equivalent to the molecules
described herein will be within the scope of the invention. The
phrase "functionally equivalent" as used herein refers to any
molecule (e.g., polypeptide and nucleic acid sequence encoding the
polypeptide) which mimics the interaction (e.g., binding, enzymatic
activity) of the WW-domain or WW-domain containing polypeptides
described herein (such as Pin1, Nedd4) or which exhibit nucleotide
or amino acid sequence identity to WW-domain containing
polypeptides such as Pin1 or Nedd4, for example. The nucleotide and
deduced amino acid of Pin1 is known. (See Hunter, T., et al., WO
97/17986, (1997), the teachings of which are incorporated herein in
their entirety.)
[0030] The invention relates to a method mediating protein-protein
interactions comprising modulating the binding of a WW-domain
containing polypeptide with a phosphorylated ligand. The ligand can
be a protein, polypeptide, peptide, or peptide mimetic with a
phosphoserine, phosphothreonine, or both a phosphoserine and
phosphothreonine residue. The ligand can be a native ligand for the
WW-domain containing polypeptide or a ligand mimic. A native ligand
is meant to refer to a phosphorylated ligand which is known to bind
a WW-domain. For example, Cdc25c is a native ligand for the
WW-domain of Pin1. A phosphorylated ligand mimic can be a protein,
polypeptide, peptide or peptide mimetic, that is a synthetic or
natural organic product, which shares structural similarity with a
native ligand for the WW-domain containing polypeptide and
interacts with a WW-domain containing polypeptide and thus
modulates the activity of the WW-domain containing polypeptide.
Native ligands or ligand mimics that have a proline residue
adjacent to a phosphorylated serine or threonine residue can bind
the WW-domain. Proline residues in native ligands can be replaced
with normative N-substituted residues to generate ligands mimics
with enhanced binding affinity according to the procedure of
Nguyan, J. T. et al., Science 282;207-211 (1998), the teachings of
which are incorporated herein in their entirety.
[0031] The interaction between a WW-domain containing polypeptide
and phosphorylated ligand can be modulated by increasing
interactions (e.g., binding) between the WW-domain and
phosphorylated ligand or inhibiting interactions (e.g., binding)
between the WW-domain and phosphorylated ligand. For example,
binding interactions between Pin1 and a subset of mitotic
phosphoproteins can be competitively inhibited by a phosphorylated
ligand mimic. For example, in the case of Pin1, the phosphorylated
peptide Pintide is a ligand mimic which competes for binding of a
native ligand to the WW-domain of Pin1 (See Example 4). Competitive
inhibition is characterized by the ability of the phosphorylated
ligand mimic to compete, alter or prevent the WW-domain containing
polypeptide from interacting with its native ligand. Likewise
binding interactions between the WW-domain of Pin1 and
phosphorylated ligands can be enhanced by phosphorylation of
specific amino acid residues in the WW-domain and target
ligand.
[0032] The term "modulated" is used herein to describe biological
activity greater (increased or enhanced or augmented activity) or
less (decreased or reduced or diminished) than the activity of the
WW-domain containing polypeptide in the absence of WW-domain/ligand
interaction. As defined herein activity encompasses binding
activity (e.g., ability to interact with a ligand) or enzymatic
(e.g., ability to isomerize phosphoserine/threonine-proline bonds
or ligase activity) activity or both. Enzymatic, catalytic or
regulatory activity are used interchangeably. The enzymatic,
catalytic or regulatory activity of the WW-domain containing
polypeptide can control the activity of a ligand or the WW-domain
containing polypeptide. For example, binding of the WW-domain of
Pin1 to phosphoserine residues in synthetic peptides such as
Pintide or mitotic cell extract proteins such as Cdc25, leads to an
increase in the peptidyl propyl cis-trans isomerase activity (e.g.,
regulatory activity) of Pin1. The phosphoprotein or phosphopeptide
specificity and affinity of WW-domain binding to ligands can be
determined using binding and regulatory assays well known to those
of skill in the art, and in vivo activity can be measured as
described in Examples 1-10. For example, in vitro regulatory
activity for Pin1 can be measured as described in Lu et al., U.S.
Ser. No. 60/058, 164 (1997), the teachings of which are
incorporated herein by reference.
[0033] The activity of ligands described herein can be modulated
following binding to WW-domains. Modulation of ligands can modulate
protein-protein interactions resulting in, for example, the
activation or deactivation of cell signaling pathways. Activation
or deactivation of a cell signaling pathway can lead to the
restoration of a biological function of the ligand. In particular,
the WW-domain of Pin1 can interact with hyperphosphorylated tau
and, thereby, allow Pin to restore microtubule function and
assembly in neurodegenerative diseases. Tau protein is associated
with several neuordegenerative diseases including Alzheimer's
disease, Corticobasal degeneration, Dementia pugilistica, Down's
syndrome, Frontotemporal dementias and Parkinsonism linked to
chromosome 17, Myotonic dystrophy, Niemann-Pick disease,
Parkinson-dementia complex of Guam, Pick's disease, postencephalic
Parkinsonism, prion disease with tangles, progressive supranuclear
palsy, subacute sclerosing panencephalistis. (Spillantini, M. G.,
et al., TINS 21:428-432 (1998)). The methods of the present
invention can be used to treat these neurodegenerative diseases.
Specifically, in Alzheimer's disease, binding of the WW-domain of
Pin1 to phosphorylated threonine 231 of tau can allow Pin1 to fully
restore the function of phosphorylated tau (e.g., to bind
microtubules and promote microtubule assembly) (Example 11). The
WW-domain of Pin1 also binds phosphorylated threonine 668 of the
amyloid precursor protein and can be used to treat
neurodegenerative diseases associated with amyloid precursor
protein. The WW-domain of WW-domain containing polypeptides can
also interact (e.g., binds) with phosphoserine or phosphothreonine
ligands thereby altering the conformation or activity of the
WW-domain polypeptide. For example, the prolyl-peptidyl cis-trans
isomerase activity of the Pin1 is altered (e.g., increased) as a
result of binding to a phosphorylated ligand such as Cdc25c. Thus,
the activity of the WW-domain containing polypeptide can be altered
(e.g., increased or decreased) after interaction (e.g., binding)
with the phosphorylated ligand.
[0034] The invention further relates to methods of regulating cell
growth by mediating the binding of the WW-domain of Pin1 to a
mitotic regulatory protein such as NIMA or Cdc25. Binding can be
mediated by regulating the phosphorylation state of a serine
residue in the WW-domain of Pin1. In particular, the serine residue
at position 16 of the WW-domain of Pin1 is dephosphorylated or
phosphorylated resulting in cell growth and cell death,
respectively. Cell growth (also referred to herein as cell
proliferation) leads to an increase in the number of cells. Cell
death can be programmed cell death such as apoptosis or the
nonprogrammed cell death such as necrosis. Techniques to assess
cell growth and cell death are well known to the skilled
artisan.
[0035] The invention also relates to a method of regulating protein
degradation comprising altering the phosphorylation state of a
WW-domain target protein. In particular, the WW-domain containing
polypeptide is Nedd4 and Nedd4 ligands are Cdc25C, amino acid
permerases, the large subunit of RNA polymerase II and
miloride-sensitive epithelial Na channel (ENaC), for example. When
the ligand is phosphorylated, the WW-domain of Nedd4 binds the
ligand and targets the ligand for protein degradation through a
ubiquitin pathway. Dephosphorylation of the WW-domain prevents
Nedd4 interaction with a ligand. Such a mechanism can be important
in regulating mitotic activators such as Cdc25 thereby regulating
cell growth. For example, modulating interactions between the
WW-domain of Nedd4 and Cdc25 by preventing Nedd4 from targeting
Cdc25 for protein degradation and results in cell death.
[0036] Also encompassed in the present invention are mutants of
WW-domain containing polypeptides with altered binding or catalytic
activity. The mutants of the present invention can be used, for
example, to further understand the mechanism of protein-protein
interactions which involve phosphoserine and phosphothreonine
binding to WW-domains. The term "mutant", as used herein, refers to
any modified nucleic acid sequence encoding a WW-domain or
WW-domain containing polypeptide. For example, the mutant can be a
polypeptide produced as a result of a point mutation or the
addition, deletion, insertion and/or substitution of one or more
nucleotides encoding the WW-domain, or any combination thereof.
Modifications can be, for example, conserved or non-conserved,
natural or unnatural. The invention also pertains to the nucleic
acid constructs encoding the mutant WW-domain containing
phosphoserine or phosphothreonine binding polypeptides and their
encoded polypeptides. Techniques to introduce mutations are well
established. Exemplary protocols are found in "Current Protocols in
Molecular Biology", Ausbel, et al., John Wiley & Co.
(1998).
[0037] As used herein a mutant also refers to the polypeptide
encoded by the mutated nucleic acid. That is, the term "mutant"
also refers to a polypeptide which is modified at one, or more,
amino acid residues from the wildtype (naturally occurring)
polypeptide. In a preferred embodiment mutants are generated by
mutations in the WW-domain of polypeptides.
[0038] In one embodiment the mutations are made to Pin1. In another
embodiment the mutations are made to Nedd4. In a particular
embodiment, the amino-WW-domain of the Pin1, as described herein,
has a mutation resulting in a altered binding or regulatory
activity. For example, in this embodiment the Pin1.sup.S16A mutant
is a mutant of Pin1 resulting from a point mutation substituting
the serine at position 16 (S16) in the WW-domain of Pin1 with an
alanine residue to generate the Pin1.sup.S16A. In the wildtype Pin1
the proline ring of the ligand is positioned in a hydrophobic
crevice between the aromatic rings of tyrosine 23 and tryptophan 34
of the WW-domain, whereas the phosphoserine residue of the ligand
fits into a cleft between serine 16 and tyrosine 23 of the
WW-domain (Macias, M. J., et al., Nature 382:646 (1996);
Ranganathan, R., et al., Cell 89:875 (1997)). The phosphate moiety
of the ligand is directed to within hydrogen binding distance of
the tyrosine 23 hydroxyl proton.
[0039] A single alanine point mutation at tyrosine 23
(Pin1.sup.Y23A) or tryptophan 34 (Pin1.sup.W34A) in the WW-domain
of Pin1 completely abolishes the ability of Pin1 to bind
phosphopeptides with high affinity, whereas a single glutamic acid
point mutation in the serine residue at position 16 (Pin1.sup.S16E)
abolishes the regulatory or isomerase activity of Pin1. Thus
different amino acid residues in the WW-domain can mediate
different activities (e.g., binding to ligands or enzymatic
activity) of the WW-domain containing polypeptide.
[0040] WW-domain containing polypeptide mutants can be made by
mutations to one, or more, amino acid residues selected from a
group consisting of serine at position 16, or arginine at position
14, or tyrosine at position 23, or tryptophan at position 34 or any
combination thereof.
[0041] Using well-known techniques to align amino acids, amino acid
residues suitable for mutation as described herein for Pin-1 can be
determined for other WW-domain containing polypeptides such as
Nedd4, YAP, FE65, formin binding protein, dystrophin, utropin,
Ess1p/Ptf1p, Rsp5, Pub1, Dodo, Msb1, ORF1, YKB2, DP71, C38D4.5,
P9659.21, Yo61, Yfx1, ZK1248.15, KO15c11, CD45AP, FBP1, FBP21,
FBP23, FBP28 and FBP30. (Rotin, D. Curr. Topics Microbiol. Immunol.
228:115 (1998)). Database accession numbers for the nucleotide and
amino acid sequences for these WW-domain-containing proteins are
known. (Rotin, D. Curr. Topics Microbiol. Immunol. 228:115 (1998)).
Nucleic acid sequences encoding the WW-domain containing
polypeptides can be mutated; the mutated nucleic acid constructs
expressed under standard experimental conditions well known to the
skilled artisan; and the resulting mutant polypeptides evaluated
for binding or enzymatic activity or both as described herein.
Appropriate amino acid residues can be substituted as described for
Pin1 using routine, art-recognized techniques. (See, for example,
Shen, M., et al., Genes & Dev 12:706 (1998)).
[0042] Techniques to assess ligand binding to a
WW-domain-containing polypeptides are known in the art. Exemplary
methods are described in Lu et al., U.S. Ser. No. 60/058,164
(1997), the teachings of which are incorporated herein by
reference.
[0043] The WW-domain containing polypeptide is preferably purified
substantially prior to use, particularly where the WW-domain or
WW-domain containing polypeptide is employed in in vitro binding
assays, in vivo treatments and in vitro screens of test substances
which alter the activity of the WW-domain containing polypeptide or
ligand. It is preferred to employ a WW-domain containing
polypeptide which is essentially pure (e.g., about 99% by weight or
to homogeneity).
[0044] WW-domain containing polypeptides can be screened for
activity using standard techniques. To screen the WW-domain
polypeptides for enzymatic activity, for example prolyl-peptidyl
cis-trans isomerase activity, before and following binding and
activation by ligands, in vitro assays with radiolabeled substrate
in the presence or absence of phosphoserine or phosphothreonine
peptides. The effects of WW-domain containing polypeptides and
mutants can be assessed in vivo employing routine transformation
techniques as described in Example 8.
[0045] The effect of WW-domain containing polypeptide interaction
with a ligand on activity of the WW-domain containing polypeptide
or the ligand can be tested. For example, particular biologic
activities such as isomerase activity, ligase activity, cell
proliferation, cell death or association with cellular targets such
as neuronal microfilaments. Protocols to evaluate these biological
activities are known to one of skill in the art. (See, for example,
Lu et al., U.S. Ser. No. 60/058,164 (1997); Lu, K. P., et al.,
Nature 380:544 (1996), the teachings of which are incorporated
herein by reference).
[0046] The present invention also provides methods of identifying a
substance that modulates the interaction of WW-domain containing
polypeptide and a phosphorylated ligand comprising the steps of
contacting the WW-domain containing polypeptide with one, or more,
test substances; maintaining the test substances and the WW-domain
containing polypeptide under conditions suitable for interaction;
and determining the interaction between the test substance and
WW-domain containing polypeptide. An interaction between the test
substance and the WW-domain containing polypeptide indicates that
the test substance modulates the interaction between the
WW-domain-containing polypeptide and the ligand. The interaction
can be determined in the presence and absence of the test
substance. One or more test substance can be evaluated
simultaneously or sequentially. The test substances identified by
the method of the invention can be used to treat disease conditions
resulting from altered WW-domain containing polypeptide/ligand
interactions.
[0047] The term "modulate" in regard to activity or "altered
activity" or "altered interaction" is defined herein as activity
different from that of the ligand or WW-domain in the absence of
the test substance.
[0048] The test substance (e.g., an inhibitor or stimulator) can be
added to the WW-domain polypeptide either before or following the
addition of the ligand under conditions suitable for maintaining
the WW-domain and ligand in a conformation appropriate for
formation of a combination. Experimental conditions for evaluating
test substances, such as buffer or media, concentration and
temperature requirements, can, initially, be similar to those
described in Examples 1-11. One of ordinary skill in the art can
determine empirically how to vary experimental conditions depending
upon the biochemical nature of the test substance. The
concentration at which the test substance can be evaluated can be
similar, more, or less than concentrations employed by the native
ligand to bind the WW-domain containing polypeptide.
