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Originally published In Press as doi:10.1074/jbc.M300648200 on July 1, 2003

J. Biol. Chem., Vol. 278, Issue 37, 34854-34863, September 12, 2003
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Interaction of SAP-1, a Transmembrane-type Protein-tyrosine Phosphatase, with the Tyrosine Kinase Lck

ROLES IN REGULATION OF T CELL FUNCTION*

Tomokazu Ito {ddagger}, Hideki Okazawa {ddagger}, Koji Maruyama §, Kyoko Tomizawa {ddagger}, Sei-ichiro Motegi {ddagger}, Hiroshi Ohnishi {ddagger}, Hiroyuki Kuwano ¶, Atsushi Kosugi § and Takashi Matozaki {ddagger} ||

From the {ddagger}Biosignal Research Center, Institute for Molecular and Cellular Regulation, Gunma University, 3-39-15 Showa-Machi, Maebashi, Gunma 371-8512, the §School of Allied Health Sciences, Faculty of Medicine, Osaka University, 1-7 Yamada-oka, Suita, Osaka 565-0871, and the Department of General Surgical Science (Surgery I), Gunma University Graduate School of Medicine, 3-39-22 Showa-Machi, Maebashi 371-8511, Japan

Received for publication, January 21, 2003 , and in revised form, June 25, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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 p62dok, 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.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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 (1, 2). The balance between protein-tyrosine phosphorylation and dephosphorylation is precisely determined by the action of protein-tyrosine kinases (PTKs)1 and protein-tyrosine phosphatases (PTPs) (3–6), although the molecular mechanisms by which activities of these enzymes are coordinately regulated remain largely unknown.

Protein-tyrosine phosphorylation plays central roles in signal transduction by the T cell antigen receptor (TCR) (7, 8). The earliest biochemical events known to be elicited by engagement of the TCR are activation of the Src family PTKs Lck and Fyn (9, 10). 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 (11). Activated Lck or Fyn mediates the phosphorylation of CD3 and the {zeta} chain, which are the signal-transducing subunits of the TCR (7, 8). 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 (12). 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-{gamma} (7, 8, 13).

In contrast to protein-tyrosine phosphorylation, the role of protein-tyrosine dephosphorylation in TCR-mediated signal transduction has been only partially resolved (14). SHP-1, a cytoplasmic PTP that contains two SH2 domains, negatively regulates the TCR-mediated signaling pathway (15, 16), and its localization in membrane rafts is required for such regulation (17). In addition, PEP and PTP-PEST, which are related cytoplasmic PTPs, also negatively regulate TCR-mediated signaling (18, 19). Whereas PEP appears to cooperate with Csk to inhibit TCR signaling, PTP-PEST dephosphorylates Shc, p130Cas, Pyk2, and focal adhesion kinase (7, 8, 14). The receptor-like PTP CD148, also known as DEP-1 (20, 21), negatively regulates the TCR-mediated signaling pathway by catalyzing the dephosphorylation of LAT (22–24). In contrast, CD45, another receptor-like PTP, dephosphorylates Tyr505 of Lck and thereby increases its activity and promotes TCR signaling (25). 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 (26). 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 (26). A "substrate-trapping" approach identified p130Cas, a prominent focal adhesion-associated component of the integrin signaling pathway, as a likely physiological substrate of SAP-1 (27). 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 (27), 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 (27, 28). Although most of these observations were made with cultured fibroblasts, together with the reduced expression of SAP-1 in advanced cancer (29), 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.2 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.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Antibodies—Rabbit polyclonal antibodies (pAbs) to SAP-1 (26), a mouse monoclonal antibody (mAb) (3G5) to SAP-1 (27), and rabbit pAbs to p62dok (30) 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 (31) 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 {zeta} 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 (27) 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 15{Delta}cat) 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 15{Delta}cat 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 (32), 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 (33). 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 {beta}-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 {beta}-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 {beta}-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 x 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 x 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 x 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 x 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 (26). Jurkat cells (~1 x 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 x 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 (27), 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 (34). Plat-E packaging cells (35) (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 x 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) (36). Expression of EcoR in human cells confers susceptibility to infection by the pMX-puro-derived retroviruses, which normally infect only rodent cells (36). 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 (26). In brief, J.EcoR cells (~1 x 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 (27). 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 x 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 (37). In brief, the cell suspension (~2 x 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.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
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 (38). 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).



