Wiskott-Aldrich syndrome is an X-linked hematopoietic disease that manifests itself in platelet deficiency and a compromised immune system. Analysis of hematopoietic cells from affected individuals reveals that mutations in the Wiskott-Aldrich syndrome protein (WASP) result in structural and functional abnormalities in the cell cortex, consistent with the suggestion that WASP is involved with regulation of the actin-rich cortical cytoskeleton. Here we report that WASP interacts with a recently described cytoskeletal-associated protein, PSTPIP, a molecule that is related to the Schizosaccharomyces pombe cleavage furrow regulatory protein, CDC15p. This association is mediated by an interaction between the PSTPIP SH3 domain and two polyproline-rich regions in WASP. Co-expression of PSTPIP with WASP in vivoresults in a loss of WASP-induced actin bundling activity and co-localization of the two proteins, which requires the PSTPIP SH3 domain. Analysis of tyrosine phosphorylation of PSTPIP reveals that two sites are modified in response to v-Src co-transfection or pervanadate incubation. One of these tyrosines is found in the SH3 domain poly-proline recognition site, and mutation of this tyrosine to aspartate or glutamate to mimic this phosphorylation state results in a loss of WASP binding in vitro and a dissolution of co-localization in vivo. In addition, PSTPIP that is tyrosine phosphorylated in the SH3 domain interacts poorly with WASPin vitro. These data suggest that the PSTPIP and WASP interaction is regulated by tyrosine phosphorylation of the PSTPIP SH3 domain, and this binding event may control aspects of the actin cytoskeleton. Wiskott-Aldrich syndrome is an X-linked hematopoietic disease that manifests itself in platelet deficiency and a compromised immune system. Analysis of hematopoietic cells from affected individuals reveals that mutations in the Wiskott-Aldrich syndrome protein (WASP) result in structural and functional abnormalities in the cell cortex, consistent with the suggestion that WASP is involved with regulation of the actin-rich cortical cytoskeleton. Here we report that WASP interacts with a recently described cytoskeletal-associated protein, PSTPIP, a molecule that is related to the Schizosaccharomyces pombe cleavage furrow regulatory protein, CDC15p. This association is mediated by an interaction between the PSTPIP SH3 domain and two polyproline-rich regions in WASP. Co-expression of PSTPIP with WASP in vivoresults in a loss of WASP-induced actin bundling activity and co-localization of the two proteins, which requires the PSTPIP SH3 domain. Analysis of tyrosine phosphorylation of PSTPIP reveals that two sites are modified in response to v-Src co-transfection or pervanadate incubation. One of these tyrosines is found in the SH3 domain poly-proline recognition site, and mutation of this tyrosine to aspartate or glutamate to mimic this phosphorylation state results in a loss of WASP binding in vitro and a dissolution of co-localization in vivo. In addition, PSTPIP that is tyrosine phosphorylated in the SH3 domain interacts poorly with WASPin vitro. These data suggest that the PSTPIP and WASP interaction is regulated by tyrosine phosphorylation of the PSTPIP SH3 domain, and this binding event may control aspects of the actin cytoskeleton. PSTPIP is a recently described coiled-coil and SH3 domain-containing protein that is homologous to CDC15p, aSchizosaccharomyces pombe