Interaction of SAP-1, a Transmembrane-type Protein-tyrosine Phosphatase, with the Tyrosine Kinase Lck
2003; Elsevier BV; Volume: 278; Issue: 37 Linguagem: Inglês
10.1074/jbc.m300648200
ISSN1083-351X
AutoresTomokazu Ito, Hideki Okazawa, Koji Maruyama, Kyoko Tomizawa, Sei‐ichiro Motegi, Hiroshi Ohnishi, Hiroyuki Kuwano, Atsushi Kosugi, Takashi Matozaki,
Tópico(s)Cytokine Signaling Pathways and Interactions
ResumoSAP-1 is a transmembrane-type protein-tyrosine phosphatase that is expressed in most tissues but whose physiological functions remain unknown. The cytoplasmic region of SAP-1 has now been shown to bind directly the tyrosine kinase Lck. Overexpression of wild-type SAP-1, but not that of a catalytically inactive mutant of SAP-1, inhibited both the basal and the T cell antigen receptor (TCR)-stimulated activity of Lck in human Jurkat T cell lines. Lck served as a direct substrate for dephosphorylation by SAP-1 in vitro. Overexpression of wild-type SAP-1 in Jurkat cells also: (i) inhibited both the activation of mitogen-activated protein kinase and the increase in cell surface expression of CD69 induced by TCR stimulation; (ii) reduced the extent of the TCR-induced increase in the tyrosine phosphorylation of ZAP-70 or that of LAT; (iii) reduced both the basal level of tyrosine phosphorylation of p62 dok , as well as the increase in the phosphorylation of this protein induced by CD2 stimulation; and (iv) inhibited cell migration. These results thus suggest that the direct interaction of SAP-1 with Lck results in inhibition of the kinase activity of the latter and a consequent negative regulation of T cell function. SAP-1 is a transmembrane-type protein-tyrosine phosphatase that is expressed in most tissues but whose physiological functions remain unknown. The cytoplasmic region of SAP-1 has now been shown to bind directly the tyrosine kinase Lck. Overexpression of wild-type SAP-1, but not that of a catalytically inactive mutant of SAP-1, inhibited both the basal and the T cell antigen receptor (TCR)-stimulated activity of Lck in human Jurkat T cell lines. Lck served as a direct substrate for dephosphorylation by SAP-1 in vitro. Overexpression of wild-type SAP-1 in Jurkat cells also: (i) inhibited both the activation of mitogen-activated protein kinase and the increase in cell surface expression of CD69 induced by TCR stimulation; (ii) reduced the extent of the TCR-induced increase in the tyrosine phosphorylation of ZAP-70 or that of LAT; (iii) reduced both the basal level of tyrosine phosphorylation of p62 dok , as well as the increase in the phosphorylation of this protein induced by CD2 stimulation; and (iv) inhibited cell migration. These results thus suggest that the direct interaction of SAP-1 with Lck results in inhibition of the kinase activity of the latter and a consequent negative regulation of T cell function. Regulation of protein-tyrosine phosphorylation contributes to many important physiological processes including cell growth, differentiation, and migration as well as glucose metabolism, synaptic transmission, and the immune response (1Hunter T. Cell. 2000; 100: 113-127Abstract Full Text Full Text PDF PubMed Scopus (2280) Google Scholar, 2Schlessinger J. Cell. 2000; 103: 211-225Abstract Full Text Full Text PDF PubMed Scopus (3557) Google Scholar). The balance between protein-tyrosine phosphorylation and dephosphorylation is precisely determined by the action of protein-tyrosine kinases (PTKs) 1The abbreviations used are: PTK, protein-tyrosine kinase; PTP, protein-tyrosine