[0049] The substances which alter the activity of the WW-domain
containing polypeptide or ligands of the invention can be
stimulators/enhancers (e.g., agonists) or inhibitors (e.g.,
antagonists) of, for example, prolyl-peptidyl cis-trans isomerase
or ubiquitin ligase activity. The substances can be polypeptides
(including post-translationally modified polypeptides), peptides,
or small molecules (including carbohydrates, steroids, lipids,
other organic molecules, anions or cations).
[0050] The term "inhibitor", as used herein, refers to a substance
which blocks, diminishes, antagonizes, hinders, limits, decreases,
reduces, restricts or interferes with WW-domain containing
polypeptide interaction with the ligand or WW-domain activity or
ligand activity or any combination thereof, or alternatively and
additionally, prevents or impedes the binding of the WW-domain
polypeptide with a ligand thereby preventing the WW-domain or
ligand from acting. By way of example, an inhibitor of Pin1 can
decrease the ability of Pin1 to bind phosphorylated ligands or
isomerize phosphoserine-/phosphothreonine-proline bonds.
[0051] The term "stimulator" or enhancer as used herein, refers to
a substance which agonizes, augments, enhances, increases,
intensifies or strengthens the interaction between a WW-domain and
ligand, or alternatively and additionally, mimics or enhances the
effect of the binding of the WW-domain polypeptide to a ligand
thereby further activating the WW-domain polypeptide or ligand. In
the case of Pin1, a substance possessing stimulatory activity can
increase peptidyl prolyl isomerase activity or can increase the
binding affinity of Pin1 to phosphorylated ligands beyond that
observed in the absence of the stimulatory substance. Likewise a
stimulator of Nedd4 ligase activity can result in augmented
targeting of polypeptides destined for protein degradation through
ubiquitin pathways.
[0052] Inhibitors or stimulators/enhancers of WW-domain containing
polypeptides or ligands of the present invention can include any
molecule that binds or interferes with (inhibitor) or facilitates
(stimulates) WW-domain interaction with its ligand or the activity
or structure of the WW-domain or ligand. Encompassed by the present
invention are inhibitor molecules that mimic the structure and
conformation of the ligand or WW-domain. The inhibitors or
stimulators of WW-domain containing polypeptides or ligands can be
naturally occurring or synthesized using standard laboratory
methods that are well known to those of skill in the art.
[0053] Another aspect of the invention relates to targeting a drug
to treat a condition in a mammal by associating a drug with a
WW-domain to form a "drug WW-domain" complex and administering the
"drug/WW-domain" complex to a mammal wherein the "drug/WW-domain"
complex interacts with a phosphorylated ligand in vivo, thereby
alleviating the condition. The condition to be treated results from
an alteration in a phosphorylated ligand which is a ligand for a
WW-domain containing polypeptide.
[0054] The invention further relates to modulating the interaction
of a WW-domain and a phosphorylated ligand by designing a drug
which interacts with a WW-domain. The drug, when administered to an
individual, binds the WW-domain thereby modulating the interaction
between the WW-domain and its phosphorylated ligand in vivo.
[0055] It is also envisioned that fragments of the WW-domain
containing polypeptides can be used in the methods of the
invention. "Fragments" of WW-domain containing polypeptides, as
used herein, refer to any part of the WW-domain capable of binding
to the phosphorylated ligand and mediating protein-protein
interactions. For example, the isolated WW-domain of a WW-domain
containing polypeptide would be considered a fragment.
[0056] In one embodiment of the present invention the WW-domains,
WW-domain containing polypeptides, ligands or test substances are
compounds comprising proteins, polypeptides and peptides. The
proteins, polypeptides and peptides of the present invention
comprise naturally-occurring amino acids (e.g., L-amino acids),
non-naturally amino acids (e.g., D-amino acids), and small
molecules that biologically and biochemically mimic the inhibitor
or stimulation peptides, referred to herein as peptide analogs,
derivatives or mimetics. (Saragovi, H. U., et al., Bio Technology,
10:773-778 (1992)). The protein, polypeptide or peptides of the
present invention can be in linear or cyclic conformation.
[0057] The WW-domains, ligands or test substances of the present
invention can be synthesized using standard laboratory methods that
are well-known to those of skill in the art, including standard
solid phase techniques. The molecules comprising polypeptides of
naturally occurring amino acids can also be produced by recombinant
DNA techniques known to those of skill, and subsequently
phosphorylated or otherwise posttranslationally modified.
[0058] The WW-domains, ligands and test substances of the present
invention can comprise either the 20 naturally occurring amino
acids or other synthetic amino acids. Synthetic amino acids
encompassed by the present invention include, for example,
naphthylalanine, L-hydroxypropylglycine,
L-3,4-dihydroxyphenylalanyl, .alpha.-amino acids such as
L-.alpha.-hydroxylysyl and D-.alpha.-methylalanyl,
L-.alpha.-methyl-alanyl, .beta. amino-acids such as .beta.-analine,
and isoquinolyl.
[0059] D-amino acids and other non-naturally occurring synthetic
amino acids can also be incorporated into the WW-domains, ligands
or test substances of the present invention. Such other
non-naturally occurring synthetic amino acids include those where
the naturally occurring side chains of the 20 genetically encoded
amino acids (or any L or D amino acid) are replaced with other side
chains, for instance with groups such as alkyl, lower alkyl, cyclic
4-, 5-, 6-, to 7-membered alkyl, amide, amide lower alkyl, amide
di(lower alkyl), lower alkoxy, hydroxy, carboxy and the lower ester
derivatives thereof, and with 4-, 5-, 6-, to 7-membered
heterocyclic.
[0060] As used herein, "lower alkyl" refers to straight and
branched chain alkyl groups having from 1 to 6 carbon atoms, such
as methyl, ethyl propyl, butyl and the like. "Lower alkoxy"
encompasses straight and branched chain alkoxy groups having from 1
to 6 carbon atoms, such as methoxy, ethoxy and the like.
[0061] Cyclic groups can be saturated or unsaturated, and if
unsaturated, can be aromatic or non-aromatic. Heterocyclic groups
typically contain one or more nitrogen, oxygen, and/or sulphur
heteroatoms, e.g., furazanyl, furyl imidazolidinyl, imidazolyl,
imidazolinyl, isothiazolyl, isoxazolyl, morpholinyl (e.g.,
morpholino), oxazolyl, piperazinyl (e.g., 1-piperazinyl), piperidyl
(e.g., 1-piperidyl, piperidino), pyranyl, pyrazinyl, pyrazolidinyl,
pyrazolinyl pyrazolyl, pyridazinyl, pyridyl, pyrimidinyl,
pyrrolidinyl (e.g., 1-pyrrolidinyl), pyrrolinyl, pyrrolyl,
thiadiazolyl, thiazolyl, thienyl, thiomorpholinyl (e.g.,
thiomorpholino), and triazolyl. The heterocyclic groups can be
substituted or unsubstituted. Where a group is substituted, the
substituent can be alkyl, alkoxy, halogen, oxygen, or substituted
or unsubstituted phenyl. (See U.S. Pat. No. 5,654,276 and U.S. Pat.
No. 5,643,873, the teachings of which are herein incorporated by
reference).
[0062] Biologically active derivatives or analogs of the
above-described WW-domains, ligands and test substances (e.g.,
inhibitors or stimulators), referred to herein as peptide mimetics,
can be designed and produced by techniques known to those of skill
in the art. (See e.g., U.S. Pat. Nos. 4,612,132; 5,643,873 and
5,654,276, the teachings of which are herein incorporated by
reference). These mimetics can be based, for example, on a specific
WW-domain sequences or known ligands and maintain the relative
positions in space of the WW-domain or ligand. These peptide
mimetics possess biologically activity (e.g., prolyl-peptidyl
cis-trans isomerase, ubiquitin ligase or microtubule binding
activity) similar to the biological activity of the corresponding
WW-domain containing polypeptide ligand or test substance, but
possess a "biological advantage" over the corresponding peptide
with respect to one, or more, of the following properties:
solubility, stability, and susceptibility to hydrolysis and
proteolysis.
[0063] Methods for preparing peptide mimetics include modifying the
N-terminal amino group, the C-terminal carboxyl group, and/or
changing one or more of the amino linkages in the peptide to a
non-amino linkage. Two or more such modifications can be coupled in
one peptide mimetic inhibitor. Modifications of peptides to produce
peptide mimetics are described in U.S. Pat. Nos. 5,643,873 and
5,654,276, the teachings of which are incorporated herein by
reference.
[0064] Where the WW-domains, ligands or test substances of present
invention comprise amino acids, the peptides can also be cyclic
proteins, peptides and cyclic peptide mimetics. Such cyclic
peptides can be produced using known laboratory techniques (e.g.,
as described in U.S. Pat. No. 5,654,276, the teachings of which are
herein incorporated in their entirety by reference).
[0065] The test substances identified as inhibitors or stimulators
as described herein can be used in vitro to study cell cycle
regulation, mitotic events, protein degradation and
neurodegenerative diseases. For example, the WW-domain of the
present invention can be used to evaluate mitotic events and
programmed cell death in mammalian cells by interacting with
specific phosphoproteins and evaluating the effects on the cell
cycle and apoptosis. By way of illustration, the WW-domain of Pin1
can bind phosphorylated tau protein or amyloid precursor protein
and restore neuronal function or promote neuronal survival in
Alzheimer's disease by preventing cell death (e.g., apoptosis).
[0066] The present invention provides methods of modulating the
activity of WW-domain containing polypeptides or their ligands
comprising modulating the interaction of the WW-domain with a
ligand, wherein the ligand contains a phosphoserine or
phosphothreonine. Ligands refer to any molecule (e.g., polypeptide,
peptide mimetic, or small organic molecule) which interacts with a
WW-domain or WW-domain containing polypeptide. Methods to detect
binding can include, for example, the use of labeled (e.g.,
fluorescent, biotin, radioactive, luminescent) activated WW-domains
or ligands and detection techniques such as solid-phase plate
assays; immunoprecipitation; Western blotting, and fluorescence
aniostropy assays. Such technologies are well established and
within the technical expertise of one of ordinary skill in the
art.
[0067] The identification of substances which alter (e.g., inhibit
or stimulate) WW-domain ligand interaction as identified herein can
be important in defining pathways which lead to carcinogenesis and
to the development of novel, specific and more effective treatment
regimens.
[0068] Certain WW-domain containing polypeptide play a key role in
transducing signaling pathways to mediate, for example, cell
division and apoptosis (e.g., Pin1), and protein degradation (e.g.,
Nedd4). It is further envisioned that the WW-domains and mutants of
the present invention and substances which alter their activity can
be used to evaluate, interfere and treat events such as cell
spreading in metastatic cancers.
[0069] As another example, because Pin1 is critical regulator for
mitosis (Lu, K. P., et al., U.S. Ser. No. 60/058,164 (1997); Shen,
M., et al., Genes & Development 12:706-720 (1998)) and
substances which alter (e.g., inhibit) the activity of a WW-domain
can be used to discern the mechanisms for certain aspects of cell
division such as embryonic development. The identification of
substrates for and substances which alter WW-domain containing
polypeptides and their ligands can be useful for the study of cell
cycle events.
[0070] The inhibitors or stimulators of interactions between
WW-domain and ligands of the present invention can be used to
interfere with eukaryotic cell growth and to treat hyperplastic and
neoplastic disorders in mammals. As defined herein, mammals include
rodents (such as rats, mice or guinea pigs), domesticated animals
(such as dogs or cats), ruminant animals (such as horses, cows) and
primates (such as monkeys or humans). For example, a
phosphorylation of the WW-domain of Pin1, which attenuates some
cell signaling pathways, can be useful in anti-neoplastic therapies
for the treatment of diseases such as leukemia. Certain neoplasms
have been attributed to an augmentation in the phosphorylation of
cellular effectors which can be offset or neutralized by wildtype
or mutant of WW-domains thereby turning off or controlling the
unregulated cellular growth or pathway.
[0071] Neoplastic and hyperplastic disorders include all forms of
malignancies, psoriasis, retinosis, atherosclerosis resulting from
plaque formation, leukemias and benign tumor growth. For example,
such disorders include lymphomas, papilomas, pulmonary fibrosis,
and rheumatoid arthritis.
[0072] The methods of the present invention can be used to modulate
protein-protein interactions in neurodegenerative diseases to
restore neuronal function or prevent neuronal cell death, and
alleviate disease symptoms. Neurodegenerative diseases that can be
treated by the methods of the present invention include Alzheimer's
disease, multiple sclerosis, muscular dystrophy Corticobasal
degeneration, Dementia pugilistica, Down's syndrome, Frontotemporal
dementias and Parkinsonism linked to chromosome 17, Myotonic
dystrophy, Niemann-Pick disease, Parkinson-dementia complex of
Guam, Pick's disease, postencephalic Parkinsonism, prion disease
with tangles, progressive supranuclear palsy, subacute sclerosing
panencephalistis. (Spillantini, M. G., et al., TINS 21:428-432
(1998)). As an example, the WW-domain of Pin1 can bind
phosphorylated tau protein or amyloid precursor protein and restore
nerve cell function, prevent apoptosis, or both.
[0073] Biologically active derivatives, analogs or mimics of the
above-described WW-domains, ligands, test substances,
drug/WW-domain complexes and drugs designed to interact with a
WW-domain can be formulated into compositions with an effective
amount of the WW-domain, ligand, drug/WW-domain complex, or drug as
the active ingredient. Such compositions can also comprise a
pharmaceutically acceptable carrier, and are referred to herein as
pharmaceutical compositions. The inhibitor or stimulation
compositions of the present invention can be administered
intravenously, parenterally, orally, nasally, by inhalation, by
implant, by injection, or by suppository. The mode of
administration is preferably at the location of the target cells.
The inhibitor or stimulation composition can be administered in a
single dose or in more than one dose over a period of time to
achieve a level of inhibitor which is sufficient to confer the
desired effect.
[0074] Suitable pharmaceutical carriers include, but are not
limited to sterile water, salt solutions (such as Ringer's
solution), alcohols, polyethylene glycols, gelatin, carbohydrates
such as lactose, amylose or starch, magnesium stearate, talc,
silicic acid, viscous paraffin, fatty acid esters,
hydroxymethylcellulose, polyvinyl pyrolidone, etc. The
pharmaceutical preparations can be sterilized and desired, mixed
with auxiliary agents, e.g., lubricants, preservatives,
stabilizers, wetting agents, emulsiers, salts for influencing
osmotic pressure, buffers, coloring, and/or aromatic substances and
the like which do not deleteriously react with the active
compounds. They can also be combined where desired with other
active substances, e.g., enzyme inhibitors, to reduce metabolic
degradation.
[0075] For parenteral application, particularly suitable are
injectable, sterile solutions, preferably oily or aqueous
solutions, as well as suspensions, emulsions, or implants,
including suppositories. Ampules are convenient unit dosages.
