A dual participation of ZAP-70 and scr protein tyrosine kinases is required for TCR-induced tyrosine...

3360 V. Lang et al. Eur. J. Immunol. 1997.27: 3360-3367

A dual participation of ZAP-70 and scr protein tyrosine kinases is required for TCR-induced tyrosine phosphorylation of Sam68 in Jurkat T cells

Valerie Lang’, Dorninique Mege2, Monique Sernichon’, Helene Gary-Gouy’ and Georges Bismuth’

’ Laboratoire d’lmmunologie Cellulaire et Tissulaire, CNRS URA 625, Centre Hospitalier Pitie-

a Laboratoire d’lmmunologie Moleculaire, Departement d’lmmunologie, lnstitut Pasteur, Paris, Salpgtriere, Paris, France

France

Sam68 has been initially described as a substrate of src kinases during mitosis in fibroblasts. Recent evidence suggests that in T lymphocytes Sam68 may act as an adaptor protein and participate in the early biochemical cascade triggered after CD3 stimulation. A direct interaction between Sam68 and the two src kinases involved in T cell activation, ~ 5 9 ~ ” and p56ICk, as well as a partnership of Sam68 with various key downstream signaling molecules, like phospholipase Cy-1 and Grb2, has been shown. In this study we analyze the contribution of p56ICk, as well as the role of ZAP-70, the second class of protein tyrosine kinase involved in T cell activation, in Sam68 tyrosine phosphorylation in the human Jurkat T cell line. Using the src inhibitor PP1 [4-amino-5-(4-methylphenyl)7-(t-butyl) pyrazolo [3,4-d] pyrymidine] and cell variants with defective expression of p56Ick or expressing a dominant negative form of ZAP-70, we demonstrate that, while both p561Ck and ZAP-70 are dispensable for the low constitutive phosphorylation of Sam68 observed in Jurkat cells, a cooperation between the two kinases is required to increase its rapid phosphorylation observed in vivo after CD3 stimulation. We also show that recombinant forms of both p56Ick and ZAP-70 phosphorylate Sam68 in vitro. However, using CD2 stimulated cells, we observe that p56ICk activation by itself does not induce Sam68 tyrosine phosphorylation. We conclude that ~ 5 9 ~ ” and p56ICk differently participate in regulating the phosphorylation state of Sam68 in T cells and that ZAP-70 may

revised Oct. 16, 1997;

contribute to Sam68 tyrosine phosphorylation and to the specific recruitment of this molecule after CD3 stimulation.

Key words: T lymphocytes I T cell receptor I Signal transduction I src kinase

1 Introduction

Sam68 was first described as a protein specifically tyrosine phosphorylated both in normal and in v-src transformed fibroblasts blocked in mitosis [1,2]. Its sequence revealed homology to heterogeneous nuclear ribonucleoproteins (hnRNP) [3], a group of RNA binding proteins essentially located in the nucleus and involved in various aspects of the RNA metabolism [4]. Like many hnRNP proteins, Sam68 has a particular domain called

[I 173181

Abbreviations: GAP: GTPase-activating protein GST: Glutathione S-transferase hnRNP: Heterogeneous nuclear ribonucleoprotein KH: hnRNP K homology PLC: Phos- pholipase C PP1: 4-Amino-5-(4-methylphenyl)7-(t-butyl) pyrazolo[3,4-d] pyrymidine PTK. Protein tyrosine kinase SH: src homology

accepted Oct. 22, 1997.

KH (hnRNP K Homology), which is necessary for RNA binding, and it has been recently demonstrated that a natural isoform of Sam68 with a deletion within the KH domain antagonized cell cycle progression [5].

