Interactions between the protein-tyrosine kinase ZAP-70, the proto-oncoprotein Vav, and tubulin in Jurkat T cells.

Two molecules involved in signal transduction via the T cell antigen receptor, namely the protein-tyrosine kinase ZAP-70 and the proto-oncoprotein Vav, were found to be constitutively associated with tubulin in Jurkat T cells. Both were able to bind to tubulin independently of one another, as determined by transient transfection into COS-7 cells. The ZAP-70 associated with tubulin was preferentially tyrosine-phosphorylated after T cell antigen receptor stimulation of Jurkat T cells, suggesting that this interaction was functionally significant. Vav was also found to co-immunoprecipitate with ZAP-70 from cell extracts depleted of tubulin. This raised the possibility that Vav might be a substrate for ZAP-70 protein-tyrosine kinase activity. However, tyrosine phosphorylation of Vav preceded that of ZAP-70, indicating that Vav was unlikely to be a downstream target of ZAP-70. The association of ZAP-70 and Vav with tubulin implies that the microtubules may be involved in the signaling function of these two molecules, perhaps by targeting them to their appropriate intracellular location.

Two molecules involved in signal transduction via the T cell antigen receptor, namely the protein-tyrosine kinase ZAP-70 and the proto-oncoprotein Vav, were found to be constitutively associated with tubulin in Jurkat T cells. Both were able to bind to tubulin independently of one another, as determined by transient transfection into COS-7 cells. The ZAP-70 associated with tubulin was preferentially tyrosine-phosphorylated after T cell antigen receptor stimulation of Jurkat T cells, suggesting that this interaction was functionally significant. Vav was also found to co-immunoprecipitate with ZAP-70 from cell extracts depleted of tubulin. This raised the possibility that Vav might be a substrate for ZAP-70 protein-tyrosine kinase activity. However, tyrosine phosphorylation of Vav preceded that of ZAP-70, indicating that Vav was unlikely to be a downstream target of ZAP-70. The association of ZAP-70 and Vav with tubulin implies that the microtubules may be involved in the signaling function of these two molecules, perhaps by targeting them to their appropriate intracellular location.
Stimulation of the T cell antigen receptor (TCR) 1 initiates a cascade of signal transduction events, the most proximal of which is the induction of PTK activity, which is essential for the signaling process (1). This involves the cytoplasmic tails of the CD3 complex (␥␦⑀) and the chain of the TCR becoming phosphorylated on specific tyrosine residues within ITAMs (1). Studies in mutant Jurkat T cells and transfected COS cells have indicated that the Src family protein-tyrosine kinase, Lck, is required for tyrosine phosphorylation of the TCR immunoreceptor tyrosine-based activation motifs (2,3). Biochemical and genetic experiments have also indicated a role for the Src family PTK, Fyn, in TCR signaling (4). However, its intracellular localization suggests that its function may be distinct from that of Lck (5).
ITAM phosphorylation results in recruitment of the Syk family PTKs, ZAP-70 and Syk, to the TCR via the binding of their two SH2 domains (6 -9). ZAP-70 and Syk are then tyrosine-phosphorylated themselves, which for ZAP-70 has been shown to activate its kinase activity (10), and tyrosine phosphorylation of multiple intracellular proteins is induced (3,6). An essential role for ZAP-70 in TCR signaling and T cell development has been revealed by genetic studies (11,12).
TCR stimulation induces tyrosine phosphorylation of a number of other intracellular proteins besides TCR subunits and ZAP-70 (13). The identity of many of these proteins is not known, and the functional consequences of the majority of the tyrosine phosphorylations are unclear. However, several phosphotyrosyl proteins have been identified which appear to play an important role in the signaling process. These include phospholipase C ␥1, ERK mitogen-activated protein kinases, and Vav (14). Tyrosine phosphorylation of phospholipase C ␥1 increases its catalytic activity, resulting in the hydrolysis of phosphatidylinositol 4,5-bisphosphate to inositol 1,4,5trisphosphate and diacylglycerol (14,15). These second messengers, in turn, induce the mobilization of cytoplasmic calcium and the activation of protein kinase C, respectively. The phosphorylation and activation of the ERK by the TCR induces the phosphorylation of a number of transcription factors that are important in the induction of gene transcription (16). The role of Vav is unknown. However, gene targeting experiments have indicated that it is required for optimal signaling via the TCR (17)(18)(19). Sequence homology suggests that Vav is a GDP/GTP exchange protein for a small G-protein of the Rho/Rac subfamily (20), but its identity is presently unclear.