phosphoprotein involved with the assembly of the cytokinetic cleavage furrow (1Spencer S. Dowbenko D. Cheng J. Li W. Brush J. Utzig S. Simanis V. Lasky L.A. J. Cell Biol. 1997; 138: 845-860Crossref PubMed Scopus (151) Google Scholar, 2Dowbenko D. Spencer S. Quan C. Lasky L.A. J. Biol. Chem. 1998; 273: 989-996Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar, 3Fankhauser C. Reymond A. Cerutti L. Utzig S. Hofmann K. Simanis V. Cell. 1995; 82: 435-444Abstract Full Text PDF PubMed Scopus (222) Google Scholar). PSTPIP is associated with protein-tyrosine phosphatase (PTP) 1The abbreviations used are: PTP, protein-tyrosine phosphatase; WASP, Wiskott-Aldrich syndrome protein; GST, glutathione S-transferase; GFP, green fluorescent protein; CHO, Chinese hamster ovary; BTK, Bruton's tyrosine kinase; aa, amino acids; HSCF, hematopoietic stem cell fraction. 1The abbreviations used are: PTP, protein-tyrosine phosphatase; WASP, Wiskott-Aldrich syndrome protein; GST, glutathione S-transferase; GFP, green fluorescent protein; CHO, Chinese hamster ovary; BTK, Bruton's tyrosine kinase; aa, amino acids; HSCF, hematopoietic stem cell fraction. HSCF, a member of the PEST family of PTPs (4Cheng J. Daimaru L. Fennie C. Lasky L.A. Blood. 1996; 88: 1156-1167Crossref PubMed Google Scholar), via a critical tryptophan in the PSTPIP coiled-coil domain (2Dowbenko D. Spencer S. Quan C. Lasky L.A. J. Biol. Chem. 1998; 273: 989-996Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar), and this association mediates the dephosphorylation of tyrosine residues in PSTPIP that are modified either by co-expression of the v-Src tyrosine kinase or in the presence of the PTP inhibitor pervanadate. Throughout interphase, the coiled-coil domain localizes PSTPIP to actin-rich regions of the cell, including the cortical cytoskeleton and lammelipodia, and the protein migrates to the cleavage furrow during cytokinesis. Overexpression of PSTPIP in mammalian cells induces filapodial extension and cell rounding, and induced expression of the mammalian protein in S. pombe results in a dominant negative inhibition of cytokinesis (1Spencer S. Dowbenko D. Cheng J. Li W. Brush J. Utzig S. Simanis V. Lasky L.A. J. Cell Biol. 1997; 138: 845-860Crossref PubMed Scopus (151) Google Scholar). These data suggest that PSTPIP is a tyrosine phosphorylated cytoskeletal regulatory protein that may be involved with the control of cytokinesis.Wiskott-Aldrich syndrome is an X-linked hematopoietic disease that presents with platelet abnormalities and immunodeficiency (5Amman A. Hong R. Stiehm E.R. Immunologic Disorders in Infants and Children. W. B. Saunders, Philadelphia1989: 257-315Google Scholar, 6Ochs H. Slichter S. Harker L. Von B. Clark R. Wedgwood R. Blood. 1980; 55: 243-252Crossref PubMed Google Scholar), and mutations in the Wiskott-Aldrich syndrome protein (WASP) cause this syndrome (7Derry J. Ochs H. Francke U. Cell. 1994; 78: 635-644Abstract Full Text PDF PubMed Scopus (821) Google Scholar, 8Kwan S.H., T. Radtke B. Blaese R. Rosen F. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4706-4710Crossref PubMed Scopus (96) Google Scholar, 9Kolluri R. Shehabeldin A. Peacocke M. Lamhonwah A. Teichert-Kuliszewska K. Weissman S. Siminovitch K. Hum. Mol. Genet. 