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• W1965627178 abstract "Although cytoskeletal regulation is critical to cell function during interphase and mitosis, the components of the cytoskeleton involved with its control are only beginning to be elucidated. Recently, we reported the identification of a cytoskeletal-associated protein,proline-serine-threoninephosphatase-interacting protein (PSTPIP), whose level of tyrosine phosphorylation was controlled by PEST-type protein-tyrosine phosphatases (PTPs) bound to a novel protein interaction site in the PSTPIP predicted coiled-coil domain. We also showed that the PSTPIP SH3 domain interacts with theWiskott-Aldrich syndromeprotein (WASP), a cytoskeletal regulatory protein, in a manner modulated by tyrosine phosphorylation. Here we describe the identification of PSTPIP 2, a widely expressed protein that is related to PSTPIP. PSTPIP 2 lacks an SH3 domain but contains a region predicted to bind to PEST-type PTPs, and structure-function analyses demonstrate that PSTPIP 2 interacts with the proline-rich C terminus of the PEST-type PTP hematopoietic stem cell factor in a manner similar to that previously demonstrated for PSTPIP. Confocal microscopy revealed that PSTPIP 2 colocalizes with PSTPIP in F actin-rich regions. PSTPIP 2 was found to be efficiently phosphorylated in v-Src-transfected or pervanadate-treated cells at two tyrosines conserved in PSTPIP, but in contrast to PSTPIP, tyrosine phosphorylated PSTPIP 2 was only weakly dephosphorylated in the presence of PTP HSCF. Finally, analysis of oligomer formation demonstrated that PSTPIP and PSTPIP 2 formed homo- but not heterodimers. These data suggest that a family of tyrosine phosphorylated, PEST PTP binding proteins may be implicated in cytoskeletal regulation. Although cytoskeletal regulation is critical to cell function during interphase and mitosis, the components of the cytoskeleton involved with its control are only beginning to be elucidated. Recently, we reported the identification of a cytoskeletal-associated protein,proline-serine-threoninephosphatase-interacting protein (PSTPIP), whose level of tyrosine phosphorylation was controlled by PEST-type protein-tyrosine phosphatases (PTPs) bound to a novel protein interaction site in the PSTPIP predicted coiled-coil domain. We also showed that the PSTPIP SH3 domain interacts with theWiskott-Aldrich syndromeprotein (WASP), a cytoskeletal regulatory protein, in a manner modulated by tyrosine phosphorylation. Here we describe the identification of PSTPIP 2, a widely expressed protein that is related to PSTPIP. PSTPIP 2 lacks an SH3 domain but contains a region predicted to bind to PEST-type PTPs, and structure-function analyses demonstrate that PSTPIP 2 interacts with the proline-rich C terminus of the PEST-type PTP hematopoietic stem cell factor in a manner similar to that previously demonstrated for PSTPIP. Confocal microscopy revealed that PSTPIP 2 colocalizes with PSTPIP in F actin-rich regions. PSTPIP 2 was found to be efficiently phosphorylated in v-Src-transfected or pervanadate-treated cells at two tyrosines conserved in PSTPIP, but in contrast to PSTPIP, tyrosine phosphorylated PSTPIP 2 was only weakly dephosphorylated in the presence of PTP HSCF. Finally, analysis of oligomer formation demonstrated that PSTPIP and PSTPIP 2 formed homo- but not heterodimers. These data suggest that a family of tyrosine phosphorylated, PEST PTP binding proteins may be implicated in cytoskeletal regulation. protein-tyrosine phosphatase hematopoietic stem cell fraction glutathione S-transferase hemagglutinin. The actin cytoskeleton is critically involved with a diversity of cell functions including cell morphology, motility, and cytokinesis (1Kreis T. Vale R. Guidebook to the Cytoskeletal and Motor Proteins. Oxford University, Oxford1994: 1-276Google Scholar). Although a number of structural and regulatory components of the cytoskeleton have been identified in both yeast and higher eukaryotic systems, the