Interaction between Sam68 and Src family tyrosine kinases, Fyn and Lck, in T cell receptor signaling.

The Src family protein-tyrosine kinase, Fyn, is associated with the T cell receptor (TCR) and plays an important role in TCR-mediated signaling. We found that a human T cell leukemia virus type 1-infected T cell line, Hayai, overexpressed Fyn. To identify the molecules downstream of Fyn, we analyzed the tyrosine phosphorylation of cellular proteins in the cells. In Hayai, a 68-kDa protein was constitutively tyrosine-phosphorylated. The 68-kDa protein was coimmunoprecipitated with various signaling proteins such as phospholipase C γ1, the phosphatidylinositol 3-kinase p85 subunit, Grb2, SHP-1, Cbl, and Jak3, implying that the protein might function as an adapter. Purification and microsequencing of this protein revealed that it was the RNA-binding protein, Sam68 (Src associated in mitosis, 68 kDa). Sam68 was associated with the Src homology 2 and 3 domains of Fyn and also those of another Src family kinase, Lck. CD3 cross-linking induced tyrosine phosphorylation of Sam68 in uninfected T cells. These data suggest that Sam68 participates in the signal transduction pathway downstream of TCR-coupled Src family kinases Fyn and Lck in lymphocytes, that is not only in the mitotic pathway downstream of c-Src in fibroblasts.

Antigen engagement of the T cell receptor (TCR) 1 induces a signal transduction cascade that leads to the expression of a number of genes and activation of T cells. One of the earliest biological events after the ligation of ligands to their receptors is the activation of protein-tyrosine kinases after the activation of PLC␥1, PI 3-kinase, and the Ras pathway via the Shc-Grb2-SOS complex (1,2). The Src family protein-tyrosine kinases, Fyn (associated with TCR (3)) and Lck (associated with coreceptors CD4 and CD8), have been shown to be responsible for T cell activation (1).
Fyn is activated on engagement of the TCR (4,5). Many reports have suggested its important role in TCR-mediated signaling. Overexpression of Fyn in transgenic mice increases the level of intracellular calcium and the proliferative response in thymocytes (6). IL-2 production was increased by the overexpression of Fyn in a T cell hybridoma (7,8). Moreover, targeting of the fyn locus results in marked suppression of the proliferation signal, at least in single positive (CD4 ϩ CD8 Ϫ or CD4 Ϫ CD8 ϩ ) mature thymocytes (9,10). Src family kinases are supposed to phosphorylate the TCR chain that then recruits ZAP-70 and Shc (11)(12)(13)(14). Ligation of the SH3 domain of Fyn to the p85 subunit of PI 3-kinase induces activation of the PI 3-kinase (15). We have demonstrated that the tyrosine phosphorylation of PLC␥1, Vav, ZAP-70, mitogen-activated protein kinase (8), HS1 (16), and Cbl (17) is enhanced after TCR stimulation by overexpression of Fyn in a T cell hybridoma. However, molecules downstream of Fyn are not fully understood yet. It is important to identify them to understand TCR signaling further.
Human T cell leukemia virus type 1 (HTLV-1) is a retrovirus that can immortalize and transform human CD4 ϩ T cells (18 -21). Several reports have suggested that HTLV-1-infected T cells exhibit altered expression or activity of tyrosine kinases: the absence of the lymphocyte-specific protein-tyrosine kinase, Lck (22), and instead, the presence of Lyn, which is not expressed in normal T cells (23). Jak3 and the downstream signal transducers and activators of transcription (STAT), which interact with IL-2 receptor signaling (24), were shown to be constitutively activated in infected T cells (25). During a study on transformation by HTLV-1, we found that a HTLV-1-infected T cell line, Hayai, overexpressed Fyn more than 10 times compared with Jurkat T cells. To examine the consequence of Fyn overexpression, we analyzed the tyrosine phosphorylation in the cell line. In Hayai, a 68-kDa protein was predominantly tyrosine-phosphorylated. The 68-kDa protein was precipitated with the SH2 and SH3 domains of Fyn and also coimmunoprecipitated with various signal-transducing molecules such as SHP-1, PLC␥1, the PI 3-kinase p85 subunit, and Grb2. Purification and microsequencing of the protein revealed that it was Sam68. Sam68 is the RNA-binding protein that has been identified to be associated with c-Src and is phosphorylated during mitosis (26 -29). In uninfected T cells, Sam68 was also coprecipitated with the SH2 and SH3 domains of Lck. Our results suggest that in T cells, Sam68 may function as an adapter linking the TCR-coupled Fyn and Lck kinases to downstream signaling molecules. In addition, we further discuss the effect of hyperphosphorylation of Sam68 on the cell cycle. scribed (16). Antibodies to phosphotyrosine (Tyr(P), 4G10), Lyn (Lyn9) (30), the regulatory subunit of PI 3-kinase (p85) (30), and PLC␥1 (31) were generous gifts from Drs. H. Nariuchi, T. Yamamoto, Y. Fukui (University of Tokyo), and Y. Homma (Fukushima Medical School), respectively. Another monoclonal antibody to Tyr(P) (6D12) was obtained from Medical Biological Laboratories Co., Inc. (Nagoya, Japan).