[0076] It will be appreciated that the actual effective amounts of
an inhibitor or stimulation in a specific case can vary according
to the specific inhibitor compound being utilized, the particular
composition formulated, the mode of administration and the age,
weight and condition of the patient, for example. As used herein,
an effective amount of inhibitor is an amount of inhibitor which is
capable of inhibiting the phosphatase activity of the phosphatase
of interest, thereby inhibiting target cell growth and resulting in
target cell death, for example. Dosages for a particular patient
can be determined by one of ordinary skill in the art using
conventional considerations, (e.g. by means of an appropriate,
conventional pharmacological protocol).
[0077] The present invention further relates to a method of
treating a WW-domain containing polypeptide-mediated condition in a
mammal, wherein the condition results from alteration in the
WW-domain or WW-domain ligand, comprising introducing into the
mammal an amount of substance effective to regulate the WW-domain
or ligand activity in the mammal, thereby alleviating the
condition. Regulation of WW-domain or ligand activity can be
up-regulation (e.g., an increase or enhancement in ligase or PPIase
activity) or down-regulation (e.g., a decrease or inhibition in
ligase or PPIase).
[0078] The WW-domains, WW-domain-containing protein (e.g., Pin1,
Nedd4), mutants, or drugs of the present invention can be used to
treat a WW-domain-mediated condition or disease in a mammal wherein
the condition results from an alteration in the regulation of
WW-domain or its ligand activity, comprising delivering to target
cells the WW-domain or mutant described herein, or a nucleic acid
sequence encoding the activated phosphatase, in vitro or in vivo,
wherein the amount of the WW-domain or mutant introduced
effectively alters the interaction between a WW-domain and its
ligand in a target cell in a mammal. The phrase "WW-domain
polypeptide-mediated disease or condition" is intended to refer to
a cellular process wherein the endogenous activity of the WW-domain
or its ligand is not sufficiently regulated, for example, as a
result of inadequate cellular levels or activity of a WW-domain or
alternatively and additionally, a condition wherein the levels or
activity of a WW-domain ligand exceeds the capacity of the
endogenous WW-domain thereby resulting in a cell in which the
delicate balance of activity is disturbed. For example, a WW-domain
of Pin1, Pin1 protein, Pin1 mimic, WW-domain, or WW-domain mimic
can be used to regulate a condition arising from
hyperphosphorylation or a protein such as tau in Alzheimer's
disease. Thus, the WW domains of the invention can be used
experimentally or therapeutically to reduce or enhance the activity
of ligands. WW-domain polypeptide mediated diseases or conditions
can be, for example, uncontrolled cell growth or proliferation such
as neoplastic disorders or cell death.
[0079] The WW-domain of the invention can be delivered to a cell by
the use of vectors comprising one or more nucleic acid sequences
encoding the WW-domain. Vectors, as used herein, can include viral
and non-viral vectors. Examples of nonviral vectors are lipids or
liposomes (U.S. Pat. No. 5,676,954, the teachings of which are
incorporated herein by reference). Alternatively, DNA can be
introduced into cells via a gene gun, as described in (Tynan, E.
F., et al., Proc. Natl. Acad. Sci. USA., 90:11478-11482 (1993)).
The nucleic acid sequence can be been incorporated into the genome
of the viral vector. In vitro, the viral vector containing the
WW-domain described herein or nucleic acid sequences encoding the
WW-domain can be contacted with a cell and infectivity can occur.
The cell can then be used experimentally to study, for example,
unrestricted cell growth in vitro or be implanted into a patient
for therapeutic use. The cell can be migratory, such as
hematopoietic cells, or non-migratory such as a solid tumor or
fibroblast. The cell can be present in a biological sample obtained
from the patient (e.g., blood, bone marrow) and used in the
treatment of disease such as Alzheimer's or muscular dystrophy, or
can be obtained from cell culture and used to dissect cell
proliferation, cell death or protein degradation pathways in in
vivo and in vitro systems. After contact with the viral vector
comprising the WW-domain or a nucleic acid sequence encoding the
WW-domain, the sample can be returned or readministered to a cell
or patient according to methods known to those practiced in the
art. In the case of delivery to a patient or experimental animal
model (e.g., rat, mouse, monkey, chimpanzee), such a treatment
procedure is sometimes referred to as ex vivo treatment or therapy.
Frequently the cell is targeted from the patient or animal and
returned to the patient or animal once contacted with the viral
vector comprising the activated mutant of the present invention. Ex
vivo gene therapy has been described, for example, in Kasid, et
al., Proc. Natl. Acad. Sci. USA 87:473 (1990); Rosenberg, et al.,
New Engl. J. Med. 323:570 (1990); Williams, et al., Nature 310476
(1984); Dick, et al., Cell 42:71 (1985); Keller, et al., Nature
318:149 (1985) and Anderson, et al., U.S. Pat. No. 5,399,346
(1994).
[0080] Where a cell is contacted in vitro, the cell incorporating
the viral vector comprising a nucleic acid sequence of the
WW-domain can be implanted into a patient or experimental animal
model for delivery or used in in vitro experimentation to study
cellular events mediated by WW-domain containing polypeptides such
as certain aspects of cell growth, cell death, protein processing,
and neuronal regulation.
[0081] Where the viral vector comprising the WW-domain phosphatase
of the invention or an isolated nucleic acid sequence encoding the
WW-domain is delivered to a patient or experimental animal, the
mode of administration is preferably at the location of the cells
which are to be treated. As such, the administration can be nasally
(e.g., as in administering a vector expressing ADA), orally (e.g.,
as in an inhalant or spray as in administering a vector expressing
the cystic fibrosis transmembrane conductance regulator (CFTR)) or
by injection (e.g., as in administering a vector expressing a
suicide gene to a tumor). Other modes of administration (e.g.,
parenteral, mucosal, systemic, implant or intraperitoneal) are
generally known in the art. The substances can, preferably, be
administered in a pharmaceutically acceptable carrier, such as
saline, sterile water, Ringer's solution, and isotonic sodium
chloride solution.
[0082] Generally, viral vectors which can be used therapeutically
and experimentally are known in the art. Examples include the
vectors described by Srivastava, A., U.S. Pat. No. 5,252,479
(1993); Anderson, W. F., et al., U.S. Pat. No. 5,399,346 (1994);
Ausubel et al., "Current Protocols in Molecular Biology", John
Wiley & Sons, Inc. (1998). Suitable viral vectors for the
delivery of nucleic acids to cells include, for example,
replication defective retrovirus, adenovirus, parvovirus (e.g.,
adeno-associated viruses), and coronavirus. Examples of
retroviruses include avian leukosis-sarcoma, mammalian C-type,
B-type viruses, lentiviruses (Coffin, J. M., "Retroviridae: The
Viruses and Their Replication", In: Fundamental Virology, Third
Edition, B. N. Fields, et al., eds., Lippincott-Raven Publishers,
Philadelphia, Pa., (1996)). Viral vectors infect cells by known
mechanisms thereby delivery the activated mutant protein tyrosine
phosphatase or the nucleic acid encoding the activated phosphatase.
The mechanism of infectivity depends upon the viral vector and
target cell. For example, adenoviral infectivity of HeLa cells
occurs by binding to a viral surface receptor, followed by
receptor-mediated endocytosis and extrachromosomally replication
(Horwitz, M. S., "Adenoviruses" In: Fundamental Virology, Third
Edition, B. N. Fields, et al., eds., Lippincott-Raven Publishers,
Philadelphia, Pa., (1996)).
[0083] The present invention describes a novel function of the
WW-domain as a phosphoserine or phosphothreonine binding module.
For example, the WW-domain mediates phosphorylation-dependent
interactions between Pin1 and a defined subset of mitosis-specific
proteins, and neuronal proteins such as tau and amyloid precursor
protein. These interactions are essential for the Pin1 mitotic
function in the cell and are highly regulated by phosphorylation of
Pin1. Thus, the WW-domain plays a crucial role in regulating the
function of the essential mitotic PPIase Pin1.
[0084] Serine phosphorylation, often on PSET sequences (rich in
Pro, Glu, Ser and Thr), controls the timing of ubiquitination of a
variety of proteins, and ubiquitin-protein ligases are responsible
for substrate recognition (Rechsteiner, M. et al., TIBS 21:267-271
(1996); Clurman, B. E. et al., Genes Dev 10:1979-1990 (1996); Won,
K. A. et al., EMBO J. 16:3797-3804 (1997); Verma, I. M., et al.,
Proc Natl Acad Sci USA 94:11758-11760 (1997)). The ligase Nedd4 has
been shown to ubiquitinate protein substrates in a
phosphorylation-dependent manner. For example, ubiquitination of
uracil permease by the budding yeast Nedd4 homologue RSP 5 depends
on phosphorylation on a PEST sequence and ubiquitination of Cdc25
by the fission yeast homologue Pub1 occurs in mitotic cells, where
Cdc25 is heavily phosphorylated (Hein, C. et al., Mol. Micro
18:77-87 (1995); Galan, J. et al., EMBO J16:5847-5854 (1997);
Marchal, C. et al. Mol Cell Biol 18:314-321 (1998); Nefsky, B. et
al., EMBO J 15:1301 (1996)).
[0085] The present invention shows that the phosphorylated form of
Cdc25 can specifically interact with Nedd4 WW-domains. These
results document a novel ubiquitination mechanism, where WW-domains
of a ubiquitin ligase bind pSer-containing sequences, targeting
catalytic domain of the ligase to phosphorylated substrates to
initiate protein degradation. This mechanism can be used to degrade
Cdc25C at the late stage of mitosis (Hein, C. et al., Mol. Micro
18:77-87 (1995); Galan, J. et al., EMBO J16:5847-5854 (1997);
Marchal, C. et al. Mol Cell Biol 18:314-321 (1998); Nefsky, B. et
al., EMBO J 15:1301 (1996)). Three mammalian Nedd4-like genes have
been identified, each containing four WW-domains (Rotin, D. Curr.
Top. Microbiol. Immunol 228:115 (1998); Pirezzi, G. et al., J.
Biol. Chem. 272:14611 -(1997)). Although the affinity of Nedd4
WW-domains for pSer sequences is not as high as that of Pin1
WW-domain, multiple WW-domains can increase the affinity of ligases
for phosphorylated substrates and/or allow enzymes to interact with
a range of the substrates.
[0086] Both NMR and X-ray structural analysis show that the overall
structures of WW-domains are almost identical whether the WW-domain
is expressed as an isolated domain or present in its native
polypeptide (Macias, M. J. et al., Nature 382:646 (1996);
Ranganathan, K. et al., Cell 89:875 (1997)), indicating that the
WW-domain-binding sequences have been identified, namely PPLP and
PPXY motifs (Rotin, D. Curr. Top. Microbiol. Immunol 228:115
(1998); Bedford, M. T. et al., EMBO J. 16:2376 (1997)).
[0087] The present invention shows the WW-domain is a tightly
regulated novel pSer binding module. The amino acids Tyr-23 and
Trp-34 in the WW-domain of Pin1 are critical for phosphoserine or
phosphothreonine binding, and Ser-16 is important for regulation of
catalytic activity. Tryptophan residues are frequently used to
mediate the interactions with the phosphate group of pSer (Copley,
R. R. et al., J. Mol. Biol. 242:321 (1994)). For example, in the
NMR structure of the pKID/KIX complex, the interactions are
stabilized by hydrogen bonding interactions between the phosphate
moiety of pSer in pKID and the hydroxyl group of a Tyr residue in
KIX (Radharkrishman, I. et al., Cell 91:741 (1997)). Furthermore,
the present invention shows that here for the WW-domain binding of
Pin1 to a ligand, and Ala substitution of the analogical Tyr, but
not Lys, disrupts the interactions between pKID and KIX, despite
the proximity of Lys to pSer. Thus, it is likely that the
interactions between the Pin1 WW-domain and phosphoproteins are
stabilized by the hydrogen bonding interactions between the
hydroxyl group of Tyr-23 and the phosphate moiety of pSer and that
these interactions are disrupted upon phosphorylation of Ser-16
because of the negatively charged phosphate group and hydrogen
bonding interactions with the Tyr-23 side chain.
[0088] The three amino acid residues critical for binding and
regulation of the Pin1 WW-domain (Ser 16; Tyr 23, Tyr 24) are found
in a subset of other WW-domains, including one in dystrophin
(Rotin, D., Curr. Top. Microbiol. Immunol. 228:115 (1998)).
Dystrophin is a protein product of the gene responsible for
Duchenne and Becker muscular dystrophy. Similar to Pin1, dystrophin
is also associated with a group of membrane proteins (Bonneman, C.
G. et al, Curr. Opin. Pediatr. 8:569); Winder, S. J., J. Muscle
Res. Cell. Motil 18:617 (1997)). Phosphorylation is suggested to
regulate the formation of the dystrophin complexes (Luise, M. et
al., Biochem J 293:243 (1993); Shemanko, C. S. et al. Mol. Cell.
Biobhem 152:63 (1995)).
[0089] PPIases catalyze rotation about the peptide bond preceding a
Pro residue, thereby regulating the confirmation of substrates
(Dolinski, K. et al., Proc. Natl. Acad sci: USA 94:13093 (1997)).
Pin1 is a unique PPIase that is required for isomerization of the
phosphorylated Ser/The-Pro peptide bond and regulated activity of
phosphoproteins (Schutkowski, M. et al. Biochemistry 37:5566
(1998); Shen, M. et al. Genes & Development 12:706 (1998)).
PPIase-negative mutants reduce the affinity of Pin1 for
phosphoproteins, suggesting that PPIase activity can affect
phosphoprotein binding. The present invention shows that the PPIase
domain alone can bind the phosphopeptide and also display the
pSer/The-Pro-specific PPIase in vitro. However, the PPIase domain
has about 10 fold lower affinity for the phosphopeptide than the
WW-domain, and, the PPIase domain alone can not interact with
protein substrates in vitro, or carry out the Pin1 function in
vivo. These results indicate that an additional targeting function
is required to confer the specificity of the PPIase domain.
Interestingly, the WW-domain displays a much higher affinity for
the phosphopeptide and directly interacts with mitotic
phosphoproteins. Furthermore, WW-domain point mutations that
disrupt its ability to bind phosphoproteins abolish the Pin1
function in the cell. These results indicate that, by interacting
with pSer-Pro motifs, the WW-domain functions as a targeting
domain, allowing the efficacious interaction between the enzyme and
substrates.
[0090] A common feature of Pin1-binding proteins (MPM-2 antigens)
is phosphorylated on multiple Ser/Thr residues clustered at the
regulatory domain of molecules during mitosis (Izumi, T. et al.,
Mol. Biol. Cell 6:215 (1995); Kumagai, A. et al., Science 273:1377
(1996); Ye, X. S. et al., EMBO J. 14:986 (1995)). Phosphorylation
on multiple sites is necessary for activity, or to mutate multiple
phosphorylation sites to disrupt the functions. For example,
multiple phosphorylation events in Cdc25C and NIMA, whose functions
are regulated by Pin1, are important for their mitotic function
(Izumi, T. et al., Mol. Biol. Cell 6:215 (1995); Kumagai, A. et
al., Science 273:1377 (1996); Ye, X. S. et al., EMBO J. 14:986
(1995)). These results suggest that multiple phosphorylation events
are required for regulating the function of Pin1 target proteins.
