Shb links SLP-76 and Vav with the CD3 complex in Jurkat T cells Cecilia K. Lindholm 1 , Maria L. Henriksson 2 , Bengt Hallberg 2 and Michael Welsh 1 1 Department of Medical Cell Biology, Biomedicum, Uppsala University, Sweden; 2 Department of Medical Biosciences/Pathology, Umea ˚ University, Sweden This study addresses the interactions between the adaptor protein Shb and components involved in T cell signalling, including SLP-76, Gads, Vav and ZAP70. We show that both SLP-76 and ZAP70 co-immunoprecipitate with Shb in Jurkat T cells and that Shb and Vav co-immunoprecipitate when cotransfected in COS cells. We also demonstrate, utilizing fusion protein constructs, that SLP-76, Gads and Vav associate independently of each other to different domains or regions, of Shb. Overexpression of an SH2 domain-defective Shb causes diminished phosphorylation of SLP-76 and Vav and consequently decreased activation of c-Jun kinase upon T cell receptor (TCR) stimulation. Shb was also found to localize to glycolipid-enriched membrane microdomains (GEMs), also called lipid rafts, after TCR stimulation. Our results indicate that upon TCR stimula- tion, Shb is targeted to these lipid rafts where Shb aids in recruiting the SLP-76–Gads–Vav complex to the T cell receptor f-chain and ZAP70. Keywords:Shb;Vav;SLP-76;Tcellreceptor;lipidrafts. The early events in T cell signalling involve the activation of Lck, which subsequently phosphorylates the TCR f-chain and the e-chain of the CD3 complex on ITAM motifs (immunoreceptor tyrosine-based activation motifs). This recruits the kinases ZAP70 or Syk that consequently induces tyrosine phosphorylation of several intracellular substrates, for example, phospholipase C-c1(PLC-c1), LAT [1], Vav and SLP-76 [2]. Functional interactions between these effector molecules are believed to be necessary for T cell maturation, proliferation and differentiation. One aspect of T cell receptor (TCR) activation relates to specific areas (microdomains) of the T cell plasma mem- brane, rich in glycosphingolipids, cholesterol and GPI- anchored proteins, but poor in phospholipids (reviewed in [3]). These glycolipid-enriched membrane microdomains (GEMs), or lipid rafts, are rich in PTKs (protein tyrosine kinases), monomeric and trimeric G proteins and several other signaling proteins. In resting T cells LAT, Lck, Fyn, Syk, Cbl and Ras are located in rafts, whereas some other proteins are recruited to the rafts upon T cell receptor stimulation. The latter group includes SLP-76, Gads, Vav, ZAP70, TCR f-chain, PLC-c1, Shc and PKC [4,5]. The adapter proteins SLP-76 and Gads and the guanine nucleotide exchange factor Vav are critical for appropriate T cell activation. SLP-76 and Vav are both phosphorylated upon CD3 stimulation in Jurkat T cells. SLP-76 is phosphorylated by ZAP70 [6] at multiple residues (Tyr113, Tyr128 and Tyr145) of which Tyr113 and Tyr128 are responsible for allowing the binding of the Vav SH2 domain [7]. SLP-76 has also been shown to associate with Lck, LAT, Grb2 and SLAP-130 in Jurkat T cells. Vav is reported to be phosphorylated by Lck on Tyr174 and this process activates the exchange activity of Vav [8,9]. However, other studies have suggested tyrosine- 174 on Vav as a putative candidate for phosphorylation by Syk and Zap70 [10]. Vav is a guanine nucleotide exchange factor for the GTPases Rac1 and Cdc42, which cause, among other things, activation of the c-Jun N-terminal kinases (JNK). Gads (Grb2-related adaptor downstream of Shc) is not phosphorylated in response to TCR stimulation, but it has been shown to associate with several proteins upon T cell activation, including Shc, SLP-76 and LAT [11,12]. The Shb adapter protein was cloned from a