Interaction between Sam68 and Src Family Tyrosine Kinases, Fyn and Lck, in T Cell Receptor Signaling*

(Received for publication, October 22, 1996, and in revised form, December 16, 1996)

Noemi Fusaki Dagger §, Akihiro Iwamatsu , Makio Iwashima par and Jun-ichi Fujisawa Dagger **

From the Dagger  Department of Microbiology, Kansai Medical University, 10-15 Fumizono-cho, Moriguchi-shi, Osaka 570, Japan,  Central Laboratories for Key Technology, Kirin Brewery Co., Ltd., 1-13-15 Fukuura, Kanazawa-ku, Yokohama-shi, Kanagawa 236, Japan, and the par  Division of Cell and Information, Precursory Research for Embryonic Science and Technology, Research Development Corporation of Japan, Mitsubishi Kasei Institute of Life Sciences, 11 Minamiooya, Machida, Tokyo 194, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
Acknowledgments
REFERENCES


ABSTRACT

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 gamma 1, 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.


INTRODUCTION

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 PLCgamma 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+CD8- or CD4-CD8+) mature thymocytes (9, 10). Src family kinases are supposed to phosphorylate the TCR zeta  chain that then recruits ZAP-70 and Shc (11-14). 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 PLCgamma 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+ 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, PLCgamma 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.


EXPERIMENTAL PROCEDURES

Antibodies

Antibodies to Jak3, Lck, SHP-1, Cbl, Grb2, and Sam68 were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). Anti-CD3 monoclonal antibody NU-T3 was purchased from Nichirei Corp. (Tokyo, Japan). Anti-Fyn and ZAP-70 antibodies were as described (16). Antibodies to phosphotyrosine (Tyr(P), 4G10), Lyn (Lyn9) (30), the regulatory subunit of PI 3-kinase (p85) (30), and PLCgamma 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).

Cells and Preparation of Soluble Cell Lysates

Jurkat and HTLV-1-infected cell lines (MT-1, MT-2, HUT-102, and Hayai) were maintained in RPMI 1640 supplemented with 10% fetal calf serum. Exponentially growing cells were collected and solubilized at 4 °C in lysis buffer (1% Nonidet P-40, 150 mM NaCl, 10 mM Tris-HCl, pH 7.4, 50 units/ml Trasylol (Bayer Leverkusen, Germany), and 1 mM Na3VO4). The lysates were cleared by centrifugation and then used for the study (16).

Stimulation of Cells

CD3 cross-linking was performed as described (8). Briefly, cells were incubated with 10 µg/ml NU-T3 for 30 min on ice, washed in medium twice, and then cross-linked with 10 µg/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 Na3VO4 and 0.1 mM H2O2 (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 × 108 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).


RESULTS

Altered Expression of Tyrosine Kinases in HTLV-1-infected Cells

Since several reports suggest that HTLV-1-infected T cells exhibit altered expression of tyrosine kinases (22, 23), we analyzed the expression of tyrosine kinases in HTLV-1-infected and -uninfected T cell lines by immunoblotting (Fig. 1). Expression of Jak3, which was reported to be constitutively activated in HTLV-1-infected cells (25), was increased in infected cells, MT-1, MT-2, HUT-102, and Hayai (lanes 2-5) compared with Jurkat (lane 1). In contrast, ZAP-70 was decreased in the infected cell lines (lanes 2-5). Since Jak3 is associated with the IL-2 receptor (24, 36, 37) and ZAP-70 with activated TCR (11, 12), these observations may be correlated with the increased expression of the IL-2 receptor (20, 21) and down-regulation of CD3 in these infected cells (38). As to the Src family kinases, the expression of Lck was absent in virus-integrated cells (lanes 2-5); instead, Lyn (both p53 and p56) (39) was expressed in the viral transactivator Tax-expressing cells (lanes 3-5), consistent with previous reports (22, 23). Note that expression of Fyn varied with the cell line. It was significantly overexpressed in Hayai (lane 5), whereas it was hardly detectable in MT-2 and HUT-102 (lanes 3 and 4).