phosphatase; TCR, T cell antigen receptor; SH, Src homology; LAT, linker of activated T cells; MAP, mitogen-activated protein; pAb, polyclonal antibody; mAb, monoclonal antibody; FITC, fluorescein isothiocyanate; FBS, fetal bovine serum; GST, glutathione S-transferase; EcoR, ecotropic receptor; pNPP, p-nitrophenyl phosphate; PMA, phorbol 12-myristate 13-acetate; WT, wild type; Mes, 4-morpholineethanesulfonic acid. PTP, protein-tyrosine phosphatase; PEST, proline (P), glutamic acid (E), serine (S), threonine (T)-rich sequence; PEP, PEST-domain phosphatase. and protein-tyrosine phosphatases (PTPs) (3Hunter T. Cell. 1995; 80: 225-236Abstract Full Text PDF PubMed Scopus (2608) Google Scholar, 4Hubbard S.R. Till J.H. Annu. Rev. 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Natl. Acad. Sci. U. S. A. 1990; 87: 4358-4362Crossref PubMed Scopus (534) Google Scholar). In human T cells, the TCR-mediated activation of Lck results from autophosphorylation of this enzyme on Tyr394. In contrast, phosphorylation of Tyr505 near the COOH terminus of Lck by the Src family kinase Csk negatively regulates Lck activity (11Bergman M. Mustelin T. Oetken C. Partanen J. Flint N.A. Amrein K.E. Autero M. Burn P. Alitalo K. EMBO J. 1992; 11: 2919-2924Crossref PubMed Scopus (274) Google Scholar). Activated Lck or Fyn mediates the phosphorylation of CD3 and the ζ chain, which are the signal-transducing subunits of the TCR (7Cantrell D. Annu. Rev. Immunol. 1996; 14: 259-274Crossref PubMed Scopus (596) Google Scholar, 8Samelson L.E. Annu. Rev. Immunol. 2002; 20: 371-394Crossref PubMed Scopus (470) Google Scholar). These modifications occur within immunoreceptor tyrosine-based activation motifs and direct the recruitment of ZAP-70, a Syk family PTK, to the TCR through interaction with its tandem Src homology 2 (SH2) domains (12Iwashima M. Irving B.A. van Oers N.S. Chan A.C. Weiss A. Science. 1994; 263: 1136-1139Crossref PubMed Scopus (2) Google Scholar). The consequent activation of ZAP-70 results in the phosphorylation of various adapter proteins including LAT (for linker of activated T cells), SLP-76, Vav, and phospholipase C-γ (7Cantrell D. Annu. Rev. Immunol. 1996; 14: 259-274Crossref PubMed Scopus (596) Google Scholar, 8Samelson L.E. Annu. Rev. Immunol. 2002; 20: 371-394Crossref PubMed Scopus (470) Google Scholar, 13Zhang W. Sloan-Lancaster J. Kitchen J. Trible R.P. Samelson L.E. Cell. 1998; 92: 83-92Abstract Full Text Full Text PDF PubMed Scopus (1071) Google Scholar). In contrast to protein-tyrosine phosphorylation, the role of protein-tyrosine dephosphorylation in TCR-mediated signal transduction has been only partially resolved (14Cloutier J.F. Veillette A. J. Exp. Med. 1999; 189: 111-121Crossref PubMed Scopus (359) Google Scholar). SHP-1, a cytoplasmic PTP that contains two SH2 domains, negatively regulates the TCR-mediated signaling pathway (15Plas D.R. Johnson R. Pingel J.T. Matthews R.J. Dalton M. Roy G. Chan A.C. Thomas M.L. Science. 1996; 272: 1173-1176Crossref PubMed Scopus (335) Google Scholar, 16Johnson K.G. LeRoy F.G. Borysiewicz L.K. Matthews R.J. J. Immunol. 1999; 162: 3802-3813PubMed Google Scholar), and its localization in membrane rafts is required for such regulation (17Kosugi A. Sakakura J. Yasuda K. Ogata M. Hamaoka T. Immunity. 2001; 14: 669-680Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). In addition, PEP and PTP-PEST, which are related cytoplasmic PTPs, also negatively regulate TCR-mediated signaling (18Gjorloff-Wingren A. Saxena M. Williams S. Hammi D. Mustelin T. Eur. J. Immunol. 