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  		FIG. 1.
Yeast two-hybrid screening for molecules that interact with the cytoplasmic region of SAP-1. A, the domain structure of SAP-1 and the region of SAP-1 used as a bait. Numbers indicate amino acid residues. FNs, fibronectin type III-like domains; TM, transmembrane domain. B, domain structure of human Lck indicating portions of the protein encoded either by clones obtained by yeast two-hybrid screening (clones 9 and 15) or by deletion mutants (Lck-cat, clone 15{Delta}cat) used for determination of the region of Lck responsible for the interaction with SAP-1 by two-hybrid analysis. Numbers indicate NH2- and COOH-terminal residue positions of each domain of Lck. L40 yeast cells containing HIS3 and LacZ reporter genes were cotransformed with plasmids encoding constructs consisting of the DNA binding domain of LexA fused to the cytoplasmic region of SAP-1 and of the trans-activation domain of GAL4 fused to either full-length Lck or indicated Lck mutants. Transformed cells that grew on histidine-deficient medium (–His) within 3 days were scored as positive. The {beta}-galactosidase ({beta}-gal) activity of each clone was tested by filter assay and scored as positive (blue) or negative (white) after incubation for 2 h. Relative levels of growth on histidine-deficient medium and of {beta}-galactosidase activity are indicated as +, ++, or +++.

 

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 SAP-1. Immunoblot analysis revealed that immunoprecipitates prepared from the transfected cells with a mAb to Lck contained the Myc epitope-tagged cytoplasmic region of SAP-1 (Fig. 2B). We next determined whether SAP-1 binds Lck in Jurkat T cells. Reverse transcription-PCR analysis revealed the presence of SAP-1 mRNA in Jurkat cells (data not shown). SAP-1 was immunoprecipitated from Jurkat cell lysates with the mAb 3G5 to SAP-1, and the resulting precipitates were subjected to immunoblot with the mAb 154A7 to SAP-1 or pAbs to Lck. Immunoblot analysis with the mAb 154A7 revealed the expression of SAP-1 protein in the lysates of Jurkat cells (Fig. 2C, upper panel). Moreover, immunoblot of immunoprecipitates from Jurkat cells with the mAb 3G5 to SAP-1 revealed the interaction of SAP-1 with Lck (Fig. 2C, lower panel). These results thus suggest that the cytoplasmic region of SAP-1 binds Lck directly both in vitro and in vivo.



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  		FIG. 2.
Interaction of the cytoplasmic region of SAP-1 with Lck in vitro (A) and in vivo (B and C). A, GST and a GST fusion protein containing the cytoplasmic region of SAP-1 (GST-SAP-1-WT) were expressed in bacteria, purified with the use of glutathione-Sepharose beads, and subjected to SDS-polyacrylamide gel electrophoresis and staining with Coomassie Blue (right panel). The position of each recombinant protein is indicated on the right, and those of molecular size standards are shown on the left. Lysates prepared from Jurkat cells were incubated for 5 h at 4 °C with GST-SAP-1-WT or GST immobilized on glutathione-Sepharose beads, after which bead-bound proteins were subjected to immunoblot analysis with pAbs to Lck ({alpha}Lck)(left panel). Cell lysates were also subjected directly to immunoblot analysis with pAbs to Lck as a positive control. The position of Lck is indicated. B, COS-7 cells were transiently transfected both with 2 µg of pCIneo containing cDNA for the cytoplasmic region of SAP-1 tagged with the Myc epitope and with 2 µg of pCLS containing Lck cDNA. Forty-eight hours after transfection, cell lysates were prepared and subjected to immunoprecipitation with a mAb to Lck or with control mouse immunoglobulin G, as indicated. The resulting precipitates were then subjected to immunoblot analysis with pAbs to SAP-1 ({alpha}SAP-1) (left panel). Duplicate samples were subjected to immunoblot analysis with pAbs to Lck to verify the amount of Lck in the immunoprecipitates (right panel). Cell lysates were also subjected directly to immunoblot analysis with pAbs to SAP-1 or to Lck. Positions of the Myc epitope-tagged cytoplasmic region of SAP-1 (SAP-1-cyto) and of Lck are indicated. All results are representative of three independent experiments. C, Jurkat cell lysates were prepared and subjected to immunoprecipitation with the mAb 3G5 to SAP-1 or with the mAb 9E10 as a control, as indicated. The resulting precipitates were then subjected to immunoblot analysis with the mAb 154A7 to SAP-1 (upper panel) or with pAbs to Lck (lower panel). The positions of Lck and SAP-1 in Jurkat cells are indicated.

