Inhibition of Cell Growth and Spreading by Stomach Cancer-associated Protein-tyrosine Phosphatase-1 (SAP-1) through Dephosphorylation of p130
2001; Elsevier BV; Volume: 276; Issue: 18 Linguagem: Inglês
10.1074/jbc.m007208200
ISSN1083-351X
AutoresTetsuya Noguchi, Masahiro Tsuda, Hitoshi Takeda, Toshiyuki Takada, Kenjiro Inagaki, Takuji Yamao, Kaoru Fukunaga, Takashi Matozaki, Masato Kasuga,
Tópico(s)Cytokine Signaling Pathways and Interactions
ResumoSAP-1 (stomach cancer-associated protein-tyrosine phosphatase-1) is a transmembrane-type protein-tyrosine phosphatase that is abundant in the brain and certain cancer cell lines. With the use of a "substrate-trapping" approach, p130cas, a major focal adhesion-associated phosphotyrosyl protein, has now been identified as a likely physiological substrate of SAP-1. Expression of recombinant SAP-1 induced the dephosphorylation of p130cas as well as that of two other components of the integrin-signaling pathway (focal adhesion kinase and p62dok) in intact cells. In contrast, expression of a substrate-trapping mutant of SAP-1 induced the hyperphosphorylation of these proteins, indicating a dominant negative effect of this mutant. Overexpression of SAP-1 induced disruption of the actin-based cytoskeleton as well as inhibited various cellular responses promoted by integrin-mediated cell adhesion, including cell spreading on fibronectin, growth factor-induced activation of extracellular signal-regulated kinase 2, and colony formation. Finally, the enzymatic activity of SAP-1, measured with an immunocomplex phosphatase assay, was substantially increased by cell-cell adhesion. These results suggest that SAP-1, by mediating the dephosphorylation of focal adhesion-associated substrates, negatively regulates integrin-promoted signaling processes and, thus, may contribute to contact inhibition of cell growth and motility. SAP-1 (stomach cancer-associated protein-tyrosine phosphatase-1) is a transmembrane-type protein-tyrosine phosphatase that is abundant in the brain and certain cancer cell lines. With the use of a "substrate-trapping" approach, p130cas, a major focal adhesion-associated phosphotyrosyl protein, has now been identified as a likely physiological substrate of SAP-1. Expression of recombinant SAP-1 induced the dephosphorylation of p130cas as well as that of two other components of the integrin-signaling pathway (focal adhesion kinase and p62dok) in intact cells. In contrast, expression of a substrate-trapping mutant of SAP-1 induced the hyperphosphorylation of these proteins, indicating a dominant negative effect of this mutant. Overexpression of SAP-1 induced disruption of the actin-based cytoskeleton as well as inhibited various cellular responses promoted by integrin-mediated cell adhesion, including cell spreading on fibronectin, growth factor-induced activation of extracellular signal-regulated kinase 2, and colony formation. Finally, the enzymatic activity of SAP-1, measured with an immunocomplex phosphatase assay, was substantially increased by cell-cell adhesion. These results suggest that SAP-1, by mediating the dephosphorylation of focal adhesion-associated substrates, negatively regulates integrin-promoted signaling processes and, thus, may contribute to contact inhibition of cell growth and motility. protein-tyrosine phosphatase receptor-like PTP stomach cancer-associated protein-tyrosine phosphatase-1 focal adhesion FA kinase Chinese hamster ovary monoclonal antibody glutathione S-transferase hemagglutinin epitope extracellular signal-regulated kinase phosphate-buffered saline lysophosphatidic acid epidermal growth factor 12-O-tetradecanoylphorbol-13-acetate p-nitrophenylphosphate extracellular matrix cytomegalovirus Dynamic changes in the extent of tyrosine phosphorylation of