1999; 29: 3845-3854Crossref PubMed Scopus (161) Google Scholar, 19Davidson D. Veillette A. EMBO J. 2001; 20: 3414-3426Crossref PubMed Scopus (100) Google Scholar). Whereas PEP appears to cooperate with Csk to inhibit TCR signaling, PTP-PEST dephosphorylates Shc, p130 Cas , Pyk2, and focal adhesion kinase (7Cantrell D. Annu. Rev. Immunol. 1996; 14: 259-274Crossref PubMed Scopus (596) Google Scholar, 8Samelson L.E. Annu. Rev. Immunol. 2002; 20: 371-394Crossref PubMed Scopus (470) Google Scholar, 14Cloutier J.F. Veillette A. J. Exp. Med. 1999; 189: 111-121Crossref PubMed Scopus (359) Google Scholar). The receptor-like PTP CD148, also known as DEP-1 (20Ostman A. Yang Q. Tonks N.K. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9680-9684Crossref PubMed Scopus (203) Google Scholar, 21Honda H. Inazawa J. Nishida J. Yazaki Y. Hirai H. Blood. 1994; 84: 4186-4194Crossref PubMed Google Scholar), negatively regulates the TCR-mediated signaling pathway by catalyzing the dephosphorylation of LAT (22Tangye S.G. Wu J. Aversa G. de Vrice J.E. Lanier L.L. Phillips J.H. J. Immunol. 1998; 161: 3803-3807PubMed Google Scholar, 23de la Fuente-Garcia M.A. Nicolas J.M. Freed J.H. Palou E. Thomas A.P. Vilella R. Vives J. Gaya A. Blood. 1998; 91: 2800-2809Crossref PubMed Google Scholar, 24Baker J.E. Majeti R. Tangye S.G. Weiss A. Mol. Cell Biol. 2001; 21: 2393-2403Crossref PubMed Scopus (71) Google Scholar). In contrast, CD45, another receptor-like PTP, dephosphorylates Tyr505 of Lck and thereby increases its activity and promotes TCR signaling (25Thomas M.L. Brown E.J. Immunol. Today. 1999; 20: 406-411Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar). The molecular mechanism by which the activity of Lck is down-regulated through protein-tyrosine dephosphorylation in vivo and the identity of the PTPs that mediate such regulation remain unknown, however. SAP-1 (for stomach cancer-associated protein-tyrosine phosphatase-1) was originally identified as a PTP expressed in a stomach cancer cell line (26Matozaki 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). It is a transmembrane-type PTP with a single catalytic domain in its cytoplasmic region and eight fibronectin type III-like domains in its extracellular region (26Matozaki 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). A “substrate-trapping” approach identified p130 Cas , a prominent focal adhesion-associated component of the integrin signaling pathway, as a likely physiological substrate of SAP-1 (27Noguchi T. Tsuda M. Takeda H. Takada T. Inagaki K. Yamao T. Fukunaga K. Matozaki T. Kasuga M. J. Biol. Chem. 2001; 276: 15216-15224Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). In addition, overexpression of SAP-1 resulted in the dephosphorylation of several additional focal adhesion-associated proteins, including focal adhesion kinase and paxillin, as well as in the impairment of reorganization of the actin-based cytoskeleton (27Noguchi T. Tsuda M. Takeda H. Takada T. Inagaki K. Yamao T. Fukunaga K. Matozaki T. Kasuga M. J. Biol. Chem. 2001; 276: 15216-15224Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar), suggesting that SAP-1 regulates the latter process. Overexpression of this PTP also inhibited cell proliferation, an effect that was mediated in part either by attenuation of growth factor-induced activation of mitogen-activated protein (MAP) kinase or by caspase-dependent apoptosis (27Noguchi T. Tsuda M. Takeda H. Takada T. Inagaki K. Yamao T. Fukunaga K. Matozaki T. Kasuga M. J. Biol. Chem. 2001; 276: 15216-15224Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 28Takada T. Noguchi T. Inagaki K. Hosooka T. Fukunaga K. Yamao T. Ogawa W. Matozaki T. Kasuga M. J. Biol. Chem. 2002; 277: 34359-34366Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). Although most of these observations were made with cultured fibroblasts, together with the reduced expression of SAP-1 in advanced cancer (29Seo Y. Matozaki T. Tsuda M. Hayashi Y. Itoh H. Kasuga M. Biochem. Biophys. Res. Commun. 