Fig. 1. Expression of tyrosine kinases in HTLV-1-infected or -uninfected T cell lines. Lysates of 1 × 106 cells of Jurkat (lane 1) or of HTLV-1-infected cell lines, MT-1, MT-2, HUT102, and Hayai (lanes 2-5), were subjected to SDS-PAGE and Western blotting with anti-Jak3, ZAP-70, Fyn, Lck, or Lyn antibodies (from top to bottom panels, respectively). Virus integration and Tax expression are indicated as + and -.
[View Larger Version of this Image (64K GIF file)]


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 H2O2 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).


Fig. 2. Enhanced tyrosine phosphorylation of cellular proteins in a HTLV-1-infected T cell line, Hayai. A, 1 × 106 cells of Jurkat, MT-2, and Hayai, respectively, were incubated with (+) or without (-) pervanadate (PV) at 37 °C for 10 min. The lysates were subjected to SDS-PAGE and Western blotting with anti-Tyr(P) antibodies (4G10). B, lysates of 1 × 107 cells of Hayai before (-) and after (+) pervanadate stimulation were affinity-precipitated (AP) with agarose-conjugated GST-fusions of Fyn-SH2, Fyn-SH3, or GST only. The precipitates were subjected to SDS-PAGE and Western blotting with 4G10.
[View Larger Version of this Image (36K GIF file)]


Since Hayai overexpressed Fyn and the expression of other kinases, Jak3, Lck, and Lyn, was similar to MT-2 (Fig. 1), we considered that these proteins might be substrates for Fyn. Thus, we examined whether the proteins were associated with the functional domains of Fyn, SH2, and SH3 (Fig. 2B). The tyrosine-phosphorylated 68-kDa protein (designated as p68, indicated by an arrow) was efficiently precipitated with both Fyn-SH2 and -SH3, regardless of stimulation (lanes 2, 3, 5, and 6). GST alone did not precipitate these proteins (lanes 1 and 4).

p68 Was Not ZAP-70 or SHP-1

The molecular weight of p68 was similar to those of SH2-containing molecules, the tyrosine kinase, ZAP-70, and the hematopoietic-specific tyrosine phosphatase, SHP-1 (40-43). These two have been reported to be phosphorylated by Lck or Fyn (11, 12, 16, 44). Therefore, we examined whether p68 was identical to these proteins or not (Fig. 3). Lysates were subjected to anti-ZAP-70 immunoprecipitation (lanes 1-4) or anti-SHP-1 immunoprecipitation (lanes 5-12), and then the phosphorylated proteins were detected by anti-Tyr(P) immunoblotting (lanes 1-8). Note that although Lck was deficient in Hayai, ZAP-70 was phosphorylated after pervanadate treatment (lane 4). However, the phosphorylation was only detected after stimulation; thus, ZAP-70 is distinct from constitutively phosphorylated p68. With anti-SHP-1, the phosphorylated 68-kDa protein was immunoprecipitated in Hayai cells regardless of stimulation (lanes 7 and 8, indicated by an open arrowhead). However, the mobility of the band was slower than that in the case of Jurkat (closed arrow). The mobility of SHP-1 expressed in Hayai was confirmed to be the same as in Jurkat (lanes 9-12, reblotting of the same filter with anti-SHP-1); therefore, p68 was different from SHP-1 but coimmunoprecipitated with SHP-1.


Fig. 3. p68 was not ZAP-70 or SHP-1 itself but was coprecipitated with SHP-1. Lysates of 1 × 107 cells of Jurkat and Hayai before (-) and after (+) pervanadate (PV) stimulation were subjected to immunoprecipitation with anti-ZAP-70 (lanes 1-4) or anti-SHP-1 (lanes 5-12) antibodies. The precipitates were then subjected to SDS-PAGE and Western blotting with 4G10 (lanes 1-8). For the immunoprecipitates of anti-SHP-1, the filter was reblotted with anti-SHP-1 antibodies to confirm the position of SHP-1 (lanes 9-12). The positions of ZAP-70 and SHP-1 are indicated by arrows. The position of p68 is indicated by open arrowheads.
[View Larger Version of this Image (39K GIF file)]


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, PLCgamma 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.