mechanisms by which the cell regulates the shape changes that occur during physiological responses remain to be determined. Of particular interest are the possible roles that tyrosine kinases and phosphatases may play in cytoskeletal regulation. Currently, there appears to be evidence for the involvement of at least three tyrosine kinases in cytoskeletal control. Early results suggested that Src family members regulated actin assembly and cell shape (2Felice G. Eason P. Nermut M. Kellie S. Eur. J. Cell Biol. 1990; 52: 47-59PubMed Google Scholar, 3Wu H. Reynolds A. Kanner S. Vines R. Parsons J.T. Mol. Cell. Biol. 1991; 11: 5113-5124Crossref PubMed Scopus (375) Google Scholar, 4Clark E. Brugge J. Mol. Cell. Biol. 1993; 13: 1863-1871Crossref PubMed Scopus (198) Google Scholar, 5Schaller M. Bouton A. Flynn D. Parsons J.T. Prog. Nucleic Acid Res. Mol. Biol. 1993; 44: 205-227Crossref PubMed Scopus (27) Google Scholar, 6Cooper J. Howell B. Cell. 1993; 73: 1051-1054Abstract Full Text PDF PubMed Scopus (495) Google Scholar, 7Chrzanowska-Wodnicka M. Burridge K. J. Cell Sci. 1994; 107: 3643-3654PubMed Google Scholar, 8Durieu-Trautmann O. Chaverot N. Cazaubon S. Strosberg A. Couraud P. J. Biol. 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EMBO J. 1995; 14: 2471-2482Crossref PubMed Scopus (163) Google Scholar, 15Banin S. Truong O. Katz D. Waterfield M. Brickell P. Gout I. Curr. Biol. 1996; 6: 981-988Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar, 16Boyer B. Roche S. Denoyelle M. Thiery J. EMBO J. 1997; 16: 5904-5913Crossref PubMed Scopus (124) Google Scholar, 17Manie S. Beck A. Astier A. Law S. Canty T. Hirai H. Druker B. Avraham H. Haghayeghi N. Sattler M. Salgia R. Griffin J. Golemis E. Freedman A. J. Biol. Chem. 1997; 272: 4230-4236Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 18Thomas S.M. Brugge J.S. Annu. Rev. Cell Dev. Biol. 1997; 13: 513-609Crossref PubMed Scopus (2166) Google Scholar). Many of these same cytoskeletal-associated proteins were hyperphosphorylated in cells derived from mice deficient in Csk, a tyrosine kinase that down-modulates the catalytic activity of Src kinases, consistent with physiological roles for Src-mediated phosphorylation (19Thomas S. Soriano P. Imamoto A. Nature. 1995; 376: 267-271Crossref PubMed Scopus (304) Google Scholar). Although the effects of Src-induced tyrosine phosphorylation on cytoskeletal-associated protein function have yet to be completely elucidated, it has been shown, for example, that this modification modulates the actin cross-linking activity of cortactin as well as the calpain-mediated proteolysis of this protein (20Huang C. Ni Y. Wang T. Gao Y. Haudenschild C. Zhan X. J. Biol. Chem. 1997; 272: 13911-13915Abstract Full Text Full Text PDF PubMed Scopus (220) Google Scholar, 21Huang C. Tandon N.N. Greco N.J. Ni Y. Wang T. Zhan X. J. Biol. Chem. 1997; 272: 19248-19252Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). The association between the extracellular matrix and the cytoskeleton is mediated by the integrin family of adhesion molecules at a subcellular site termed the focal adhesion, and the focal adhesion kinase, p125Fak, is another tyrosine kinase proposed to be involved with cytoskeletal regulation. Interestingly, this kinase appears to mediate the tyrosine phosphorylation of both p130cas and paxillin in response to integrin-mediated adhesion, and these phosphorylation events are induced by the formation of complexes with Src kinases (22Kaplan K. Bibbins K. Swedlow J. Arnaud M. Morgan D. Varmus H. EMBO J. 1994; 13: 4745-4756Crossref PubMed Scopus (220) Google Scholar, 23Flinn H. Ridley A.J. J. Cell Sci. 1996; 109: 1133-1141PubMed Google Scholar, 24Schlaepfer D.D. Hunter T. Cell Struct. Funct. 1996; 21: 445-450Crossref PubMed Scopus (73) Google Scholar, 25Tachibana K. Urano T. Fujita H. Ohashi Y. Kamiguchi K. Iwata S. Hirai H. Morimoto C. J. Biol. Chem. 1997; 272: 29083-29090Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar, 26Hanks S. Polte T. Bioessays. 