1997; 231: 705-711Crossref PubMed Scopus (40) Google Scholar), they suggest that this enzyme functions as a suppressor of cell growth. SAP-1 mRNA has been detected in most tissues examined but is especially abundant in the spleen. 2H. Okazawa and T. Matozaki, unpublished data. To explore the biological role of SAP-1 in the immune system, we have attempted to identify molecules that interact with the cytoplasmic region of this protein. We now show that this region of SAP-1 binds directly to Lck. Furthermore, overexpression of wild-type SAP-1 resulted in down-regulation of the kinase activity of Lck and thereby negatively regulated TCR-mediated T cell functions. Antibodies—Rabbit polyclonal antibodies (pAbs) to SAP-1 (26Matozaki 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), a mouse monoclonal antibody (mAb) (3G5) to SAP-1 (27Noguchi T. Tsuda M. Takeda H. Takada T. Inagaki K. Yamao T. Fukunaga K. Matozaki T. Kasuga M. J. Biol. Chem. 2001; 276: 15216-15224Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar), and rabbit pAbs to p62 dok (30Noguchi 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 (104) Google Scholar) were generated as described previously. The mouse mAb (154A7) to SAP-1 was also generated by using a recombinant immunoglobulin-Fc fusion protein, which contained three fibronectin-type III-like domains of SAP-1 in its extracellular region (amino acids 1–250), as an antigen. The detail for the preparation of this recombinant protein will be described elsewhere. The mAb was purified from culture supernatants of the hybridoma by column chromatography on protein A-Sepharose 4FF (Amersham Biosciences). Two mouse mAbs (anti-T112 and anti-T113) to CD2 (31Meuer S.C. Hussey R.E. Fabbi M. Fox D. Acuto O. Fitzgerald K.A. Hodgdon J.C. Protentis J.P. Schlossman S.F. Reinherz E.L. Cell. 1984; 36: 897-906Abstract Full Text PDF PubMed Scopus (921) Google Scholar) were kindly provided by E. L. Reinherz (Dana-Faber Cancer Institute, Boston, MA). Mouse mAbs to CD3 (OKT3), or to the Myc epitope tag (9E10) were purified from the culture supernatants of hybridoma cells. A mouse mAb to Lck, rabbit pAbs to Lck, rabbit pAbs to LAT, and a mouse mAb (4G10) to phosphotyrosine were obtained from Upstate Biotechnology. Rabbit pAbs to human Src autophosphorylated on Tyr416 that also recognize the autophosphorylation sites of other Src family PTKs, including Lck, were from Cell Signaling Technology. Rabbit pAbs to MAP kinase and to active MAP kinase (pTEpY) were from Promega; rabbit pAbs to ZAP-70 and a horseradish peroxidase-conjugated mouse mAb (PY20) to phosphotyrosine were from Santa Cruz Biotechnology; and a mouse mAb to CD247 (TCR ζ chain) was from COSMO BIO. A fluorescein isothiocyanate (FITC)-conjugated mouse mAb to CD69 for flow cytometry was obtained from BD Pharmingen. Goat antibodies to mouse immunoglobulins were obtained from Southern Biotechnology Associates, Inc. Plasmids for Yeast Two-hybrid Screening and Transient Transfection—The expression vectors pBTM116HA and pCIneo-myc were kindly provided by Y. Takai (Osaka University, Osaka, Japan). To