Fig. 4. p68 was coprecipitated with various signaling molecules. Lysates of 1 × 107 cells of Hayai before (-) and after (+) pervanadate (PV) stimulation were subjected to immunoprecipitation with anti-PLCgamma 1, PI 3-kinase p85 subunit, Grb2, Cbl, or Jak3 antibodies. The control was precipitated with Protein G-Sepharose only. The precipitates were then subjected to SDS-PAGE and Western blotting with 4G10. Asterisks indicate the positions of PLCgamma 1, Cbl, Jak3, p85, and Grb2, from top to bottom, respectively, as determined by reblotting of the filter with each antibody (data not shown). H, heavy chains of the precipitating antibodies; IP, immunoprecipitates.
[View Larger Version of this Image (54K GIF file)]


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 p21ras 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).


Fig. 5. p68 was identified as Sam68. A, sequence alignment of peptides derived from the purified p68 with Sam68 (human GTPase-activating protein-associated p62) (28). B, 1 × 107 cells of Jurkat, MT-2, and Hayai were stimulated as described above. The lysates were subjected to immunoprecipitation with anti-Sam68 antibodies and immunoblotting with 4G10 (upper panel) or anti-Sam68 (lower panel) antibodies. PV, pervanadate. C, lysates of Jurkat cells were precipitated with GST fusions of Lck-SH2, Lck-SH3, or each domain with a mutation (R154K in SH2, P112L in SH3, indicated by asterisks) and immunoblotted with anti-Sam68 antibodies.
[View Larger Version of this Image (30K GIF file)]


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-PLCgamma 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).


Fig. 6. Biological effect of tyrosine phosphorylation of Sam68 in T cells. A, 1 × 107 cells of Jurkat were incubated with (CD3, lane 2) or without (-, lane 1) anti-CD3 antibodies and then cross-linked with goat anti-mouse IgG at 37 °C for 2 min. Pervanadate treatment was performed as in Fig. 2 (PV, lane 3). The lysates were subjected to anti-Sam68 immunoprecipitation and 4G10 immunoblotting. B, lysates of Hayai were subjected to immunoprecipitation with anti-Cbl (lanes 3 and 4), anti-Jak3 (lanes 5 and 6), or anti-Sam68 (lanes 9 and 10) antibodies. The precipitates were then subjected to Western blotting with anti-Sam68 (lanes 1-6) or anti-Jak3 (lanes 7-10) antibodies. Control precipitates were also shown in lanes 1, 2, 7, and 8. C, lysates of Jurkat and Hayai were subjected to immunoprecipitation with anti-Grb2 (lanes 1-4 and 9-12) or anti-Sam68 (lanes 5-8 and 13-16) antibodies. The precipitates were then subjected to Western blotting. Upper portion of the membrane was blotted with anti-Sam68 antibodies (lanes 1-8) and the lower portion with anti-Grb2 antibodies (lanes 9-16). Association is indicated in lanes 1-4 and 13-16. The precipitation efficiency of the samples is shown in lanes 5-8 and 9-12. IP, immunoprecipitate; H, heavy chains of the precipitating antibodies.
[View Larger Version of this Image (26K GIF file)]



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 PLCgamma 1 and the SH2 domains of PLCgamma 1, Grb2, and Fyn in HeLa cells coexpressing Sam68 and Fyn. Another report showed that in addition to PLCgamma 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 G2/M phase, whereas 13.1% of Jurkat and 12.2% of MT-2 were in the G2/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.


FOOTNOTES

*   This study was supported in part by the Sagawa Foundation for the Promotion of Cancer Research and a grant-in-aid from the Ministry of Education, Science and Culture.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§   Present address: Dept. of Biotechnology, Research Inst. for Biological Sciences, Science University of Tokyo, 2669 Yamazaki, Noda-shi, Chiba 278, Japan. Tel.: 81-471-23-9779, Fax: 81-471-22-4131.
**   To whom correspondence should be addressed. Tel.: 81-6-992-1001, Fax: 81-6-993-1668.
1   The abbreviations used are: TCR, T cell receptor; PLC, phospholipase C; PI, phosphatidylinositol; HTLV-1, human T cell leukemia virus type-1; IL-2, interleukin-2; PAGE, polyacrylamide gel electrophoresis; Sam68, Src associated in mitosis, 68 kDa; SH2/3, Src homology 2/3; Tyr(P), phosphotyrosine; GST, glutathione S-transferase.