Stimulation of Cells-CD3 cross-linking was performed as described (8). Briefly, cells were incubated with 10 g/ml NU-T3 for 30 min on ice, washed in medium twice, and then cross-linked with 10 g/ml goat anti-mouse IgG (Cappel, Organon Teknika Corp., West Chester, PA) for 2 min at 37°C. Pervanadate treatment was performed with 0.1 mM Na 3 VO 4 and 0.1 mM H 2 O 2 (32) for 10 min at 37°C.
Immunoprecipitation and Immunoblotting-Immunoprecipitation and immunoblotting were performed as described previously (16). Detection of immunoblotted proteins was performed using an ECL Western blotting detection set (Amersham, Buckinghamshire, United Kingdom). Preparation of glutathione S-transferase (GST) fusions and affinity precipitation of proteins with agarose-conjugated GST fusions were performed as described (16). The DNAs encoding GST-SH2 and -SH3 of Fyn were provided by H. Umemori (University of Tokyo). SH2 of Lck contains amino acid residues 120 -229 of murine Lck. SH3 of Lck contains residues 62-127 (33). For the SH2 mutant, Arg-154 was changed to Lys (12). For the SH3 mutant, Pro-112 was changed to Lys (33).
Purification and Microsequencing of p68 -Lysate from 1 ϫ 10 8 cells of Hayai was immunodepleted by incubation with anti-ZAP-70 antibodies coupled to Sepharose beads (16). The unbound fraction was then batch-absorbed to anti-Tyr(P) antibodies (6D12) immobilized with Protein G-Sepharose (Pharmacia Biotech Inc.). For purification, we used anti-Tyr(P) antibody 6D12 instead of 4G10 because the elution was more efficient using with the former (data not shown). Immune complexes were thoroughly washed with lysis buffer and then with phosphate-buffered saline and eluted by the addition of phenyl phosphate (final concentration, 50 mM) (34). Then, the eluate was separated by SDS-PAGE and transferred to polyvinylidene difluoride membrane (Applied Biosystems). The immobilized protein was reduced, S-carboxymethylated, followed by in situ digestion with Achromobacter protease I, and then subjected to reverse phase high performance liquid chromatography (Wakosil-II AR C18 300Å, Wako Pure Chemicals, Osaka, Japan). Amino acid sequencing was performed with a gas phase sequencer (model PPSQ-10, Shimadzu) as described (35).
p68 Was Constitutively Tyrosine-phosphorylated in Fyn-overexpressing Hayai Cells-To determine the consequence of overexpression of Fyn in Hayai, we examined the tyrosine phosphorylation of cellular proteins. Another HTLV-1-infected cell line, MT-2, and an uninfected cell line, Jurkat, were also examined as controls. Since the expression of CD3 was down-regulated in HTLV-1-infected cells (38), cells were incubated with pervanadate, a combination of vanadate and H 2 O 2 that mimics the effect of TCR ligation (32). Compared with Jurkat ( Fig. 2A, lane  1), the infected cell line, Hayai, showed enhanced tyrosine phosphorylation of several proteins of around 75-85, 68, and 60 kDa even before stimulation (lane 5), and phosphorylated proteins were further increased after stimulation (lane 6). The 60-kDa protein was Fyn itself, revealed by reblotting of the filter with anti-Fyn antibodies (data not shown). In another infected cell line, MT-2, constitutive phosphorylation was observed but not as prominently as in Hayai (lane 3).