cellular proteins are fundamental to signal transduction pathways that regulate cell growth and differentiation, the cell cycle, tissue morphogenesis, and cytoskeletal organization. Tyrosine phosphorylation is tightly controlled by coordination of the activities of protein-tyrosine kinases and protein-tyrosine phosphatases (PTPs)1 (1Hunter T. Cell. 1995; 80: 225-236Abstract Full Text PDF PubMed Scopus (2584) Google Scholar). Although much progress has been made in our understanding of the structure, function, and regulation of protein-tyrosine kinases, PTPs are less well characterized. Similar to protein-tyrosine kinases, PTPs can be divided into two structurally distinct subgroups: cytoplasmic PTPs and transmembrane-type (receptor-like) PTPs (RPTPs) (2Charbonneau H. Tonks N.K. Annu. Rev. Cell Biol. 1992; 8: 463-493Crossref PubMed Scopus (296) Google Scholar). Despite their marked diversity in overall structure, all PTPs possess a core sequence (I/V)HCXAGXXR(S/T)G that contains conserved cysteine and arginine residues critical for enzymatic activity (3Barford D. Jia Z. Tonks N.K. Nat. Struct. Biol. 1995; 12: 1043-1053Crossref Scopus (181) Google Scholar). The cellular functions of PTPs are thought to be determined by their substrate specificity, subcellular localization, and interaction with other signaling molecules. Although initial observations of the effects of overexpression of PTPs suggested that these enzymes might simply counteract the signaling events elicited by protein-tyrosine kinases, more recent genetic and biochemical evidence indicates that PTPs play more complex roles in a wide range of cellular activities (4Matozaki T. Kasuga M. Cell. Signal. 1996; 8: 13-19Crossref PubMed Scopus (47) Google Scholar, 5Neel B.G. Tonks N.K. Curr. Opin. Cell Biol. 1997; 9: 193-204Crossref PubMed Scopus (732) Google Scholar). For example, SHP-1 and SHP-2, two Src homology 2 domain-containing cytoplasmic PTPs play negative and positive regulatory roles, respectively, in protein-tyrosine kinase signaling (6Adachi M. Fischer E.H. Ihle J. Imai K. Jirik F. Neel B. Pawson T. Shen S.H. Thomas M. Ullrich A. Zhao Z. Cell. 1996; 85: 15Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar). Members of the band 4.1 family of cytoplasmic PTPs, including PTPH1 and PTPMEG, are thought to regulate cytoskeletal reorganization (7Yang Q. Tonks N.K. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 5949-5953Crossref PubMed Scopus (171) Google Scholar, 8Gu M.X. York J.D. Warshawsky I. Majerus P.W. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 5867-5871Crossref PubMed Scopus (155) Google Scholar). In addition, the RPTP PTPα affects cell-substratum adhesion and cellular transformation by regulating the activity of the protein-tyrosine kinase Src (9Su J. Muranjan M. Sap J. Curr. Biol. 1999; 9: 505-511Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar). Finally, members of the LAR family of RPTPs, including PTPς (10Wallace M.J. Batt J. Fladd C.A. Henderson J.T. Skarnes W. Rotin D. Nat. Genet. 1999; 21: 334-338Crossref PubMed Scopus (118) Google Scholar) and PTPδ (11Sommer L. Rao M. Anderson D.J. Dev. Dyn. 1997; 208: 48-61Crossref PubMed Scopus (53) Google Scholar) as well as Drosophila DLAR and the related DPTP69D (12Desai C.J. Gindhart Jr., J.G. Goldstein L.S. Zinn K. Cell. 1996; 84: 599-609Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar, 13Krueger N.X. Van-Vactor D. Wan H.I. Gelbart W.M. Goodman C.S. Saito H. Cell. 1996; 84: 611-622Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar), are essential for axon guidance or neuronal differentiation in the developing nervous system. We have previously cloned a human RPTP termed SAP-1 (stomach cancer-associated PTP-1) (14Matozaki T. Suzuki T. Uchida T. Inazawa J. Ariyama T. Matsuda K. Horita