This laboratory has demonstrated previously that ␣-tubulin is constitutively tyrosine-phosphorylated in human T cells (21). In this study, it is shown that TCR stimulation of Jurkat T cells induced the tyrosine phosphorylation of two proteins that coprecipitated with tubulin. These proteins were identified as ZAP-70 and Vav and suggest a role for the microtubule cytoskeleton in the signaling functions of these two proteins.

EXPERIMENTAL PROCEDURES
Cells and Antibodies-J6 cells were cultured in RPMI containing 3% FCS. COS-7 cells (from the European Collection of Animal Cell Cultures, Porton Down, UK) were grown in DMEM containing 10% FCS. CD3 antibody, OKT3, was obtained from the American Type Tissue Collection (Rockville, MD), and (FabЈ) 2 fragments kindly prepared by A. Tutt and M. Glennie (Tenovus, Southampton, UK). Other antibodies were 4G10, anti-Tyr(P) monoclonal antibody (from Brian Druker, Oregon Health Sciences University, Portland, OR), TAT-1 antibody, ␣-tubulin (from Keith Gull, University of Manchester, Manchester, UK), KMX antibody, ␤-tubulin (from Keith Gull, University of Manchester), and monoclonal anti-actin (Amersham International). Rabbit anti-Vav antiserum, Vav-1, was raised against a synthetic peptide corresponding to residues 575-590 of human proto-Vav, and was used for immunoprecipitation of Vav. A monoclonal anti-Vav antibody was used for immunoblotting (Upstate Biotechnology, Inc., Lake Placid, New York). Anti-ZAP-70 antiserum, ZAP-4, was raised against a synthetic peptide corresponding to residues 271-290 of human ZAP-70. For immunoblotting, the ZAP-4 antibody was affinity-purified using the immunizing peptide immobilized on Sulfolink coupling gel (Pierce).
DNA Constructs-To generate a GST fusion protein of the Vav SH2 domain, the polymerase chain reaction was used to generate a cDNA fragment of base pairs 2377-2669 of human Vav cDNA (from Shulamit Katzav, Jewish General Hospital, Montreal). This product was then subcloned into the EcoRI site of the pGEX 4T-3 vector (Pharmacia) while maintaining the correct reading frame. The fidelity and orientation of the polymerase chain reaction product was confirmed by DNA sequencing. GST and the GST-Vav SH2 fusion protein were purified from lysates of transfected Escherichia coli DH5␣ cells using glutathione-Sepharose (Pharmacia) following a standard Pharmacia protocol. The purity of these proteins exceeded 80%. Purified proteins were dialyzed against 50% glycerol, 50 mM Tris, pH 8.
ZAP-70 cDNA (from Arthur Weiss, Howard Hughes Medical Institute, San Francisco, CA) and vav cDNA were subcloned into the eukaryotic expression vector pcDNA3neo (InVitrogen) for COS-7 transfection experiments.
Immunoprecipitation of proteins from cell lysates and Western blotting was carried out as described previously (21). To analyze the interaction of ZAP-70 with Vav SH2 domain, 5 g of GST-SH2(Vav) fusion protein or GST were prebound to 10 l of glutathione-Sepharose (Pharmacia) and were washed in IPB, prior to addition to cell lysates. Polyvinylidene difluoride membranes were stripped of bound antibody using the Amersham ECL protocol in experiments in which blots were probed for multiple antigens.
COS Cell Transfection-2 g of pcDNA3neo expression vector, containing the appropriate cDNA insert, was added to 1.5 ml of Optimem (Life Technologies, Inc.) and mixed with 1.5 ml of Tris-buffered saline, pH 7.4, containing 1 mg⅐ml Ϫ1 DEAE-Dextran (Pharmacia). The resulting suspension was added to PBS-washed COS-7 cells at 50% confluence in 90-mm Petri dishes for 45 min at 37°C. Following aspiration, cells were treated with DMEM containing 10% FCS and 100 g⅐ml Ϫ1 of chloroquine for 3 h at 37°C, then incubated in DMEM containing 10% FCS for 48 h, washed once in PBS, and lysed by addition of 1 ml of IPB. Immunoprecipitations were carried out as described above.