By its association with numerous intracellular proteins through src homology (SH)2- and SH3-type interactions, Sam68 also has the features of an adaptor protein. Inter- actions with phospholipase C (PLC)y-1, Grb2, Grap (a Grb2-like protein), the regulatory subunit of phosphatidylinositol-3 (P13) kinase, p47phox, Itk, SHP-1, Cbl, Jak3, and Nck have been reported [6-121. Mainly, Sam68 can also form stable complexes with the SH3 domain of src [1,2,9] and, in its tyrosine phosphorylated form, with the SH2 domain of the kinase [1,13]. Since Sam68 tyrosine phosphorylation and association with src is increased in mitotic cells, it has been concluded that Sam68 was a privileged target for src [14]. Interac-

001 4-2980/97/1212-3360$17.50 + .50/0 0 WILEY-VCH Verlag GmbH, D-69451 Weinheim, 1997

Eur. J. Immunol. 1997.27: 3360-3367 Sam68 tyrosine phosphorylation in T lymphocytes 3361

tions of Sam68 with two other src kinases expressed in T cells and essential for T cell activation, ~ 5 9 ' ~ " and ~56 ' '~ , have also been uncovered. This has been documented in IL-2-activated human peripheral blood T cells where Sam68 can be found associated with the SH2 domain of p56ICk [15] and in HeLa cells co-transfected with Sam68 and ~ 5 9 ~ ' where an interaction of Sam68 with the SH3 and the SH2 domains of the kinase was reported [6]. The same kind of interaction was more recently observed in various human T cell lines, such as HTLV-1-infected T cells overexpressing ~59'"" or in the Jurkat T cell line where T cell activation initiated by specific triggering of the CD3 complex induced Sam68 tyrosine phosphorylation [ l l ] .

Both ~ 5 9 ~ " and ~ 5 6 ' ' ~ have been shown to be activated after TCR triggering [16-231. The observation that Sam68 tyrosine phosphorylation increased after CD3 stimulation therefore suggests that, in T cells, Sam68 may also be a preferred substrate of src kinases linking the TCR to downstream effector molecules. However, additional observation showed that Sam68 was constitutively tyrosine phosphorylated in T cell lines overexpressing ~ 5 9 ~ ' [ l l ] . It is, therefore, not clear whether p5gV', ~ 5 6 " ~ or both are involved in constitutive and CD3-induced Sam68 phosphorylation. Moreover, another class of tyrosine kinases, ZAP--/O/Syk protein tyrosine kinases (PTK), has been shown necessary to observe most of the early tyrosine phosphorylations induced after the engagement of CD3TTCR [24-281. Its role in Sam68 phosphorylation has not yet been investigated. In the present report we used the Jurkat T cell line as well as cell variants with defective activity in ~ 5 6 ' ' ~ and ZAP-70 PTK to investigate Sam68 phosphorylation in T cells.

2 Results and discussion

2.1 ~ 5 6 ' ' ~ is dispensable for basal but not CD3- induced tyrosine phosphorylation of Sam68 in Jurkat T cells

A close partnership between Sam68 and src kinases has been reported in different cell systems, including T cells [1,2,6,9,11,13,15]. We first investigated the contribution of the two src tyrosine kinases involved in TCR-mediated signaling, ~ 5 6 " ~ and p59@', in tyrosine phosphorylation of Sam68 in Jurkat T cells.

As recently reported, Sam68 is phosphorylated in Jurkat cells after CD3 triggering [ l l ] . Fig. 1A shows the anti- phosphotyrosine immunoblot obtained when the NP40 cell lysates of cells incubated with or without UCHT1, a CD3c-specific mAb, were immunoprecipitated with a

polyclonal antiserum against Sam68. A significant amount of phospho-Sam68 was regularly present in the unstimulated cells. However, within 30 s of incubation with the mAb, a clear increase in the tyrosine phosphorylation of Sam68 was observed. Then, the labeling prog- ressively declined as usually found in this cell line for many other tyrosine phosphorylated proteins. To assess

