(Received for publication, October 22, 1996, and in revised form, December 16, 1996)
From the 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
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
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 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.
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 PLC1, 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
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 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+ 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, PLC1, 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.
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 PLC1 (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).
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 CellsCD3 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 ImmunoblottingImmunoprecipitation 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 p68Lysate 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).
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).
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).
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-1The 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.
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, PLC1, 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 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).
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-PLC1, 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).
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 PLC1 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 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.
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.