b-cell library in 1994 [13], but it has since been found to be ubiquitously expressed [14]. Shb is involved in FGFR-1 and PDGFR signaling, and also associates with several signaling proteins, including CrkII, Eps8, Grap and Src [14,15]. Shb is also of importance for the TCR-dependent immune response in Jurkat cells. TCR-mediated activation of NFAT was totally abolished in Jurkat cells expressing a mutant form of Shb with a defective SH2 domain, and endogenous IL-2 production was also decreased in these cells [16]. Overex- pression of wild-type Shb, however, had no effects on the CD3 mediated responses in Jurkat cells [14,16]. In Jurkat cells, Shb has previously been found to exhibit domain- dependent interactions with Grb2, LAT, PLC-c1andthe f-chain of the T cell receptor [14–16]. The SH2 domain of Shb bound the f-chain, the PTB domain bound LAT and the proline-rich regions associate with PLC-c1 and Grb2. There are also four putative tyrosine phosphorylation sites in Shb that show extensive homology with similar sequences in other adapter proteins [17], and conform with the consensus sequence Y-X-D/E/T/Q-P-F/Y/W-D/E. Correspondence to C. Lindholm, Department of Medical Cell Biology, Box 571, Biomedicum, 75123, Uppsala, Sweden. Fax: + 46 18 556401, Tel.: + 46 18 4714033, E-mail: Cecilia.Lindholm@medcellbiol.uu.se Abbreviations: TCR, T cell receptor; SH2, Src homology 2; PTB, phosphotyrosine binding; ITAM, immunoreceptor tyrosine-based activation motif; PTK, protein tyrosine kinase; ECL, enhanced chemiluminescence system; JNK, c-Jun N-terminal kinase; GST, glutathione-S-transferase; PDGF, platelet-derived growth factor; FGF, fibroblast growth factor; P-Y, phosphotyrosine; MAPK, mitogen-activated protein kinase; GEMs, glycolipid-enriched membrane microdomains; LAT, Linker for Activation of T cells. (Received 13 February 2002, revised 16 May 2002, accepted 21 May 2002) Eur. J. Biochem. 269, 3279–3288 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03008.x In this study, we describe interactions between Shb and SLP-76, Gads, Vav and ZAP70 and how a functional Shb SH2 domain is important for phosphorylation of SLP76 and Vav, possibly by ZAP70. The Shb SH2 domain is also of importance for the activation of the JNK, downstream of Vav in the T cell signalling cascade. We have also observed the recruitment of Shb to lipid rafts upon TCR stimulation in Jurkat T cells. MATERIALS AND METHODS Antibodies Anti-phosphotyrosine Ig (4G10) and anti-Vav Ig were purchased from Upstate Biotechnology (Lake Placid, NY, USA). CD3 antibody (CD3 pure) was from Becton- Dickinson (San Jose, CA). Anti-JNK Ig and anti-(phos- pho-JNK) Ig were from New England BioLabs (Beverly, MA). Anti-HA antibody was from Santa Cruz. Anti- ZAP70Ig,anti-SLP-76Igandanti-Rac1Igwerefrom Transduction Laboratories (Lexington, KY). Rabbit poly- clonal anti-Gads Ig was a gift from Jane McGlade, Hospital for Sick Children Research Institute, Toronto, Canada. Affinity purified anti-Shb Ig has been described previously [18]. DNA constructs The Shb–SH2–GST fusion protein plasmid has been described previously [13]. The Shb–PTB-Pro–GST plasmid was described previously under the name p55 ShbDSH2 [14] and produces a fusion protein corresponding to the PTB domain and two proline-rich sequences. The p55 Shb–pET plasmid has been described previously [14] and produces the full-length p55 Shb with an additional His-tag for purifica- tion on Ni-beads according to the manufacturer (Novagen). The Shb–PTB–GST plasmid was constructed using the primers 5¢-GGGATCCTTCCAGGACCCCTAC-3¢ and 5¢-AGAATTCAGGGCTCCCATGTTT-3¢ corresponding to the Shb cDNA nucleotides 840–1740. The amplified fragment was digested using EcoRI, and ligated into