K. Noguchi H. Mizuno H. Sakamoto C. Kasuga M. J. Biol. Chem. 1994; 269: 2075-2081Abstract Full Text PDF PubMed Google Scholar). This enzyme contains a single catalytic domain in the cytoplasmic region, a single transmembrane domain, and eight fibronectin type III-like domains in the extracellular region. SAP-1 belongs to the class 2 subfamily of RPTPs (15Krueger N.X. Streuli M. Saito H. EMBO J. 1990; 9: 3241-3252Crossref PubMed Scopus (372) Google Scholar), which includes HPTPβ (15Krueger N.X. Streuli M. Saito H. EMBO J. 1990; 9: 3241-3252Crossref PubMed Scopus (372) Google Scholar), DEP-1 (16Ostman A. Yang Q. Tonks N.K. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9680-9684Crossref PubMed Scopus (200) Google Scholar), PTP-U2 (17Seimiya H. Sawabe T. Inazawa J. Tsuruo T. Oncogene. 1995; 10: 1731-1738PubMed Google Scholar), and DrosophilaDPTP10D (18Yang X.H. Seow K.T. Bahri S.M. Oon S.H. Chia W. Cell. 1991; 67: 661-673Abstract Full Text PDF PubMed Scopus (134) Google Scholar, 19Tian S.S. Tsoulfas P. Zinn K. Cell. 1991; 67: 675-680Abstract Full Text PDF PubMed Scopus (156) Google Scholar). The observations that the fibronectin type III-like domain is present in many neural cell adhesion molecules and that SAP-1 is abundant in the brain suggest that this PTP plays a role in neural cell-cell adhesion signaling (14Matozaki T. Suzuki T. Uchida T. Inazawa J. Ariyama T. Matsuda K. Horita K. Noguchi H. Mizuno H. Sakamoto C. Kasuga M. J. Biol. Chem. 1994; 269: 2075-2081Abstract Full Text PDF PubMed Google Scholar). SAP-1 is also abundant in a subset of pancreatic and colorectal cancer cell lines and tissues but not in their normal counterparts (14Matozaki T. Suzuki T. Uchida T. Inazawa J. Ariyama T. Matsuda K. Horita K. Noguchi H. Mizuno H. Sakamoto C. Kasuga M. J. Biol. Chem. 1994; 269: 2075-2081Abstract Full Text PDF PubMed Google Scholar, 20Seo Y. Matozaki T. Tsuda M. Hayashi Y. Itoh H. Kasuga M. Biochem. Biophys. Res. Commun. 1997; 231: 705-711Crossref PubMed Scopus (40) Google Scholar). Furthermore, the SAP-1 gene is located on the long arm of human chromosome 19, at position q13.4, which is close to the locus of the gene for carcinoembryonic antigen at 19q13.2 (14Matozaki T. Suzuki T. Uchida T. Inazawa J. Ariyama T. Matsuda K. Horita K. Noguchi H. Mizuno H. Sakamoto C. Kasuga M. J. Biol. Chem. 1994; 269: 2075-2081Abstract Full Text PDF PubMed Google Scholar). Expression of SAP-1 may thus also be associated with cancer development. However, the biological roles of this PTP remain unknown. The identification of physiological substrates of PTPs provides important insight into the functions of these enzymes. However, substrate identification for this class of enzymes has proved problematic because of the low affinity and transient nature of the interaction of PTPs with their substrates. A recently developed substrate-trapping strategy has overcome this limitation and greatly facilitated isolation of specific substrates of PTPs (21Garton A.J. Flint A.J. Tonks N.K. Mol. Cell. Biol. 1996; 16: 6408-6418Crossref PubMed Scopus (231) Google Scholar, 22Flint A.J. Tiganis T. Barford D. Tonks N.K. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1680-1685Crossref PubMed Scopus (677) Google Scholar, 23Tiganis T. Bennett A.M. Ravichandran K.S. Tonks N.K. Mol. Cell. Biol. 1998; 18: 1622-1634Crossref PubMed Google Scholar, 24Zhang S.H. Liu J. Kobayashi R. Tonks N.K. J. Biol. Chem. 1999; 274: 17806-17812Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). With the use of this strategy, we have now identified p130cas, a major phosphotyrosyl protein that is localized to focal adhesions (FAs), as a potential substrate of SAP-1. We also provide evidence that SAP-1 negatively regulates various integrin-promoted cellular responses and that the enzymatic