Acknowledgments

We thank Drs. H. Nariuchi, T. Yamamoto, Y. Fukui, Y. Homma, and H. Umemori and Medical Biological Laboratories for providing the antibodies and plasmids. We also thank Dr. H. Doi (Kansai Medical University) for help with cell cycle analysis and Dr. T. Kurosaki (Kansai Medical University) for the helpful discussions and critical reading of this manuscript.


REFERENCES

  1. Weiss, A., and Littman, D. R. (1994) Cell 76, 263-274 [Medline] [Order article via Infotrieve]
  2. Buday, L., and Downward, J. (1993) Cell 73, 611-620 [Medline] [Order article via Infotrieve]
  3. Samelson, L. E., Phillips, A. F., Luong, E. T., and Klausner, R. D. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 4358-4362 [Abstract]
  4. Da Silva, A., Yamamoto, M., Zalvan, C. H., and Rudd, C. E. (1992) Mol. Immunol. 29, 1417-1425 [CrossRef][Medline] [Order article via Infotrieve]
  5. Tsygankov, A. Y., Broker, B. M., Fargnoli, J., Ledbetter, J. A., and Bolen, J. B. (1992) J. Biol. Chem. 267, 18259-18262 [Abstract/Free Full Text]
  6. Cooke, M. P., Abraham, K. M., Forbush, K. A., and Perlmutter, R. M. (1991) Cell 65, 281-291 [Medline] [Order article via Infotrieve]
  7. Davidson, D., Chow, L. M., Fournel, M., and Veillette, A. (1992) J. Exp. Med. 175, 1483-1492 [Abstract]
  8. Fusaki, N., Semba, K., Katagiri, T., Suzuki, G., Matsuda, S., and Yamamoto, T. (1994) Int. Immunol. 6, 1245-1255 [Abstract]
  9. Stein, P. L., Lee, H. M., Rich, S., and Soriano, P. (1992) Cell 70, 741-750 [Medline] [Order article via Infotrieve]
  10. Appleby, M. W., Gross, J. A., Cooke, M. P., Levin, S. D., Qian, X., and Perlmutter, R. M. (1992) Cell 70, 751-763 [Medline] [Order article via Infotrieve]
  11. Chan, A. C., Iwashima, M., Turck, C. W., and Weiss, A. (1992) Cell 71, 649-662 [Medline] [Order article via Infotrieve]
  12. Iwashima, M., Irving, B. A., van Oers, N. S. C., Chan, A. C., and Weiss, A. (1994) Science 263, 1136-1139 [Medline] [Order article via Infotrieve]
  13. Timuson Gauen, L. K., Zhu, Y., Letourneur, F., Hu, Q., Bolen, J. B., Matis, L. A., Klausner, R. D., and Shaw, A. S. (1994) Mol. Cell. Biol. 14, 3729-3741 [Abstract]
  14. Ravichandran, K. S., Lee, K. K., Songyang, Z., Cantley, L. C., Burn, P., and Burakoff, S. J. (1993) Science 262, 902-905 [Medline] [Order article via Infotrieve]
  15. Pasad, K. V. S., Janssen, O., Kapeller, R., Raab, M., Cantley, L. C., and Rudd, C. E. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 7366-7370 [Abstract]
  16. Fusaki, N., Matsuda, S., Nishizumi, H., Umemori, H., and Yamamoto, T. (1996) J. Immunol. 156, 1369-1377 [Abstract]
  17. Tezuka, T., Umemori, H., Fusaki, N., Yagi, T., Takata, M., Kurosaki, T., and Yamamoto, T. (1996) J. Exp. Med. 183, 675-680 [Abstract]
  18. Miyoshi, I., Kubonishi, I., Yoshimoto, S., Akagi, T., Ohtsuki, Y., Shiraishi, Y., Nagata, K., and Hinuma, Y. (1981) Nature 294, 770-771 [Medline] [Order article via Infotrieve]
  19. Yamamoto, N., Okada, M., Koyanagi, Y., Kannagi, M., and Hinuma, Y. (1982) Science 217, 737-739 [Medline] [Order article via Infotrieve]