p68 Was Coprecipitated with Various Signaling Molecules-Phosphorylated p68 was associated with Fyn-SH2 and SH3 (Fig. 2B) and coimmunoprecipitated with SHP-1 (Fig. 3); thus, we examined whether p68 was associated with other SH2-or SH3-containing proteins, PLC␥1, the PI 3-kinase regulatory subunit p85, and Grb2. Hayai was solubilized and subjected to immunoprecipitation with antibodies for the respective proteins (Fig. 4). Phosphorylated p68 was detected by 4G10 immunoblotting with immunoprecipitates of all of them (lanes 2-7). The coprecipitation with Grb2 was most prominent (lanes 6 and 7). In addition, since Jak3 was constitutively activated in HTLV-1-infected cells, it was of interest as to whether p68 was associated with Jak3. Furthermore, the association with Cbl (45) was also examined because Cbl was reported to form a complex with Src family kinases and signaling molecules after stimulation of T and B cells (17, 46 -52). Both proteins were coprecipitated with phosphorylated p68 (lanes 8 -11). p68 was not detected in the control precipitate (lane 1). These results suggest that p68 may function as an adapter for multiple proteins. Thus, we performed purification and microsequencing of the protein. p68 Was Sam68, a Mitotic Target of c-Src-Hayai cells were solubilized, and p68 was affinity-purified on an anti-Tyr(P) column. Microsequencing revealed that the sequences of two peptides derived from p68 were identical to those of Sam68 (26,27), a RNA-binding protein with proline-and tyrosine-rich regions that was identical to the protein previously called p21 ras GTPase-activating protein (GAP)-associated p62 (28,29) (Fig.  5A). For confirmation, cell lysates with or without stimulation were subjected to anti-Sam68 immunoprecipitation and 4G10 immunoblotting (Fig. 5B, upper panel). Sam68 showed increased tyrosine phosphorylation after stimulation in Jurkat and MT-2 (lanes 2 and 4). In contrast, Sam68 showed significant phosphorylation regardless of stimulation in Hayai (lanes 5 and 6). The level of expression of Sam68 was similar in these cells as judged by reblotting of the same filter with anti-Sam68 antibodies (lower panel). Thus, we concluded that the hyperphosphorylation of Sam68 in Hayai was due to the enhanced kinase activity in the cells, not the amount of Sam68. In addition, phosphorylated proteins around 60 kDa were detected in immunoprecipitates of Sam68 (lanes 2, 4, and 6), which appeared to be dominantly expressed Src family kinases, Lck in Jurkat, Lyn in MT-2, and Fyn in Hayai, respectively, because these kinases were detected at the same positions by reblotting with specific antibodies for each kinase (data not shown). The result with GST fusions of SH2 and SH3 domains Lck also supported the association of Src family kinases with Sam68 in uninfected cells (Fig. 5C). The association via SH3 domain was constitutive, but association via SH2 domain was only detected after stimulation in Jurkat. The mutation in the domain almost abolished the association (asterisks). Similar results were obtained using GST fusions with Fyn (data not shown).
Biological Effect of the Phosphorylation of Sam68 in T Cells-To determine if Sam68 is involved in TCR signaling in normal T cells, we first performed CD3 cross-linking of Jurkat and detected the subsequent tyrosine phosphorylation of Sam68. The phosphorylation of Sam68 was increased after CD3 cross-linking (Fig. 6A). Then, we performed a coprecipitation experiment to confirm that Sam68 was associated with the signaling molecules in response to TCR stimulation, since the results in Fig. 4 imply p68 functions as an adapter. Consistent with Fig. 4, Sam68 was detected constitutively in Hayai, with immunoprecipitates of anti-PLC␥1, the PI 3-kinase p85, Grb2, and SHP-1 (data not shown), and also of anti-Jak3 and Cbl (Fig. 6B, lanes 3-6). Association of Jak3 with Sam68 was further confirmed by anti-Sam68 immunoprecipitation (Fig.  6B, lanes 9 and 10). Among these proteins, anti-Grb2 coprecipitated Sam68 most efficiently, as shown in Fig. 4. In Jurkat cells, the association was slightly increased after stimulation (Fig. 6C, lanes 1 and 2). In Hayai cells, Sam68 was coimmunoprecipitated with anti-Grb2, regardless of pervanadate stimulation (lanes 3 and 4). The same results were obtained in a reverse experiment. Grb2 was constitutively coimmunoprecipitated with anti-Sam68 antibodies in Hayai (lanes 15 and 16). The level of coprecipitation was much higher in Hayai cells than in Jurkat cells (compare lane 2 with 4). The difference seemed to be due to the degree of phosphorylation of the protein (Fig. 5B) as the expression of Sam68 and Grb2 in both types of cells was confirmed to be similar (Fig. 6C, lanes 5-12). DISCUSSION We demonstrated in this study that Sam68, a mitotic target of c-Src, was constitutively tyrosine-phosphorylated in a HTLV-1-infected T cell line, Hayai, which overexpressed the Src family kinase, Fyn. Sam68 was constitutively associated with various signaling molecules, especially with Grb2 in Hayai, suggesting its involvement in the Ras pathway (Fig.  6C). Sam68 seemed to be the preferential substrate for Fyn and Lck in T cells because the association with ZAP-70 was much less significant as confirmed by a coprecipitation experiment (Fig. 3, and data not shown). These findings suggested that Sam68 participated in the signal transduction pathway downstream of TCR-coupled Src family kinases.