activity of this PTP is up-regulated by cell-cell adhesion. The cDNAs encoding a catalytically inactive mutant of SAP-1 (SAP-1C/S), in which Cys1022 is replaced by Ser, and a substrate-trapping mutant of SAP-1 (SAP-1D/A), in which Asp988 is replaced by Ala, were generated by site-directed mutagenesis with human SAP-1 cDNA as a template and a transformer site-directed mutagenesis kit (CLONTECH). The full-length wild-type and mutant SAP-1 cDNAs were then inserted separately into theHindIII site of the pRc/CMV expression vector (Invitrogen). The pSRα vector encoding hemagglutinin (HA)-tagged p130caswas kindly provided by H. Hirai (University of Tokyo), the pBabe vector encoding Myc-tagged paxillin-α was provided by H. Sabe (Osaka Bioscience Institute), the pRc/CMV vector encoding HA-tagged focal adhesion kinase (FAK) was provided by S. K. Hanks (Vanderbilt University, Nashville, TN), and the pcDNA3 vector encoding HA-tagged extracellular signal-regulated kinase 2 (ERK2) was provided by J. S. Gutkind (National Institutes of Health, Bethesda, MD). The pRc/CMV vector encoding HA-tagged mouse p62dok was generated as described (25Noguchi T. Matozaki T. Inagaki K. Tsuda M. Fukunaga K. Kitamura Y. Kitamura T. Shii K. Yamanashi Y. Kasuga M. EMBO J. 1999; 18: 1748-1760Crossref PubMed Scopus (103) Google Scholar). Chinese hamster ovary (CHO) cell lines stably expressing wild-type SAP-1 (SAP-1WT) or SAP-1C/S were generated as described previously (26Noguchi T. Matozaki T. Horita K. Fujioka Y. Kasuga M. Mol. Cell. Biol. 1994; 14: 6674-6682Crossref PubMed Scopus (348) Google Scholar), and the expression level of each SAP-1 protein was determined by immunoblot analysis with polyclonal antibodies to SAP-1 as described below. CHO-K1 cells and the established cell lines were maintained in Ham's F-12 medium supplemented with 10% fetal bovine serum. 293 human embryonic kidney cells, Panc-1 cells, WiDr cells, Swiss 3T3 cells, and NIH 3T3 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. Transfection of CHO-K1 cells or 293 cells (∼2 × 105 cells/60-mm dish) was performed with the use of a CellPhect Transfection Kit (Amersham Pharmacia Biotech). Rabbit polyclonal antibodies to SAP-1 (14Matozaki T. Suzuki T. Uchida T. Inazawa J. Ariyama T. Matsuda K. Horita K. Noguchi H. Mizuno H. Sakamoto C. Kasuga M. J. Biol. Chem. 1994; 269: 2075-2081Abstract Full Text PDF PubMed Google Scholar) and the mouse monoclonal antibody (mAb) 3G5 to SAP-1 were generated in response to a glutathioneS-transferase (GST) fusion protein containing the cytoplasmic region of SAP-1 in our laboratory. Rabbit polyclonal antibodies to p62dok were described previously (25Noguchi T. Matozaki T. Inagaki K. Tsuda M. Fukunaga K. Kitamura Y. Kitamura T. Shii K. Yamanashi Y. Kasuga M. EMBO J. 1999; 18: 1748-1760Crossref PubMed Scopus (103) Google Scholar). The mAb 12CA5 to the HA epitope tag and the mAb 9E10 to the Myc tag were purified from the culture supernatants of mouse hybridoma cells. Mouse mAbs to p130cas, to paxillin, and to β-catenin were obtained from Transduction Laboratories; horseradish peroxidase-conjugated mouse mAb PY20 to phosphotyrosine and rabbit polyclonal antibodies to p130cas or to FAK were from Santa Cruz Biotechnology; rabbit polyclonal antibodies that react specifically with tyrosine-phosphorylated ERK or with total ERK protein were from New England BioLabs; a mouse mAb to vinculin was from Sigma; fluorescein isothiocyanate-conjugated sheep polyclonal antibodies to mouse immunoglobulin and Texas red-conjugated donkey polyclonal antibodies to rabbit immunoglobulin were from Amersham Pharmacia Biotech. Cells were thawed on ice in 1 ml of ice-cold lysis buffer (20 mmTris-HCl (pH 7.6), 140 mm NaCl, 1 mm EDTA, 1% (v/v) Nonidet P-40) containing 5 mm NaF, 1 mmphenylmethylsulfonyl