Title: Critical Role of WW Domain Phosphorylation in Regulating Phosphoserine Binding Activity and Pin1 Function
Abstract: Phosphoserine-binding modules help determine the specificity of signal transduction events. One such module, the group IV WW domain, plays an essential role in targeting the phosphorylation-specific prolyl isomerase Pin1 to its substrates. These modules require Ser/Thr phosphorylation of their ligands for binding activity. However, phosphorylation of these modules and its functional significance have not been described, nor is it known whether the function of Pin1 is regulated. Here we show that Pin1 WW domain is phosphorylated on Ser16 both in vitro and in vivo. Further, this phosphorylation regulates the ability of the WW domain to mediate Pin1 substrate interaction and cellular localization. Moreover, both Pin1 and WW domain mutants refractory to Ser16 phosphorylation act as dominant-negative mutants to induce mitotic block and apoptosis and increase multinucleated cells with 8 N DNA content. Thus, phosphorylation is a new mechanism critical for regulating WW domain phosphoserine binding activity and Pin1 function. Phosphoserine-binding modules help determine the specificity of signal transduction events. One such module, the group IV WW domain, plays an essential role in targeting the phosphorylation-specific prolyl isomerase Pin1 to its substrates. These modules require Ser/Thr phosphorylation of their ligands for binding activity. However, phosphorylation of these modules and its functional significance have not been described, nor is it known whether the function of Pin1 is regulated. Here we show that Pin1 WW domain is phosphorylated on Ser16 both in vitro and in vivo. Further, this phosphorylation regulates the ability of the WW domain to mediate Pin1 substrate interaction and cellular localization. Moreover, both Pin1 and WW domain mutants refractory to Ser16 phosphorylation act as dominant-negative mutants to induce mitotic block and apoptosis and increase multinucleated cells with 8 N DNA content. Thus, phosphorylation is a new mechanism critical for regulating WW domain phosphoserine binding activity and Pin1 function. Src homology 2 Trp-Trp peptidyl-prolyl isomerase mitotic phosphoprotein monoclonal-2 glutathione S-transferase green fluorescent protein red fluorescent protein 4–6-diamidino-2-phenylindole protein kinase A protein kinase C phosphorylated serine or threonine residue Compelling evidence indicates that phosphorylation on Ser or Thr residues (pSer/Thr) also promotes the formation of protein-protein (1Pawson T. Scott J.D. Science. 1997; 278: 2075-2080Crossref PubMed Scopus (1887) Google Scholar, 2Yaffe M.B. Cantley L.C. Nature. 1999; 402: 30-31Crossref PubMed Scopus (70) Google Scholar, 3Sudol M. Hunter T. Cell. 2000; 103: 1001-1004Abstract Full Text Full Text PDF PubMed Scopus (295) Google Scholar). Small pSer/Thr-binding protein modules reminiscent of SH21 domains involved in recognition of phosphorylated Tyr have been recently described, which include group IV WW domains and FHA domains (2Yaffe M.B. Cantley L.C. Nature. 1999; 402: 30-31Crossref PubMed Scopus (70) Google Scholar, 3Sudol M. Hunter T. Cell. 2000; 103: 1001-1004Abstract Full Text Full Text PDF PubMed Scopus (295) Google Scholar, 4Lu P.J. Zhou X.Z. Shen M. Lu K.P. Science. 1999; 283: 1325-1328Crossref PubMed Scopus (587) Google Scholar, 5Durocher D. Henckel J. Fersht A.R. Jackson S.P. Mol. Cell. 1999; 4: 387-394Abstract Full Text Full Text PDF PubMed Scopus (328) Google Scholar, 6Li J. Smith G.P. Walker J.C. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7821-7826Crossref PubMed Scopus (99) Google Scholar, 7Verdecia M.A. Bowman M.E. Lu K.P. Hunter T. Noel J.P. Nat. Struct. Biol. 2000; 7: 639-643Crossref PubMed Scopus (423) Google Scholar). However, the role of phosphorylation of these modules themselves has not been described.The best characterized pSer/Thr-binding WW domain is one in the peptidyl-prolyl isomerase (PPIase) Pin1 (4Lu P.J. Zhou X.Z. Shen M. Lu K.P. Science. 