Sam68 was first identified as a tyrosine-phosphorylated protein with a RNA binding property in c-Src-overexpressing mitotic fibroblasts (26,27). Sam68 is composed of five proline-rich regions and a C-terminal tyrosine-rich region, in addition to a KH domain for RNA binding (53). This structure implies that Sam68 recruits proline-or Tyr(P)-binding proteins, such as SH3-or SH2-containing proteins (54). Using a yeast two-hybrid system, Richard et al. (55) showed that Fyn-SH3 was associated with mouse Sam68 (previously termed GTPase-activating protein-associated p62) via its proline-rich regions. They also showed the association of Sam68 with the SH3 domain of PLC␥1 and the SH2 domains of PLC␥1, Grb2, and Fyn in HeLa cells coexpressing Sam68 and Fyn. Another report showed that in addition to PLC␥1 and Grb2, Sam68 was associated with the SH2 and SH3 domains of p85 in mitotic NIH3T3 cells overexpressing c-Src (56). These results are consistent with our observations in Hayai cells (Figs. 4 and 6C). In addition to these SH2-and SH3-containing molecules, we suggested that Sam68 was associated with SHP-1, Jak3, and Cbl in T cells (Figs. 3, 4, and 6B). Since Jak3 or Cbl contains neither SH2 nor SH3, the association is mediated by an unknown mechanism or an indirect association via Src family kinases that interact with the IL-2 receptor (57) or Cbl (17,48,49,51). Our results and others described above imply the function of Sam68 as an adapter recruiting these signaling molecules to Src family kinases in T cells (Fyn and Lck) and fibroblasts (Fyn and Src).
The constitutive association of Sam68 with Fyn and Grb2 was prominent in Hayai cells (Fig. 6C). A recent report (58) suggests that the catalytic activity of guanine nucleotide exchanger mSOS was enhanced by Fyn in T cells. Therefore, this observation suggests that the Ras pathway and the following mitogen-activated protein kinase cascade may be constitutively activated through the association of signaling molecules linked by Sam68 in the HTLV-1-infected cell line. Consistently, a member of the mitogen-activated protein kinase family, c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) was constitutively activated in HTLV-1-infected cells (59). These observations may serve as examples that account for the deregulated proliferative response of HTLV-1-infected T cells.
The functional significance of the RNA binding property of Sam68 has yet to be determined. Although Sam68 binds to poly(U) homopolymer in vitro, the physiological target is unknown. In contrast, the regulation of RNA-binding ability has been studied. Sam68 is tyrosine-phosphorylated during mitosis, and binding of phosphorylated Sam68 to RNA is impaired (60). The above observations suggest it may be involved in cell cycling. To determine the effect of hyperphosphorylation of Sam68 in Hayai on the cell cycle, we examined the DNA contents of T cell lines. By propidium iodide staining of DNA and flow cytometric analysis, 21.5% of the Hayai cells were found to be in the G 2 /M phase, whereas 13.1% of Jurkat and 12.2% of MT-2 were in the G 2 /M phase. Furthermore, Hayai was more efficiently arrested at the M phase by Nocodazol treatment than the other cells (data not shown). It is suggested that overexpression of Fyn or constitutive phosphorylation of Sam68 may affect the cell cycle. However, further study is required for a definite conclusion. Analysis of the function of Sam68 in the cell cycle may provide an insight into the roles of Src family kinases in mitotic control.