In Vitro Polymerization of Tubulin-Tubulin was polymerized from cytosolic extracts using a modification of the method described by Vallee and Collins (30). 5 ϫ 10 8 J6 cells were washed three times in Ca 2ϩ /Mg 2ϩ -free PBS then once with ice-cold PEM buffer (0.1 M PIPES-NaOH, pH 6.6, 1 mM EGTA, 1 mM MgSO 4 ). The cells were divided into two aliquots and swollen in 5 ml of ice-cold hypotonic buffer (1 mM EGTA, 1 mM MgSO 4 , 10 mM PIPES, pH 6.6) for 15 min. Phenylmethylsulfonyl fluoride was added to 1 mM, Na 3 VO 4 to 1 mM, and small peptide inhibitors (chymostatin, leupeptin, and pepstatin) to 1 g⅐ml Ϫ1 each, final concentration. Cells were lysed on ice using a tight fitting Dounce homogenizer (50 strokes), then PIPES (1 M, pH 6.9) added to give a final concentration of 0.1 M. Effective cell breakage was confirmed by trypan blue exclusion. The suspension was cleared by sequential centrifugation at 2°C at 1,500 ϫ g for 5 min at 37,000 ϫ g for 10 min and finally at 100,000 ϫ g for 90 min. The resulting supernatant was the cytosolic fraction. To polymerize tubulin, taxol (20 M final concentration; Calbiochem) and GTP (1 mM final concentration; Sigma) were added to this cytosolic extract, incubated at 37°C for 30 min, and then cooled on ice for 15 min. No taxol or GTP was added to controls, which were kept on ice. Extracts were underlaid with PEM buffer containing 10% sucrose (plus 20 M taxol and 1 mM GTP as appropriate). Polymerized tubulin was recovered by centrifugation at 100,000 ϫ g for 30 min at 4°C, and the tubulin-depleted cytosol was aspirated to analyze by SDS-polyacrylamide gel electrophoresis or for immunoprecipitation. The "tubulin" pellet was washed twice in ice-cold PEM, containing taxol and GTP, and resuspended in reducing Laemmli sample buffer. Immunoprecipitation from tubulin-depleted or control cytosol was carried out as described above.

RESULTS AND DISCUSSION
Tyrosine phosphorylation of two proteins which co-precipitated with ␣-tubulin was strongly induced after CD3 stimulation in Jurkat T cells (Fig. 1a). These proteins had relative molecular massesS of 70 and 100 kDa. On longer exposure, constitutive tyrosine-phosphorylated ␣-tubulin (a doublet with an approximate relative molecular mass of 55 KDa) was detected, as reported previously (21). In some experiments, a weak Tyr(P) band was also detected at 75 kDa. Immunoprecipitation with other anti-␣-tubulin antibodies and an anti-␤tubulin antibody produced qualitatively similar results (data not shown). Comparison of anti-␣ tubulin immunoprecipitates with anti-Tyr(P) immunoprecipitates indicated that the interaction between tubulin and the two Tyr(P) proteins was highly selective (Fig. 1a).
A panel of antibodies was used to investigate by immunoblotting whether the tubulin-associated proteins corresponded to previously identified Tyr(P) proteins. This analysis indicated that both ZAP-70 (9) and Vav (22) co-precipitated with tubulin in Jurkat T cells (Fig. 1a) and also in human T lymphoblasts (data not shown). These proteins had identical mobilities to the 70-and 100-kDa tubulin-associated Tyr(P) proteins, respectively. Several other proteins, which are tyrosine-phosphorylated in activated T cells, including the chain (23), Lck (24), HS-1 (25), CD5 (26), and cbl (27), were not detected in anti-␣tubulin immunoprecipitates (data not shown). Syk (6) was also undetectable in anti-␣ tubulin immunoprecipitates from Jurkat T cells. However, this was probably due to its low expression levels in these cells as Syk was the major tubulin-associated Tyr(P) band in WEHI 231 B cells (data not shown). In reciprocal experiments, both ␣and ␤-tubulin co-immunoprecipitated with ZAP-70 and Vav (Fig. 1b). Competition with the immunizing ZAP-4 and Vav-1 peptides in the cell lysates inhibited the co-immunoprecipitation of both ␣and ␤-tubulin by anti-ZAP and anti-Vav antibodies, respectively (Fig. 1b). Thus the detected associations of tubulin with ZAP-70 and Vav were specific. ZAP-70-tubulin and Vav-tubulin interactions were constitutive and did not alter following CD3 stimulation.