EcoRI digested pGEX-2T vector. The SLP-76-Pro–GST plasmid was constructed using the primers 5¢-CGAGGGATCCCT GCAGAACTCCATCCTGCCTG-3¢ and 5¢-CATTTAAT GAATTCTCTTCCTCCGC-3¢ corresponding to SLP-76 nucleotides 466–1245. The amplified fragment was cut using BamH1 and EcoR1 and ligated into pGEX-2TK. The SLP-76-SH2-GST plasmid was constructed using the primers 5¢-GGAAGGATCCAATTCATTAAATGAAGA GTG-3¢ and 5¢-GGCTATAACGAATTCTGGGTACCC TGCAGCATG-3¢. This fragment was also cut with BamH1 and EcoR1 and ligated into pGEX-2TK. The PAK-CD- GST (PAK Crib domain) plasmid has previously been described previously [19]. The Shb wild-type plasmid contains the Shb cDNA inserted in pcDNA1 vector, and has been described previously. The Shb R522K plasmid has also been described previously [14]. Briefly, the arginine at position 522 was converted to a lysine, in full-length Shb cDNA, and inserted in pcDNA1 expression vector. The Shb DPTB-Tyr plasmid was constructed by the deletion of the nucleotides 880–1603 in the Shb cDNA (containing the four tyrosine phosphory- lation sites and the PTB domain), using the restriction enzymes FspIandBstEII. The hemeagglutinin-tagged SLP-76 vectors were constructed as follows. The SLP-76 fragment (1–534), the SLP-76-Tyr fragment (1–155) and the SLP-76-SH2 fragment (415–534) were cut out from the above mentioned pGEX-2TK vectors using BamH1 and EcoR1 and ligated into pSG5A vector, containing an HA tag. The Vav expression plasmid was a kind gift from A. Weiss, San Francisco, CA, USA. Transient transfections COS cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FcII and antibiotics. The cells were washed three times in serum-free DMEM and transfected with 2 lgofeach plasmid or emply vector, as indicated in the figures, using Lipofectamine as recommended by the manufac- turer. Jurkat T cells were washed twice in NaCl/P i and transfected by electroporation (380 V, 950 lFina1-cm cuvette) with 15 lg of each plasmid as indicated in figure 3. Binding experiments and immunoprecipitations Jurkat cells or Jurkat cells overexpressing Shb R522K [16], were maintained in RPMI medium supplemented with 10% FcII (Hyclone) and antibiotics. COS cells were maintained in DMEM medium supplemented with 10% FcII. Jurkat cells were collected by centrifugation and suspended in RPMI 1640 medium lacking serum before stimulation with the CD3 antibody at 37 °C for 2 min. COS cells were either unstimulated or stimulated with pervanadate for 20 min at 37 °C. Jurkat cells were pelleted by centrifugation and COS cells were scraped together using a rubber policeman. The cells were lysed in Triton lysis buffer (0.15 M NaCl, 0.05 M Tris pH 7.5, 0.5% Triton X-100, 1 m M NaF, 0.1 m M orthovanadate, 100 UÆmL )1 trasylol, 2 m M phenylmethane- sulfonyl fluoride) for 10 min. Nuclei were pelleted by centrifugation and cell extracts were incubated with the immobilized fusion proteins on glutatione Sepharose beads (Amersham-Pharmacia Biotech, Uppsala, Sweden) or Ni-beads (Novagen) for 30 min on ice and then washed three times with NaCl/P i /1% Triton. Alternatively, the cell extracts were preincubated with protein A Sepharose for 15 min (Jurkat cell extracts only) and then immunoprecipi- tated for 1 h with antibodies against Shb, SLP-76, Vav, ZAP70, HA or preimmune sera (IgG). Immunoprecipitates were pelleted using either protein A or protein G Sepharose (50 lL) and then washed three times with NaCl/P i /1% Triton. The fusion protein complexes, cell extracts or the immuno-complexes were then resolved on SDS/PAGE and subjected to Western blotting onto Immobilon filters (Millipore) in 20% methanol, 190 m M glycine, 23 m M Tris and 0.02% SDS. The blots were subsequently incubated with blocking solution (5% BSA in NaCl/P i /0.5% Tween) and primary antibody as indicated. Immuno-reactivity was detected using horseradish peroxidase-conjugated secondary antibodies and ECL (Amersham–Pharmacia Biotech, Uppsala, Sweden) according to the manufacturer’s instructions. For detection of Rac1 activity, the amount of Rac1 bound to the PAK-CD fusion protein was determined using Rac1 antisera, and normalized to total Rac1, in cell lysate. 