Figure 1. ~ 5 6 " ~ is dispensable for basal but not CD3- induced tyrosine phosphotylation of Sam68 in Jurkat T cells (A) Jurkat T cells (1 0 x 1 06) were stimulated with UCHTl , a CD3~-specific mAb, for the indicated period of time. Cells were then lysed in NP40 lysis buffer and Sam68 immunoprecipitated with a specific polyclonal antiserum and protein A-Sepharose. Immune complexes were resolved by SDS- PAGE on an 8% polyacrylamide gel. After blotting, the membrane was probed with the anti-phosphotyrosine mAb 4G10. (B) Prior to stimulation with the CD3~-specific mAb UCHTl for 2 min, Jurkat T cells were incubated with increas- ing concentrations of the src inhibitor PP1 for 15 min at 37" C. Cells were then lysed in NP40 lysis buffer and 60 pg of the solubilized proteins subjected to Western blot analysis with the anti-phosphotyrosine mAb. Note that the cell viabil- ity was not affected by PP1 treatment (not shown). (C) After a 15-min incubation with the indicated concentrations of PP1, Sam68 was irnmunoprecipitated as in (A) from Jurkat cells and from the ~56"~-defective Jurkat cell variant JCaM1 stimulated for 2 min with the CD3~-SpeCifiC mAb UCHTl. The anti-phosphotyrosine imrnunoblots with 4G10 are shown.


kDa 66-

~ 6 2 ~ ~ ~ -

49.5-

Stimulation

CD2 CD3 - CD2 CD3

t- Sam68

i.p.p. p21 rasGAP Sam68

Figure 2. Sam68 is not tyrosine-phosphorylated after CD2 Stimulation. Jurkat cells (10 x lo6) were stimulated with the CD3c-specific mAb UCHT1 or the CD2-specific mAb pair X11+D66 for 2 min and 5 min, respectively. After lysis in NP40 lysis buffer, equal amount of proteins were immunoprecipitated either with a p21 rasGAP-specific mAb or with the polyclonal antiserum specific for Sam68 and protein A- Sepharose. Immune complexes were resolved by SDS- PAGE on an 8 % polyacrylamide gel, blotted and the membrane probed with the anti-phosphotyrosine mAb 4G10.

the role of p561Ck and p5gWn and their respective contribution in Sam68 phosphorylation, we performed the same type of experiments with the src inhibitor PP1 and the ~ 5 6 " ~ defective Jurkat variant JCaM1. PP1 has been described as a potent and selective inhibitor of src kinases in vitro and also as an inhibitor of phosphorylation on intact peripheral blood T cells [29]. In Jurkat cells, as shown in Fig. lB, PP1 inhibits CD3-induced tyrosine phosphorylations in a dose-dependent fashion. At the same time, PPl also inhibits Sam68 phosphorylation as shown in Fig. lC, upper panel, after immunoprecipitating the molecule. No residual phosphorylation of Sam68 was usually observed with PPl concentrations above 2.5pM showing that both constitutive and CD3-triggered phosphorylation of the molecule were sensitive to the effect of the src inhibitor. It is useful to mention here that in Jurkat T cells, Sam68, which has an apparent molecular mass of 64-65 kDa, migrates closely to other phos- phoproteins induced after CD3 stimulation. Sam68 was therefore difficult to localize in the whole pattern of tyrosine-phosphorylated proteins (see Fig. 1 B). In JCaM1 cells, as in the wildtype, we observed a constitutive tyrosine phosphorylation of Sam68 (Fig. lC, lower panel). However, in this cell variant, no increase was triggered after CD3 stimulation. Tyrosine phosphorylation of Sam68 in JCaM1 cells was inhibited by PP1 in a dose- dependent fashion. Taken together, these results demonstrate that the basal tyrosine phosphorylation of Sam68 does not involve ~ 5 6 " ~ but likely ~ 5 9 ~ " . They also demonstrate that the induction of Sam68 phosphorylation after CD3 stimulation requires ~ 5 6 " ~ , suggesting that both kinases could be involved in regulating the phosphorylation of Sam68 in T cells.

2.2 pSkk activation does not induce Sam68 tyrosine phosphorylation by itself

CD2 is an alternative T cell activation pathway that can be triggered using two mAb specific for distinct regions of the molecule [30]. Most of the early biochemical events observed after CD3 stimulation, i.e. tyrosine phosphorylation of intracellular proteins including PLCy- 1, and calcium response are observed after CD2 stimulation [31, 321. Mainly, we and others have reported that CD2 activation of Jurkat T cells strongly activates p56"" [22, 33, 341. To further investigate the commitment of p56Ick in Sam68 phosphorylation, we therefore compared in Fig. 2 the tyrosine phosphorylation of Sam68 immunoprecipitated from cells incubated with mAb UCHT1 or with mAb X11+ D66, a mitogenic pair of CD2-specific antibodies which stimulates ~ 5 6 " ~ [34]. p21 rasGAP immunoprecipitates from the same lysates were run in parallel as a control of CD2 stimulation since we reported, in a previous work, the specific tyrosine phos-