To test whether ZAP-70 and Vav required each other, or distinct hematopoietic-specific proteins, to interact with tubu-  (Fig. 1c). Co-transfection of ZAP-70 and Vav did not alter the level of interaction of these proteins with tubulin (data not shown). Thus the interactions of ZAP-70 and Vav with tubulin were independent and did not require other proteins which were hematopoietic cell-specific.
A 100-kDa Tyr(P) band, with the same mobility as Vav, co-immunoprecipitated with ZAP-70 in CD3-stimulated Jurkat T cells (Fig. 2a, top panel). Immunoblotting with anti-Vav antibody demonstrated that Vav was constitutively co-purified in anti-ZAP-70 immunoprecipitates and probably corresponded to the 100-kDa Tyr(P) protein (Fig. 2a, middle panel). In some experiments, it was also possible to detect very low levels of ZAP-70 specifically co-purifying in anti-Vav immunoprecipitates, also in a constitutive fashion (data not shown). Although co-immunoprecipitation of Vav with ZAP-70 was constitutive, a GST fusion protein of the Vav-SH2 domain precipitated ZAP-70 only after TCR stimulation (Fig. 2b), as has been reported previously by Katzav et al. (28). Our data suggest that a constitutive interaction between Vav and ZAP-70 existed which was not mediated via the Vav SH2 domain. One possible explanation for this is that TCR stimulation induces a conformational change allowing the SH2 domain of Vav, which is already complexed with ZAP-70, to bind to its tyrosinephoshorylated target sequence on ZAP-70. However, it cannot be excluded that a small fraction of total Vav may be induced to associate with ZAP-70 after TCR stimulation via its SH2 domain, but this increase may be below the detection limit of the assay. The possibility that tubulin mediated the interaction between Vav and ZAP-70 is considered below.
In contrast to the present study, Katzav et al. (28) found that the interaction between ZAP-70 and Vav was induced by CD3 stimulation using an anti-Vav antibody for immunoprecipitation. This may reflect the use of an anti-Vav antibody which cannot recognize Vav that is constitutively associated with ZAP-70. Katzav et al. (28) also did not detect Vav in immunoprecipitates using the 1222 anti-ZAP-70 antibody. A direct comparison of 1222 anti-ZAP-70 antibody with ZAP-4 anti-ZAP-70 antibody, which is used in the present study, has demonstrated that only ZAP-4 antibody co-immunoprecipitated Vav (data not shown). These results suggest that there are qualitative differences between the two anti-ZAP-70 antibodies, such that Vav associated with ZAP-70 is only immunoprecipitated by ZAP-4 antibody and not by 1222 antibody in detectable quantities.
Microtubule-associated proteins can be operationally defined as proteins which co-purify with tubulin polymerized in vitro (29). The presence of ZAP-70 and Vav in anti-␣ tubulin immunoprecipitates suggested that these proteins might also copurify with polymerized tubulin. To test this hypothesis, taxol and GTP were added to Jurkat cytosolic extracts to promote in vitro tubulin polymerization (30). Taxol/GTP treatment removed over 80 -90% of tubulin from cytosolic extracts, and large amounts of tubulin were detected in the pellet, as expected (Fig. 3a). The pellet fraction contained only trace amounts of actin, and actin was not depleted from the cytosol taxol/GTP treatment, confirming the specificity of this method for polymerization of tubulin (Fig. 3a). Immunoblotting demonstrated that 35% (Ϯ5% S.E.; n ϭ 4) of ZAP-70 and 6% (Ϯ2% S.E.; n ϭ 4) of Vav were depleted from cytosolic extracts by taxol/GTP treatment, and both of these molecules were detected in the pellet fraction (Fig. 3a). Two other cytosolic Tyr(P) proteins, HS-1 and cbl, were not depleted from the cytosol or detected in the pellet fraction after taxol/GTP treatment (data not shown), suggesting that the co-purification of ZAP-70 and Vav with polymerized tubulin was specific. The chain, Lck, and CD5 were not present in the cytosolic fraction, as they are membrane-associated. These data indicated that both ZAP-70 and Vav could interact with polymerized tubulin in vitro. Extraction of Jurkat T cells with PM2G buffer, which maintains the microtubule