3280 C. K. Lindholm et al. (Eur. J. Biochem. 269) Ó FEBS 2002 Peptide synthesis and peptide inhibition experiments Peptides corresponding to tyrosine-phosphorylation sites in SLP-76 and Vav were synthesized. The peptides used in this paper include: SLP-Y113: WSSFEEDDYESPND, SLP- Y128: QDGEDDGDYESPNE, SLP-Y145: APVEDDA DYEPPPS, Vav-Y174: NEEAEGDEIYEDLMRL and Vav-Y692: ILANRSDGTYLVRQRV, where Y indicates phosphotyrosine. For peptide inhibition experiments, cell lysates prepared as above were incubated with Shb–PTB- Pro–GST, Shb–PTB–GST or p55 Shb–pET fusion protein in the presence of 100 l M phosphorylated peptide or phospho- tyrosine (10 m M )at4 °C for 30 min. After three washes with NaCl/P i /1% Triton, the samples were separated by SDS/ PAGE and transferred onto Immobilon filter as described above. Anti-SLP76 Ig and anti-Vav Ig were used to detect the amount of these proteins bound to the Shb fusion protein. Isolation of GEM fractions Jurkat cells (5 · 10 7 ) where either left unstimulated or stimulated with CD3 antibody at 37 °C for 3 min, followed by lysis at 4 °C for 30 min in 1 mL of Mes-buffered saline (25 m M Mes, pH 6.5, 150 m M NaCl, 5 m M EDTA, 1% Triton X-100, 0.05 m M orthovanadate, 100 U mL )1 trasy- lol, 10 m M NaF, 1 m M Pefablock). The lysates where then mixed with 1 mL 80% sucrose in Mes-buffered saline and transferred to ultracentrifuge tubes. The samples were overlaid with 2 mL of 30% sucrose in Mes-buffered saline, followed by 5% sucrose in Mes-buffered saline. The Triton- insoluble fractions were separated from the cell lysates by ultracentrifugation for 22 h at 250 000 g in a Beckman SW50.1 rotor at 4 °C. Fractions (400 lL) were removed sequentially starting from the top of the gradient. The proteins in each fraction were precipitated using 20% trichloroacetic acid. The precipitates were washed with acetone, dried and resuspended in 40 lLofSDS-sample buffer. The fractions (fraction 1–6 contain the lipid rafts) were then resolved on SDS/PAGE and subjected to Western blotting as described above. The blots were subsequently incubated with blocking solution (5% BSA in NaCl/P i / 0.5% Tween) and primary antibody as indicated. Immuno- reactivity was detected as described above. RESULTS Expression of Shb with an R522K mutation in the SH2 domain affects the co-immunoprecipitation of several tyrosine phosphorylated proteins To further investigate the protein-interactions of the Shb adapter protein, we attempted to identify additional binding partners for Shb in Jurkat T cells. Shb immunoprecipitation in Jurkat T cells revealed several coimmunoprecipitated proteins (Fig. 1). We detected phosphoproteins correspond- ing to the previously identified proteins PLC-c1 at 160 kDa, Linker for Activation of T cells (LAT) at 35 kDa and the TCR f-chain at 22 kDa, but also proteins of 100, 75 and 70 kDa. The association between Shb and PLC-c1was previously shown to be independent of TCR activation, whereas the interactions between LAT or the f-chain and Shb were found to be dependent on TCR activation [14,16]. We have also described the R522K mutation in the SH2 domain of Shb [14,16], and how expression of this Shb mutant decreases the tyrosine phosphorylation of PLC-c1, LAT and the TCR f-chain. Jurkat R522K-2 cell lysates displayed