Figure 3. Expression of a dominant negative form of ZAP- 70 inhibits CD3-induced tyrosine phosphorylation of Sam68. (A) Jurkat J05.2 and FF18-22 cells (10 x lo6) were Stimulated or not with the CD3~specific mAb UCHTl for 2 min. After lysis in NP40 lysis buffer, 60 pg of the solubilized proteins were subjected to Western blot analysis with the anti- phosphotyrosine mAb. (B) Remaining cell lysates from A were immunoprecipitated with the polyclonal antiserum specific for Sam68 and protein A-Sepharose. Immune complexes were resolved by SDS-PAGE on an 8 % polyacrylamide gel, blotted and the membrane probed with the anti- phosphotyrosine mAb 4G10. (C) The blot obtained after probing the different cell lysates obtained in (A) with the antiserum specific for Sam68 is shown.


tyrosine phosphorylated after CD2 triggering [34], as found, in the present report, for Sam68 (see Fig. 2). Thus, a relationship between ZAP-70 activation and Sam68 phosphorylation is conceivable. To further assess this hypothesis, we used Jurkat cells stably transfected with a dominant negative form of ZAP-70. Jurkat FF18-22 cells stably overexpress a ZAP-70 mutant (fivefold excess of ZAP-70 mutant over endogeneous ZAP-70), in which both Tyr-492 and Tyr-493 from the positive regulatory kinase loop were mutated to Phe [40]. These muta- tions have been shown to inhibit CD3-TCR-induced activation events (calcium increase, IL-2 production) by interfering with the tyrosine phosphorylation of specific downstream signaling effectors such as pp36-38 (see Fig. 3A and [40]) and a 145-kDa band probably corresponding to PLCy-1 (see Fig. 3A). As shown in Fig. 3B after Sam68 immunoprecipitation, we found a similar level of constitutive tyrosine phosphorylation of the molecule in J05.2, a Jurkat cell transfected with the empty vector, and in FF18-22. Thus both ~ 5 6 " ~ and ZAP-70 are dispensable for constitutive phosphorylation of Sam68 in Jurkat cells. However, only a poor increase in the labeling of Sam68 was observed after CD3 stimulation of FF18-22. Note that both transfectants expressed the same amount of Sam68 as shown by probing the different cell lysates with the anti-Sam68 antiserum (Fig. 3C). We also controlled by kinetic studies that the tyrosine phosphorylation of Sam68 was not delayed in FF18-22 (data not shown).

A B kDa

GST-Sam68 + - 97.4

GST-[ASHZ]ZAP-'IO + - 66.2 p56Ick+

1 2 3 1 2 3

Figure 4. Sam68 is phosphorylated in vitro by ZAP-70 and ~56"~ . (A) GST-Sam68 recombinant fusion proteins (1 pg), immobilized on sepharose beads, were subjected to in vitro kinase assays in the presence of [Y-~'P]ATP and a purified GST fusion protein of the catalytic domain of ZAP-70 (GST-[ASHP]ZAP-70; 80 ng) (lane 2) or a wild-type form of ~ 5 6 " ~ (1/1500 final dilution of a crude lysate of SF9 insect cells infected with a p56"k-recombinant baculovirus) (lane 3). Lane 1 contains a control without kinase addition. Autora- diography of a 10 % SDS-polyacrylamide gel is shown. (B) Coomassie blue staining of the gel shown in (A).