  20. Yodoi, J., and Uchiyama, T. (1992) Immunol. Today 13, 405-411 [CrossRef][Medline] [Order article via Infotrieve]
  21. Yoshida, M. (1993) Trends Microbiol. 1, 131-135 [Medline] [Order article via Infotrieve]
  22. Koga, Y., Oh-Hori, N., Sato, H., Yamamoto, N., Kimura, G., and Nomoto, K. (1989) J. Immunol. 142, 4493-4499 [Abstract/Free Full Text]
  23. Yamamashi, Y., Mori, S., Yoshida, M., Kishimoto, T., Inoue, K., Yamamoto, T., and Toyoshima, K. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 6538-6542 [Abstract]
  24. Schindler, C., and Darnell, J. E. (1995) Annu. Rev. Biochem. 64, 621-651 [CrossRef][Medline] [Order article via Infotrieve]
  25. Migone, T.-S., Lin, J.-X., Cereseto, A., Mulloy, J. C., O'Shea, J. J., Franchini, G., and Leonard, W. J. (1995) Science 269, 79-81 [Medline] [Order article via Infotrieve]
  26. Taylor, S., and Shalloway, D. (1994) Nature 368, 867-871 [CrossRef][Medline] [Order article via Infotrieve]
  27. Fumagalli, S., Totty, N. F., Hsuan, J. J., and Courtneidge, S. A. (1994) Nature 368, 871-874 [CrossRef][Medline] [Order article via Infotrieve]
  28. Wong, G., Müller, O., Clark, R., Conroy, L., Moran, M. F., Polakis, P., and McCormick, F. (1992) Cell 69, 551-558 [Medline] [Order article via Infotrieve]
  29. Lock, P., Fumagalli, S., Polakis, P., McCormick, F., and Courtneidge, S. A. (1996) Cell 84, 23-24 [Medline] [Order article via Infotrieve]
  30. Yamanashi, Y., Fukui, Y., Wongsasant, B., Kinoshita, Y., Ichimori, Y., Toyoshima, K., and Yamamoto, T. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 1118-1122 [Abstract]
  31. Homma, Y., Emori, Y., Shibasaki, K., Suzuki, K., and Takenawa, T. (1990) Biochem. J. 269, 13-18 [Medline] [Order article via Infotrieve]
  32. O'Shea, J. J., McVicar, D. W., Bailey, T. L., Burns, C., and Smyth, M. J. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 10306-10310 [Abstract]
  33. Clark, S. G., Stern, M. J., and Horvitz, H. R. (1992) Nature 356, 340-344 [CrossRef][Medline] [Order article via Infotrieve]
  34. Yamanashi, Y., Okada, M., Senba, T., Yamori, T., Umemori, H., Tsunasawa, S., Toyoshima, K., Kitamura, D., Watanabe, T., and Yamamoto, T. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 3631-3635 [Abstract]
  35. Iwamatsu, A. (1992) Electrophoresis 13, 142-147 [Medline] [Order article via Infotrieve]
  36. Johnston, J. A., Kawamura, M., Kirken, R. A., Chen, Y.-Q., Blake, T. B., Shibuya, K., Ortaldo, J. R., McVicar, D. W., and O'Shea, J. J. (1994) Nature 370, 151-153 [CrossRef][Medline] [Order article via Infotrieve]
  37. Witthuhn, B. A., Silvennoinen, O., Miura, O., Lai, K. S., Cwik, C., Liu, E. T., and Ihle, J. N. (1994) Nature 370, 153-157 [CrossRef][Medline] [Order article via Infotrieve]
  38. Matsuoka, M., Hattori, T., Chosa, T., Tsuda, H., Kuwata, S., Yoshida, M., Uchiyama, T., and Takatsuki, K. (1986) Blood 4, 1070-1076
  39. Yamanashi, Y., Miyasaka, M., Takeuchi, M., Ilic, D., Mizuguchi, J., and Yamamoto, T. (1991) Cell Regul. 2, 979-987 [Medline] [Order article via Infotrieve]