1995; 4: 1119-1126Crossref PubMed Scopus (86) Google Scholar). WASP is a multi-domain protein containing pleckstrin homology, CDC42-binding, polyproline, and actin regulatory motifs (7Derry J. Ochs H. Francke U. Cell. 1994; 78: 635-644Abstract Full Text PDF PubMed Scopus (821) Google Scholar). Hematopoietic cells from Wiskott-Aldrich syndrome patients have abnormalities in their cortical cytoskeletons (6Ochs H. Slichter S. Harker L. Von B. Clark R. Wedgwood R. Blood. 1980; 55: 243-252Crossref PubMed Google Scholar, 10Molina I. Kenney D. Rosen F. Remold-Odonnell E. J. Exp. Med. 1992; 176: 867-874Crossref PubMed Scopus (118) Google Scholar), and overexpression of WASP induces bundling of F actin, which may be regulated by CDC42, a GTPase involved with cytoskeletal modulation (11Symons M. Derry J. Karlak B. Jiang S. Lamahieu V. McCormick F. Francke U. Abo A. Cell. 1996; 84: 723-734Abstract Full Text Full Text PDF PubMed Scopus (740) Google Scholar,12Lamarche N. Tapon N. Stowers L. Burbelo P. Aspenstorm P. Bridges T. Chant J. Hall A. Cell. 1996; 87: 519-529Abstract Full Text Full Text PDF PubMed Scopus (524) Google Scholar). A WASP homologue in Saccharomyces cerevisiae is involved with cortical actin assembly and cytokinesis (13Lechler T. Li R. J. Cell Biol. 1997; 138: 95-103Crossref PubMed Scopus (50) Google Scholar, 14Li R. J. Cell Biol. 1997; 136: 649-658Crossref PubMed Scopus (219) Google Scholar). In addition, WASP binds to various tyrosine kinases as well as the Nck and GRB 2 adaptor proteins, suggesting a link with cellular signal transduction pathways (15Cory G. McCarthy-Morrogh L. Banin S. Gout I. Brickell P. Levinsky R. Kinnon C. Lovering R. J. Immunol. 1996; 157: 3791-3796PubMed Google Scholar, 16Kinnon C. Cory G.O.C. MacCarthy-Morrogh L. Banin S. Gout I. Lovering R.C. Brickell P.M. Biochem. Soc. Trans. 1997; 25: 648-650Crossref PubMed Scopus (10) Google Scholar, 17Miki H. Nonoyama S. Zhu Q. Aruffo A. Ochs H. Takenawa T. Cell Growth Differ. 1997; 8: 195-202PubMed Google Scholar, 18Rivero-Lezcano O. Marcilla A. Sameshima J. Robbins K. Mol. Cell. Biol. 1995; 15: 5725-5731Crossref PubMed Scopus (280) Google Scholar, 19She H. Rockow S. Tang J. Nishimura R. Skolnik E. Chen M. Margolis B. Li W. Mol. Biol. Cell. 1997; 8: 1709-1721Crossref PubMed Scopus (104) Google Scholar). These data suggest that WASP is a scaffold that mediates the assembly of various signaling and structural components involved with cytoskeletal regulation.Together, these data suggest the involvement of both PSTPIP and WASP in the control of the cytoskeleton. Here we show a physical interaction between the SH3 domain of PSTPIP and proline-rich regions of WASP. This interaction appears to modulate the actin bundling activity localized to the C terminus of WASP (11Symons M. Derry J. Karlak B. Jiang S. Lamahieu V. McCormick F. Francke U. Abo A. Cell. 1996; 84: 723-734Abstract Full Text Full Text PDF PubMed Scopus (740) Google Scholar). Analysis of the tyrosine phosphorylation of PSTPIP reveals that one of the two phosphotyrosines in this protein is within the polyproline-binding pocket of the SH3 domain, and this modification appears to regulate the binding between PSTPIP and WASP. This study describes a novel mechanism for the modulation of the interaction between two components involved with regulation of the cytoskeleton. PSTPIP is a recently described coiled-coil and SH3 domain-containing protein that is homologous to CDC15p, aSchizosaccharomyces pombe phosphoprotein involved with the assembly of the cytokinetic cleavage furrow (1Spencer S. Dowbenko D. Cheng J. Li W. Brush J. Utzig S. Simanis V. Lasky