generate a yeast bait vector (pBTM116HA-SAP-1-cyto) encoding the cytoplasmic region of SAP-1 (amino acids 778–1117), we performed the polymerase chain reaction (PCR) with the pRC/CMV vector containing the full-length SAP-1 cDNA (27Noguchi T. Tsuda M. Takeda H. Takada T. Inagaki K. Yamao T. Fukunaga K. Matozaki T. Kasuga M. J. Biol. Chem. 2001; 276: 15216-15224Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar) as a template, the sense primer 5′-AAAGAATTCAAGAGGAGGAATAAGAG-3′, and the antisense primer 5′-AAAGTCGAC TTAGACCTCCTCCAAC-3′. The PCR product was digested with EcoRI and SalI and then inserted into pBTM116HA. To generate a yeast prey vector (pACT clone 15Δcat) that encoded a central portion of human Lck (amino acids 154–242), we performed PCR with a partial Lck cDNA (clone 15) as a template, the sense primer 5′-CTATTCGATGATGAAGATACCCCACCAAACCC-3′, and the antisense primer 5′-AAACTCGAGTCACGTCTCCCTGGGAACC-3′. The PCR product was digested with EcoRI and XhoI and then inserted into the vector pACT2. The plasmid pACT Lck-cat, which encodes the catalytic domain of Lck (amino acids 243–509), was also prepared in the same manner as was pACT clone 15Δcat but with the sense primer 5′-AAAGAATTCGGGAGAGCGAGAGC-3′ and the antisense primer 5′-GTGAACTTGCGGGGTTTTTCAGTACGA-3′. To generate the pCIneo-myc-SAP-1-cyto vector, which encodes the Myc epitope-tagged cytoplasmic region of SAP-1, for transient transfection, we excised the cDNA fragment encoding this region of SAP-1 from pBTM116HA-SAP-1-cyto by digestion with EcoRI and SalI and subcloned it into the corresponding sites of pCIneo-myc. A pCLS vector containing the full-length human Lck cDNA (32Ohta M. Morita T. Shimotohno K. Jpn. J. Cancer Res. 1990; 81: 440-444Crossref PubMed Scopus (6) Google Scholar), which was kindly provided by K. Shimotohno (Kyoto University, Kyoto, Japan), was also used for transient transfection of COS-7 cells. Yeast Two-hybrid Screening and Interaction Assays—For yeast two-hybrid screening, a human spleen Matchmaker library in pACT2 (Clontech) was screened as described previously (33Hata Y. Butz S. Südhof T.C. J. Neurosci. 1996; 16: 2488-2494Crossref PubMed Google Scholar). In brief, Saccharomyces cerevisiae strain L40 was sequentially transfected with the bait vector pBTM116HA-SAP-1-cyto and the cDNA library with the use of lithium acetate. Transformants were selected on plates lacking histidine, uracil, tryptophan, and leucine. Positive clones were then picked after incubation for 4–6 days at 30 °C and assayed for β-galactosidase activity by the filter method. Extrachromosomal DNA was isolated by the glass bead method from the yeast clones that grew in the absence of histidine and were also β-galactosidase-positive. Prey plasmids were rescued in Escherichia coli HB101 cells, which were selected on M9 plates containing proline (50 μg/ml) and ampicillin (100 μg/ml). Interaction of the prey and bait was examined again by retransfection of yeast cells with bait and prey vectors together, followed by selection on plates lacking histidine and assay of β-galactosidase. Cell Culture, Transient Transfection, and Stimulation—COS-7 cells were maintained under a humidified atmosphere of 5% CO2 and 95% air at 37 °C in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (FBS) (Invitrogen), penicillin (100 units/ml), and streptomycin (100 μg/ml). Jurkat cells were maintained in RPMI 1640 supplemented with 10% FBS, penicillin (100 units/ml), and streptomycin (100 μg/ml). COS-7 cells (∼1 × 106 in a 100-mm dish) were subjected to transient