 

Generation of Jurkat Cell Lines Overexpressing SAP-1 and Effects of SAP-1 Overexpression on the TCR-mediated Activation of Lck—Stimulation of the TCR induces the activation of Lck by autophosphorylation on Tyr394 (39). Given that our data showed that Lck binds to the cytoplasmic region of SAP-1, we next determined whether SAP-1 regulates the TCR-mediated activation of Lck using Jurkat T cell line. To generate Jurkat cell lines that overexpress either wild-type SAP-1 (SAP-1-WT) or the catalytically inactive mutant SAP-1-C/S (27), we subjected cells expressing EcoR (J.EcoR cells) to retrovirus-mediated transfection (36). We obtained several independent cell lines that expressed either SAP-1-WT (J-SAP-1-WT cells) or SAP-1-C/S (J-SAP-1-C/S cells). A J-SAP-1-WT cell line (clone A4) and a J-SAP-1-C/S cell line (clone D2) were chosen for further characterization because they expressed the recombinant proteins at a high level (Fig. 3A). The level of endogenous Lck was not changed by the expression of SAP-1-WT or that of SAP-1-C/S (Fig. 3A). These cell lines were subjected to immunoprecipitation with the mAb 3G5 to SAP-1, and the resulting precipitates were assayed for PTP activity. The PTP activity derived from J-SAP-1-WT cells was markedly greater than that from J-SAP-1-C/S cells, the latter of which was similar to that derived from mock-transfected Jurkat cells (Fig. 3B). Moreover, no significant change of the PTP activity by TCR stimulation was observed in J-SAP-1-WT cells (Fig. 3B). To determine the interaction of SAP-1 with Lck in J-SAP-1-WT cells, lysates prepared from unstimulated J-SAP-1-WT cells were subjected to immunoprecipitation with either pAbs to Lck or the mAb 3G5 to SAP-1 (Fig. 3C). The immunoblotting of the precipitate with either the mAb 154A7 to SAP-1 or pAbs to Lck revealed that SAP-1 interacted with Lck in unstimulated J-SAP-1-WT cells (Fig. 3C). Furthermore, the extent of interaction of SAP-1 with Lck was not markedly changed by TCR stimulation (Fig. 3D).



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  		FIG. 3.
Establishment of Jurkat cell lines stably expressing wild-type SAP-1 or the catalytically inactive mutant SAP-1-C/S and the interaction of SAP-1 with Lck. A, J.EcoR cells were infected with culture supernatants obtained from Plat-E cells that had been transfected either with retrovirus vectors encoding SAP-1-WT or SAP-1-C/S or with the empty vector alone (Mock). Lysates prepared from resulting Jurkat cell lines stably expressing SAP-1-WT (J-SAP-1-WT cells) or SAP-1-C/S (J-SAP-1-C/S cells) was subjected to immunoblot analysis with the mAb to SAP-1 (upper panel) or with pAbs to Lck (lower panel). B, mock-transfected or SAP-1-overexpressing Jurkat (J-SAP-1-WT or J-SAP-1-C/S) cells were either left untreated (–) or stimulated with OKT3 and cross-linking antibodies (+) as described under "Experimental Procedures." Cell lysates were then subjected to immunoprecipitation with the mAb 3G5 to SAP-1, and the resulting precipitates were assayed for PTP activity with pNPP as substrate. Data are expressed as absorbance units at 410 nm and are means of triplicates from a representative experiment. C, J-SAP-1-WT cell lysates were prepared and subjected to immunoprecipitation with pAbs to Lck or with control rabbit immunoglobulin G (left panels), or with the mAb 3G5 to SAP-1 or with control mouse immunoglobulin G (right panels), as indicated. The resulting precipitates were then subjected to immunoblot analysis with the mAb 154 A7 to SAP-1 (upper panels). Same samples were subjected to immunoblot analysis with a mAb to Lck (left lower panel) or with pAbs to Lck (right lower panel). Cell lysates were also subjected directly to immunoblot analysis with the mAb to SAP-1 or pAbs to Lck. Positions of the SAP-1 and Lck are indicated. D, J-SAP-1-WT cell lysates were prepared in the absence (–) or the presence (+) of OKT3 stimulation and subjected to immunoprecipitation with pAbs to Lck. The resulting precipitates were then subjected to immunoblot analysis with the mAb 154A7 to SAP-1 (upper panel). Same samples were subjected to immunoblot analysis with a mAb to Lck (lower panel).