L.A. J. Cell Biol. 1997; 138: 845-860Crossref PubMed Scopus (151) Google Scholar, 2Dowbenko D. Spencer S. Quan C. Lasky L.A. J. Biol. Chem. 1998; 273: 989-996Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar, 3Fankhauser C. Reymond A. Cerutti L. Utzig S. Hofmann K. Simanis V. Cell. 1995; 82: 435-444Abstract Full Text PDF PubMed Scopus (222) Google Scholar). PSTPIP is associated with protein-tyrosine phosphatase (PTP) 1The abbreviations used are: PTP, protein-tyrosine phosphatase; WASP, Wiskott-Aldrich syndrome protein; GST, glutathione S-transferase; GFP, green fluorescent protein; CHO, Chinese hamster ovary; BTK, Bruton's tyrosine kinase; aa, amino acids; HSCF, hematopoietic stem cell fraction. 1The abbreviations used are: PTP, protein-tyrosine phosphatase; WASP, Wiskott-Aldrich syndrome protein; GST, glutathione S-transferase; GFP, green fluorescent protein; CHO, Chinese hamster ovary; BTK, Bruton's tyrosine kinase; aa, amino acids; HSCF, hematopoietic stem cell fraction. HSCF, a member of the PEST family of PTPs (4Cheng J. Daimaru L. Fennie C. Lasky L.A. Blood. 1996; 88: 1156-1167Crossref PubMed Google Scholar), via a critical tryptophan in the PSTPIP coiled-coil domain (2Dowbenko D. Spencer S. Quan C. Lasky L.A. J. Biol. Chem. 1998; 273: 989-996Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar), and this association mediates the dephosphorylation of tyrosine residues in PSTPIP that are modified either by co-expression of the v-Src tyrosine kinase or in the presence of the PTP inhibitor pervanadate. Throughout interphase, the coiled-coil domain localizes PSTPIP to actin-rich regions of the cell, including the cortical cytoskeleton and lammelipodia, and the protein migrates to the cleavage furrow during cytokinesis. Overexpression of PSTPIP in mammalian cells induces filapodial extension and cell rounding, and induced expression of the mammalian protein in S. pombe results in a dominant negative inhibition of cytokinesis (1Spencer S. Dowbenko D. Cheng J. Li W. Brush J. Utzig S. Simanis V. Lasky L.A. J. Cell Biol. 1997; 138: 845-860Crossref PubMed Scopus (151) Google Scholar). These data suggest that PSTPIP is a tyrosine phosphorylated cytoskeletal regulatory protein that may be involved with the control of cytokinesis. Wiskott-Aldrich syndrome is an X-linked hematopoietic disease that presents with platelet abnormalities and immunodeficiency (5Amman A. Hong R. Stiehm E.R. Immunologic Disorders in Infants and Children. W. B. Saunders, Philadelphia1989: 257-315Google Scholar, 6Ochs H. Slichter S. Harker L. Von B. Clark R. Wedgwood R. Blood. 1980; 55: 243-252Crossref PubMed Google Scholar), and mutations in the Wiskott-Aldrich syndrome protein (WASP) cause this syndrome (7Derry J. Ochs H. Francke U. Cell. 1994; 78: 635-644Abstract Full Text PDF PubMed Scopus (821) Google Scholar, 8Kwan S.H., T. Radtke B. Blaese R. Rosen F. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4706-4710Crossref PubMed Scopus (96) Google Scholar, 9Kolluri R. Shehabeldin A. Peacocke M. Lamhonwah A. Teichert-Kuliszewska K. Weissman S. Siminovitch K. Hum. Mol. Genet. 1995; 4: 1119-1126Crossref PubMed Scopus (86) Google Scholar). WASP is a multi-domain protein containing pleckstrin homology, CDC42-binding, polyproline, and actin regulatory motifs (7Derry J. Ochs H. Francke U. Cell. 1994; 78: 635-644Abstract Full Text PDF PubMed Scopus (821) Google Scholar). Hematopoietic cells from Wiskott-Aldrich syndrome patients have abnormalities in their cortical cytoskeletons (6Ochs H. Slichter S. Harker L. Von B. Clark R. Wedgwood R. Blood. 