phorylation of the p21 rasGAP-associated protein p62, now referred as ~ 6 2 ~ ' ~ [35, 361, after CD2 stimulation of Jurkat T cells [37]. Moreover, Sam68 and ~ 6 2 ~ ' ~ were considered for a long time to be related proteins [38]. As shown in Fig. 2, no significant increase in the tyrosine phosphorylation of Sam68 could be detected in the CD2-stimulated cells. At the same time, an extensive labeling of the band co-immunoprecipitating with p21 rasGAP and probably corresponding to ~ 6 2 ~ ' ~ was visualized in the CD2 extract. It was poorly induced by UCHT1, confirming our previous results (371. Stimulation times were choosen according to the maximal level of tyrosine phosphorylation of cellular proteins induced by CD3 or CD2 mAb [37], but kinetic experiments gave identical results (data not shown). An association between Sam68 and ~ 5 6 " ~ molecules in T cells has been previously demonstrated [ l 1 , 151. Since CD2 activates ~ 5 6 " ~ , one should expect to observe a significant tyrosine phosphorylation of Sam68 after CD2 ligation. This was not the case, a result which further suggests that ~ 5 6 " ~ in vivo stimulation alone does not support Sam68 tyrosine phosphorylation in Jurkat T cells.

2.3 ZAP-70 contributes to Sam68 tyrosine phosphorylation in CD3-activated cells

A second PTK, ZAP-70, is involved in CD3-TCR signaling after its recruitment to the tyrosine-phosphorylated ITAM (intracellular tyrosine activation motifs) of the or E

polypeptides of activated receptors [39]. We recently demonstrated that ZAP-70 was poorly recruited and

To further assess the participation of ZAP-70 in Sam68 phosphorylation, we constructed a GST fusion protein with Sam68 and performed in vitro kinase assays using this protein and ZAP-70. We used a recombinant form of ZAP-70 containing the active catalytic domain of the kinase fused to GST and deleted of its two SH2 domains. A recombinant wild-type form of p5SJCk was used in parallel. As shown in Fig. 4A, a strong autophosphoryla- tion was observed with the two recombinant PTK. Impor- tantly, both also phosphorylate GST-Sam68 which migrates with an apparent molecular mass of 98 kDa as also shown in Fig. 48 after Coomassie blue staining of the same gel.

The mechanisms by which ZAP-70 controls the tyrosine phosphorylation events after CD3-TCR ligation is not fully understood. It is currently accepted that TCR- induced activation of ~ 5 6 " ~ or ~ 5 9 ~ ' ' is required not only for the ITAM phosphorylaton, but also for ZAP-70 kinase activation by phosphorylation of Tyr-492 and/or Tyr-493, two critical residues in the activation loop of the kinase domain [40,41]. Moreover, once bound to the ITAM regions, ~ 5 6 " ~ and ZAP-70 kinases physically stay associated via the SH2 domain of ~56"" which probably has important synergistic functional consequences in coupl-


ing TCR stimulation to downstream signaling pathways [42,43]. Our results strongly suggest that this kind of syn- ergy between the two PTK is required to achieve Sam68 phosphorylation in CD3-activated Jurkat T cells.

2.4 Concluding remarks

Sam68 has been previously described as a favorite substrate of PTK from the src family. Accordingly, a strong constitutive tyrosine phosphorylation of Sam68 in T cells overexpressing ~ 5 9 ~ " has been shown, suggesting a role for ~ 5 9 ~ " in this phenomenon [I I]. However, we show in the present report that the phosphorylation status of Sam68 in T cells is presumably a more complex issue and that, depending on the cell stimulation conditions, its phosphorylation can be controlled by other PTK such as ~ 5 6 ' " ~ and ZAP-70. Sam68 has the capacity to bind src kinases but also various SH2 and SH3-domain- containing signaling molecules like PLCy-1 or Grb2. Both are essential mediators in coupling TCR activation to calcium increase and p21 ras activation [44]. Although it is too early to ascribe a function for Sam68 in these signaling pathways, it has been suggested that tyrosine- phosphorylated Sam68 may serve as an adaptor protein linking TCR-associated src kinases to these downstream effectors [6,11]. If true, this could also suggest that different adaptor proteins like Sam68 on the one hand and ~ 6 2 ~ " ~ on the other could have some redundant func- tions in the CD3 and CD2 activation pathways. Besides, Sam68 also has features of RNA binding proteins. A KH domain and an RGG box-like region, both predictive of RNA-binding proteins, are present in the molecule. Accordingly, RNA-binding properties of Sam68 have been reported [1,3]. Interestingly, a modification in the ability of Sam68 to bind RNA upon phosphorylation of tyrosine residues has also been shown [45]. Thus Sam68 may belong to this class of proteins which are substrates of tyrosine kinases and may link cell stimulation with some aspects of RNA metabolism. As reported in src- transformed fibroblasts [3], using fractionation studies we found that, in T cells, Sam68 is localized both on the cell membrane and in the nucleus (data not shown). We do not know at the present time whether Sam68 can translocate from one compartment to another. However, as has been proposed for hnRNP proteins [46], once phosphorylated on the cell membrane after CD3 stimulation, Sam68 could move into the nucleus to control RNA localization and alternative splicing regulation. This hypothesis, as well as the contribution of Sam68 in the early metabolic events triggered by TCR stimulation, are now under investigation.