Little is known how to coordinate these multiply phosphorylated
events into "all-or-nothing" activity.
[0091] SH2 domains have been demonstrated to be critical for
generating processive phosphorylation by nonreceptor tyrosine
kinases (Songyang, Z. et al. Nature 373:536 (1995); Mayer, B. J. et
al., Curr. Biol. 5:296 (1995)). SH2 domains in these kinases prefer
to bind phosphotyrosine residues that have been phosphorylated by
its own catalytic domain. The resulting high phosphorylation of
substrates on multiple sites (Songyang, Z. et al. Nature 373:536
(1995); Mayer, B. J. et al., Curr. Biol. 5:296 (1995)). WW-domains
can facilitate the processive isomerization of proteins that have
been phosphorylated by mitotic kinases at multiple sites. The
processive isomerization is triggered by binding of the higher
affinity WW-domain of Pin1 to a Ser-phosphorylated site on a
substrate protein. Once bound, the high local concentration drives
isomerization of all sites that are sterically accessible to the
lower affinity catalytic PPIase domain. This can provide a means by
which to generate coordinate "all-or-nothing" activity of mitotic
phosphoproteins and subsequently sequential mitotic events.
[0092] The following Examples are offered for the purpose of
illustrating the present invention and are not to be construed to
limit the scope of this invention. The teachings of all references
cited herein are hereby incorporated by reference.
EXAMPLE 1
[0093] WW-Domains Interact with Phosphorylated Ligands
[0094] Pin1 WW-Domains
[0095] GST-fusion proteins containing the WW-domain, PPIase-domain
or the entire Pin 1 protein were prepared and incubated with
interphase (G1/S arrested; Control) or dividing (M phase) HeLa cell
extracts using well-known procedures (Lu, K. P., et al., Nature
380:544 (1996); (Shen, M., et al., Genes & Devl. 12:706
(1998)). Briefly, HeLa cells were arrested at the G1/S boundary or
mitosis by incubation with thymidine and aphidicolin or nocodazole
for 16 h, respectively. The cells were lysed and supernatants
incubated with 10 .mu.l of agarose beads containing GST-Pin1;
GST-WW-domain of Pin1; GST-PPIase domain of Pin1; or control GST
for 2 h at 4.degree. C. The phosphorylated precipitated proteins
were washed 5 times in buffer containing 1% Triton X-100 before
subjecting to immunoblotting analysis using MPM-2 antibody, as
described previously (Yaffe, M. B. et al., Science 278:1957 (1997);
Schukowski, et al., Biochemistry 37:5566 (1998); Shen, M. et al.,
Genes & Dev 12:706 (1998)). MPM-2 recognizes a subset of
mitotic phosphoproteins including Pin1-binding proteins such as
cdc25.
[0096] Intense signal, indicative of strong binding, was detected
in extracts from mitotic HeLa cell extracts incubated with the
entire Pin1 protein or its WW-domain, but not when mitotic extracts
were incubated with the PPIase domain. These data show that the
WW-domain of Pin1 is responsible for Pin1 binding to phosphorylated
ligands. No specific binding was observed in interphase extracts
incubated with WW-domain PPIase domain or the entire Pin1 protein.
Similar results were also obtained with the isolated WW-domain from
Ess1/Ptf1, the yeast Pin1 homologue. In contrast, no specific
binding was observed for the isolated PPIase domain of Pin1 or when
control GST was incubated with either interphase or mitotic HeLa
cell extracts. These results show that the WW-domain not the
catalytic PPIase domain, is responsible for phosphoprotein binding
of Pin1, a property which is highly conserved in humans (Pin 1) and
yeast (Ess1/Ptf1).
[0097] NEDD4 WW-Domains
[0098] NEDD4 and its yeast homologues Rsp5 and Publ are ubiquitin
protein ligases containing three or four WW-domains (Rotin, D.
Curr. Top. Microbiol. 228:115-133 (1998)). The Nedd4 yeast
homologues ubiquitinate the phosphoproteins uracil permease and
Cdc25C (Hein, C., et al., Mol. Micro. 18:77-87 (1995)), which do
not contain the typical Pro-rich motif (Rotin, D., Curr. Top.
Microbiol. Immunol. 228:115-133 (1998)). In contrast to the Pin1
WW-domain, the Nedd4 WW-domain-2 bound only a few MPM-2 antigens in
GST pulldown experiments with HeLa cell extracts. To detect
interactions with other phosphoproteins, Nedd4 WW-domain-1 and -2
were used to bind .sup.32P or .sup.35S-labeled cell lysates. HeLa
cells were labeled overnight with .sup.32P orthophosphate or
.sup.35S-Met, as described (Lu, K. P., et al, J. Biol. Chem.
268:8769 (1993)). Cells were lysed in lysis buffered with or
without phosphatase inhibitors (40 mM glycerol phosphate, 50 mM
NaF, 10 mM Na V04 and 2 .mu.M okadeic acid) (Shen, M., et al.,
Genes & Dev. 12:706 (1998)). For dephosphorylation experiments,
three Ser phosphatases (CIP, PP1 and PP2A) were added to lysates
for 30 min at 30.degree. C. in the absence of presence of the
phosphatase inhibitors, as described previously (Lu, K. P., et al.,
J. Biol. Chem. 268:8769 (1993)).
[0099] Control GST bound only few minor labeled proteins, whereas
both Nedd4 WW-domains bound a similar subset of proteins from
labeled lysates. When cell lysates were pretreated with Ser
phosphatases, the ability of the WW-domains to bind most cellular
proteins was reduced by approximately 10 fold. Binding was restored
to approximately half of that observed with controls when
phosphatase inhibitors were included. Similar results were also
obtained between Pin1 or dystrophin WW-domain but with different
subsets of phosphoproteins. These results indicate that different
WW-domains interact with distinct subsets of phosphoproteins in a
phosphorylation-dependent manner.
[0100] To confirm that Nedd4 WW-domains bind a specific
phosphoprotein in a phosphorylation-dependent manner interactions
between Nedd4 WW-domains and Cdc25C were examined. To various
degrees, all three Nedd4 WW-domains bound the mitotically
phosphorylated form, but not the interphase phosphorylated form of
both HeLa Cdc25C and in vitro synthesized Xenopus Cdc25C. Peptide
binding assays showed that the Nedd4 WW-domain-2 also exhibited a
significant phosphorylation-dependent affinity towards both Pintide
and the Cdc25C peptide (Table 1). The Kd values for the
phosphopeptides were also lower than those for the Pro-rich peptide
that was thought to be a Nedd4 WW-domain-binding site (Table 1)
(Chen, H. I., et al., Proc. Natl. Acad. Sci. USA 92:7819 (1995);
Staub, O., et al., EMBO J. 15:2371 (1996); Bedford, M. J., et al.,
EMBO J, 16:2376 (1997)). These results demonstrate that, like the
Pin1 WW-domain, Nedd4 WW-domains also bind pSer-containing
sequences.
1TABLE 1 Binding constants of WW-domains and peptides Pintide Cdc25
Peptide Pro-Rich WFYpSPFLE WFYSPFLE EQPLpTPVTDL EQPLTPVTDL
IPGTPPPNYD WW-domain Kd (.mu.M) Kd (.mu.M) Kd (.mu.M) Kd (.mu.M) Kd
(.mu.M) Pin1 WW-domain 1.0 N.B. 2.2 N.B.* N.B. Nedd4 WW-domain 10.0
N.B. 20.0 N.B.* >40.dagger.(47-118.dagger-dbl.) The N-terminus
of peptides was labeled with fluorescein and purified by TLC.
Different concentrations of GST-WW-domains and control GST were
incubated with the labeled peptides (WFYpSPFLE, SEQ ID NO: 8;
WFYSPFLE, SEQ ID NO: 9; EQPLpTPVTDL, # SEQ ID NO: 10; EQPLTPVTDL,
SEQ ID NO: 11; and IPGTPPPNYD, SEQ ID NO: 12) and dissociation
constants were measured by fluorescence aniostropy assay. Each
value represents the average of three independent experiments. No
binding was detected between GST and all peptides used. N.B., not
binding detected; *not binding detected by incubating the
GST-WW-domain with the peptide immortalized on a membrane, followed
by immunoblotting analysis using GST antibody; .dagger.an estimated
Kd since binding did not reach the plateau even when the WW-domain
was used at 100 .mu.M, the highly concentration that could be used
in this assay; .dagger-dbl.previously reported Kds for the
interaction between the Yap WW-domain and various Pro-Rich peptides
(Macias, M. J., et al., Nature 382: 646 (1996); Ranganathan, K. P.,
et al., Cell 89: 875 (1997)).
EXAMPLE 2
[0101] WW-Domain Binding Depends Upon Phosphorylation of Ligands
and Protects the Ligand from Dephosphorylation
[0102] Interactions between the WW-domain of Pin1 and specific
phosphorylated ligands were examined. To detect
phosphorylation-dependent interaction, Cdc25C, Plk1 and Pin1
ligands, were synthesized by in vitro transcription and translation
in the presence of .sup.35S-Met and incubated with Xenopus
interphase or mitotic extracts or mitotic extracts followed by
treatment with calf intestine phosphatase (M+CIP). Protein
complexes were separated on SDS-gels either directly (input) or
first subjected to GST pull down with the N-terminal WW-domain
(amino acids 1-54) or C-terminal PPIase domain (amino acids 47-163)
or the entire Pin1 protein (Shen, M. et al., Genes & Dev 12:706
(1998)). The labeled protein-GST bead complexes were washed
extensively and bound proteins analyzed by SDS-PAGE and
autoradiography using standard techniques.
[0103] To determine whether WW-domain binding protects
dephosphorylation of its targets .sup.35S labeled (His).sub.6
epitope tagged Cdc25C was phosphorylated by mitotic extracts and
precipitated by GST fusion protein beads or Ni-NTA beads. The
isolated Cdc25C was then incubated with control buffer or CIP,
followed by separation on SDS-containing gels and
autoradiography.
[0104] The isolated WW-domain of Pin1 and Pin1 bound the
phosphorylated Cdc25C in mitotic cell extracts, but not interphase
extracts. The WW-domain did not bind Cdc25C when the mitotically
phosphorylated Cdc25C was dephosphorylated by calf intestine
phosphatase (CIP) prior to the binding. When mitotically
phosphorylated Cdc25C was precipitated using GST beads containing
Pin1 or its WW-domain, CIP failed to dephosphorylate Cdc25C. In
contrast, CIP was able to dephosphorylate Cdc25C almost completely
when precipitated by Ni-NTA beads against the N-terminal His tag.
Similar results were obtained with another Pin1-binding protein
Plk1. These results demonstrate that WW-domain binding depends on
phosphorylation of target proteins and when bound to a protein
ligand the WW-domain protects the target protein from
dephosphorylation.
EXAMPLE 3
[0105] Identification of Pin1 WW-Domain Binding Sites in Cdc25C by
Peptide Scan
[0106] Arrays of thirteen amino acids with ten amino acid overlaps
corresponding to protein sequences in Cdc25C were synthesized and
their C-termini linked through a .beta.-Ala residue and
decaethyleneglycol to a cellulose matrix (Rudiger et al., (EMBO J.,
16:1501 (1997)). A total of 270 thirteen amino acid peptide
sequences were analyzed.
[0107] Positions 1-155 represent a complete peptide scan of human
Cdc25C with all conserved Ser/Thr-Pro motifs in phosphorylated
form, whereas positions 156-270 represent nonphosphorylated peptide
scan, which covers regions of Cdc25C that contain Ser/Thr-Pro
motifs. The peptide bond cellulose membranes were incubated with
Pin1 or GST-Pin1 WW-domain, and washed, followed by immunoblotting
using anti-Pin1 antibodies or anti-GST antibodies, as described
Rudiger et al., (EMBO J., 16:1501 (1997)). Similar results were
obtained with either Pin1 or Pin1 WW-domain. High affinity Pin1
WW-domain-binding sites were located at Thr48 and Thr67 in
Cdc25C.
EXAMPLE 4
[0108] WW-Domain Binding to the Ligand is Inhibited by
Phosphorylated Peptides
[0109] To examine the ability of a phosphopeptide to compete with
phosphoproteins for binding to the WW-domain, the Pin1 binding
phosphopeptide Pintide (WFYpSPRLKK, SEQ ID NO: 13) (Lu, K. P. et
al., U.S. Ser. No. 60/058,164 (1997)) was used in competition
assays. A nonphosphorylated counterpart of Pintide (WFYSPRLKK, SEQ
ID NO: 14) (C-Pintide) was used as a control.
[0110] When Pin1 or its WW-domain were incubated with various
concentrations (0, 25, 50, 125, 250, 500 .mu.M) of Pintide or
control peptide (C-Pintide) before incubation with mitotic
extracts, the phosphoprotein-binding activity was significantly
reduced in a concentration dependent manner by Pintide, but not
with the nonphosphorylated peptide. (FIG. 1)
[0111] Pintide prevented Pin1 and its WW-domain from binding to
phosphopeptide MPM2 antigens with similar affinity (Pin 1+pser;
WW+pSer; FIG. 1). Significant competition was detected at 50 .mu.M,
with a complete competition observed at 250-500 .mu.M (FIG. 1). No
competition between Pintide and WW-domain phosphopeptide binding
was observed with increasing concentrations of proline-rich
peptides (WW+Pro FIG. 1) or nonphosphorylated peptides (Pin1+Ser;
WW+Ser; FIG. 1). These results demonstrate that a small
phosphoserine-containing peptide, such as Pintide, can compete with
phosphoproteins, not proline rich, binding to Pin1 or its WW-domain
in a phosphorylation-dependent manner.
EXAMPLE 5
[0112] WW-Domains Bind Phosphopeptides with High Affinity
[0113] To determine the affinity of Pin1, and its WW or PPIase
domain for phosphopeptides, peptides were labeled with fluorescein
and their interactions with Pin1 measured using quantitative
fluorescence anisotrophy. To prevent nonspecific labeling, a
Pintide analogue (WFYpSPFLE) was used, which binds Pin1 with a high
affinity based on the peptide library screen as described by (Lu,
K. P. et al., U.S. Ser. No. 60/058,164 (1997), the teachings of
which are incorporated herein in their entirety.