1999; 283: 1325-1328Crossref PubMed Scopus (587) Google Scholar, 7Verdecia M.A. Bowman M.E. Lu K.P. Hunter T. Noel J.P. Nat. Struct. Biol. 2000; 7: 639-643Crossref PubMed Scopus (423) Google Scholar). Pin1 specifically isomerizes the pSer/Thr–Pro bond (8Yaffe M.B. Schutkowski M. Shen M. Zhou X.Z. Stukenberg P.T. Rahfeld J. Xu J. Kuang J. Kirschner M.W. Fischer G. Cantley L.C. Lu K.P. Science. 1997; 278: 1957-1960Crossref PubMed Scopus (674) Google Scholar, 9Ranganathan R. Lu K.P. Hunter T. Noel J.P. Cell. 1997; 89: 875-886Abstract Full Text Full Text PDF PubMed Scopus (605) Google Scholar, 10Zhou X.Z. Lu P.J. Wulf G. Lu K.P. Cell. Mol. Life Sci. 1999; 56: 788-806Crossref PubMed Scopus (123) Google Scholar). Pin1 is essential for mitotic progression (11Lu K.P. Hanes S.D. Hunter T. Nature. 1996; 380: 544-547Crossref PubMed Scopus (794) Google Scholar, 12Zhou X.Z. Kops O. Werner A. Lu P.J. Shen M. Stoller G. Küllertz G. Stark M. Fischer G. Lu K.P. Mol. Cell. 2000; 6: 873-883Abstract Full Text Full Text PDF PubMed Scopus (477) Google Scholar, 13Wu X. Wilcox C.B. Devasahayam G. Hackett R.L. Arevalo-Rodriguez M. Cardenas M.E. Heitman J. Hanes S.D. EMBO J. 2000; 19: 3727-3738Crossref PubMed Scopus (140) Google Scholar) and is required for the DNA replication checkpoint (14Winkler K.E. Swenson K.I. Kornbluth S. Means A.R. Science. 2000; 287: 1644-1647Crossref PubMed Scopus (159) Google Scholar). Pin1 substrates are a defined subset of phosphorylated proteins, including many MPM-2 antigens (8Yaffe M.B. Schutkowski M. Shen M. Zhou X.Z. Stukenberg P.T. Rahfeld J. Xu J. Kuang J. Kirschner M.W. Fischer G. Cantley L.C. Lu K.P. Science. 1997; 278: 1957-1960Crossref PubMed Scopus (674) Google Scholar,13Wu X. Wilcox C.B. Devasahayam G. Hackett R.L. Arevalo-Rodriguez M. Cardenas M.E. Heitman J. Hanes S.D. EMBO J. 2000; 19: 3727-3738Crossref PubMed Scopus (140) Google Scholar, 15Shen M. Stukenberg P.T. Kirschner M.W. Lu K.P. Genes Dev. 1998; 12: 706-720Crossref PubMed Scopus (302) Google Scholar, 16Crenshaw D.G. Yang J. Means A.R. Kornbluth S. EMBO J. 1998; 17: 1315-1327Crossref PubMed Scopus (167) Google Scholar, 17Lu P.J. Wulf G. Zhou X.Z. Davies P. Lu K.P. Nature. 1999; 399: 784-788Crossref PubMed Scopus (624) Google Scholar, 18Patra D. Wang S.X. Kumagai A. Dunphy W.G. J. Biol. Chem. 1999; 274: 36839-36842Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 19Albert A. Lavoie S. Vincent M. J. Cell Sci. 1999; 112: 2493-2500PubMed Google Scholar, 20Morris D.P. Phatnani H.P. Greenleaf A.L. J. Biol. Chem. 1999; 274: 31583-31587Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). Pin1-catalyzed prolyl isomerization regulates the conformation and function of phosphoproteins (15Shen M. Stukenberg P.T. Kirschner M.W. Lu K.P. Genes Dev. 1998; 12: 706-720Crossref PubMed Scopus (302) Google Scholar, 17Lu P.J. Wulf G. Zhou X.Z. Davies P. Lu K.P. Nature. 1999; 399: 784-788Crossref PubMed Scopus (624) Google Scholar) and also facilitates dephosphorylation (12Zhou X.Z. Kops O. Werner A. Lu P.J. Shen M. Stoller G. Küllertz G. Stark M. Fischer G. Lu K.P. Mol. Cell. 2000; 6: 873-883Abstract Full Text Full Text PDF PubMed Scopus (477) Google Scholar, 15Shen M. Stukenberg P.T. Kirschner M.W. Lu K.P. Genes Dev. 1998; 12: 706-720Crossref PubMed Scopus (302) Google Scholar, 17Lu P.J. Wulf G. Zhou X.Z. Davies P. Lu K.P. Nature. 