cytoskeleton intact (21,31), indicated that both ZAP-70 and Vav were associated with soluble and polymerized tubulin in vivo (data not shown). Cytosolic extracts were prepared from unstimulated (Ϫ) or OKT3-stimulated (ϩ) Jurkat T cells. In a, tubulin was polymerized in vitro from cytosol by addition of taxol and GTP (Tax) and the centrifuged to generate the "pellet" fraction. No taxol and GTP was added to the control cytosol (cont.). Cytosol and pellet fractions were Western blotted and probed sequentially for Vav, ZAP-70, ␣-tubulin, and actin, as indicated on the left-hand side of the panels. The amount of each fraction loaded was adjusted to give the same number of cell equivalents. In b, Vav (Vav I.P.) and ZAP-70 (ZAP-70 I.P.) were immunoprecipitated from cytosolic extracts prepared as in a, with and without taxol/GTP treatment. Immunopurified proteins were Western blotted and probed for the antigens indicated beside the panels. In c, control and taxol-treated cytosol were immunoprecipitated with anti-ZAP-70 antiserum. The immunoprecipitated proteins were Western blotted and probed for ZAP-70 and Vav (left-hand panels). In the right-hand panel, total cytosol (control and taxol-treated) were Western blotted and probed for ␣-tubulin content.
Similar amounts of ZAP-70 were immunoprecipitated from the control and taxol-treated cytosolic extracts under conditions where ZAP-70 antibody was limiting (Fig. 3b). Under these conditions, taxol/GTP reduced the relative abundance of tyrosine-phosphorylated ZAP-70 in the cytoplasm by 70% (Ϯ5%; n ϭ 4), as judged by anti-Tyr(P) immunoblotting (Fig.  3b). As expected, a tyrosine-phosphorylated band with identical mobility to ZAP-70 preferentially accumulated in the microtubule pellet from activated cell extracts (data not shown). These data indicate that ZAP-70 that was tyrosine-phosphorylated after TCR stimulation was preferentially associated with tubulin polymerized in vitro. This further suggests that the interaction of ZAP-70 with tubulin may be important for the activation of this kinase. In contrast, cytosolic tubulin depletion had little effect on the amount of tyrosine-phosphorylated Vav (Fig. 3b) detected in anti-Vav immunoprecipitates (reduction of 13% Ϯ 5% S.E.; n ϭ 4).
The association of both ZAP-70 and Vav with tubulin ( Fig.  2a) suggested that the presence of Vav in ZAP-70 immunoprecipitates might be mediated via tubulin. However, taxol/GTP depletion of the majority of cytosolic tubulin only slightly reduced the level of Vav in ZAP-70 immunoprecipitates (Fig. 3c). These data indicated that Vav interaction with ZAP-70 was largely independent of tubulin.
The interactions of ZAP-70 and Vav with components of the microtubule cytoskeleton and with each other raised the possibility that these proteins might be functionally interlinked in TCR signal transduction. As Vav is tyrosine-phosphorylated after TCR stimulation (32,33), this suggested that Vav might be a downstream substrate of ZAP-70 PTK. To investigate this, the kinetics of phosphorylation of ZAP-70 and Vav were analyzed after CD3 stimulation in Jurkat T cells. Tyrosine phosphorylation of Vav was extremely rapid, reaching a maximum at 0.5 to 1 min and then falling to base-line levels by 10 min (Fig. 4). In contrast the tyrosine phosphorylation of ZAP-70 was slower, peaking at 5-10 min and falling to base-line levels by 30 min. The kinetics of tyrosine phosphorylation of ZAP-70 and Vav that co-immunoprecipitated with ␣-tubulin were very similar to that detected in anti-ZAP-70 and anti-Vav immunoprecipitates (Fig. 4, bottom panel). ZAP-70 PTK is activated by tyrosine phosphorylation (10). These data (Fig. 4) suggested that Vav was tyrosine-phosphorylated before ZAP-70 was activated and, therefore, that Vav was unlikely to be a downstream target of ZAP-70.
In conclusion, these data demonstrate that ZAP-70 and Vav interact with tubulin and each other in T lymphocytes. A functional link, therefore, is likely to exist between these signaling molecules and the tubulin cytoskeleton. The possibility that the intracellular targeting of ZAP-70 and Vav is dependent on interactions with tubulin is currently being investigated.