1997; 19: 137-145Crossref PubMed Scopus (440) Google Scholar, 27Cary L.A. Han D.C. Polte T.R. Hanks S.K. Guan J.L. J. Cell Biol. 1998; 140: 211-221Crossref PubMed Scopus (416) Google Scholar). In addition, Rho, a small GTPase that stimulates focal adhesion and stress fiber formation, stimulates the tyrosine phosphorylation of p125Fak, p130cas, and paxillin (23Flinn H. Ridley A.J. J. Cell Sci. 1996; 109: 1133-1141PubMed Google Scholar, 28Barry S.T. Flinn H.M. Humphries M.J. Critchley D.R. Ridley A.J. Cell Adhes. Commun. 1996; 4: 387-398Crossref Scopus (118) Google Scholar). These data thus provide a further link between the cytoskeleton and Src-mediated tyrosine phosphorylation. Finally, the Abl tyrosine kinase, which has a C-terminal actin binding motif, has been found to induce striking cytoskeletal abnormalities when expressed as an oncogenic hybrid with the BCR protein, and these cytoskeletal changes were dependent upon a functional Abl tyrosine kinase domain (29Salgia R. Li J.L. Ewaniuk D.S. Pear W. Pisick E. Burky S.A. Ernst T. Sattler M. Chen L.B. Griffin J.D. J. Clin. Invest. 1997; 100: 46-57Crossref PubMed Scopus (149) Google Scholar, 30Kadlec L. Pendergast A.M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12390-12395Crossref PubMed Scopus (55) Google Scholar). In addition, some of the proteins that are tyrosine phosphorylated by Abl are likely to function as regulators of the cytoskeleton (31De Jong R. Ten Hoeve J. Heisterkamp N. Groffen J. Oncogene. 1997; 14: 507-513Crossref PubMed Scopus (79) Google Scholar, 32Wang B.L. Golemis E.A. Kruh G.D. J. Biol. Chem. 1997; 272: 17542-17550Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar, 33Comer A.R. Ahern-Djamali S.M. Juang J.L. Jackson P.D. Hoffmann F.M. Mol. Cell. Biol. 1998; 18: 152-160Crossref PubMed Scopus (65) Google Scholar). Although the functional reasons for these various tyrosine phosphorylation events remain elusive, it is likely that one important consequence of these modifications is the assembly of signaling complexes via the recognition of phosphotyrosine binding sites by various SH2 domains. Protein-tyrosine phosphatases (PTPs)1 have also been implicated in cytoskeletal regulation. Treatment of cells with PTP inhibitors such as phenylarsine oxide and pervanadate resulted in profound changes in cell morphology and actin distribution (34Bennett P. Dixon R. Kellie S. J. Cell Sci. 1993; 106: 891-901PubMed Google Scholar, 35Retta S.F. Barry S.T. Critchley D.R. Defilippi P. Silengo L. Tarone G. Exp. Cell Res. 1996; 229: 307-317Crossref PubMed Scopus (72) Google Scholar). This type of analysis was greatly enhanced by Choquet and colleagues (36Choquet D. Felsenfeld D. Sheetz M. Cell. 1997; 88: 39-48Abstract Full Text Full Text PDF PubMed Scopus (1070) Google Scholar), who showed that phenylarsine oxide modulated the ability of the cell to strengthen cytoskeletal linkages through integrins adhering to the extracellular matrix protein fibronectin. The tyrosine phosphorylation states of Src and p125Fak tyrosine kinases appeared to be modulated by PTPs in response to extracellular matrix binding to integrin receptors, again consistent with a role for these enzymes in cytoskeletal regulation (35Retta S.F. Barry S.T. Critchley D.R. Defilippi P. Silengo L. Tarone G. Exp. Cell Res. 1996; 229: 307-317Crossref PubMed Scopus (72) Google Scholar, 37Ezumi Y. Takayama H. Okuma M. J. Biol. Chem. 1995; 270: 11927-11934Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). The most convincing data regarding the participation of a known PTP in the regulation of a cytoskeletal-associated protein are found in the case of the regulation of p130cas tyrosine phosphorylation by PTP PEST (38Garton A. Flint A. Tonks N. Mol. Cell. Biol. 1996; 16: 6408-6418Crossref PubMed Scopus (232) Google Scholar, 39Garton A.J. Burnham M.R. Bouton A.H. Tonks N.K. Oncogene. 