3 Materials and methods

3.1 Cells

The Jurkat T cell line (clone J77) was grown in RPMl 1640 medium (Flow Laboratories, Irvine, GB) supplemented with 10 % FCS, antibiotics (50 U/ml penicillin, 50 yg/ml streptomycin), L-glutamine (2 mM) and sodium pyruvate (1 mM). The p56lCk-defective Jurkat variant, JCaM1, was purchased from ATCC (Rockville, MD) and was grown in the same culture conditions. Jurkat J05.2 and FF18-22 cells were previously described [29]. FF18- 22 cells were stably transfected with the pSRapuro vector containing a ZAP-70 construct in which both Tyr-492 and Tyr-493 were mutated to the Phe.JO5.2 was transfected with the empty vector. Both were cultured in com- plete medium supplemented with 10 pg/ml of puromycin (Sigma, Saint Quentin Fallavier, France).

3.2 Antibodies

The CDSespecific mAb UCHT1 was kindly provided by Dr. P.L.C. Beverley (Imperial Cancer Research, London, GB). mAb specific for CD2 were a generous gift from Dr. L. Boumsell (INSERM U448, Centre Hospitalier Henri Mondor, Creteil, France): X11 recognizes the TI 1.1 epitope of CD2, whereas D66 is directed to the so-called CD2R cryptic epitope of the molecule. The anti- phosphotyrosine mAb 4G10 was purchased from Upstate Biotechnology Incorporated (Lake Placid, NY). The anti-p21 rasGAP (GAP) mAb was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). A polyclonal antibody specific for Sam68 was prepared by immuniz- ing rabbits with a recombinant form of human Sam68 protein (residues 49 to 445) fused to GST.

3.3 Cell stimulation and irnmunoprecipitation

T cells (1 0 x 1 06) were washed once and resuspended in 1 ml of RPMl medium containing 10 mM Hepes, pH 7.2, and were then equilibrated for 15 min at 37" C. Cell stimulation was achieved by incubation at 37" C in the presence of the CD3~-specific mAb UCHTl , or of the CD2- specific mAb pair X I 1+D66, or in medium alone. All stim- ulating mAb were used at a 1/500 dilution of ascitic fluids. Activation was stopped by brief centrifugation and lysis at 4" C for 1 h in lysis buffer (20 mM Tris-HCI pH 7.5, 140 mM NaCI, 1 mM EDTA, 50 U/ml aprotinin, 1 mM PMSF, 1 mM sodium orthovanadate) containing 1 % (v/v) NP40 detergent. Nuclei and cellular debris were removed by centrifugation at 10000 x g for 10 min, and the amount of proteins present in each postnuclear supernatant was determined. Samples were then diluted


in Laemmli sample buffer (500 mM Tris-HCI pH 6.8, 10 % SDS, 10 % glycerol, 5 % 2-ME, 10 % bromophenol blue), boiled for 3 min and used for Western blotting experiments. For Sam68 immunoprecipitation, lysates containing equal amounts of proteins were incubated for 2 h at 4" C with 20 pl of protein A-Sepharose beads previously incubated 1 h at 4" C with the anti-Sam68 rabbit antiserum. After four washes in the lysis buffer, immune complexes were recovered by boiling 3 min in Laemmli sample buffer. The src kinase inhibitor PP1 (Calbiochem, France Biochem, Meudon, France) was used as previously described [30]. Cells were incubated for 15 min at 37" C with PP1 in RPMl medium prior to stimulation.