1999; 399: 784-788Crossref PubMed Scopus (624) Google Scholar). Thus, Pin1-dependent peptide bond isomerization is a critical post-phosphorylation regulatory mechanism (12Zhou X.Z. Kops O. Werner A. Lu P.J. Shen M. Stoller G. Küllertz G. Stark M. Fischer G. Lu K.P. Mol. Cell. 2000; 6: 873-883Abstract Full Text Full Text PDF PubMed Scopus (477) Google Scholar). However, it is likely that Pin1 is regulated by a post-translational modification.The first and essential step in Pin1-dependent regulation of targets is the substrate binding activity mediated by its WW domain (10Zhou X.Z. Lu P.J. Wulf G. Lu K.P. Cell. Mol. Life Sci. 1999; 56: 788-806Crossref PubMed Scopus (123) Google Scholar). The WW domain functions as a pSer/Thr-Pro-binding module, with the binding pocket including the side chains of Ser16, Arg17, Tyr23, and Trp34 (4Lu P.J. Zhou X.Z. Shen M. Lu K.P. Science. 1999; 283: 1325-1328Crossref PubMed Scopus (587) Google Scholar, 7Verdecia M.A. Bowman M.E. Lu K.P. Hunter T. Noel J.P. Nat. Struct. Biol. 2000; 7: 639-643Crossref PubMed Scopus (423) Google Scholar). Interestingly, Ser16 is located at the center of the shadow pSer/Thr-Pro-binding pocket. Here we show an essential role for phosphorylation of Ser16 in the regulation of the WW domain pSer/Thr binding activity and subsequent Pin1 cell cycle function. Compelling evidence indicates that phosphorylation on Ser or Thr residues (pSer/Thr) also promotes the formation of protein-protein (1Pawson T. Scott J.D. Science. 1997; 278: 2075-2080Crossref PubMed Scopus (1887) Google Scholar, 2Yaffe M.B. Cantley L.C. Nature. 1999; 402: 30-31Crossref PubMed Scopus (70) Google Scholar, 3Sudol M. Hunter T. Cell. 2000; 103: 1001-1004Abstract Full Text Full Text PDF PubMed Scopus (295) Google Scholar). Small pSer/Thr-binding protein modules reminiscent of SH21 domains involved in recognition of phosphorylated Tyr have been recently described, which include group IV WW domains and FHA domains (2Yaffe M.B. Cantley L.C. Nature. 1999; 402: 30-31Crossref PubMed Scopus (70) Google Scholar, 3Sudol M. Hunter T. Cell. 2000; 103: 1001-1004Abstract Full Text Full Text PDF PubMed Scopus (295) Google Scholar, 4Lu P.J. Zhou X.Z. Shen M. Lu K.P. Science. 1999; 283: 1325-1328Crossref PubMed Scopus (587) Google Scholar, 5Durocher D. Henckel J. Fersht A.R. Jackson S.P. Mol. Cell. 1999; 4: 387-394Abstract Full Text Full Text PDF PubMed Scopus (328) Google Scholar, 6Li J. Smith G.P. Walker J.C. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7821-7826Crossref PubMed Scopus (99) Google Scholar, 7Verdecia M.A. Bowman M.E. Lu K.P. Hunter T. Noel J.P. Nat. Struct. Biol. 2000; 7: 639-643Crossref PubMed Scopus (423) Google Scholar). However, the role of phosphorylation of these modules themselves has not been described. The best characterized pSer/Thr-binding WW domain is one in the peptidyl-prolyl isomerase (PPIase) Pin1 (4Lu P.J. Zhou X.Z. Shen M. Lu K.P. Science. 1999; 283: 1325-1328Crossref PubMed Scopus (587) Google Scholar, 7Verdecia M.A. Bowman M.E. Lu K.P. Hunter T. Noel J.P. Nat. Struct. Biol. 2000; 7: 639-643Crossref PubMed Scopus (423) Google Scholar). Pin1 specifically isomerizes the pSer/Thr–Pro bond (8Yaffe M.B. Schutkowski M. Shen M. Zhou X.Z. Stukenberg P.T. Rahfeld J. Xu J. Kuang J. Kirschner M.W. Fischer G. Cantley L.C. Lu K.P. Science. 1997; 278: 1957-1960Crossref PubMed Scopus (674) Google Scholar, 9Ranganathan R. Lu K.P. Hunter T. Noel J.P. Cell. 1997; 89: 875-886Abstract Full Text Full Text PDF PubMed Scopus (605) Google Scholar, 10Zhou X.Z. Lu P.J. Wulf G. Lu K.P. Cell. Mol. Life Sci. 1999; 56: 788-806Crossref PubMed Scopus (123) Google Scholar). Pin1 is essential for mitotic progression (11Lu K.P. Hanes S.D. Hunter T. Nature. 1996; 380: 544-547Crossref PubMed Scopus (794) Google Scholar, 12Zhou X.Z. Kops O. Werner A. Lu P.J. Shen M. Stoller G. Küllertz G. Stark M. Fischer G. Lu K.P. Mol. Cell. 2000; 6: 873-883Abstract Full Text Full Text PDF PubMed Scopus (477) Google Scholar, 13Wu X. Wilcox C.B. Devasahayam G. Hackett R.L. Arevalo-Rodriguez M. Cardenas M.E. Heitman J. Hanes S.D. EMBO J. 2000; 19: 3727-3738Crossref PubMed Scopus (140) Google Scholar) and is required for the DNA replication checkpoint (14Winkler K.E. Swenson K.I. Kornbluth S. Means A.R. Science. 