1997; 15: 877-885Crossref PubMed Scopus (144) Google Scholar). As described above, p130cas, which is a focal adhesion-associated protein containing an SH3 domain and a number of phosphotyrosines capable of potentially interacting with SH2 domains, is phosphorylated in response to integrin-mediated adhesion and in v-Src expressing cells. Analysis of substrates for PTP PEST using “substrate trap” catalytic domain mutants (40Flint A.J. Tiganis T. Barford D. Tonks N.K. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1680-1685Crossref PubMed Scopus (681) Google Scholar) demonstrated that p130cas was indeed a substrate for the tyrosine phosphatase activity of this PTP (38Garton A. Flint A. Tonks N. Mol. Cell. Biol. 1996; 16: 6408-6418Crossref PubMed Scopus (232) Google Scholar). The recognition of this substrate by PTP PEST appeared due to a combination of direct substrate binding to the catalytic domain as well as an interaction between the p130cas SH3 region and a proline-rich site in the phosphatase (39Garton A.J. Burnham M.R. Bouton A.H. Tonks N.K. Oncogene. 1997; 15: 877-885Crossref PubMed Scopus (144) Google Scholar). Although the functional significance of pp130cas dephosphorylation by PTP PEST remains to be determined, it is likely that the removal of phosphates results in the termination of downstream signaling events, some of which are likely to regulate the cytoskeleton (27Cary L.A. Han D.C. Polte T.R. Hanks S.K. Guan J.L. J. Cell Biol. 1998; 140: 211-221Crossref PubMed Scopus (416) Google Scholar). Recently, we demonstrated yet another potential role for PEST-type PTPs in cytoskeletal control. In these studies, we showed that PTP HSCF, a PEST-type PTP expressed in hematopoietic progenitor cells, bound to a novel SH3 domain containing protein, termed PSTPIP (proline-serine-threoninephosphatase-interacting protein) that is homologous to CDC15p, an Schizosaccharomyces pombeprotein required for assembly of the cytokinetic cleavage furrow (41Spencer S. Dowbenko D. Cheng J. Li W.L. Brush J. Utzig S. Simanis V. Lasky L.A. J. Cell Biol. 1997; 138: 845-860Crossref PubMed Scopus (153) Google Scholar). PSTPIP was tyrosine phosphorylated in pervanadate-treated or v-Src-transfected cells, and PTP HSCF dephosphorylated these tyrosines only when the two proteins interacted with each other through the C-terminal proline-rich domain of the PTP. Confocal microscopy revealed that PSTPIP was localized to the cortical cytoskeleton, lamellipodia, and cytokinetic cleavage furrow, and overexpression of mammalian PSTPIP in the yeast S. pombe resulted in a dominant negative inhibition of cytokinesis. Analysis of the binding interaction between PTP HSCF and PSTPIP suggested that the C-terminal proline-rich domain conserved in all PEST-type PTPs mediated binding to a tryptophan-containing site in the PSTPIP potential coiled-coil domain, consistent with the proposal that PSTPIP tyrosine phosphorylation is generally regulated by diverse PEST-type PTPs (42Dowbenko 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). The potential importance of PSTPIP tyrosine phosphorylation was highlighted by studies aimed at determining the function of the SH3 domain of this protein (43Wu Y. Spencer S.D. Lasky L.A. J. Biol. Chem. 1998; 273: 5765-5770Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). This work revealed that the SH3 domain of PSTPIP interacted with two proline-rich regions in theWiskott-Aldrich syndromeprotein (WASP), an X-linked, cdc42 GTPase regulated cytoskeletal-associated protein whose mutation in humans results in immunodeficiency and thrombocytopenia by causing defects in the structure of the cortical actin cytoskeleton (44Derry J. Ochs H. Francke U. Cell. 1994; 78: 635-644Abstract Full Text PDF PubMed Scopus (836) Google Scholar, 45Gallego M.D. Santamarı́a M. Peña J. Molina I.J. Blood. 