fluoride, aprotinin (10 μg/ml), and 1 mm sodium vanadate. The cell lysates were centrifuged at 10,000 × g for 15 min at 4 °C, and the resulting supernatants were subjected to immunoprecipitation and immunoblot analysis. For immunoprecipitation, the supernatants were incubated for 3 h at 4 °C with antibody-coupled protein G-Sepharose beads (20 μl of beads) (Amersham Pharmacia Biotech). The beads were washed three times with 1 ml of WG buffer (50 mm Hepes-NaOH (pH 7.6), 150 mm NaCl, 0.1% (v/v) Triton X-100) and then suspended in Laemmli sample buffer. Immunoblot analysis with various antibodies was performed with the use of the ECL detection system (Amersham Pharmacia Biotech). The cytoplasmic regions of SAP-1WT, SAP-1C/S, and SAP-1D/A were produced as GST fusion proteins. The polymerase chain reaction was performed with wild-type or mutant SAP-1 cDNA as template and with 5′-TAGGATCCCCAGGGGACATCCCAGCTGAAG (nucleotides 2436–2457 of SAP-1 cDNA) and 5′-TCGAATTCGGGCTGCCGACCCAGCCCCCTCG (nucleotides 3400–3422) as sense and antisense primers, respectively. The amplification products were digested with BamHI andEcoRI and inserted in-frame into the BamHI andEcoRI sites of pGEX-2T (Amersham Pharmacia Biotech). The encoded GST fusion proteins were then expressed in Escherichia coli and purified with the use of glutathione-Sepharose beads (Amersham Pharmacia Biotech). The recombinant full-length SHP-2 was prepared as described previously (26Noguchi T. Matozaki T. Horita K. Fujioka Y. Kasuga M. Mol. Cell. Biol. 1994; 14: 6674-6682Crossref PubMed Scopus (348) Google Scholar). Panc-1 cells in one 100-mm dish were treated with 100 μm pervanadate for 30 min and then lysed in 1 ml of buffer A (20 mm Tris-HCl (pH 7.4), 100 mm NaCl, 1 mm EDTA, 10% (v/v) glycerol, 1% (v/v) Triton X-100) containing 1 mm phenylmethylsulfonyl fluoride, 1 mm benzamidine, 10 μm leupeptin, aprotinin (10 μg/ml), and 5 mm iodoacetic acid. After incubation of the lysate for 30 min at 4 °C with gentle agitation, dithiothreitol was added to a final concentration of 10 mmto inactivate any unreacted iodoacetic acid. The lysate was then centrifuged at 100,000 × g for 30 min at 4 °C, and the resulting supernatant was incubated for 2 h at 4 °C with glutathione-Sepharose beads conjugated with 10 μg of recombinant SAP-1 protein. The beads were washed three times with 1 ml of buffer A, and bound material was subjected to immunoblot analysis. In some experiments, 1 mm orthovanadate was included during the incubation of beads with lysate. Lysates from pervanadate-treated Panc-1 cells were subjected to immunoprecipitation; the resultant precipitates containing tyrosine-phosphorylated proteins were fractionated by SDS-polyacrylamide gel electrophoresis, and the separated proteins were transferred to a nitrocellulose filter. The dephosphorylation reaction was performed by incubating the filter in the absence or presence of recombinant SAP-1 or SHP-2 for 20 min at 30 °C in a solution containing 50 mm Hepes-NaOH (pH 7.1), 150 mm NaCl, 10 mm dithiothreitol, and 2 mm EDTA. The filter was then washed for 5 min at room temperature with phosphate-buffered saline (PBS) containing 500 mm NaCl, 0.5% (w/v) SDS, and 0.1% (v/v) Triton X-100. The extent of tyrosine phosphorylation of each protein was determined by immunoblot analysis with mAb PY20 to phosphotyrosine. Cells were detached from the culture dish by treatment with 0.025% trypsin, collected by centrifugation, and washed once with serum-free Ham's F-12 medium. The cells were replated in serum-free Ham's F-12 medium at a density of 1 × 105 cells/ml on 60-mm culture dishes coated with fibronectin (10 μg/ml) (Sigma) or poly-l-lysine (10 μg/ml) (Sigma). The cells were then incubated for 2 h at 37 °C in a humidified incubator containing 5% CO2, after which they