 

We determined the activation state of Lck by immunoblot analysis with pAbs specific for autophosphorylated tyrosine residues of Src family PTKs including Lck; autophosphorylation of these residues results in the activation of these PTKs (40). Lck was thus immunoprecipitated from Jurkat cell lysates, and the resulting precipitates were subjected to such immunoblot analysis. The autophosphorylation of Lck was observed even in unstimulated Jurkat cells, and a small increase of this parameter was observed in TCR-stimulated mock-transfected cells (Fig. 4A). This result was consistent with the previous observation (24). In contrast, the autophosphorylation of Lck was markedly reduced in both unstimulated and TCR-stimulated J-SAP-1-WT cells (Fig. 4A). Overexpression of SAP-1-C/S did not change both basal and TCR-stimulated Lck activities. The PTP activity of SAP-1 thus appeared to be required for the inhibition of TCR-mediated Lck activation by this protein.



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  		FIG. 4.
Effect of SAP-1 overexpression on the activation of Lck in response to TCR stimulation (A) and dephosphorylation of Lck by SAP-1 in vitro (B). A, mock-transfected or SAP-1-overexpressing Jurkat (J-SAP-1-WT or J-SAP-1-C/S) cells were either left untreated (–) or stimulated with OKT3 and cross-linking antibodies (+) as described under "Experimental Procedures." Cell lysates were then subjected to immunoprecipitation with mAbs to Lck, and the resulting precipitates were subjected to immunoblot analysis either with pAbs to the autophosphorylation sites of Src family kinases ({alpha}pSrc) or with pAbs to Lck, as indicated. B, lysates of pervanadate-stimulated Jurkat cells were subjected to immunoprecipitation with mAbs to Lck, and the resulting precipitates were incubated first for 1 h at 4 °C and then for 30 min at 30 °C with GST-SAP-1-WT, GST-SAP-1-C/S, or GST. Reaction mixtures were then subjected to immunoblot analysis either with pAbs to the autophosphorylation sites of Src family kinases or with pAbs to Lck. Densitometric analysis was performed, ratio of the band intensity of pSrc to that of Lck for each lane was calculated, and results were expressed as a percentage of the value with unstimulated mock-transfected cells (A) or of that with GTS alone (B). All results are representative of three independent experiments.

 

To examine whether SAP-1 directly dephosphorylates Lck in vitro, we immunoprecipitated Lck from lysates of pervanadate-stimulated Jurkat cells. The resulting precipitates were then incubated either with GST alone or with GST fusion proteins of wild-type SAP-1 or SAP-1-C/S, after which the reaction mixtures were subjected to immunoblot analysis with pAbs to the autophosphorylated tyrosine residues of Src family PTKs. Incubation with GST-SAP-1-WT, but not with either GST or GST-SAP-1-C/S, completely abolished the autophosphorylation of Lck (Fig. 4B), suggesting that autophosphorylated Lck is a direct substrate of SAP-1.