1997; 90: 3089-3097Crossref PubMed Google Scholar). In addition, mutation of Bee-1, an Saccharomyces cerevisiae WASP homologue, resulted in defects in the cortical cytoskeleton and cytokinesis, consistent with roles for both the mammalian and yeast proteins in actin regulation (46Li R. J. Cell Biol. 1997; 136: 649-658Crossref PubMed Scopus (219) Google Scholar). Analysis of the phosphorylated tyrosines in PSTPIP demonstrated that one of two modified residues was found within the polyproline binding pocket of the SH3 domain (43Wu Y. Spencer S.D. Lasky L.A. J. Biol. Chem. 1998; 273: 5765-5770Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). Mutation of this tyrosine to acidic residues (either glutamate or aspartate) to mimic the incorporation of a negatively charged phosphate resulted in an almost complete loss of WASP binding. These data suggested that tyrosine phosphorylation of PSTPIP modulated its interaction with WASP and that one function of the bound PTP was to maintain PSTPIP in a dephosphorylated and, hence, WASP-associated, state. PSTPIP is a tyrosine phosphorylated, cytoskeletal-associated protein that may be involved with the regulation of cytokinesis and the cortical cytoskeleton through its SH3-induced interactions with WASP. Here we describe a widely expressed, PSTPIP-related protein, termed PSTPIP 2, that is tyrosine phosphorylated but that lacks an SH3 domain. PSTPIP 2 interacts with PTP HSCF in a manner reminiscent of PSTPIP, but it is only weakly dephosphorylated by bound PTP HSCF. Both PSTPIP and PSTPIP 2 homo-oligomerize, and PSTPIP 2, like PSTPIP, is associated with the actin cytoskeleton. These data suggest that PEST-type PTPs interact with at least two tyrosine phosphorylated cytoskeletal-associated proteins, and these interactions may regulate diverse cytoskeletal functions. Small regions of homology with the PSTPIP protein sequence (41Spencer S. Dowbenko D. Cheng J. Li W.L. Brush J. Utzig S. Simanis V. Lasky L.A. J. Cell Biol. 1997; 138: 845-860Crossref PubMed Scopus (153) Google Scholar) were identified by Blast scanning of the public data bases. Nucleotide probes derived from these homologous regions were used to screen several libraries using standard procedures. Full-length cDNA clones were isolated and sequenced using standard procedures. GST fusion proteins were constructed as described previously (41Spencer S. Dowbenko D. Cheng J. Li W.L. Brush J. Utzig S. Simanis V. Lasky L.A. J. Cell Biol. 1997; 138: 845-860Crossref PubMed Scopus (153) Google Scholar, 42Dowbenko 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, 43Wu Y. Spencer S.D. Lasky L.A. J. Biol. Chem. 1998; 273: 5765-5770Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). These proteins were used to immunize rabbits, and the resultant antibodies were purified by affinity chromatography using the original GST fusion proteins as affinity matrices. Coprecipitation was done as described previously (41Spencer S. Dowbenko D. Cheng J. Li W.L. Brush J. Utzig S. Simanis V. Lasky L.A. J. Cell Biol. 1997; 138: 845-860Crossref PubMed Scopus (153) Google Scholar, 42Dowbenko 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, 43Wu Y. Spencer S.D. Lasky L.A. J. Biol. Chem. 1998; 273: 5765-5770Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). Briefly, nontransfected Baf3 or transfected COS cells were lysed in Nonidet P-40 buffer, and the resultant lysates were immunoprecipitated either with affinity purified polyclonal antibodies (Baf3) or anti-epitope tag monoclonal antibodies (transfected cells). The resultant precipitates were electrophoresed on SDS-polyacrylamide gels, electrophoretically blotted, and probed with either polyclonal or monoclonal antibodies. GST constructs containing either wild type or mutant PSTPIP 2 were produced as described previously (41Spencer S. Dowbenko D. Cheng J. Li W.L. Brush J. Utzig S. Simanis V. Lasky L.A. J. Cell Biol. 1997; 138: 845-860Crossref PubMed Scopus (153) Google Scholar, 42Dowbenko 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, 43Wu Y. Spencer S.D. Lasky L.A. J. Biol. Chem. 1998; 273: 5765-5770Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). In vitro translation and GST precipitation were performed as described previously (41Spencer S. Dowbenko D. Cheng J. Li W.L. Brush J. Utzig S. Simanis V. Lasky L.A. J. Cell