  40. Shen, S.-H., Bastein, L., Posner, B. I., and Chrétien, P. (1991) Nature 352, 736-739 [CrossRef][Medline] [Order article via Infotrieve]
  41. Plutzky, J. B., Neel, B. G., and Rosenberg, R. D. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 1123-1127 [Abstract]
  42. Yi, T., Cleveland, J. L., and Ihle, J. N. (1992) Mol. Cell. Biol. 12, 836-846 [Abstract]
  43. Matthews, R. J., Bowne, D. B., Flores, E., and Thomas, M. L. (1992) Mol. Cell. Biol. 12, 2396-2403 [Abstract]
  44. Lorenz, U., Ravichandran, K. S., Pei, D., Walsh, C. T., Burakoff, S. J., and Neel, B. G. (1994) Mol. Cell. Biol. 14, 1824-1834 [Abstract]
  45. Blake, T. J., Shapiro, M., Morse, H. C., III, and Langdon, W. Y. (1991) Oncogene 6, 653-657 [Medline] [Order article via Infotrieve]
  46. Donovan, J. A., Wange, R. L., Langdon, W. Y., and Samelson, L. E. (1994) J. Biol. Chem. 269, 22921-22924 [Abstract/Free Full Text]
  47. Fukazawa, T., Reedquist, K. A., Trub, T., Soltoff, S., Panchamoorthy, G., Druker, B., Cantley, L., Shoelson, S. E., and Band, H. (1995) J. Biol. Chem. 270, 19141-19150 [Abstract/Free Full Text]
  48. Panchamoorthy, G., Fukazawa, T., Miyake, S., Soltoff, S., Reedquist, K., Druker, B., Shoelson, S., Cantley, L., and Band, H. (1996) J. Biol. Chem. 271, 3187-3194 [Abstract/Free Full Text]
  49. Giles, O., Cory, C., Lovering, R. C., Hinshelwood, S., MacCarthy-Morrogh, L., Levinsky, R. J., and Kinnon, C. (1995) J. Exp. Med. 182, 611-615 [Abstract]
  50. Panchamoorthy, G., Fukazawa, T., Stolz, T., Payne, G., Reedquist, K., Shoelson, S., Zhou, S., Cantley, L., Walsh, C., and Band, H. (1994) Mol. Cell. Biol. 14, 6372-6385 [Abstract]
  51. Sattler, M., Salgia, R., Okuda, K., Uemura, N., Durstin, M. A., Pisick, E., Xu, G., Li, J.-L., Prasad, K. V., and Griffin, J. D. (1996) Oncogene 12, 839-846 [Medline] [Order article via Infotrieve]
  52. Ribon, V., Hubbell, S., Herrera, R., and Saltiel, A. R. (1996) Mol. Cell. Biol. 16, 45-52 [Abstract]
  53. Gibson, T. J., Thompson, J. D., and Heringa, J. (1993) FEBS Lett. 324, 361-366 [CrossRef][Medline] [Order article via Infotrieve]
  54. Pawson, T. (1995) Nature 373, 573-580 [CrossRef][Medline] [Order article via Infotrieve]
  55. Richard, S., Yu, D., Blumer, K. J., Hausladen, D., Olszowy, M. W., Connelly, P. A., and Shaw, A. S. (1995) Mol. Cell. Biol. 15, 186-197 [Abstract]
  56. Taylor, S. J., Anafi, M., Pawson, T., and Shalloway, D. (1995) J. Biol. Chem. 270, 10120-10124 [Abstract/Free Full Text]
  57. Taniguchi, T., and Minami, Y. (1993) Cell 73, 5-8 [Medline] [Order article via Infotrieve]
  58. Li, B.-Q., Subleski, M., Fusaki, N., Yamamoto, T., Copeland, T., Princler, G. L., Kung, H.-F., and Kamata, T. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 1001-1005 [Abstract/Free Full Text]
  59. Xu, X., Heidenreich, O., Kitajima, I., McGuire, K., Li, Q., Su, B., and Nerenberg, M. (1996) Oncogene 13, 135-142 [Medline] [Order article via Infotrieve]
  60. Wang, L. L., Richard, S., and Shaw, A. S. (1995) J. Biol. Chem. 270, 2010-2013 [Abstract/Free Full Text]

©1997 by The American Society for Biochemistry and Molecular Biology, Inc.