reduced tyrosine phosphorylation of proteins migrating as 160, 100, 75 and 35 kDa (Fig. 1) upon CD3 stimulation. Shb immunoprecipitation of the R522K cell lysates revealed decreased phosphotyrosine content of the bands corresponding to PLC-c1 (160 kDa), LAT (36 kDa) and the f-chain (22 kDa). In addition, the Shb immuno- precipitations exhibited reduced tyrosine phosphorylation of the 75 and 100 kDa proteins, in the Jurkat R522K-2 cells, unlike the 70-kDa product, which was not, or only slightly, affected. Probing the blot with an antibody reactive with SLP-76 revealed a weak band at 75 kDa that was detected in the CD3-stimulated control cells. The R522K-2 cells exhibited the presence of SLP-76 regardless of whether the cells were stimulated with CD3 or not. The reduced phosphorylation of the 160-, 100-, 75-, 35- and 22-kDa proteins in Jurkat cell lysates has previously been verified in another clone overexpressing R522K Shb and after tran- sient transfection of the R522K Shb cDNA [14,16]. Fig. 1. Shb coimmunoprecipitates with several tyrosine phosphorylated proteins. Jurkat-neo or Jurkat R522K-2 cells (10 7 ) were left unstimu- lated (–) or were stimulated with anti-CD3 Ig for 2 min in 37 °C(+). The cells were then lysed in triton lysis buffer and cell extracts were precleared with protein A Sepharose and then subjected to a-Shb immunoprecipitation. The precipitates and cell extracts were resolved on SDS/PAGE and blotted for SLP-76, Shb and phosphotyrosine using the 4G10 antibody. The positions of SLP-76, Shb, LAT and molecular mass markers are indicated. Ó FEBS 2002 Association of Shb, SLP-76 and Vav in Jurkat cells (Eur. J. Biochem. 269) 3281 Shb associates with SLP-76, ZAP70 and Vav in CD3 stimulated Jurkat T cells To further elucidate the identity of the 75-kDa protein seen after a-Shb immunoprecipitation, another blot was probed with anti-(SLP-76) Ig. SLP-76 was present in a-Shb immunoprecipitates after CD3 stimulation, but not after immunoprecipitation with preimmune serum (Fig. 2A). Likewise, the 70-kDa protein that coimmunoprecipitated with Shb was identified as ZAP70 (Fig. 2A). To assess the mode of interactions between Shb and SLP- 76 or ZAP70, GST fusion proteins comprising p55 ShbDSH2 (Shb–PTB-Pro–GST) [14], Shb–PTB–GST, Shb–SH2–GST [13] or GST (as control) were incubated with cell extracts from CD3 stimulated or unstimulated Jurkat cells (Fig. 2B). We also tested if phosphotyrosine could inhibit the binding between fusion protein and cell protein. ZAP70 was found to associate with both the Shb– PTB-Pro-GST fusion protein and the Shb–SH2–GST fusion protein, and these associations were slightly increased after CD3 stimulation. The interaction between the SH2 domain and ZAP70 was completely blocked with phospho- tyrosine, whereas binding of ZAP70 to the Shb–PTB-Pro– GST fusion protein was only partially inhibited under these conditions. The blot was also stained with amidoblack to verify that equal amounts of Shb–PTB-Pro–GST fusion protein were used. SLP-76 was found to associate with the Shb–PTB-Pro– GST fusion protein, containing both the PTB domain and two of the N-terminal proline-rich domains. This interac- tion was only slightly inhibited by phosphotyrosine. To test if the Shb PTB domain could be involved in this interaction we searched the protein-sequence of SLP-76 for D-D-X-Y, which is the consensus sequence for the Shb PTB domain Fig. 2. Association of Shb with SLP-76 and ZAP70 in CD3 stimulated T cells and pervanadate stimulated COS cells. Jurkat cells (10 7 )were unstimulated (–) or stimulated by CD3 cross-linking for 2 min in 37 °C (+) before lysis in triton lysis buffer. (A) Cell extracts were subjected to immunoprecipitation using normal