3.4 Western blotting

For analysis of tyrosine-phosphorylated proteins, 60 pg of cellular lysates or immunoprecipitated proteins were loaded onto an 8 % SDS-polyacrylamide gel, and elec- trophoretically transferred for 1 h at 65 V to a nitrocellu- lose membrane (Schleicher and Schuell, Dassel, Ger- many). The quality of the transfer was always checked with Ponceau S (Sigma, Saint Quentin Fallavier, France) staining of the membrane. lmmunoblotting was then performed with the anti-phosphotyrosine mAb 4G10 (0.2 pg/ml) or Sam68 rabbit antiserum (1/2000 dilution) followed by peroxidase-labeled goat anti-mouse or goat anti-rabbit antisera (Bio-Rad, lvry Sur Seine, France), respectively. Reaction was revealed with an enhanced chemiluminescence system (ECL; Amersham, Paris, France) according to the supplier's instructions.

3.5 GST fusion protein of Sam68

The p62-KL1 plasmid, containing the full-length Sam68 coding sequence [3], was kindly provided by Dr. F. McCormick (Onyx Pharmaceuticals, Richmond, CA). The Sam68 insert was isolated by digesting p62-KL1 with EcoRl and Not1 . The DNA fragment was blunted with the Klenow fragment of DNA polymerase I and subcloned in- frame into the bacterial expression vector pGEX-2T to generate a GST fusion protein. The recombinant plasmid was used to transform fscherichia coli strain DH5a. Cul- tures were grown at 37°C and induced with 100 yM isopropyl 6-D-thiogalactopyranoside for 3 h. Cells were harvested by centrifugation. The pellet was washed once in PBS and resuspended in the same buffer. Cells were lysed using sonication, and lysates were centrifuged for 10 min at 10000 x g at 4"C, then stored at -80" C.

3.6 In vitro kinase assay

A wild-type form of ~ 5 6 " ~ (provided as a crude lysate of SF9 insect cells infected with a p56'ck-recombinant baculovirus) was kindly provided by Dr. S. Fischer (INSERM U 363, lnstitut Cochin de Genetique Moleculaire, Paris, France). Purified recombinant ZAP-70 molecules (a GST fusion protein of the catalytic domain of the kinase) were kindly provided by Dr. A. lsacchi (Department of Molecu- lar Biology, Pharmacia & Upjohn, Milano, Italy). For kinase assays, GST-Sam68 fusion proteins were purified by affinity chromatography on glutathione-sepharose beads (Pharmacia Biotech, Uppsala, Sweden). Fusion protein complexes were washed three times in a buffer (20 mM Tris-HCI pH 7.5,140 mM NaCI, 1 mM EDTA) containing 1 % (v/v) NP40 detergent and once with a phosphorylation buffer containing 50 mM Pipes, pH 6.8, 10 mM MnCI,, 10 mM MgCI,, 50 pM sodium orthovanadate, 50 unitdm1 aprotinin and 1 mM PMSF. The beads were then resuspended with 30 pI of phosphorylation buffer supplemented with 10 pM cold ATP and 10 pCi of [Y-~'P]ATP (> 4000 Ci/mmole, Isotopchim, Ganagobie- Peyrus, France) and the reaction was started by the addition of ~ 5 6 " ~ or ZAP-70 in a volume of 1 pl. After 10 min at room temperature, the reaction was stopped by the addition of Laemmli buffer and boiling. Proteins were separated by 10 % SDS-PAGE, and the gel was dried and subjected to autoradiography on X-ray film.

Acknowledgments: The authors are indebted to Drs. Oreste Acuto and Vincenzo Di Bartolo for helpful discus- sions during this work. This work was supported by grants from the Association de la Recherche contre le Cancer and the Centre National de la Recherche Scientifique.

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Correspondence: Georges Bismuth, Laboratoire d'lmmunologie Cellulaire, CNRS URA 625, Centre Hospitalier Pitie- SalpGtriere, Cervi, 83, Blvd de I'Hijpital, F-75013 Paris, France Fax: +33-1-42177490 e-mail: [email protected]

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