transfection with 2 μg of pCIneo-myc-SAP-1-cyto and with 2 μg of pCLS containing Lck cDNA by exposure to the FuGENE 6 reagent (Roche). After 48 h, cell lysates were prepared and subjected to immunoprecipitation and immunoblot analysis as described below. For stimulation of the TCR, Jurkat cells (∼1 × 107) were incubated first for 30 min on ice with the mAb OKT3 (10 μg/ml) and then for 5 min at 37 °C with goat antibodies to mouse immunoglobulins (20 μg/ml). For CD2 stimulation, Jurkat cells (∼1 × 107) were incubated for 10 min at 37 °C with the combination of anti-T112 and anti-T113 (1:100 dilution of ascites fluid). Cell lysates were then prepared and subjected to immunoprecipitation and immunoblot analysis. Immunoprecipitation and Immunoblot Analysis—COS-7 cells were frozen in liquid nitrogen and then lysed on ice in 1 ml of an ice-cold lysis buffer (20 mm Tris-HCl (pH 7.6), 140 mm NaCl, 2.6 mm CaCl2, 1 mm MgCl2, 1% Nonidet P-40, 10% glycerol) containing 1 mm phenylmethylsulfonyl fluoride, aprotinin (10 μg/ml), and 1 mm sodium vanadate. Jurkat cells were lysed on ice in 500 μl of an ice-cold lysis buffer identical to that used for COS-7 cells with the exception that 1% Nonidet P-40 was replaced by 1% Brij 97 also containing 1 mm phenylmethylsulfonyl fluoride, 10 μg/ml aprotinin, and 1 mm sodium vanadate. 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. In brief, the supernatants were incubated for 4 h at 4 °C with protein G-Sepharose beads (20 μl of beads) (Amersham Biosciences) conjugated with various antibodies. The beads were then washed three times with 1 ml of lysis buffer, resuspended in SDS sample buffer, and subjected to SDS-polyacrylamide gel electrophoresis and immunoblot analysis with various antibodies. Immune complexes were detected with an ECL detection kit (Amersham Biosciences). In Vitro Protein Binding Assay—For assay of the interaction of SAP-1 with Lck in vitro, a glutathione S-transferase (GST) fusion protein containing the cytoplasmic region of wild-type SAP-1 (GST-SAP-1-WT) was generated and purified as described previously (26Matozaki 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). Jurkat cells (∼1 × 107) were lysed on ice in 1 ml of ice-cold lysis buffer containing 1 mm phenylmethylsulfonyl fluoride, 10 μg/ml aprotinin, and 1 mm sodium vanadate, and the lysates were centrifuged at 10,000 × g for 15 min at 4 °C. The resulting supernatants were incubated for 5 h at 4 °C with GST-SAP-1-WT or GST, each of which was immobilized on glutathione-Sepharose beads (10 μg of protein/15 μl of packed beads; Amersham Biosciences). The beads were then washed three times with 1 ml of ice-cold lysis buffer, suspended in SDS sample buffer, and subjected to immunoblot analysis with pAbs to Lck. Retrovirus Production and Infection—Full-length cDNAs for wild-type SAP-1 or a catalytically inactive SAP-1 mutant (SAP-1-C/S), in which Cys1022 was replaced by serine (27Noguchi T. Tsuda M. Takeda H. Takada T. Inagaki K. Yamao T. Fukunaga K. Matozaki T. Kasuga M. J. Biol. Chem. 2001; 276: 15216-15224Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar), were inserted into the EcoRI site of the pMX-puro vector (kindly provided by T. Kitamura, University of Tokyo, Tokyo, Japan). The production of retroviruses encoding the SAP-1 proteins and infection of cells with these viruses were performed as described (34Onishi M. Nosaka T. Misawa K. Mui A.L. Gorman D. McMahon M. Miyajima A. Kitamura T. Mol. Cell Biol. 1998; 18: 3871-3879Crossref PubMed Scopus (349) Google Scholar). Plat-E packaging