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.

What is claimed is:  
     
         1 . A method of mediating protein-protein interaction comprising modulating the binding of a WW-domain containing polypeptide with a phosphorylated ligand.  
     
     
         2 . The method of  claim 1  wherein the WW-domain containing polypeptide is selected from the group consisting of 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.  
     
     
         3 . The method of  claim 1  wherein the ligand is selected from the group consisting of: NIMA, Cdc25C, Cdc27, Plk1, Myt1, Rab4, tau protein, amyloid precursor protein, Wee1, Mos, Sox3, Xbr-1b, MP75 (E-MAP-115), MP110 (Cdc5), MP68, and MP30.  
     
     
         4 . The method of  claim 1  wherein the binding of the WW-domain containing polypeptide to a ligand is inhibited.  
     
     
         5 . The method of  claim 4  wherein the inhibition comprises competitive inhibition wherein a phosphorylated ligand mimic binds to the WW-domain containing polypeptide, thereby inhibiting the binding of the WW-domain polypeptide to the ligand.  
     
     
         6 . The method of  claim 4  wherein the phosphorylated ligand comprises a phosphoserine or phosphothreonine peptide, or peptide mimetic or small organic molecule.  
     
     
         7 . The method of  claim 6  wherein the phosphorylated ligand comprises an amino acid sequence selected from the group consisting of: SEQ ID NOS: 8, 10, 13, 20 and 29.  
     
     
         8 . The method of  claim 4  wherein the inhibition comprises phosphorylating the WW-domain containing polypeptide or inhibiting the dephosphorylation of the WW-domain containing polypeptide, thereby inhibiting the binding of the WW-domain containing polypeptide to the ligand.  
     
     
         9 . The method of  claim 1  wherein the binding of the WW containing polypeptide to a ligand is enhanced.  
     
     
         10 . The method of  claim 9  wherein enhancement comprises phosphorylating the ligand thereby enhancing binding of a WW-domain containing polypeptide to the phosphorylated ligand.  
     
     
         11 . The method of  claim 9  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 . A method of regulating cell growth comprising-mediating the binding of the Pin1 WW-domain to a mitotic regulatory protein.  
     
     
         13 . The method of  claim 12  wherein the regulated cell growth is cell proliferation.  
     
     
         14 . The method of  claim 13  wherein the Pin1 WW-domain binds to phosphorylated NIMA, thereby resulting in cell proliferation.  
     
     
         15 . The method of  claim 13  wherein the Pin1 WW-domain is dephosphorylated whereby the Pin1 WW-domain binds to a phosphorylated mitotic regulatory protein, thereby resulting in cell proliferation.  
     
     
         16 . The method of  claim 12  wherein cell growth is inhibited.  
     
     
         17 . The method of  claim 16  wherein the Pin1 WW-domain binding to phosphorylated Cdc25C is inhibited, thereby inhibiting cell growth.  
     
     
         18 . The method of  claim 16  wherein the Pin1 WW-domain is phosphorylated whereby the Pin1 WW-domain does not bind to a phosphorylated mitotic regulatory protein, thereby resulting in cell death.  
     
     
         19 . A method of inhibiting Pin1 prolyl-peptidyl cis-trans isomerase activity comprising inhibiting the binding of the Pin1 WW-domain to as phosphorylated ligand.  
     
     
         20 . The method of  claim 19  wherein the Pin1 WW-domain is phosphorylated, thereby inhibiting the binding of the WW-domain to the phosphorylated ligand.  
     
     
         21 . The method of  claim 20  wherein Ser 16 of SEQ ID NO: 33 is phosphorylated.  
     
     
         22 . A method of regulating protein degradation comprising mediating the binding of the NEDD4 WW-domain to a phosphorylated ligand.  
     
     
         23 . The method of  claim 22  wherein protein degradation is inhibited comprising inhibiting the binding of the NEED4 WW-domain to a phosphoserine or phosphothreonine ligand, thereby inhibiting ubiquitin ligase activity.  
     
     
         24 . A method of regulating the interaction of a WW-domain of dystrophin to a phosphorylated ligand.  
     
     
         25 . A method of regulating a neurodegenerative disease in a mammal comprising modulating the interaction between a WW-domain-containing protein modulating interaction and a phosphorylated ligand.  
     
     
         26 . The method of  claim 25 , wherein the phosphoryland ligand is selected from the group consisting of tau protein and amyloid precursor protein.  
     
     
         27 . A method of regulating the function of tau in Alzheimer's disease comprising mediating the binding of the Pin1 WW-domain to phosphorylated tau.  
     
     
         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, 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.  
     
     
         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, Wee1, 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 . A substance identified by the method of  claim 29 .  
     
     
         35 . A method of identifying a test substance that modulates the interaction between a WW-domain 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 thereby producing a combination;    b) maintaining the combination of step a) under conditions suitable for interaction between the ligands and the WW-domain;    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 interaction in the absence of the test substance, under conditions suitable for interaction between the ligand and the WW-domain,    wherein the difference in the interaction indicates that the test substance modulates the interaction between the ligand and the WW-domain.    
     
     
         36 . The method of  claim 35 , wherein the WW-domain is 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, Yfx1, ZK1248.15, KO15c11, CD45AP, FBP11, FBP21, FBP23, FBP28 and FBP30 WW-domains  
     
     
         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, Wee1, Mos, Sox3, Xbr-1b, MP75 (E-MAP-115), MP110 (Cdc5), MP68, and MP30.  
     
     
         38 . A substance identified by the method of  claim 35 .  
     
     
         39 . A mutant Pin1 WW-domain, wherein the mutation comprises the 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.  
     
     
         40 . The mutant of  claim 39 , wherein the modified amino acid is replaced with an amino acid residue selected from the group consisting of alanine, glutamic acid or phenylalanine.  
     
     
         41 . A method of targeting a drug to treat a condition in a mammal, wherein the condition results from an alteration in a phosphorylated ligand which is a ligand for a WW-domain containing polypeptide, comprising the steps of: 
 a) combining the drug and the WW-domain containing polypeptide or a fragment thereof under conditions suitable to form a drug WW-domain complex; and    b) administering the drug/WW-domain complex of step a) to the mammal, wherein the drug/WW-domain complex and phosphorylated ligand interact, thereby alleviating the condition.    
     
     
         42 . A method of modulating the interaction between a WW-domain-containing protein and a phosphorylated ligand in an individual comprising the steps of: 
 a) providing a drug that interacts with the WW-domain;    b) administering the drug to the individual, wherein the drug binds the WW-domain, thereby modulating the interaction between the WW-domain and the phosphorylated ligand.