Biol. 1997; 138: 845-860Crossref PubMed Scopus (153) Google Scholar, 42Dowbenko 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). GST precipitation of proteins from transfected cell lysates was performed as described previously (41Spencer S. Dowbenko D. Cheng J. Li W.L. Brush J. Utzig S. Simanis V. Lasky L.A. J. Cell Biol. 1997; 138: 845-860Crossref PubMed Scopus (153) Google Scholar, 42Dowbenko 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, 43Wu Y. Spencer S.D. Lasky L.A. J. Biol. Chem. 1998; 273: 5765-5770Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). PSTPIP 2 tyrosine residues were mutated to phenylalanine using polymerase chain reaction mutagenesis as described previously (41Spencer S. Dowbenko D. Cheng J. Li W.L. Brush J. Utzig S. Simanis V. Lasky L.A. J. Cell Biol. 1997; 138: 845-860Crossref PubMed Scopus (153) Google Scholar, 42Dowbenko 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, 43Wu Y. Spencer S.D. Lasky L.A. J. Biol. Chem. 1998; 273: 5765-5770Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). Tyrosine phosphorylation of wild type and mutant PSTPIP 2 was determined by immunoprecipitation of Myc epitope-tagged proteins from lysates of cells that were either incubated with the tyrosine phosphatase inhibitor pervanadate or transfected with the constitutively activated v-Src tyrosine kinase. The resultant precipitated proteins were analyzed by Western blotting with anti-phosphotyrosine antibody. Myc or FLAG epitope-tagged forms of PSTPIP and PSTPIP 2 were transfected in various combinations into COS cells, and lysates were immunoprecipitated with antibodies against either the Myc or FLAG epitopes and Western blotted with either antibody as described previously (41Spencer S. Dowbenko D. Cheng J. Li W.L. Brush J. Utzig S. Simanis V. Lasky L.A. J. Cell Biol. 1997; 138: 845-860Crossref PubMed Scopus (153) Google Scholar, 42Dowbenko 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, 43Wu Y. Spencer S.D. Lasky L.A. J. Biol. Chem. 1998; 273: 5765-5770Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). A form of PSTPIP with the green fluorescent protein at its N terminus was constructed by polymerase chain reaction. This form of the protein shows an identical cortical distribution with the FLAG epitope-tagged form. 2S. Spencer and L. Lasky, unpublished observations. This protein and Myc epitope-tagged PSTPIP 2 were cotransfected into Chinese hamster ovary cells, and the cells were analyzed by confocal microscopy as described previously (41Spencer S. Dowbenko D. Cheng J. Li W.L. Brush J. Utzig S. Simanis V. Lasky L.A. J. Cell Biol. 1997; 138: 845-860Crossref PubMed Scopus (153) Google Scholar, 42Dowbenko 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, 43Wu Y. Spencer S.D. Lasky L.A. J. Biol. Chem. 1998; 273: 5765-5770Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). In addition, Myc epitope-tagged PSTPIP 2 was transfected into COS cells, and the colocalization of this protein with F actin was determined using fluorescein isothiocyanate-labeled phalloidin as described previously Myc epitope-tagged PSTPIP 2. In silico scanning of the public data bases with the PSTPIP protein (41Spencer S. Dowbenko D. Cheng J. Li W.L. Brush J. Utzig S. Simanis V. Lasky L.A. J. Cell Biol. 1997; 138: 845-860Crossref PubMed Scopus (153) Google Scholar) resulted in the identification of several related sequences that were utilized to isolate cDNAs encoding PSTPIP-related proteins from human and murine sources. Fig. 1 A illustrates that the murine sequence (predicted molecular mass of 38,948 Da) was approximately 41% similar to PSTPIP throughout the potential coiled-coil region of the protein, but the related protein lacked the SH3 domain that was previously shown to interact with the WASP (43Wu Y. Spencer S.D. Lasky L.A. J. Biol. Chem. 1998; 273: 5765-5770Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). Analysis of two different human clones revealed approximately 41% similarity with PSTPIP and a much higher level