Effects of SAP-1 Overexpression on MAP Kinase Activation and CD69 Expression Induced by TCR Stimulation—Given that expression of SAP-1-WT markedly inhibited TCR-induced Lck activation and that activated Lck mediates signaling that leads sequentially to the activation of MAP kinase and upregulation of cell surface expression of CD69 (7, 41), we next determined effects of overexpression of SAP-1 on these latter two manifestations of TCR stimulation. Immunoblot analysis with pAbs specific for activated MAP kinase revealed that TCR stimulation induced the activation of MAP kinase in mock-transfected Jurkat cells and that this response was markedly inhibited in J-SAP-1-WT cells but not in J-SAP-1-C/S cells (Fig. 5A). Similarly, flow cytometry with a mAb to CD69 revealed that expression of SAP-1-WT, but not that of SAP-1-C/S, greatly inhibited the increase in surface expression of CD69 induced by TCR stimulation (Fig. 5B). The combination of activation of protein kinase C by PMA and Ca2+ mobilization by ionomycin also up-regulates the surface expression of CD69 in a manner independent of TCR-mediated early tyrosine phosphorylation events (41). The increase in the surface expression of CD69 induced by the combination of PMA and ionomycin in J-SAP-1-WT cells was similar to that apparent in mock-transfected Jurkat cells or in J-SAP-1-C/S cells (Fig. 5B), suggesting that overexpression of SAP-1 affects upstream events in the signaling pathway responsible for TCR-induced activation of MAP kinase.



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  		FIG. 5.
Effects of SAP-1 overexpression on MAP kinase activation (A) and CD69 expression (B) induced by TCR stimulation. A, mock-transfected or SAP-1-overexpressing Jurkat (J-SAP-1-WT or J-SAP-1-C/S) cells were either left untreated or stimulated with OKT3 and cross-linking antibodies, after which cell lysates were subjected to immunoblot analysis with pAbs specific for active MAP kinase ({alpha}pMAPK) or with pAbs to MAP kinase ({alpha}MAPK), as indicated. Positions of p44 and p42 isoforms of MAP kinase are indicated. B, mock-transfected or SAP-1-overexpressing Jurkat cells were stimulated for 16 h either with immobilized OKT3 (left panels) or with 1 µM PMA plus 0.5 µM ionomycin (PMA + Iono) (right panels). Stimulated (continuous trace) and unstimulated (dotted trace) cells were then stained with FITC-conjugated mAbs to CD69 and analyzed by flow cytometry. All results are representative of three independent experiments.

 

Effects of SAP-1 Overexpression on Tyrosine Phosphorylation of TCR{zeta}, ZAP-70, and LAT Induced by TCR Stimulation— Activation of either Lck or Fyn results in the tyrosine phosphorylation of immunoreceptor tyrosine-based activation motifs within the TCR {zeta} chain (7–10). ZAP-70 then binds to tyrosine-phosphorylated TCR{zeta} through its SH2 domains (12), thereby tyrosine-phosphorylated by Lck, and activated to catalyze the tyrosine phosphorylation of LAT (13). We therefore examined whether overexpression of SAP-1 affected the tyrosine phosphorylation of TCR{zeta}. TCR stimulation resulted in the tyrosine phosphorylation of TCR{zeta} in mock-transfected Jurkat cells (Fig. 6A). The TCR-stimulated tyrosine phosphorylation of this protein was not substantially affected by expression of SAP-1-WT or SAP-1-C/S (Fig. 6A). In contrast, the extent of tyrosine phosphorylation of ZAP-70 induced by TCR stimulation was reduced in J-SAP-1-WT cells, compared with that apparent in mock-transfected cells and J-SAP-1-C/S cells (Fig. 6B). Furthermore, the extent of tyrosine phosphorylation of LAT induced by TCR stimulation was markedly reduced in J-SAP-1-WT cells, compared with that apparent in mock-transfected cells and J-SAP-1-C/S cells (Fig. 6C).