[0114] Pintide and its nonphosphorylated counterpart were
synthesized and incubated with GST-Pin1 or the GST-WW-domain of
Pin1 in a binding buffer, using established procedures (Shen, M. et
al., Genes & Dev 12:706 (1998)). After a 1 hr incubation,
mitotic HeLa cell extracts were added and subjected to GST pull
down experiments, followed by immunoblotting analysis using the
MPM-2 antibody. To obtain semi-quantitative data, films of
immunoblots were scanned at the region of 55 kDa, the major
Pin1-binding protein, and data analyzed using ImageQuan (ScanJet II
CX). The peptide binding constants were measured using a
fluorescence polarization assay (Jiskoot, W. et al., Anal Biochem
196:421 (1991)). Peptides were fluorescein labeled at the
N-terminus using the Fluorescein Amine Labeling Kit (Pan Vera
Corp.) and purified by TLC according to the manufacturer's
interactions. To prevent nonspecific labeling, a Pintide analogue
(WFYpSPFLE) and the nonphosphorylated control were used. Various
concentrations of Pin1 and its mutant proteins were incubated with
0.1 .mu.M of the labeled peptides in a binding buffer containing 50
mM HEPES, pH 7.4, 100 mM NaCl, 2% glycerol. Fluorescence
polarization values were obtained using a Pan Vera Beacon 2000
system, as described by the manufacturer.
[0115] No binding was detected between Pintide and the PPIase
domain or the nonphosphorylated control peptide and Pin1, its
WW-domain or PPIase domain. Pin1 and its WW-domain bound Pintide
(Tables 1 and 2). The WW-domain of Nedd4 bound Pintide with low
affinity (Kd=10 .mu.M) and did not bind the nonphosphorylated
central peptide.
2TABLE 2 Binding Constants of Mutant Proteins and Peptides
WFYpSPFLE Kd(.mu.M) WFYSPFLE Pin1 Protein High affinity Low
affinity Kd Pin1* 1.2 11.0 Not binding WW-domain* 1.0 -- Not
binding PPlase Domain* -- 15.0 Not binding GST-Pin1 1.2 13.0 Not
binding GST-Pin1.sup.Y23A -- 13.5 N.D. GST-Pin1.sup.W34A -- 14.0
N.D. GST-Pin1.sup.R14A 2.0 13.5 N.D. GST-Pin1.sup.S16A 1.2 10.5
N.D. GST-Pin1.sup.S16E -- 10.5 N.D. GST-Pin1.sup.S18E 1.0 12.0 N.D.
The N-terminus of peptides (WFYpSPFLE, SEQ ID NO: 8; WFYSPFLE, SEQ
ID NO: 9) was labeled with fluorescein-C6-amine labeling kit and
purified by TLC (Pan Vera). Different concentrations of proteins as
indicated as well as control GST were incubated with the labeled #
peptides and binding was measured by fluorescence aniostropy assay.
Each value represents the average of three independent experiments.
No binding was detected between Pin1 and the nonphosphorylated
peptide or between GST and either peptide. *the N-terminal tag was
cleaved from these proteins by thrombin. N.D., not determined
[0116] Pin1 displayed two binding sites for Pintide with high
(Kd=1.2 .mu.M) and low (Kd=11.0 .mu.M) affinities (Table 2). The
isolated WW-domain contained the high affinity binding site (Kd=1.2
.mu.M) and the PPIase domain contained a low affinity (Kd=15.0
.mu.M) binding site. These results demonstrate that both the
WW-domain and the PPIase domain can bind the phosphopeptide;
however, the binding affinity of the WW-domain is significantly
higher (Kd=1.2 .mu.M) than the binding affinity of the PPIase
domain (Kd=15.0 .mu.M). These data show that the WW-domain binds
with high affinity to phosphopeptides and, specifically, a defined
set of mitotic phosphoproteins. The interactions between WW-domains
and target phosphoproteins are mediated by phosphoserine residues
and protect dephosphoiylation of ligands when bound to WW-domains.
Therefore, the Pin1 WW-domain is a phosphoserine-binding
module.
EXAMPLE 6
[0117] WW-Domain Mutants--Effects on Phosphoprotein Binding
[0118] To determine the structural basis for WW-domain-binding
specificity, site-directed mutagenesis, followed by molecular
modeling, was performed based on the Pin1 crystal structure
(Macias, M. J. et al., Nature 382:646 (1996); Ranganathan, R. et
al., Cell 89:875 (1997)). The PPIase domain, not the WW-domain of
Pin1, contains a conserved basic patch in the active site, which is
critical for recognition of phosphoserine (Yaffe, M. B. et al.,
Science 278:1957 (1997); Schutkowski, M. et al., Biochemistry
37:5566 (1998)). The WW-domain contains a hydrophobic cleft. A
hydrophobic patch at the surface of a molecule often suggests a
protein-protein interaction surface (Janin, J. et al., J. Biol.
Chem. 265:16027 (1990); Clackson, T. et al., Science 267:383
(1995); Young, L. et al., Protein Sci. 3:717 (1994)). The
hydrophobic cluster in the WW-domain of Pin1 sequesters a PEG
molecule, which forms close contacts with Ser-16, Tyr-23 and Trp-34
located at three different strands of the anti-parallel .beta.
sheet, respectively (FIG. 2) (Macias, M. J. et al., Nature 382:646
(1996); Ranganathan, R. et al., Cell 89:875 (1997)).
[0119] A statistical analysis of phosphate binding sites in
proteins ranks the propensity of Tyr to bind phosphate next only to
that of Arg (Copley, R. R. et al., J. Mol. Biol. 242:321 (1994)).
Thus, it is likely that Tyr-23 is important for WW-domain binding
to phosphoserine. To examine whether this is the case, the
WW-domain of Pin1 was mutated, using standard PCR mutagenesis
techniques (Shen, M., et al., Genes & Dev. 12:706 (1998)), at
Tyr-23 and Trp-34, as well as Arg-14, a residue close to Tyr-23 in
the structure. Pin1 mutants were generated using PCR mutagenesis
procedures (Shen, M. et al., Genes & Dev 12:706 (1998)). GST
and (His).sub.6 fusion proteins containing Pin1 and various mutants
were produced and tags cleaved using thrombin (Shen, M. et al.,
Genes & Dev 12:706 (1998)). The mutated Pin1 proteins were
examined for their ability to bind phosphoproteins and peptides
(Tables 2, 3 and 4).
[0120] Substitution of Arg-14 with Ala (Pin1.sup.R14A) did not
appear to cause a significant change in WW-domain to binding
phosphopeptide or phosphoproteins, indicating that electrostatic
interactions between Arg-14 in Pin1 are not essential for binding
(Tables 2 and 3). In contrast, a single Ala point mutation of
either Tyr-23 (Pin1.sup.Y23A) or Trp-34 (Pin1.sup.W34A) completely
abolished the ability of Pin1 to bind either phosphoproteins or the
phosphopeptide with high affinity, similar to the isolated PPIase
domain (Tables 2, 3 and 4). These data indicate that Tyr-23 and
Trp-34 are critical amino acids for the pSer-binding activity of
the WW-domain.
3TABLE 3 Binding Constants of WW-domain Mutants Kd for Pintide
Protein (.mu.M) Pin1 1.2 Pin1.sup.R14A 2.0 Pin1.sup.S16A 1.2
Pin1.sup.W34A N.B. Pin1.sup.Y23A N.B. Pin1.sup.Y23F 5.0 Different
concentrations of various Pin1 proteins were incubated with the
fluorescein labeled Pintide and binding constants measured by
fluorescence aniostrophy assay. Each value represents the average
of three independent experiments. # Pin1 mutations only affected
the Kd of the high affinity pSer-binding site in the WW-domain, not
the low affinity pSer-binding site in the PPIase domain.
[0121]
4TABLE 4 Functional Properties of the WW-domain Mutants
Phosphoprotein PPlase Pin1 Protein binding activity Activity (%) In
vivo function Pin1 + 100 + WW-domain + 0 - PPlase Domain - 90 -
Pin1.sup.Y23A - 85 - Pin1.sup.W34A - 94 - Pin1.sup.R14A + 92 +
Pin1.sup.S16A + 96 + Pin1.sup.S16E - 95 - Pin1.sup.S18E + 98 +
Pin1.sup.Y23F +/- 94 - Pin 1 and Pin1 mutant proteins were
expressed and purified as GST fusion proteins. The
phosphoprotein-binding activity was assayed by incubating
GST-fusion proteins with mitotic extracts, followed by
immunoblotting analysis using the MPM2 antibody. +, binding was
detected;, no binding was detected. PPIase activity was assayed
using the peptide substrate (Schutkowski, M., et al., Biochemistry
37: 5566 (1998)) and represented relative to the activity of the
wild-type protein defined as 100%. The in vivo function of Pin1 and
its mutants was assayed by rescuing the temperature-sensitive ptf1
yeast mutant.
[0122] Tyrosine-mediated phosphorylation-dependent interactions
have been reported between the phosphorylated KID domain of CREB
and the KIX domain of the coactivator CBP (Radhakrishnan, I. et
al., Cell 91:741-752 (1997)).
[0123] A pSer-Pro dipeptide was modeled into the hydrophobic
cluster of the WW-domain in the place of the PEG molecule. Computer
assisted molecular modeling based on co-ordinates of the Pin1
structure reported by Ranganathan et al., (Cell 89:875 (1997)), was
performed using QUANTA on an SGI Indigo II workstation. Placement
of the pSer-Pro dipeptide into the hydrophobic cleft of the
WW-domain was determined by hydrophobic, hydrogen bonding and Van
der Waals interactions. The Pro ring sits in a hydrophobic crevice
stacked between the aromatic rings of Tyr-23 and Trp-34, whereas
the pSer fits into a space between Ser-16 and Tyr-23, with the
phosphate moiety being directed within hydrogen bonding distance of
the Tyr-23 hydroxyl proton.
EXAMPLE 7
Pin1 Phosphorylation is Regulated In Vivo
[0124] To determine whether Pin1 phosphoprotein-binding activity is
regulated by Pin1 phosphorylation Pin1 mutants were constructed in
regions of the WW-domain predicted to form the hydrophobic cleft.
For example, if Ser-16 in the pSer-binding pocket was
phosphorylated, a negatively charged residue can be introduced into
the binding pocket and the phosphate group can form hydrogen
bonding interactions with the side chain of Tyr-23. Phosphorylation
of Ser-16 could prevent the Pin1 WW-domain from interacting with
its ligand. To test this hypothesis, experiments were performed to
determine whether Pin1 is a phosphoprotein and whether Pin1
phosphorylation is regulated during the cell cycle in vivo.
[0125] To detect in vivo phosphorylation of Pin1, HeLa cells were
arrested at the G1/S boundary or at mitosis in the presence of
.sup.32P orthophosphate (10 .mu.Ci/ml) (Shen, M. et al., Genes
& Dev 12:706 (1998)). The cells were lysed in RIPA buffer and
subjected to immunoprecipitation using Pin1-specific antibodies,
followed by separation on modified SDS-containing gels. For
detecting a molecular weight shift of Pin1 during the cell cycle
indicative of a change in the phosphorylation state of Pin1, HeLa
cells were released from G1/S arrest for various times, the cell
cycle analyzed by FACS and total lysates prepared in RIPA buffer
were subjected to immunoblotting analysis using Pin1 antibodies, as
previously described (Shen, M. et al., Genes & Dev 12:706
(1998)).
[0126] In vivo .sup.32P-labeling experiments showed that Pin1 was
hyperphosphorylated when cells were arrested at the G1/S boundary,
mainly exhibiting as a single slow migrating species on SDS-gels.
Pin1 was dephosphorylated when cells were arrested at mitosis, as
indicated by the appearance of a fast migrating, lower molecular
weight species of Pin1 on SDS-gels. To further determine the
kinetics of Pin1 dephosphorylation during the cell cycle, HeLa cell
lysates were collected at different times after release from the
G1/S arrest and subjected to high resolution SDS-PAGE, followed by
immunoblotting analysis using Pin1 antibody as described in Example
2. As shown previously (Shen, M. et al., Genes & Dev 12:706
(1998)), total Pin1 levels did not fluctuate during the cell cycle.
However, two different molecular weight forms of Pin1 were
detected. The faster migrating, lower molecular weight form of Pin1
was cell cycle-dependent, appearing only when cells were
progressing through mitosis or when arrested at mitosis by
nocodazole. These kinetic data are strongly correlated with the
ability of Pin1 to bind phosphoproteins (Shen, M. et al., Genes
& Dev 12:706 (1998)). These results show that the appearance of
the fast migrating species of Pin1 is the dephosphorylated form of
Pin1 and that Pin1 is phosphorylated in a cell cycle-regulated
manner. Phosphorylation prevents Pin1 from interacting with
phosphoserine ligands.
EXAMPLE 8
[0127] Phosphorylation of the WW-Domain Prevents Interaction with
Ligands
[0128] To examine the effect of Pin1 phosphorylation on Pin1
binding to ligands, Pin1 and Pin1 mutant proteins were incubated
with the catalytic subunit of PKA and PKC (a mixture of .alpha.,
.beta. and .gamma., UBI) in a kinase reaction buffer containing 500
.mu.M cold ATP at 30.degree. C. for 15 min (Lu, K. P. et al., Anal.
Biochem. 196:421 (1991)). The reactions were stopped by adding SDS
sample buffer and reaction products separated on SDS-gels, followed
by autoradiography. Pin1 proteins were isolated and used to bind
MPM-2 antigens from mitotic extracts from HeLa cells, as previously
described (Shen, M., et al., Genes & Dev 12:706 (1998)).
Experiments were also performed with PKA and PKC, casein kinase,
cyclin B/Cdc2 and SRPK1 kinases.
[0129] The kinases readily phosphorylated Pin1 and its WW-domain.
More importantly, phosphorylation by PKA, but not PKC, completely
abolished the interactions between Pin1 and MPM2 antigens or
between WW-domain and MP2 antigens. This is especially significant
because Ser-16 in Pin1 is located in the PKA consensus
phosphorylation site (KRXS) (Pearson, R. B. et al., Methods in
Enzymol. 200:62-81 (1991)). These results indicate that
phosphorylation of the WW-domains of Pin1 can prevent Pin1 from
interacting with phosphorylated ligands.
[0130] To pinpoint the regulatory phosphorylation site in the Pin1
WW-domain, Ser-16 was mutated to Glu, a phosphorylatable amino acid
residue. The resulting mutant (Pin1.sup.S16E) protein failed to
bind mitotic phosphoproteins. Furthermore, no high affinity-binding
site for the phosphoserine peptide was detected in Pin1.sup.S16E
(Tables 2, 3 and 4). These results indicate that the S16E mutation
completely abolishes the ability of the Pin1 WW-domain to bind its
ligands, as is the case of PKA phosphorylation. As a control, a
nearby Ser residue, Ser-18, was mutated to Glu (Pin1.sup.S18E). The
Pin1.sup.S18E mutation did not affect the ability of Pin1 to bind
phosphoproteins or Pintide peptide (Tables 2, 3 and 4). These
results indicate that Ser-16 is a critical phosphorylation site
that regulates interactions between Pin1 and phosphoproteins.