cells (35Morita S. Kojima T. Kitamura T. Gene Ther. 2000; 7: 1063-1066Crossref PubMed Scopus (1377) Google Scholar) (kindly provided by T. Kitamura) were maintained under a humidified atmosphere of 5% CO2 and 95% air at 37 °C in Dulbecco's modified Eagle's medium supplemented with 10% FBS, puromycin (1 μg/ml) (Sigma), blasticidin (10 μg/ml) (Invitrogen), penicillin (100 units/ml), and streptomycin (100 μg/ml). Cells (∼2 × 106) were transiently transfected with 3 μg of pMX-puro vectors with the use of FuGENE 6. Fresh medium (Dulbecco's modified Eagle's medium supplemented with 10% FBS) was added to the cells 16 h after transfection, and supernatants (6 ml) were harvested after incubation for an additional 24 h. Jurkat cells expressing the ecotropic receptor (J.EcoR cells) were kindly supplied by T. Saito (Chiba University, Chiba, Japan) (36Yamasaki S. Nishida K. Hibi M. Sakuma M. Shiina R. Takeuchi A. Ohnishi H. Hirano T. Saito T. J. Biol. Chem. 2001; 276: 45175-45183Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). Expression of EcoR in human cells confers susceptibility to infection by the pMX-puro-derived retroviruses, which normally infect only rodent cells (36Yamasaki S. Nishida K. Hibi M. Sakuma M. Shiina R. Takeuchi A. Ohnishi H. Hirano T. Saito T. J. Biol. Chem. 2001; 276: 45175-45183Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). Parental J.EcoR cells were infected with each retrovirus-containing culture supernatant supplemented with Polybrene (10 μg/ml) (hexadimethren bromide, Sigma). The culture medium was refreshed 24 h after infection and was replaced by serum-supplemented RPMI 1640 containing puromycin (2 μg/ml) after incubation for an additional 24 h. Colonies were then isolated after 14 days. Several cell lines expressing SAP-1-WT or SAP-1-C/S were identified by immunoblot analysis of cell lysates with pAbs to SAP-1. Assay of PTP Activity—The PTP activity of SAP-1 immunoprecipitated from Jurkat cells with the mAb 3G5 was assayed with p-nitrophenyl phosphate (pNPP) as a substrate as described previously (26Matozaki 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). In brief, J.EcoR cells (∼1 × 107) stably expressing SAP-1-WT or SAP-1-C/S were lysed on ice in 500 μl of ice-cold lysis buffer containing 1 mm phenylmethylsulfonyl fluoride and aprotinin (10 μg/ml). Postnuclear cell lysates were subjected to immunoprecipitation with 2 μg of mAb 3G5 prebound to protein G-Sepharose beads, after which the beads were washed twice with 1 ml of WG buffer and twice with 1 ml of PTP assay buffer (40 mm Mes-NaOH (pH 5.0), 1.6 mm dithiothreitol) before incubation for 30 min at 30 °C with 200 μl of PTP assay buffer containing 25 mm pNPP. The reaction was terminated by addition of 200 μl of 1 m NaOH, and absorbance at 410 nm was measured. Duplicate samples were subjected to immunoblot analysis with pAbs to SAP-1 to determine the amount of SAP-1 protein in the immunoprecipitates. In Vitro Dephosphorylation Assay—Jurkat cells were stimulated with pervanadate (100 μm Na3VO4, 10 μm H2O2 in phosphate-buffered saline) for 10 min at 37 °C, after which postnuclear cell lysates were prepared and subjected to immunoprecipitation with pAbs to Lck as described above. The resulting precipitates were washed three times with ice-cold dephosphorylation buffer (100 mm Hepes-NaOH (pH 7.6), 150 mm NaCl, 2 mm dithiothreitol, 2 mm EDTA). A GST fusion protein of the catalytically inactive mutant SAP-1-C/S was generated and purified as previously described (27Noguchi T. Tsuda M. Takeda H. Takada