1980; 55: 243-252Crossref PubMed Google Scholar, 10Molina I. Kenney D. Rosen F. Remold-Odonnell E. J. Exp. Med. 1992; 176: 867-874Crossref PubMed Scopus (118) Google Scholar), and overexpression of WASP induces bundling of F actin, which may be regulated by CDC42, a GTPase involved with cytoskeletal modulation (11Symons M. Derry J. Karlak B. Jiang S. Lamahieu V. McCormick F. Francke U. Abo A. Cell. 1996; 84: 723-734Abstract Full Text Full Text PDF PubMed Scopus (740) Google Scholar,12Lamarche N. Tapon N. Stowers L. Burbelo P. Aspenstorm P. Bridges T. Chant J. Hall A. Cell. 1996; 87: 519-529Abstract Full Text Full Text PDF PubMed Scopus (524) Google Scholar). A WASP homologue in Saccharomyces cerevisiae is involved with cortical actin assembly and cytokinesis (13Lechler T. Li R. J. Cell Biol. 1997; 138: 95-103Crossref PubMed Scopus (50) Google Scholar, 14Li R. J. Cell Biol. 1997; 136: 649-658Crossref PubMed Scopus (219) Google Scholar). In addition, WASP binds to various tyrosine kinases as well as the Nck and GRB 2 adaptor proteins, suggesting a link with cellular signal transduction pathways (15Cory G. McCarthy-Morrogh L. Banin S. Gout I. Brickell P. Levinsky R. Kinnon C. Lovering R. J. Immunol. 1996; 157: 3791-3796PubMed Google Scholar, 16Kinnon C. Cory G.O.C. MacCarthy-Morrogh L. Banin S. Gout I. Lovering R.C. Brickell P.M. Biochem. Soc. Trans. 1997; 25: 648-650Crossref PubMed Scopus (10) Google Scholar, 17Miki H. Nonoyama S. Zhu Q. Aruffo A. Ochs H. Takenawa T. Cell Growth Differ. 1997; 8: 195-202PubMed Google Scholar, 18Rivero-Lezcano O. Marcilla A. Sameshima J. Robbins K. Mol. Cell. Biol. 1995; 15: 5725-5731Crossref PubMed Scopus (280) Google Scholar, 19She H. Rockow S. Tang J. Nishimura R. Skolnik E. Chen M. Margolis B. Li W. Mol. Biol. Cell. 1997; 8: 1709-1721Crossref PubMed Scopus (104) Google Scholar). These data suggest that WASP is a scaffold that mediates the assembly of various signaling and structural components involved with cytoskeletal regulation. Together, these data suggest the involvement of both PSTPIP and WASP in the control of the cytoskeleton. Here we show a physical interaction between the SH3 domain of PSTPIP and proline-rich regions of WASP. This interaction appears to modulate the actin bundling activity localized to the C terminus of WASP (11Symons M. Derry J. Karlak B. Jiang S. Lamahieu V. McCormick F. Francke U. Abo A. Cell. 1996; 84: 723-734Abstract Full Text Full Text PDF PubMed Scopus (740) Google Scholar). Analysis of the tyrosine phosphorylation of PSTPIP reveals that one of the two phosphotyrosines in this protein is within the polyproline-binding pocket of the SH3 domain, and this modification appears to regulate the binding between PSTPIP and WASP. This study describes a novel mechanism for the modulation of the interaction between two components involved with regulation of the cytoskeleton. We thank Dr. K. Siminovitch for graciously making results about WASP-PSTPIP interactions available before publication and for the human WASP cDNA clone. We also thank Dr. Clifford Quan for peptide synthesis, Dr. Wenlu Li for help with confocal microscopy, and Lorraine Sarullo for help with figures. We also thank Dr. Emma Lasky for fruitful discussions during the course of this project.
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