[0131] Since PKA phosphorylated Pin1 on multiple sites as detected
by phosphopeptide analysis, further experiments were performed to
determine whether Ser-16 is the critical phosphorylation site that
regulates phosphoprotein binding. Ser-16 was substituted with Ala,
a nonphosphorylatable amino acid residue, and the mutant protein
was used to bind MPM-2 antigens and the Pintide analogue. Similar
to wild-type Pin1, the Pin1.sup.S16A mutant interacted with all
Pin1 ligands and the Pintide peptide (Tables 2, 3 and 4),
indicating that Ala is able to substitute for Ser-16 to fulfill the
spatial requirement for the binding. More importantly, the
interactions of Pin1.sup.S16A with phosphoproteins or the Pintide
analogue were not affected by PKA phosphorylation (Tables 2, 3 and
4), although the mutant protein could still be phosphorylated by
PKA. These results confirm that phosphorylation on Ser-16 is both
necessary and sufficient to regulate the interaction between Pin1
and phosphoproteins. Thus, the interaction between Pin1 and its
ligands is tightly regulated, depending on phosphorylation of
ligands as well as dephosphorylation of the pSer-binding pocket of
its WW-domain.
EXAMPLE 9
[0132] Phosphoprotein-Binding Activity of the WW-Domain OF Pin1 is
Essential for the In Vivo Function of Pin1
[0133] Given the essential role of the WW-domain in conferring
Pin1-binding specificity in vitro, a critical question is whether
this domain is important in vivo. To address this question,
experiments using the PIN1 yeast homologue, ESS1/PTF1 were
performed. ESS1/PTF1 is essential for cell growth and human Pin1
can carry out this essential function when transfected into yeast
cells (Lu, K. P. et al., Nature 380:544 (1996); Hanes, S. D. et
al., Yeast 5:55 (1989); Hani, J. et al., FEBS Lett. 365:198
(1995)). A temperature-sensitive ptf1 mutant strain, YPM2, grows at
the permissive temperature (23.degree. C.), but not at the
restrictive temperature (30.degree. C.) (Hanes, S. D. et al., Yeast
5:55 (1989); Hani, J. et al., FEBS Lett. 365:198 (1995)). This
phenotype is completely rescued by a 1.5 kb PTF1 genomic fragment,
which also contains the promoter and the 3' processing sequence
(FIG. 3). To insure that all human Pin1 proteins were expressed at
physiological levels under normal regulation, the coding sequence
of the fully functional ESS1/PTF1 gene in a Yepvector was replaced
with the coding sequence of the human PIN1 (or Pin1 mutant) cDNA
(FIG. 3) and transformed into a temperature-sensitive ptf1 strain.
Transformants were selected on minimal media minus Leu at the
permissive temperature (23.degree. C.) and protein expression was
detected by immunoblotting analysis using 12CA5 monoclonal antibody
specific for the HA epitope tag inserted at the N-terminus. The HA
tag does not affect the Pin1 function (Lu, K. P. et al., Nature
380:544 (1996)). Those strains expressing similar levels of Pin1
and Pin1 mutants were grown at permissive and nonpermissive
temperature. At least 34 strains were tested for each construct,
with similar results.
[0134] When transformed into YPM2 cells, the human Pin1 fully
complemented the temperature-sensitive phenotype, indicating that
human Pin1 is fully functional when expressed under the endogenous
promoter. To determine whether the WW-domain is important for Pin1
to exert its essential function, the WW-domain and the PPIase
domain of Pin1 were individually expressed at a similar level to
the whole length protein (Table 4). These results indicate that the
WW-domain is indispensable in vivo. To further confirm this
observation, various WW-domain point mutants were introduced into
YPM2 strains using the same expression vector and expressed at
levels similar to that of wild type protein in cells. The WW-domain
mutants that were able to bind phosphoproteins rescued the ptf1
phenotype (Table 4). However, Pin1 mutations, including S16E, Y23A,
W34A, which disrupt interactions between the WW-domain and
phosphoproteins, abolish the ability of Pin1 to support cell
growth. These results demonstrate that phosphoprotein-binding
activity of the WW-domain is essential for the in vivo
propyl-peptidyl cis-trans isomerase activity of Pin1.
EXAMPLE 10
[0135] Interaction between PIN1 WW-Domain and Phosphorylated Tau
And Amyloid Precursor Protein Peptides
[0136] The interaction between Pin1 and tau proteins, which are
heavily phosphorylated at mitosis and in Alzheimer's disease, were
examined. Pin1 bound phosphorylated tau and colocalized with tau at
paired helical filaments in brain sections of patients with
Alzheimer's disease. To map the Pin1-binding site in tau or amyloid
proteins, Pin1 or its WW-domain mutants were incubated with
phosphorylated (pT, pS) or nonphosphorylated (S,T) peptides derived
from tau or amyloid protein, followed by measuring peptide binding
using ELISA assay. Pin1 bound with high affinity (Kd=25 nM) only
the phosphorylated Thr-231 tau peptide, an interaction mediated by
the Pin1 WW-domain as the Pin1.sup.R14A, but not Pin1.sup.Y23A
Table 5; FIG. 4B. The Pin1 WW-domain also specifically bind
phosphorylated Thr-668 amyloid precursor protein peptide (Table
5).
[0137] A lower affinity binding constant was obtained with ELISA
assays compared to fluorescence aniostropy assays. This might be
due to the following reasons: 1) peptides are oriented at the same
direction in ELISA assay, but not in aniostrophy assay; 2) ELISA
assay is more sensitive than aniostrophy assay; and/or 3) different
peptides have different affinities. In any case, the Pin1 WW-domain
mediates specific interaction between Pin1 and tau or amyloid
proteins.
5TABLE 5 Specific Interaction between the Pin 1 WW-domain and a
Phosphorylated Tau Peptide SEQ NO. Tau Peptides Binding (OD @ 405
nm) .vertline. 15 DAGLKESPLQTPTE (pS-46) 0.00 .vertline. 16
TRIPAKTPPAPKT (pT-175) 0.00 .vertline. 17 GYSSPGSPGTPGSR (pS-202)
0.08 .vertline. 18 SRSRTPSLPTPPT (pS-214) 0.00 .vertline. 19
KVAVVRTPPKSPS (T-231) 0.00 .vertline. 20 KVAVVRTIPPKSPS (pT-231)
1.46 .vertline. 21 VRTPPKSPSSAKSR (pS-235) 0.11 Pin 1 .vertline. 22
VQSKIGSLDNITH (pS-356) 0.00 .vertline. 23 GSLDNITHVPGGG (pT-361)
0.00 .vertline. 24 TSPRHLSNVSSTG (pS-409) 0.00 .vertline. 25
PRHLSNVSSTGSIDMV (pS-412) 0.02 .vertline. 26 PRHLSNVSSTGSIDMV
(pS-413) 0.00 .vertline. 27 NVSSTGSIDMVDS (pS-416) 0.00 .vertline.
28 SIDMVDSPQLATL (pS-422) 0.00 Mutant Pin 1.sup.R14A 29
KVAVVRTPPKSPS (pT-231) 1.30 Pin 1.sup.Y23A 30 KVAVVRTPPKSPS
(pT-231) 0.00 Amyloid Precursor Protein Peptide 31 KEVDAAVTPEERHLS
(T-668) 0.00 32 KEVDAAVTPEERHLS (pT-668) 1.81
EXAMPLE 11
[0138] Functional Restoration of Alzheimer Phosphorylated Tau by
the WW-Domain of Pin1
[0139] A neuropathological hallmark in Alzheimer's disease is the
neurofibrillary tangle, the main components of which are paired
helical filaments (PHFs) composed of the microtubule-associated
protein tau (Lee, V. M. Curr Opin Neurobiol 5:663-668 (1995);
Mandelkow, E. et al., Neurobiol Aging 16:347-354 (1995); Kosik, K.
S. et al., Ann NY Acad Sci 777:114-120 (1996); Spillantini, M. G.
and Goedert, M. Trends Neurosci 21:428-433 (1998) and Iqbal, K. et
al., J Neural Transm Suppl 53:169-180 -(1998)). Tau is
hyperphosphorylated in PHFs (Lee, V. M. et al., Science 251:675-678
(1991); Goedert, M. et al., Neuron 8:159-168 (1992); Greenberg, S.
G. et al., J Biol Chem 267:564-569 (1992)) and phosphorylation of
tau causes loss of its ability to bind microtubules and promote
microtubule assembly (Bramblett, G. T. et al., Neuron 10:1089-1099
(1993); Yoshida, H. and Ihara, Y. J Neurochem 61:1183-1186 (1993);
Iqbal, K. et al., FEBS Lett 349:104-108 (1994)). Restoring the
function of phosphorylated tau could prevent or reverse PHF
formation in Alzheimer's disease.
[0140] Phosphorylation on serines or threonines that precede
proline (Ser/Thr-Pro) alter the prolyl isomerization rate and
create a binding site for the prolyl isomerase Pin1 (Lu, K. P. et
al., Nature 380:544-547 (1996); Yaffe, M. B. et al., Science
278:1957-1960 (1997); Shen, M. et al., Genes Dev. 12:706-720
(1998); Schutkowski, M. et al., Biochemistry 37:5566-5575 (1998);
Crenshaw, D. G. et al., S. Embo J 17:1315-1327 (1998)). Pin1
specifically isomerizes phosphorylated Ser/Thr-Pro bonds and
regulates the function of several mitotic phosphoproteins (Lu, K.
P. et al., Nature 380:544-547 (1996); Yaffe, M. B. et al., Science
278:1957-1960 (1997); Shen, M. et al., Genes Dev. 12:706-720
(1998)).
[0141] The following data show that Pin1 binds a specific
phosphorylated Thr-Pro motif in tau. Pin1 colocalizes and
copurifies with PHFs, and soluble Pin1 is significantly depleted in
brains of patients with Alzheimer disease. Furthermore, Pin1 fully
restores the ability of phosphorylated tau to bind microtubules and
promote microtubule assembly in vitro. Thus, Pin1 is the first
molecule that can restore the biological activity of phosphorylated
tau without dephosphorylation. In addition, since depletion of Pin1
induces mitotic arrest and apoptosis (Shen, M. et al., Genes Dev.
12:706-720 (1998)), sequestration of Pin1 into PHFs in Alzheimer's
disease can contribute to neuronal loss.
[0142] Pin1 Binds and Regulates Mitotic Phosphoproteins
[0143] Pin1 binds and regulates the function of a defined subset of
mitotic phosphoproteins by interacting with conserved
phosphorylated Ser/Thr-Pro motifs that are also recognized by
MPM-2, a mitosis-specific, phosphorylation-dependent monoclonal
antibody (mAb) (Yaffe, M. B. et al., Science 278:1957-1960 (1997);
Shen, M. et al., Genes Dev. 12:706-720 (1998)). Tau is an MPM-2
antigen phosphorylated on multiple Ser/Thr-Pro motifs during
mitosis (Illenberger, S. et al., Mol Biol Cell 9:1495-1512 (1998)).
Experiments were undertaken to determine whether Pin1 binds tau.
Tau isoform was either synthesized by in vitro transcription and
translation in the presence of .sup.35S-Met or produced in bacteria
as an N-terminal His-tagged protein, followed by purification using
NTA-Ni columns (Yaffe, M. B. et al., Science 278:1957-1960 (1997);
Shen, M. et al., Genes Dev. 12:706-720 (1998)). To generate
interphase- and mitosis-specific phosphorylated form of tau, tau
was incubated with Xenopus interphase and mitotic extracts,
respectively (Shen, M. et al., Genes Dev. 12:706-720 (1998)). To
prepare Cdc2 phosphorylated tau, purified recombinant tau was
incubated with purified cyclin B/Cdc2 (UBI) for 6 to 12 hr at room
temperature in a buffer containing 500 .mu.M cold ATP, plus trace
[.sup.32P]-ATP in some experiments, (Vincent, I. et al., J Neurosci
17:3588-3598 (1997)).
[0144] Pin1 did not bind tau incubated with interphase Xenopus
extracts, but did bind tau that was phosphorylated by mitotic
extracts. Mitotic binding between Pin1 and tau was abolished when
mitotically phosphorylated tau was dephosphorylated by alkaline
phosphatase. These results indicate that Pin1 binds phosphorylated
tau in a mitosis-specific and phosphorylation-dependent manner, as
shown for many other Pin1-binding proteins (Shen, M. et al., Genes
Dev. 12:706-720 (1998)), including Cdc25.
[0145] Mitotic events are aberrently activated in the Alzheimer's
disease brain, including re-expression of Cdc2 kinase and cyclin B
(Vincent, I. et al., J Cell Biol 132:413-425 (1996); Vincent, I. et
al., J Neurosci 17:3588-3598 (1997); Nagy, Z. et al., Acta
Neuropathol 94:6-15 (1997); Nagy, Z. et al., Acta Neuropathol
(Berl) 93:294-300 (1997)). The phosphorylation pattern of tau in
mitotic cells is strikingly similar to that in Alzheimer's disease
(AD) brains, as detected by phosphorylation site-specific tau mAbs
(Illenberger, S. et al., Mol Biol Cell 9:1495-1512 (1998); Vincent,
I. et al., J Cell Biol 132:413-425 (1996); Vincent, I. et al., J
Neurosci 17:3588-3598 (1997); Kondratick, C. M. and Vandre, D. D. J
Neurochem 67:2405-2416 (1996); Vincent, I. et al., Neurobiol Aging
19:287-296 (1998); Preuss, U. and Mandelkow, E. M. Eur J Cell Biol
76: 176-184 (1998)). Mitotically phosphorylated tau is recognized
by AD-specific, phosphorylation-dependent tau mAbs, including CP9,
TG3 and PHF1 (Illenberger, S. et al., Mol Biol Cell 9:1495-1512
(1998); Vincent, I. et al., J Cell Biol 132:413-425 (1996);
Vincent, I. et al., J Neurosci 17:3588-3598 (1997)); Kondratick, C.
M. and Vandre, D. D. J Neurochem 67:2405-2416 (1996); Vincent, I.
et al., Neurobiol Aging 19:287-296 (1998); Preuss, U. and
Mandelkow, E. M. Eur J Cell Biol 76: 176-184 (1998)). These results
indicate that common Ser/Thr-Pro motifs of tau are phosphorylated
in normal mitotic cells and in Alzheimer brains. Thus, Pin1 can
bind and regulate the function of tau in AD.
[0146] Pin1 Interactions with Tau in Extracts of Brains from
Alzheimer's Patients
[0147] Pin1 interactions with tau in AD brains were examined using
a GST-Pin1 pulldown assay (Shen, M. et al., Genes Dev. 12:706-720
(1998)). Glutathione beads containing GST or GST-Pin1 were
incubated with normal or AD brain extracts, or PHFs purified
(Vincent, I. J. and Davies, P. Proc Natl Acad Sci USA 89:2878-2882
(1992)), and proteins associated with the beads were subjected to
immunoblotting analysis using CP27, which recognizes all forms of
tau. Recombinant and mutant Pin1 proteins were produced as
N-terminal GST or His-tagged fusion proteins (Shen, M. et al.,
Genes Dev. 12:706-720 (1998)). PHFs were purified by immunoaffinity
chromatography (Vincent, I. J. and Davies, P. Proc Natl Acad Sci
USA 89:2878-2882 (1992)). Pin1 antibodies and tau mAbs (CP27, TG3,
PHF1 and CP9) were used as previously described (Shen, M. et al.,
Genes Dev. 12:706-720 (1998); Jicha, G. A. et al., J Neurochem
69:2087-2095 (1997)).