T. Inagaki K. Yamao T. Fukunaga K. Matozaki T. Kasuga M. J. Biol. Chem. 2001; 276: 15216-15224Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). Immunoprecipitates were incubated with 20 μg of GST-SAP-1-WT, GST-SAP-1-C/S, or GST in 400 μl of dephosphorylation buffer first for 1 h at 4 °C and then for 30 min at 30 °C. Reaction mixtures were then subjected to immunoblot analysis with pAbs to phosphorylated Tyr416 of Src. CD69 Expression—For the assay of CD69 expression, Jurkat cells (1 × 106) in 24-well plates were stimulated for 16 h either with immobilized OKT3 or with the combination of 1 μm phorbol 12-myristate 13-acetate (PMA) and 0.5 μm ionomycin. Cells were then stained with an FITC-conjugated mAb to CD69 and analyzed by flow cytometry with an Epics XL instrument (Beckman Coulter). Cell Migration Assay—Cell migration was assayed with a Transwell apparatus (Corning) as described previously (37Weber K.S.C. Ostermann G. Zernecke A. Schroder A. Klickstein L.B. Weber C. Mol. Biol. Cell. 2001; 12: 3074-3086Crossref PubMed Scopus (43) Google Scholar). In brief, the cell suspension (∼2 × 106 cells in 100 μl) was transferred to a polycarbonate filter (pore size, 8 μm; Corning) in the upper compartment of the apparatus, and 500 μl of culture medium were placed in the lower compartment. The apparatus was then placed for 3 h at 37 °C in a humidified incubator containing 5% CO2. The number of cells that had migrated into the lower compartment was then counted in triplicate with a hemocytometer. Each experiment was performed in triplicate wells. Quantitative Image Analysis—Intensity of an immunoblot band was determined by densitometric analysis that was performed using NIH Image version 1.62. Interaction of the Cytoplasmic Region of SAP-1 with Lck— To identify proteins that interact with the cytoplasmic region of SAP-1, we screened a human spleen cDNA library by the yeast two-hybrid method with this region of SAP-1 (residues 778–1117) as the bait (Fig. 1A). Nine positive clones were obtained, among which three contained Lck cDNA and one contained a cDNA for CrkII, an adapter protein that is tyrosine-phosphorylated by Src family PTKs (38Matsuda M. Tanaka S. Nagata S. Kojima A. Kurata T. Shibuya M. Mol. Cell Biol. 1992; 12: 3482-3489Crossref PubMed Scopus (247) Google Scholar). Among the Lck clones, one (clone 9) contained a full-length cDNA whereas the other two (one of which is clone 15) encoded a COOH-terminal region of Lck (amino acids 154–509) containing a portion of the SH2 domain and the kinase domain (Fig. 1B). We further analyzed the precise region of Lck that was responsible for binding to the cytoplasmic region of SAP-1 by yeast two-hybrid analysis. We found that the region of Lck comprising residues 154–242 bound to the cytoplasmic region of SAP-1, whereas the region comprising amino acids 243–509 did not, suggesting that the middle portion of Lck (residues 154–242) is responsible, at least in part, for binding to SAP-1 (Fig. 1B). We next examined whether the cytoplasmic region of SAP-1 interacts with Lck directly in vitro. Lysates of human Jurkat T cells, which express endogenous Lck, were incubated with an immobilized GST fusion protein containing the cytoplasmic region of SAP-1 (GST-SAP-1-WT) or with GST alone. Lck specifically bound to GST-SAP-1-WT but not to GST (Fig. 2A). We then examined the binding of Lck to SAP-1 in COS-7 cells transfected with expression vectors for full-length Lck and the Myc epitope-tagged cytoplasmic region of SA
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