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.

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.