[0148] For determining the level of soluble Pin1, brain tissues
were sliced, cut into fine pieces and homogenized in buffer A (50
mM Hepes, pH 7.4, 150 mM NaCl, 10% glycerol, 1% Triton X-100, 5 mM
MgCl.sub.2, 1 mM EGTA, 1 mM DTT, 100 mM NaF, 2 mM Na.sub.3Vo.sub.4
and various protease inhibitors). The homogenates were centrifuged
at 100,000 g at 4.degree. C. for 30 min and the supernatants were
directly used for immunoprecipitations or immunoblotting analysis
using Pin1 antibodies described (Shen, M. et al., Genes Dev.
12:706-720 (1998)) or stored in aliquots at -80.degree. C. before
assays.
[0149] GST-Pin1, but not control GST, bound tau present in AD brain
extracts or PHFs. In contrast, Pin1 did not bind tau in age-matched
normal brain extracts. These results indicate that Pin1 interacts
with the AD-specific tau in vitro. To determine whether Pin1 forms
a stable complex with AD tau in vivo, PHFs were purified using
affinity chromatography (Vincent, I. J. and Davies, P. Proc Natl
Acad Sci U S A 89:2878-2882 (1992)), and dissolved in SDS sample
buffer, following by immunoblotting analysis using anti-Pin1
antibodies. Pin1 was detected in PHFs purified from all 6 AD brains
examined. These results indicate that Pin1 co-purifies with
PHFs.
[0150] Immunocytochemical Localization of Pin1 in Alzheimer and
Normal Brains
[0151] To further confirm that Pin1 has specific affinity for PHFs,
recombinant Pin1 was added onto brain sections, washed, and then
subjected to immunostaining using affinity purified Pin1 antibodies
to localize bound Pin1. To localization exogenously added Pin1 in
brain sections, 50 .mu.m sections were cut from formalin fixed
frontal cortex or hippocampus of human brains, endogenous
peroxidase activity blocked with H.sub.2O.sub.2, followed by
incubation with Pin1 at 0.5 .mu.M. The sections were incubated with
the mAb TG3 or anti-Pin1 antibodies that had been purified using
GST-Pin1 glutathione beads, and visualized by the immunoperoxidase
staining protocol, to detect endogenous Pin1, fixed brain sections
were first microwaved in an antigen retrieval buffer (Biogenex), as
described by the manufacturer, then subjected to immunostaining
procedure.
[0152] When recombinant Pin1 was not added to normal or AD brain
sections, no immunoreactive signal was observed, indicating that
the Pin1 antibodies do not recognize endogenous Pin1. However, if
Pin1 was added to normal and AD brain sections, dramatically
different results were observed. Although Pin1 binding signal was
not detected in normal brain sections, Pin1 binding signals were
detected in the cytoplasm of neurons in AD brain sections.
Specifically, Pin1 strongly bound neurofibrillary tangles and
neurites, as shown by co-immunostaining with TG3, which recognizes
the AD-specific conformation of tau phosphorylated on threonine-231
(T231) (Jicha, G. A. et al., J Neurochem 69:2087-2095 (1997)).
These results demonstrate that exogenous Pin1 specifically binds
the neurofibrillary tangles in neurons.
[0153] Given that Pin1 has a high affinity for the tangles and
purifies with PHFs, it is critical to examine the in vivo
relationship between Pin1 and PHFs. To address this question, fixed
brain sections were subjected to an antigen retrieval procedure.
Strong immunoreactivity was observed with Pin1 antibodies in both
normal and AD brain sections. To ensure that these signals
represent Pin1, the Pin1-specific antibodies were first depleted
using GST-Pin1 beads and then used for immunostaining.
Pin1-depleted antibodies showed no specific immunoreactivity with
either normal or AD brain sections. Strikingly different patterns
of Pin1 localization were observed in normal and AD brain sections.
Pin1 was localized primarily in nuclei of neurons in normal brain
sections and in neuronal nuclei in AD brain sections. These results
are consistent with the findings that both ectopically expressed
and endogenous Pin1 is primarily localized in the nucleus in HeLa
cells (Lu, K. P. et al., Nature 380:544-547 (1996)).
[0154] However, in AD brains, intense Pin1 immunostaining were
observed in the cytoplasm of neurons, specifically at the tangle
structure that was also recognized by TG3 (Jicha, G. A. et al., J
Neurochem 69:2087-2095 (1997)). These results indicate that both
exogenous and endogenous Pin1 specifically localize to the
neurofibrillary tangles in AD brains.
[0155] Binding of Pin1 to PBFs could trap Pin1 in the tangles,
eventually leading to depletion of the soluble Pin1 in neurons. To
test this possibility, the levels of Pin1 and two tau kinases,
GSK3b and Cdc2, were compared in AD and normal brain tissues. Brain
tissues were homogenized and soluble proteins were directly
subjected to immunoblotting analysis, followed by
semi-quantification of protein levels using ImageQuan. When
compared with 6 age-matched normal brains, GSK3b levels were
slightly reduced (40.+-.11%), and Cdc2 levels were significantly
increased by approximately 5 fold in AD brains (547.+-.87%, n=6,
P<0.01). These findings are consistent with previous studies
showing that levels of Cdc2, but not GSK3b, are abnormally elevated
in Alzheimer's disease brains (Vincent, I. et al., J Neurosci
17:3588-3598 (1997)).
[0156] The levels of soluble Pin1 in AD brains was lower than in
normal brains, with the average reduced by approximately 5 fold
(22.4.+-.3.4%). This decrease in Pin1 levels was confirmed by Pin1
immunoprecipitation analysis. These data show that soluble Pin1 is
significantly reduced in brains from human suffering from
Alzheimer's disease. Therefore, Pin1 can be a potential gene
therapy target.
[0157] Identification of Pin1 Binding Sites in Tau
[0158] The interaction between Pin1 and mitotic phosphoproteins is
mediated by the Pin1 N-terminal WW-domain, which acts as a
phosphoserine-binding module interacting with specific
phosphorylated Ser/Thr residues in ligands (Examples 1-10). To
identify the Pin1 binding site(s) in tau, phosphorylated and
nonphosphorylated peptides that cover previously identified tau
phosphorylation sites, were assayed for their ability to bind Pin1
by ELISA (Jicha, G. A. et al., J Neurochem 69:2087-2095 (1997)).
Pin1 exhibited specific and high affinity binding to a tau peptide
containing phosphorylated threonine-231 (pT231 tau peptide), with
the dissociation constant of .apprxeq.40 nM (FIG. 4A). No binding
was observed between Pin1 and the non-phosphorylated counterpart
(FIG. 4A), demonstrating an absolute requirement of T231
phosphorylation for Pin1 binding. To determine whether the
N-termiinal WW-domain of Pin1 is responsible for binding, the
mutant Pin1.sup.Y23A (Example 6) was used. The Pin1.sup.Y23A mutant
contains a single Ala substitution at the critical Tyr-23 in the
WW-domain, resulting in a complete lose of the
phosphoserine-binding activity (Example 6). No binding between
Pin1.sup.Y23A and pT231 tau peptide was detected (Table 5).
Collectively, these results show that Pin1 specifically binds the
motif containing the pT231 residue in tau through its
WW-domain.
[0159] Phosphorylation of tau on T231 (pT231-tau) has been well
documented in AD brains and can be recognized by several mAbs,
including CP9 (Illenberger, S. et al., Mol Biol Cell 9:1495-1512
(1998); Vincent, I. et al., J Neurosci 17:3588-3598 (1997); Preuss,
U. and Mandelkow, E. M. Eur J Cell Biol 76: 176-184 (1998); Jicha,
G. A. et al., J Neurochem 69:2087-2095 (1997); Billingsley, M. L.
and Kincaid, R. L. Biochem J 323:577-591 (1997)). To determine
whether Pin1 interacts with pT231-tau, GST-Pin1 beads were used to
isolate tau from AD brain extracts or PHFs and T231 phosphorylation
detected using CP9. Tau isolated by Pin1 beads was strongly
immunoreactive with CP9. These result indicate that phosphorylation
of tau on T231 results in tau binding to Pin1 and that Pin1 binding
does not result in dephosphorylation of pT231-tau. Since T231 in
tau is readily phosphorylated by Cdc2 kinase in vitro (Vincent, I.
et al., J Cell Biol 132:413-425 (1996); Vincent, I. et al., J
Neurosci 17:3588-3598 (1997); Jicha, G. A. et al., J Neurochem
69:2087-2095 (1997)), experiments were performed to determine
whether Pin1 binds tau that is phosphorylated by Cdc2 in vitro.
Pin1 and its WW-domain, but not its PPIase domain, bound Cdc2
phosphorylated tau. Thus, Pin1 binds pT231-tau through its
WW-domain. These data are consistent with Pin1 binding to
mitotically phosphorylated tau and sequestration of Pin1 in PHFs of
AD brains where Cdc2 is abnormally upregulated.
[0160] Pin1 Interactions with Tau Promote Binding of Tau to
Microtubules
[0161] The high affinity interaction between Pin1 and
phosphorylated tau can affect the biological activity of tau. Upon
phosphorylation by many protein kinases, including Cdc2, tau loses
its ability to bind microtubules (MTs) and promote MT assembly
(Bramblett, G. T. et al., Neuron 10:1089-1099 (1993); Iqbal, K. et
al., FEBS Lett 349:104-108 (1994); Yoshida, H. and Ihara, Y. J
Neurochem 61:1183-1186 (1993); Alonso, A. C. et al., Proc Natl Acad
Sci USA 91:5562-5566 (1994); Busciglio, J. et al., Neuron
14:879-888 (1995)) although the exact mechanism is not fully
understood. To determine whether Pin1 can restore the ability of
phosphorylated tau to bind MTs, phosphorylated tau was produced
using purified Cdc2 (Vincent, I. et al., J Cell Biol 132:413-425
(1996); Vincent, I. et al., J Neurosci 17:3588-3598 (1997)) and
assessed for its ability to bind Taxol-stabilized MTs in the
presence or absence of Pin1. Phosphorylation of tau by Cdc2
prevented tau from binding MTs, whereas binding was restored by
incubation with Pin1. Pin1 was detected in the fraction of
tau-bound MTs confirming interaction between Pin1 and
phosphorylated tau. These data demonstrate that Pin1 binds
phosphorylated tau and restores its ability to bind MTs.
[0162] The effect of Pin1 on the ability of phosphorylated tau to
promote MT assembly was determined using light-scattering assays
(Bramblett, G. T. et al., Neuron 10:1089-1099 (1993); Alonso, A. C.
et al, Proc Natl Acad Sci USA 91:5562-5566 (1994); Busciglio, J. et
al., Neuron 14:879-888 (1995)). Briefly, MTs were assembled from
phosphocellulose purified bovine tubulin (Cytoskeleton, Inc) and
stabilized by Taxol. The nonphosphorylated or Cdc2 phosphorylated
recombinant tau (0.1 mg/ml) was incubated with Pin1 (0.1 mg/ml) at
35.degree. C. for 5 min before adding to the MTs. Bound tau was
isolated by centrifugation (50,000.times.g) at 25.degree. C. for 20
min, followed by immunoblotting analysis using CP27 and Pin1
antibodies. The ability of tau to promote MT assembly was
determined using well established light-scattering assays. Briefly,
the assembly of MTs was initiated by incubating tubulin (2 mg/ml)
with or without tau (0.05 mg/ml) in 80 mM PIPES, pH 6.8, 1 mM EGTA,
1 mM MgCl.sub.2, 1 mM GTP, 20% glycerol at 35.degree. C. for 2 min.
The mixture was then transferred to a 100 .mu.l cuvet and the rate
of the MT assembly was monitored at room temperature using the
turbidity increase at 350 nm. To examine the effect of Pin1, Pin1
or its mutant (0.05 mg/ml) was pre-incubated with tau or Cdc2
phosphorylated tau (0.05 mg/ml) at 35.degree. C. for 5 min before
the MT assembly assays. Each experiment was repeated at least three
times, with similar results being observed. Results using GST-Pin1
or His-Pin1 were similar, indicating that the N-terminal tags have
no effect on the MT assembly assayed.
[0163] The rate of the turbidity change was minimal in the absence
of tau, but was dramatically increased when recombinant tau was
added to the mixture (FIG. 5A). However, this rate of the increase
was substantially abolished if tau was phosphorylated by Cdc2 (FIG.
5B). These results show that phosphorylation of tau by Cdc2
disrupts its ability to promote MT assembly. Although Pin1 had no
effect on the ability of nonphosphorylated tau to promote MT
assembly, Pin1 restored the ability of Cdc2 phosphorylated tau to
promote MT assembly (FIG. 5B). In contrast, the Pin1.sup.Y23A
mutant did not have any effect on the microtubule assembly
promoting effects of phosphorylated tau, indicating that the
interaction is essential for Pin1 to regulate the function of
phosphorylated tau. The MT assembly rate induced by phosphorylated
tau in the presence of Pin1 was slightly higher than that induced
by recombinant tau consistent with previous studies demonstrating
that a certain degree of tau phosphorylation is required for its
maximal activity to promote tubulin assembly (Iqbal, K. et al.,
FEBS Lett 349:104-108 (1994); de Ancos, J. G. et al., J Biol Chem
268:7976-7982 (1993)). Therefore, that Pin1 not only binds
phosphorylated tau, but also functionally restores its biological
activity.
[0164] Tau protein normally stabilizes the internal microtubular
structure of neurons that functions to transport proteins and other
molecules through the cells (Lee, V. M. Curr Opin Neurobiol
5:663-668 (1995); Mandelkow, E. et al., Neurobiol Aging 16:347-354
(1995); Kosik, K. S. et al., Ann N Y Acad Sci 777:114-120 (1996);
Spillantini, M. G. and Goedert, M. Trends Neurosci 21:428-433
(1998); Iqbal, K. et al., J Neural Transm Suppl 53:169-180 (1998)).
The importance of tau for neural function has been demonstrated by
the recent findings that mutations in tau cause hereditary forms of
frontal-temporal dementia (FTDP-17) (Clark, L. N. et al., Proc Natl
Acad Sci U S A 95:13103-13107 (1998); Spillantini, M. G. and
Goedert, M. Trends Neurosci 21:428433 (1998); Hutton, M. et al.,
Nature 393:702-705 (1998); Poorkaj, P. et al., Ann Neurol
43:815-825 (1998)). The signature lesions in FTDP-17 brains are
aggregates composed of hyperphosphorylated tau, similar to those in
brains of AD patients (Spillantini, M. G. et al., Brain Pathol
8:387402 (1998); Reed, L. A. et al., J Neuropathol Exp Neurol
57:588-601 (1998)). Certain FTDP-17 mutations also disrupt the
ability of tau to bind MTs and promote MT assembly (Hong, M. et
al., Science 282:1914-1917 (1998); Hasegawa, M. et al., FEBS Lett
437:207-210 (1998)), suggesting that the interaction between tau
and MTs is critical for the normal function of neurons.