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  		FIG. 6.
Effects of overexpression of SAP-1 on the tyrosine phosphorylation of TCR{zeta}, ZAP-70, and LAT induced by TCR stimulation. Mock-transfected or SAP-1-overexpressing Jurkat (J-SAP-1-WT or J-SAP-1-C/S) cells were left untreated or stimulated with OKT3 and cross-linking antibodies, after which cell lysates were subjected to immunoprecipitation with mAbs to TCR{zeta} ({alpha}TCR{zeta}) (A), pAbs to ZAP-70 ({alpha}ZAP-70) (B), or pAbs to LAT ({alpha}LAT) (C). Resulting precipitates were then subjected to immunoblot analysis either with horseradish peroxidase-conjugated mAb PY20 to phosphotyrosine ({alpha}PY) (A and C, upper panels) or with mAb 4G10 to phosphotyrosine ({alpha}PY) (B, upper panel). Duplicate samples were subjected to immunoblot analysis with mAbs to TCR{zeta} (A, lower panel), pAbs to ZAP-70 (B, lower panel), or pAbs to LAT (C, lower panel. Densitometric analysis was performed, ratio of the band intensity of PY to that of TCR{zeta}/ZAP-70/LAT for each lane was calculated, and results were expressed as a percentage of the value with OKT3-stimulated mock-transfected cells. All results are representative of three independent experiments.

 

Effects of SAP-1 Overexpression on Basal and CD2-induced Tyrosine Phosphorylation of p62dok—Given that overexpression of SAP-1 reduced the autophosphorylation of Lck and the tyrosine phosphorylation of LAT induced by TCR stimulation, we next examined the effect of SAP-1 on overall tyrosine phosphorylation in lysates prepared from unstimulated or TCR-stimulated Jurkat cells. Several tyrosine-phosphorylated proteins were detected even in unstimulated mock-transfected Jurkat cells, whereas the tyrosine phosphorylation of various proteins, including 150-, 120-, and 40-kDa molecules, was increased in response to TCR stimulation in these cells (data not shown). Among these tyrosine-phosphorylated proteins, the phosphorylation of a 60-kDa protein was markedly reduced in J-SAP-1-WT cells, in the absence or presence of TCR stimulation, compared with that apparent in mock-transfected cells or J-SAP-1-C/S cells (data not shown). Given that p62dok (42, 43) is a putative substrate of SAP-1 (27), we examined whether the 60-kDa tyrosine-phosphorylated protein might be p62dok. Immunoprecipitation of p62dok with specific antibodies revealed that the extent of its tyrosine phosphorylation, in the absence and presence of TCR stimulation, was markedly reduced in J-SAP-1-WT cells compared with that apparent in mock-transfected cells (Fig. 7A). The tyrosine phosphorylation of p62dok is increased by CD2 stimulation, and Lck has been implicated in this phosphorylation event (44, 45). We therefore examined the effect of SAP-1 overexpression on the tyrosine phosphorylation of p62dok induced by CD2 stimulation. Stimulation of CD2 resulted in a small increase in the extent of tyrosine phosphorylation of p62dok in mock-transfected Jurkat cells. Both the basal and CD2-stimulated tyrosine phosphorylation of p62dok were markedly reduced in J-SAP-1-WT cells (Fig. 7B). The tyrosine phosphorylation of p62dok was slightly but consistently inhibited in unstimulated or TCR-stimulated J-SAP-1-C/S cells compared with that apparent in mock-transfected cells (Fig. 7A). Such inhibition was also observed in CD2-stimulated J-SAP-1-C/S cells compared with that apparent in mock-transfected cells (Fig. 7B).



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  		FIG. 7.
Effects of overexpression of SAP-1 on the tyrosine phosphorylation of p62dok. Mock-transfected or SAP-1-overexpressing Jurkat (J-SAP-1-WT or J-SAP-1-C/S) cells were left untreated or stimulated either with OKT3 and cross-linking antibodies for 5 min (A) or with mAbs (anti-T112 and anti-T113) to CD2 (1:100 dilution) for 10 min (B). Cell lysates were then prepared and subjected to immunoprecipitation with pAbs to p62dok ({alpha}Dok), and the resulting precipitates were subjected to immunoblot analysis with horseradish peroxidase-conjugated mAb PY20 to phosphotyrosine. Duplicate samples were subjected to immunoblot analysis with pAbs to p62dok. Densitometric analysis was performed, ratio of the band intensity of PY to that of p62dok for each lane was calculated, and results were expressed as a percentage of the value with unstimulated mock-transfected cells. All results are representative of three independent experiments.