were examined under a light microscope equipped with phase-contrast optics (model IX 70; Olympus), and random fields were photographed at a total magnification of 200×. The cDNAs encoding SAP-1WT or SAP-1C/S were cloned separately into the SwaI site of pAxCAwt (27Kanegae Y. Lee G. Sato Y. Tanaka M. Nakai M. Sakaki T. Sugano S. Saito I. Nucleic Acids Res. 1995; 23: 3816-3821Crossref PubMed Scopus (598) Google Scholar), which contains the CAG promoter, and the resulting constructs were introduced together with DNA-terminal protein complex by transfection into 293 cells. The resulting recombinant adenoviruses were screened by immunoblot analysis and cloned by limiting dilution. Adenoviral vectors were propagated by a standard procedure, purified by two rounds of CsCl density gradient centrifugation, and then titrated with a limiting dilution assay in 293 cells. Swiss 3T3 cells seeded on glass coverslips in a 6-well plate were infected with viruses at 37 °C for 1 h, with brief agitation every 20 min, and were then exposed to normal growth medium. Cells were washed with PBS, fixed with 3.7% formaldehyde for 20 min, permeabilized with 0.5% (v/v) Triton X-100 in PBS for 5 min, and incubated for 2 h with TBS-T (20 mm Tris-HCl (pH 7.6), 150 mm NaCl, 0.05% (v/v) Tween 20) containing 5% (w/v) nonfat dry milk, 10% fetal bovine serum, and 1% (w/v) bovine serum albumin. The cells were then incubated for 1 h at room temperature with mAb 3G5 to SAP-1 (1 μg/ml) or with polyclonal antibodies to SAP-1 (1 μg/ml), together with either rhodamine-labeled phalloidin (0.1 μg/ml) (Sigma) or a mAb to vinculin (20 μg/ml). Cells were then washed twice with PBS and incubated with fluorescein isothiocyanate- or Texas red-conjugated secondary antibodies for 30 min. After washing three times with PBS, the cells were examined under a laser-scanning confocal microscope (model MRC-1024; Bio-Rad), and built-up images were constructed. Transfected 293 cells (60-mm dishes) were deprived of serum for 12 h and then incubated for 5 min with 10 μm lysophosphatidic acid (LPA) (Sigma) or epidermal growth factor (EGF) (100 ng/ml) (Calbiochem) or for 10 min with 1 μm 12-O-tetradecanoylphorbol-13-acetate (TPA) (Sigma). Cells were then lysed in 400 μl of a solution containing 50 mm Hepes-NaOH (pH 7.8), 150 mmNaCl, 1.5 mm MgCl2, 1 mm EDTA, 0.1% (v/v) Triton X-100, 20 mm β-glycerophosphate, 100 mm NaF, 10 mm sodium pyrophosphate, 1 mm phenylmethylsulfonyl fluoride, aprotinin (10 μg/ml), and 1 mm sodium vanadate. Recombinant ERK2 was immunoprecipitated with mAb 12CA5 to the HA tag and then subjected to immunoblot analysis with antibodies specific for activated ERK or for total ERK protein. The extent of ERK2 phosphorylation was quantified by scanning densitometry with the NIH Image program. CHO-K1 cells or NIH 3T3 cells (∼2 × 105 cells/60-mm dish) were transiently transfected with 3 μg of pRc/CMV containing (or not) SAP-1WT or SAP-1C/S cDNA. After 48 h, transfected cells were diluted 1:20 with normal growth medium supplemented with G418 (400 μg/ml) and cultured in 100-mm dishes. The medium was changed every 3 days, and, after 14 days, cells that had formed colonies were fixed with 3.7% formaldehyde and stained with crystal violet. The efficiency of colony formation was quantified by scanning densitometry with the NIH Image program. The enzymatic activity of recombinant SAP-1 was assayed with p-nitrophenylphosphate (pNPP) as a substrate as described previously (14Matozaki T. Suzuki T. Uchida T. Inazawa J. Ariyama T. Matsuda K. Horita K. Noguchi H. Mizuno H. Sakamoto C. Kasuga M. J. Biol. Chem. 1994; 269: 2075-2081Abstract Full Text PDF PubMed Google Scholar). Cellular SAP-1 activity immunopurified with mAb 3G5 was assayed with pNPP as described previously (28Streuli M. Krueger N.X. Thai