of conservation with the murine PSTPIP-related sequence (∼87% sequence similarity). Comparison of the two human clones with PSTPIP and the murine PSTPIP-related sequence revealed that one human clone, 2A (predicted molecular mass of 36,097 Da), spliced out 26 residues near the N terminus, whereas a second clone, 2B (predicted molecular mass of 34,948 Da), spliced out 33 amino acids (Fig. 1 B). Interestingly, the region spliced out from clone 2B contains a tryptophan residue (Trp-232 in the murine clone and Trp-206 in the human clone) that was previously shown to be critical for interaction with the PEST-type tyrosine phosphatase PTP HSCF and, likely, other PEST PTPs (see below) (42Dowbenko 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). Examination of the region surrounding this tryptophan (murine residues 228–253) shows a high degree of conservation between PSTPIP and the murine and human PSTPIP-related proteins. Analysis of the two tyrosines (Tyr-344 and Tyr-367) in PSTPIP that were previously shown to be phosphorylated in v-Src-transfected or vanadate-treated cells showed that they were both conserved in the murine and human PSTPIP-related proteins (Fig. 1 A) (43Wu Y. Spencer S.D. Lasky L.A. J. Biol. Chem. 1998; 273: 5765-5770Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). Finally, two concensus SH3 binding sites (PXXP, murine PSTPIP residues 278–281 and 323–326) are conserved in all of these sequences (47Feng S. Chen J., Yu, H. Simon J. Schreiber S. Science. 1994; 266: 1241-1247Crossref PubMed Scopus (745) Google Scholar). These data suggest that the murine and human clones encode PSTPIP-related proteins, and the molecules will hereafter be referred to as PSTPIP 2. Previous Northern blot analysis demonstrated that PSTPIP was expressed at relatively low levels in a restricted set of tissues including brain, spleen, lung, and testis (41Spencer S. Dowbenko D. Cheng J. Li W.L. Brush J. Utzig S. Simanis V. Lasky L.A. J. Cell Biol. 1997; 138: 845-860Crossref PubMed Scopus (153) Google Scholar). Fig. 1 C illustrates that PSTPIP 2 is more broadly expressed in every tissue examined. In addition, it is likely that PSTPIP 2 is expressed at significantly higher levels than PSTPIP, because the blot shown here was exposed for a few hours versus the previously published Northern blot of PSTPIP expression, which was exposed for approximately 1 week. Affinity-purified polyclonal antibodies against murine PSTPIP 2 and PTP HSCF (48Cheng J. Daimaru L. Fennie C. Lasky L.A. Blood. 1996; 88: 1156-1167Crossref PubMed Google Scholar) were produced against GST fusion proteins. These antibodies efficiently precipitated PSTPIP 2 and PTP HSCF from murine BaF3 hematopoietic progenitor cells (data not shown). To examine the interaction between endogenous PSTPIP 2 and PTP HSCF, coprecipitation studies were performed. Fig. 2illustrates that anti-PSTPIP 2 antibodies efficiently coprecipitated PTP HSCF from BaF3 cells, consistent with an in vivointeraction between these proteins. Interestingly, although antibodies to PTP HSCF efficiently precipitated the phosphatase, the immunoprecipitate did not contain PSTPIP 2. Because the anti-PTP HSCF antibodies are directed against the C-terminal region of the PTP (48Cheng J. Daimaru L. Fennie C. Lasky L.A. Blood. 1996; 88: 1156-1167Crossref PubMed Google Scholar), including the C-terminal site that binds to PSTPIP 2 (see below), it is possible that the polyclonal antibodies disrupt the interaction between these two proteins (42Dowbenko 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). In summary, these data are consistent with an interaction between endogenous PTP HSCF and PSTPIP 2 in vivo. Previous studies examined the interaction between PTP HSCF and PSTPIP in great detail (41Spencer S. Dowbenko D. Cheng J. Li W.L. Brush J. Utzig S. Simanis V. Lasky L.A. J. Cell Biol. 1997; 138: 845-860Crossref PubMed Scopus (153) Google Scholar, 42Dowbenko D. Spencer" @default.
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