 

Effect of Overexpression of SAP-1 on Jurkat Cell Migration—Tyrosine phosphorylation of p62dok is thought to contribute to the positive regulation of cell migration in fibroblasts, melanoma cells, and leukemia cells, and this regulation also appears to require either the Ras GTPase-activating protein or the adapter protein Nck (30, 46, 47). Previous studies also indicate that Lck and the activation of MAP kinase are important for the positive regulation of T cell migration (37, 48, 49). We therefore finally examined the effect of overexpression of SAP-1 on Jurkat cell migration with the use of a Transwell apparatus. The migratory activity of J-SAP-1-WT cells was markedly reduced compared with that of mock-transfected cells, whereas that of J-SAP-1-C/S cells appeared slightly reduced but this effect was not significant (Fig. 8).



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  		FIG. 8.
Effect of overexpression of SAP-1 on Jurkat cell migration. Mock-transfected or SAP-1-overexpressing Jurkat (J-SAP-1-WT or J-SAP-1-C/S) cells (2 x 106) were applied to polycarbonate filters in upper compartments of a Transwell apparatus. After incubation for 3 h at 37 °C, the number of cells that had migrated into lower compartments was determined. Data are means ± S.E. of triplicates from an experiment that was performed three times with similar results. *, p < 0.05 for the indicated comparison; NS, not significant (p > 0.05) (analysis of variance and Fisher's PLSD (protected least significance difference) test).

 


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
We have demonstrated that the cytoplasmic region of SAP-1, a transmembrane-type PTP, directly binds Lck, a PTK that has previously been shown to interact with the cytoplasmic tails of CD4 and CD8 (50, 51). Whereas the NH2-terminal region of Lck (amino acids 1–67) mediates its binding to CD4 or CD8, we have now shown that a middle portion of this protein (amino acids 154–242, corresponding to part of the SH2 domain and the region between the SH2 and catalytic domains) is responsible, at least in part, for its association with SAP-1. Lck thus appears to associate with multiple signaling molecules via distinct molecular regions. The cytoplasmic regions of several transmembrane-type PTPs function as molecular scaffolds. For example, the Drosophila transmembrane-type PTP Dlar binds the cytoplasmic PTK Abl as well as the tyrosine-phosphorylated protein Ena, and the formation of this complex plays an important role in axonal guidance (52). RPTPµ, another transmembrane-type PTP, forms a complex with p120ctn (53), whereas RPTP{kappa} interacts with {beta}-or {gamma}-catenin (54). In addition, the transmembrane-type PTP CD148 (DEP-1) interacts with p120ctn (55). These transmembrane-type PTPs are thus implicated in the regulation of cadherin-mediated cell adhesion (52–55). In addition to Lck, our yeast two-hybrid screening analysis revealed that SAP-1 also binds CrkII (38), an adapter protein that is tyrosine-phosphorylated by Src family PTKs; further analysis is required to determine whether this interaction is physiologically relevant. Thus, like other transmembrane-type PTPs, SAP-1 may function not only as a PTP but also as a scaffolding protein.

We have also demonstrated that overexpression of SAP-1 inhibits both the basal activity of Lck and the activation of this PTK in response to TCR stimulation. Given that SAP-1 mediated the dephosphorylation of autophosphorylated Lck in vitro, it might negatively regulate the kinase activity of Lck through direct dephosphorylation of this PTK. Consistent with this notion, overexpression of SAP-1 markedly reduced the activation of MAP kinase and the subsequent surface expression of CD69 induced by TCR stimulation. Moreover, we also found that the TCR-induced tyrosine phosphorylation of ZAP-70 and that of LAT were reduced by SAP-1 overexpression. In contrast, the tyrosine phosphorylation of TCR{zeta} induced by TCR stimulation was not affected by overexpression of SAP-1. Although Lck plays a primary role in the tyrosine phosphorylation of TCR{zeta} induced by TCR stimulation (9), Fyn also contributes to this process (10). Fyn might thus be responsible for the TCR-induced tyrosine phosphorylation of TCR{zeta} apparent in J-SAP-1-WT cells. The marked reduction in the tyrosine phosphorylation of LAT apparent in these cells might be attributable either to down-regulation of Lck activity or to direct dephosphorylation of LAT by SAP-1. In either case, it is likely that SAP-1 overexpression inhibited the TCR-induced activation of MAP kinase and surface expression of CD69 through the dephosphorylation of LAT.