T. Tang M. Saito H. EMBO J. 1990; 9: 2399-2407Crossref PubMed Scopus (268) Google Scholar). All data presented are representative of at least three independent experiments. To identify physiological substrates of SAP-1, we first generated two types of SAP-1 mutant: a catalytically inactive mutant, in which the invariant Cys1022 residue is replaced by Ser, and a substrate-trapping mutant, in which the invariant Asp988 residue is replaced by Ala. The latter mutant was expected to be a more powerful tool than was the former because it should not only be catalytically inactive but also retain the ability to bind SAP-1 substrates (22Flint A.J. Tiganis T. Barford D. Tonks N.K. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1680-1685Crossref PubMed Scopus (677) Google Scholar). We then prepared GST fusion proteins that contain the catalytic domains of wild-type SAP-1 (GST-SAP-1WT), catalytically inactive SAP-1 (GST-SAP-1C/S), or the substrate-trapping mutant of SAP-1 (GST-SAP-1D/A). Each recombinant protein was expressed in and purified from bacteria as a 60-kDa polypeptide, as revealed by SDS-polyacrylamide gel electrophoresis and Coomassie Blue staining (Fig. 1 A). Whereas GST-SAP-1WT exhibited substantial catalytic activity with the artificial substrate pNPP, GST-SAP-1C/S and GST-SAP-1D/A possessed virtually no activity (Fig.2 A). Each of the three recombinant proteins bound to glutathione-Sepharose beads was then incubated with lysates prepared from pervanadate-treated Panc-1 cells, which express SAP-1 (14Matozaki T. Suzuki T. Uchida T. Inazawa J. Ariyama T. Matsuda K. Horita K. Noguchi H. Mizuno H. Sakamoto C. Kasuga M. J. Biol. Chem. 1994; 269: 2075-2081Abstract Full Text PDF PubMed Google Scholar). Immunoblot analysis with the mAb PY20 to phosphotyrosine revealed that GST-SAP-1D/A, but not GST-SAP-1WT or GST alone, precipitated several phosphotyrosyl proteins with molecular sizes of 190, 125–135, 75–85, and 55 kDa (hereafter referred to as p190, p125–135, p75–85, and p55, respectively) (Fig. 1 B). GST-SAP-1C/S precipitated a similar group of proteins, although to a much lesser extent than did GST-SAP-1D/A (Fig. 1, B andC). The amounts of p190, p125–135, p75–85, and p55 bound to GST-SAP1D/A were markedly reduced by incubation of cell lysates with the recombinant protein in the presence of vanadate, a potent inhibitor of PTPs; in contrast, the amounts of these proteins bound to GST-SAP-1C/S were substantially increased by incubation with vanadate (Fig. 1 C). Vanadate ions interact directly with the thiolate anion of the cysteine residue in the catalytic core sequence of PTPs and thereby block enzyme-substrate association (29Denu J.M. Lohse D.L. Vijayalakshmi J. Saper M.A. Dixon J.E. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 2493-2498Crossref PubMed Scopus (249) Google Scholar, 30Huyer G. Liu S. Kelly J. Moffat J. Payette P. Kennedy B. Tsaprailis G. Gresser M.J. Ramachandran C. J. Biol. Chem. 1997; 272: 843-851Abstract Full Text Full Text PDF PubMed Scopus (716) Google Scholar). These results thus suggest that all of the detected phosphotyrosyl proteins might bind, either directly or indirectly, to the active site of SAP-1. To identify p125–135, we subjected the proteins precipitated by GST-SAP-1D/A to immunoblot analysis with antibodies specific for either FAK, Ras GTPase-activating protein, or p130cas, all of which are in the size range of 125–135 kDa and are phosphorylated on tyrosine residues. Of these antibodies, the polyclonal antibodies to p130cas specifically recognized a 125- to 135-kDa protein that bound to GST-SAP-1D/A (Fig. 1 D). A mAb to p130casthat recognizes a distinct epitope of the protein also reacted with p125–135 (data not shown). These results suggest that p125–135 might be p130cas. We also tested the