Furthermore, the absence of senile plaques and Lewy bodies in
FTDP-17 (Spillantini, M. G. et al., Brain Pathol 8:387-402 (1998);
Reed, L. A. et al., J Neuropathol Exp Neurol 57:588-601 (1998))
suggests that the tau pathology in AD may not be simply a secondary
effect of the disease process, but rather can directly lead to
neuronal loss.
[0165] Although it is established that most neurons in normal adult
brains are postmitotic and lack mitotic kinase activity (Rakie, P.
Ann. NY. Acad. Sci. 457:193-211 (1985); Nagy, Z. et al.,
Neuroscience 87:731-739 (1998)), several studies have shown that
mitotic events are abnormally activated in neurons in AD brains
(Vincent, I. et al., J Cell Biol 132:413-425 (1996); Vincent, I. et
al., J Neurosci 17:3588-3598 (1997); Nagy, Z. et al., Acta
Neuropathol 94:6-15 (1997); Nagy, Z. et al., Acta Neuropathol
(Berl) 93:294-300 (1997)). Similar patterns of phosphoepitopes are
observed in mitotic cells and AD neurons and mitotic
phosphoepitopes appear before paired helical filaments
(Illenberger, S. et al., Mol Biol Cell 9:1495-1512 (1998); Vincent,
I. et al., J Cell Biol 132:413-425 (1996); Vincent, I. et al., J
Neurosci 17:3588-3598 (1997); Kondratick, C. M. and Vandre, D. D. J
Neurochem 67:2405-2416 (1996); Vincent, I. et al., Neurobiol Aging
19:287-296 (1998); Preuss, U. and Mandelkow, E. M. Eur J Cell Biol
76: 176-184 (1998)). Therefore, it is proposed that aberrant
activation of mitotic events in neurons can contribute to
hyperphosphorylation of tau and formation of PHFs (Nagy, Z. et al.,
Neuroscience 87:731-739 (1998)). This hypothesis is further
supported by the above identified described data which show that
the essential mitotic regulator Pin1 binds the common
phosphorylated motif of tau present in mitotic cells and AD
brains.
[0166] Pin1 can restore the ability of phosphorylated tau to bind
MTs and promote MT assembly. This binding provides the first
example of restoration of the biological activity of phosphorylated
tau without dephosphorylation. Since Pin1 is able to bind
phosphorylated Ser/Thr-Pro motifs as well as to isomerize the
phosphorylated Ser/Thr-Pro peptide bonds using its N-terminal and
C-terminal domains, respectively, it is conceivable that Pin1
regulates the tau function by altering the conformation of the
phosphorylated Ser/Thr-Pro motif(s).
[0167] Pin1 inhibits entry into mitosis and directly inhibits
activation of Cdc25 (Lu, K. P. et al., Nature 380:544-547 (1996);
Shen, M. et al., Genes Dev. 12:706-720 (1998)), a key
mitosis-inducing phosphatase that removes the inhibitory phosphates
from Cdc2 (Nurse, P. Cell 79:547-550 (1994); King, R. W. et al.,
Cell 79:563-571 (1994)). Thus, Pin1 can prevent abnormal activation
of mitotic events in neurons and control the function of
phosphoproteins, such as tau, in case they are phosphorylated due
to transient and aberrant activation of Pro-directed kinases.
However, a long-term and sustained activation of mitotic events
would result in continuous hyperphosphorylation of tau, which binds
and sequesters Pin1, as seen during the development of AD. This
leads to at least two potential consequences. First,
hyperphosphorylation of tau may create more binding sites than the
capacity of the available Pin1, as suggested by the finding that
PBFs have extra binding sites for exogenous Pin1. In this case
hyperphosphorylated tau is not able to bind MTs and subsequently
forms PHFs, affecting the normal function of neurons (Lee, V. M.
Curr Opin Neurobiol 5:663-668 (1995); Mandelkow, E. et al.,
Neurobiol Aging 16:347-354 (1995); Kosik, K. S. et al., Ann N Y
Acad Sci 777:114-120 (1996); Spillantini, M. G. and Goedert, M.
Trends Neurosci 21:428-433 (1998); Iqbal, K. et al., J Neural
Transm Suppl 53:169-180 (1998)). At the same time, since depletion
of Pin1 induces mitotic arrest and apoptosis (Lu, K. P. et al.,
Nature 380:544-547 (1996)), sequestration of Pin1 to PHFs itself
might also have a deleterious effect on neurons. Therefore, both
depletion of Pin1 and formation of PHFs can contribute to neuronal
loss in AD. Since the aggregates of hyperphosphorylated tau are
also a common neuropathological feature of several other neuronal
degenerative diseases, such FTDP-17 (Spillantini, M. G. et al.,
Brain Pathol 8:387402 (1998); Reed, L. A. et al., J Neuropathol Exp
Neurol 57:588-601 (1998)) Pin1 can potentially be involved in these
diseases. Therefore, Pin1 can be a target for administration
utilizing gene therapy. The administration of Pin1, its WW-domain
or WW-domain mimic can protect and prevent neurons from undergoing
cell death (apoptosis, necrosis) or restore neuronal function in
disease states (e.g., Alzheimer's, corticob degeneration, Myotonic
dystrophy).
[0168] Equivalents
[0169] While this invention has been particularly shown and
described with references to preferred embodiments thereof, it will
be understood by those skilled in the art that various changes in
form and details may be made therein without departing from the
spirit and scope of the invention as defined by the appended
claims.
Sequence CWU 1
1
42 1 31 PRT Homo sapien 1 Leu Pro Pro Gly Trp Glu Lys Arg Met Ser
Arg Ser Ser Gly Arg Val 1 5 10 15 Tyr Tyr Phe Asn His Thr Thr Asn
Ala Ser Gln Trp Glu Arg Pro 20 25 30 2 31 PRT Unknown synthetic
peptide 2 Leu Pro Thr Pro Trp Thr Val Arg Tyr Ser Lys Ser Lys Lys
Arg Glu 1 5 10 15 Tyr Phe Phe Asn Pro Glu Thr Lys His Ser Gln Trp
Glu Glu Pro 20 25 30 3 30 PRT mouse 3 Leu Pro Pro Gly Trp Glu Glu
Lys Gln Asp Asp Arg Gly Arg Ser Tyr 1 5 10 15 Tyr Val Asp His Asn
Ser Lys Thr Thr Thr Trp Ser Lys Pro 20 25 30 4 30 PRT Homo sapien 4
Val Gln Gly Pro Trp Glu Arg Ala Ile Ser Pro Asn Lys Val Pro Tyr 1 5
10 15 Tyr Thr Asn His Glu Thr Gln Thr Thr Cys Trp Asp His Pro 20 25
30 5 30 PRT mouse 5 Ala Lys Ser Met Trp Thr Glu His Lys Ser Pro Asp
Gly Arg Thr Tyr 1 5 10 15 Tyr Tyr Asn Thr Glu Thr Lys Gln Ser Thr
Trp Glu Lys Pro 20 25 30 6 29 PRT rat 6 Leu Pro Ala Gly Trp Met Arg
Val Gln Asp Thr Ser Gly Thr Tyr Tyr 1 5 10 15 Trp His Ile Pro Thr
Gly Thr Thr Gln Trp Glu Pro Pro 20 25 7 30 PRT mouse 7 Leu Pro Ala
Gly Trp Glu Met Ala Lys Thr Ser Ser Gly Gln Arg Tyr 1 5 10 15 Phe
Leu Asn His Ile Asp Gln Thr Thr Thr Trp Gln Asp Pro 20 25 30 8 8
PRT synthetic peptide PHOSPHORYLATION (4)...(4) 8 Trp Phe Tyr Ser
Pro Phe Leu Glu 1 5 9 8 PRT synthetic peptide 9 Trp Phe Tyr Ser Pro
Phe Leu Glu 1 5 10 10 PRT synthetic peptide PHOSPHORYLATION
(5)...(5) 10 Glu Gln Pro Leu Thr Pro Val Thr Asp Leu 1 5 10 11 10
PRT synthetic peptide 11 Glu Gln Pro Leu Thr Pro Val Thr Asp Leu 1
5 10 12 10 PRT synthetic peptide 12 Ile Pro Gly Thr Pro Pro Pro Asn
Tyr Asp 1 5 10 13 9 PRT synthetic peptide PHOSPHORYLATION (4)...(4)
13 Trp Phe Tyr Ser Pro Arg Leu Lys Lys 1 5 14 9 PRT synthetic
peptide 14 Trp Phe Tyr Ser Pro Arg Leu Lys Lys 1 5 15 14 PRT
synthetic peptide 15 Asp Ala Gly Leu Lys Glu Ser Pro Leu Gln Thr
Pro Thr Glu 1 5 10 16 13 PRT synthetic peptide 16 Thr Arg Ile Pro
Ala Lys Thr Pro Pro Ala Pro Lys Thr 1 5 10 17 14 PRT synthetic
peptide 17 Gly Tyr Ser Ser Pro Gly Ser Pro Gly Thr Pro Gly Ser Arg
1 5 10 18 13 PRT synthetic peptide 18 Ser Arg Ser Arg Thr Pro Ser
Leu Pro Thr Pro Pro Thr 1 5 10 19 13 PRT synthetic peptide 19 Lys
Val Ala Val Val Arg Thr Pro Pro Lys Ser Pro Ser 1 5 10 20 14 PRT
synthetic peptide 20 Lys Val Ala Val Val Arg Thr Ile Pro Pro Lys
Ser Pro Ser 1 5 10 21 14 PRT synthetic peptide 21 Val Arg Thr Pro
Pro Lys Ser Pro Ser Ser Ala Lys Ser Arg 1 5 10 22 13 PRT synthetic
peptide 22 Val Gln Ser Lys Ile Gly Ser Leu Asp Asn Ile Thr His 1 5
10 23 13 PRT synthetic peptide 23 Gly Ser Leu Asp Asn Ile Thr His
Val Pro Gly Gly Gly 1 5 10 24 13 PRT synthetic peptide 24 Thr Ser
Pro Arg His Leu Ser Asn Val Ser Ser Thr Gly 1 5 10 25 16 PRT
synthetic peptide 25 Pro Arg His Leu Ser Asn Val Ser Ser Thr Gly
Ser Ile Asp Met Val 1 5 10 15 26 16 PRT synthetic peptide 26 Pro
Arg His Leu Ser Asn Val Ser Ser Thr Gly Ser Ile Asp Met Val 1 5 10
15 27 13 PRT synthetic peptide 27 Asn Val Ser Ser Thr Gly Ser Ile
Asp Met Val Asp Ser 1 5 10 28 13 PRT synthetic peptide 28 Ser Ile
Asp Met Val Asp Ser Pro Gln Leu Ala Thr Leu 1 5 10 29 13 PRT
synthetic peptide 29 Lys Val Ala Val Val Arg Thr Pro Pro Lys Ser
Pro Ser 1 5 10 30 13 PRT synthetic peptide 30 Lys Val Ala Val Val
Arg Thr Pro Pro Lys Ser Pro Ser 1 5 10 31 15 PRT synthetic peptide
31 Lys Glu Val Asp Ala Ala Val Thr Pro Glu Glu Arg His Leu Ser 1 5
10 15 32 15 PRT synthetic peptide 32 Lys Glu Val Asp Ala Ala Val
Thr Pro Glu Glu Arg His Leu Ser 1 5 10 15 33 39 PRT Homo sapien 33
Glu Lys Leu Pro Pro Gly Trp Glu Lys Arg Met Ser Arg Ser Ser Gly 1 5
10 15 Arg Val Tyr Tyr Phe Asn His Ile Thr Asn Ala Ser Gln Trp Glu
Arg 20 25 30 Pro Ser Gly Asn Ser Ser Ser 35 34 39 PRT Unknown
synthetic peptide 34 Thr Gly Leu Pro Thr Pro Trp Thr Val Arg Tyr
Ser Lys Ser Lys Lys 1 5 10 15 Arg Glu Tyr Phe Phe Asn Pro Glu Thr
Lys His Ser Gln Trp Glu Glu 20 25 30 Pro Glu Gly Thr Asn Lys Asp 35
35 38 PRT Homo sapien 35 Val Pro Leu Pro Ala Gly Trp Glu Met Ala
Lys Thr Ser Ser Gly Gln 1 5 10 15 Arg Tyr Phe Leu Asn His Ile Asp
Gln Thr Thr Thr Trp Gln Asp Pro 20 25 30 Arg Lys Ala Met Leu Ser 35
36 38 PRT mouse 36 Ser Pro Leu Pro Pro Gly Trp Glu Glu Arg Gln Asp
Val Leu Gly Arg 1 5 10 15 Thr Tyr Tyr Val Asn His Glu Ser Arg Arg
Thr Gln Trp Lys Arg Pro 20 25 30 Ser Pro Asp Asp Asp Leu 35 37 38
PRT Unknown synthetic peptide 37 Gly Arg Leu Pro Pro Gly Trp Glu
Arg Arg Thr Asp Asn Phe Gly Arg 1 5 10 15 Thr Tyr Tyr Val Asp His
Asn Thr Arg Thr Thr Thr Trp Lys Arg Pro 20 25 30 Thr Leu Asp Gln
Thr Glu 35 38 38 PRT Homo sapien 38 Thr Ser Val Gln Gly Pro Trp Glu
Arg Ala Ile Ser Pro Asn Lys Val 1 5 10 15 Pro Tyr Tyr Ile Asn His
Glu Thr Gln Thr Thr Cys Trp Asp His Pro 20 25 30 Lys Met Thr Glu
Leu Tyr 35 39 37 PRT rat 39 Ser Asp Leu Pro Ala Gly Trp Met Arg Val
Gln Asp Thr Ser Gly Thr 1 5 10 15 Tyr Tyr Trp His Ile Pro Thr Gly
Thr Thr Gln Trp Glu Pro Pro Gly 20 25 30 Arg Ala Ser Pro Ser 35 40
31 PRT Unknown synthetic peptide 40 Leu Xaa Xaa Gly Trp Thr Xaa Xaa
Xaa Xaa Xaa Xaa Gly Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa His Xaa Xaa
Xaa Xaa Thr Xaa Trp Xaa Xaa Pro Xaa 20 25 30 41 31 PRT Unknown
synthetic peptide 41 Leu Pro Xaa Gly Trp Glu Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Gly Xaa Xaa 1 5 10 15 Tyr Tyr Xaa Asn His Xaa Thr Xaa Xaa
Thr Xaa Trp Xaa Xaa Pro 20 25 30 42 14 PRT Unknown synthetic
peptide 42 Leu Pro Gly Trp Glu Gly Tyr Tyr Asn His Thr Thr Trp Pro
1 5 10
* * * * *