CD148 (DEP-1) negatively regulates TCR-mediated T cell responses (22–24). This transmembrane-type PTP contains 8–10 fibronectin type III-like domains in its extracellular region and a single PTP domain in its cytoplasmic region (20, 21). It is therefore structurally similar to SAP-1. The expression of CD148 in T cells is up-regulated in response to cell activation (22, 24). However, whereas this PTP directly dephosphorylates LAT, its overexpression does not inhibit the activation of Lck in response to TCR stimulation (24). Thus, despite their structural similarity and the fact that they both negatively regulate TCR-mediated T cell responses, SAP-1 and CD148 dephosphorylate distinct signaling molecules in the TCR signaling pathway.

Overexpression of SAP-1 inhibited both the basal and CD2-induced tyrosine phosphorylation of p62dok. Given that Lck is thought to be responsible for this effect of CD2 stimulation (43, 44), SAP-1 might reduce the tyrosine phosphorylation of p62dok through down-regulation of Lck activity. However, with the use of a substrate-trapping mutant of SAP-1, we have previously shown that p62dok is a putative substrate of SAP-1 (27). It is thus also possible that SAP-1 directly dephosphorylates p62dok in Jurkat cells. The overexpression of a catalytically inactive mutant of SAP-1 slightly inhibited the basal and OKT3- or CD2-stimulated tyrosine phosphorylation of p62dok. Although the mechanism underlying the inhibition remains unclear, the inhibition of this parameter by overexpression of SAP-1-WT may not depend entirely on the catalytic activity of SAP-1.

The physiological relevance of tyrosine phosphorylation of p62dok in T cells remains unknown, although putative roles in CD3- or CD2-mediated T cell signaling pathways have been proposed (44, 45, 56). We have now shown that overexpression of SAP-1 markedly reduced the migratory activity of Jurkat cells. Lymphocyte migration from the blood into tissues is an essential step in the immune response (57). Lck and the activation of MAP kinase are thought to be required for T cell migration (37, 48, 49). Tyrosine phosphorylation of p62dok as a result of its recruitment to a site near the plasma membrane also contributes to the positive regulation of cell migration in fibroblasts and melanoma cells (30, 46, 47). SAP-1 might thus negatively regulate the migratory activity of Jurkat cells by mediating the dephosphorylation of p62dok.


   FOOTNOTES
 
* This work was supported by a grant-in-aid for scientific research on priority areas (cancer), a grant-in-aid for scientific research (B), and a grant-in-aid for the 21st Century COE Program from the Ministry of Education, Culture, Sports, Science, and Technology of Japan; a grant from the Yamanouchi Foundation for Research on Metabolic Disorders; a grant from the Novartis Foundation (Japan) for the Promotion of Science; a grant from ONO Medical Research Foundation; a grant from the Cosmetology Research Foundation; a grant from Mitsui Life Social Welfare Foundation; and a grant from the Public Trust Haraguchi Memorial Cancer Research Fund. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

|| To whom correspondence should be addressed. Tel.: 81-27-220-8865; Fax: 81-27-220-8897; E-mail: matozaki{at}showa.gunma-u.ac.jp.

1 The 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. Back

2 H. Okazawa and T. Matozaki, unpublished data. Back


   ACKNOWLEDGMENTS
 
We thank Ellis L. Reinherz for providing mAbs to CD2; Yoshimi Takai for pBTM116HA and pCIneo-myc; Kunitada Shimotohno for human Lck cDNA; Sho Yamasaki and Takashi Saito for J.EcoR cells; Toshio Kitamura for pMX-puro and Plat-E cells; Tetsuya Noguchi for helpful discussion; and Akiko Hayashi, Hisae Kobayashi, and Kyoko Shimofure for technical assistance.



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