ability of antibodies to paxillin, cortactin, or SHP-2 to recognize p75–85. A mAb to paxillin specifically reacted with a 75–85-kDa protein that bound to GST-SAP-1D/A (Fig. 1 D), suggesting that p75–85 might be paxillin. However, we cannot exclude the possibility that the p125–135 and p75–85 bands contain additional unidentified proteins. The identities of p190 and p55 remain unknown. We next examined whether SAP-1 dephosphorylates p130cas and paxillin in vitro. The catalytic activity of GST-SAP-1WT with pNPP as substrate was markedly greater than that of the recombinant SHP-2 used as a control (Fig. 2 A). Tyrosine-phosphorylated forms of p130cas and paxillin immunopurified from pervanadate-treated Panc-1 cells were also incubated with equal amounts of activity of GST-SAP-1WT and GST-SHP-2, after which their phosphorylation status was determined. GST-SAP-1WT effectively dephosphorylated p130cas and, to a lesser extent, paxillin; in contrast, GST-SHP-2 or GST alone had virtually no effect on the extent of phosphorylation of these proteins (Fig.2 B). The human RPTP PTPκ dephosphorylates β-catenin (31Fuchs M. Muller T. Lerch M.M. Ullrich A. J. Biol. Chem. 1996; 271: 16712-16719Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar); however, SAP-1 did not exhibit detectable catalytic activity with β-catenin as substrate (Fig. 2 B). Together, these results indicate that SAP-1 selectively dephosphorylates p130cas and, to a lesser extent, paxillin in vitro. We also examined whether SAP-1 dephosphorylates p130cas and paxillin in intact cells. We subjected 293 cells to transient transfection with expression vectors encoding SAP-1 and either HA-tagged p130cas (HA-Cas) or Myc epitope-tagged paxillin-α and then evaluated the extent of tyrosine phosphorylation of each tagged recombinant protein. Wild-type SAP-1 (SAP-1WT) expressed in these cells possessed substantial catalytic activity, whereas the catalytically inactive mutant (SAP-1C/S) or the substrate-trapping mutant (SAP-1D/A) did not, as revealed by an immunocomplex phosphatase assay using mAb 3G5 to SAP-1 (data not shown). Coexpression of SAP-1WT, but not that of SAP-1C/S, markedly reduced the extent of tyrosine phosphorylation of HA-Cas (Fig.3 A), suggesting that SAP-1 dephosphorylates p130cas in intact cells. In contrast, coexpression of SAP-1D/A markedly increased the extent of tyrosine phosphorylation of HA-Cas. The presence of equal amounts of recombinant SAP-1 proteins in the different transfected cells was confirmed by immunoblot analysis with antibodies to SAP-1 (Fig. 3 F). Given that substrate-trapping mutants of PTPs often induce hyperphosphorylation of the corresponding substrates (21Garton A.J. Flint A.J. Tonks N.K. Mol. Cell. Biol. 1996; 16: 6408-6418Crossref PubMed Scopus (231) Google Scholar, 22Flint A.J. Tiganis T. Barford D. Tonks N.K. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1680-1685Crossref PubMed Scopus (677) Google Scholar, 23Tiganis T. Bennett A.M. Ravichandran K.S. Tonks N.K. Mol. Cell. Biol. 1998; 18: 1622-1634Crossref PubMed Google Scholar, 24Zhang S.H. Liu J. Kobayashi R. Tonks N.K. J. Biol. Chem. 1999; 274: 17806-17812Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar), these results indicate that p130cas serves as a direct substrate of SAP-1 in vivo. Coexpression of SAP-1WT also reduced the extent of tyrosine phosphorylation of paxillin-α; however, the substrate-trapping mutant did not increase the extent of tyrosine phosphorylation of this protein (Fig. 3 B). Thus, paxillin might not be a physiological substrate of SAP-1. p130casundergoes tyrosine phosphorylation during integrin-mediated cell adhesion and mediates the assembly of FAs (32Nojima Y. Morino N. Mimura T. Hamasaki K. Furuya H. Sakai
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