2000; 287: 1644-1647Crossref PubMed Scopus (159) Google Scholar). Pin1 substrates are a defined subset of phosphorylated proteins, including many MPM-2 antigens (8Yaffe M.B. Schutkowski M. Shen M. Zhou X.Z. Stukenberg P.T. Rahfeld J. Xu J. Kuang J. Kirschner M.W. Fischer G. Cantley L.C. Lu K.P. Science. 1997; 278: 1957-1960Crossref PubMed Scopus (674) Google Scholar,13Wu X. Wilcox C.B. Devasahayam G. Hackett R.L. Arevalo-Rodriguez M. Cardenas M.E. Heitman J. Hanes S.D. EMBO J. 2000; 19: 3727-3738Crossref PubMed Scopus (140) Google Scholar, 15Shen M. Stukenberg P.T. Kirschner M.W. Lu K.P. Genes Dev. 1998; 12: 706-720Crossref PubMed Scopus (302) Google Scholar, 16Crenshaw D.G. Yang J. Means A.R. Kornbluth S. EMBO J. 1998; 17: 1315-1327Crossref PubMed Scopus (167) Google Scholar, 17Lu P.J. Wulf G. Zhou X.Z. Davies P. Lu K.P. Nature. 1999; 399: 784-788Crossref PubMed Scopus (624) Google Scholar, 18Patra D. Wang S.X. Kumagai A. Dunphy W.G. J. Biol. Chem. 1999; 274: 36839-36842Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 19Albert A. Lavoie S. Vincent M. J. Cell Sci. 1999; 112: 2493-2500PubMed Google Scholar, 20Morris D.P. Phatnani H.P. Greenleaf A.L. J. Biol. Chem. 1999; 274: 31583-31587Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). Pin1-catalyzed prolyl isomerization regulates the conformation and function of phosphoproteins (15Shen M. Stukenberg P.T. Kirschner M.W. Lu K.P. Genes Dev. 1998; 12: 706-720Crossref PubMed Scopus (302) Google Scholar, 17Lu P.J. Wulf G. Zhou X.Z. Davies P. Lu K.P. Nature. 1999; 399: 784-788Crossref PubMed Scopus (624) Google Scholar) and also facilitates dephosphorylation (12Zhou X.Z. Kops O. Werner A. Lu P.J. Shen M. Stoller G. Küllertz G. Stark M. Fischer G. Lu K.P. Mol. Cell. 2000; 6: 873-883Abstract Full Text Full Text PDF PubMed Scopus (477) Google Scholar, 15Shen M. Stukenberg P.T. Kirschner M.W. Lu K.P. Genes Dev. 1998; 12: 706-720Crossref PubMed Scopus (302) Google Scholar, 17Lu P.J. Wulf G. Zhou X.Z. Davies P. Lu K.P. Nature. 1999; 399: 784-788Crossref PubMed Scopus (624) Google Scholar). Thus, Pin1-dependent peptide bond isomerization is a critical post-phosphorylation regulatory mechanism (12Zhou X.Z. Kops O. Werner A. Lu P.J. Shen M. Stoller G. Küllertz G. Stark M. Fischer G. Lu K.P. Mol. Cell. 2000; 6: 873-883Abstract Full Text Full Text PDF PubMed Scopus (477) Google Scholar). However, it is likely that Pin1 is regulated by a post-translational modification. The first and essential step in Pin1-dependent regulation of targets is the substrate binding activity mediated by its WW domain (10Zhou X.Z. Lu P.J. Wulf G. Lu K.P. Cell. Mol. Life Sci. 1999; 56: 788-806Crossref PubMed Scopus (123) Google Scholar). The WW domain functions as a pSer/Thr-Pro-binding module, with the binding pocket including the side chains of Ser16, Arg17, Tyr23, and Trp34 (4Lu P.J. Zhou X.Z. Shen M. Lu K.P. Science. 1999; 283: 1325-1328Crossref PubMed Scopus (587) Google Scholar, 7Verdecia M.A. Bowman M.E. Lu K.P. Hunter T. Noel J.P. Nat. Struct. Biol. 2000; 7: 639-643Crossref PubMed Scopus (423) Google Scholar). Interestingly, Ser16 is located at the center of the shadow pSer/Thr-Pro-binding pocket. Here we show an essential role for phosphorylation of Ser16 in the regulation of the WW domain pSer/Thr binding activity and subsequent Pin1 cell cycle function. We thank L. Cantley, B. Neel, and T. Hunter for constructive suggestions.
Publication Year: 2002
Publication Date: 2002-01-01
Language: en
Type: article
Indexed In: ['crossref', 'pubmed']
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Cited By Count: 234
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