rabbit serum (IgG) or anti-Shb antibody (Shb). (B) Cell extracts were incubated for 30 min on ice with the indicated immobilized fusion proteins in the presence (+) or absence (–) of 10 m M phosphotyrosine (P-Y). (C) Cell extracts were incubated for 30 min on ice with immobilized p55 Shb–pET fusion protein in the absence or presence of tyrosine-phosphorylated peptides or phosphotyrosine (P-Y). The peptides correspond to tyrosines 113, 128 and 145 in SLP-76 and tyrosines 174 and 692 in Vav. SLP-3Y contains all three SLP-peptides. (D) Cell extracts were incubated for 30 min on ice with immobilized SLP-76-Pro-GST or SLP-76-SH2-GST fusion proteins. (E) COS cells, transfected with Shb and different HA-tagged SLP-76 constructs or vector, as indicated, were stimulated, or not, with pervanadate, for 15 min in 37 °C, before lysis. The lysates were subjected to immunoprecipitations using anti-HA Ig. (F) Jurkat cell extracts were incubated with immobilized p55 Shb–pET fusion protein in the absence (–) or presence (+) of phosphotyrosine (P-Y). The samples in (A–F) were resolved on SDS/PAGE. The blots were probed with the antibodies indicated and the blot in (B) was stained using amido-black. The positions of SLP-76, ZAP70, Gads and Shb are indicated with arrows. 3282 C. K. Lindholm et al. (Eur. J. Biochem. 269) Ó FEBS 2002 binding site [14]. Three such sequences were found, all previously reported to be involved in cell signalling [7,20]. Three synthetic phosphopeptides were made corresponding to these three sites (SLP-Y113, SLP-Y128 and SLP-Y145). However, no inhibition of SLP-76 binding to the Shb fusion protein could be seen using these peptides (Fig. 2C). In addition, the Shb PTB domain fusion protein does not allow binding of SLP-76 (results not shown). The two LAT- peptides previously reported to inhibit the LAT–Shb association [16] were also tested, and had no inhibitory effect on SLP-76, ZAP70 or Vav association to Shb (results not shown). To characterize the Shb–SLP-76 interaction, we utilized fusion proteins corresponding to the SH2 domain of SLP76 and the proline-rich regions of SLP-76. We found that Shb associates with the SH2 domain of SLP-76, but not to the proline-rich regions of SLP-76 (Fig. 2D), and that this association was increased by CD3-stimulation. To test this further, we overexpressed HA-tagged SLP-76 or different domains of SLP-76, together with wild type Shb in COS cells, and performed HA-immunoprecipitations. Figure 2E shows that Shb is associated with wild-type SLP-76 and the SH2 domain of SLP-76 upon pervanadate stimulation (to increase the degree of protein tyrosine phosphorylation) in COS cells. We then hypothesized that the interaction between Shb and SLP-76 might be of a trimeric nature, where the SH2 domain of SLP-76 asso- ciates with a phosphotyrosine motif in Shb, and the proline rich sequences of Shb interact with some other adaptor, which also has the ability to bind SLP-76 (Fig. 8). After investigating possible SLP-76-interacting adaptor proteins, we considered the Grb2-related adaptor Gads a candidate. Gads was found to associate with the p55 Shb fusion protein, in a phosphotyrosine-independent manner (Fig. 2F), which was very similar to our findings concern- ing the association of SLP-76 to the Shb–PTB-Pro–GST fusion protein. The same blot was also probed with anti- (SLP-76) Ig, and the pattern of association was found to be quite similar to that of Gads. The significance of the SLP-76–Shb interaction was further verified by transient transfections of Jurkat cells with different Shb mutants, to study the pattern of tyrosine phosphorylation. Cells cotransfected with SLP-76 and wild-type Shb, exhibited a normal patern of tyrosine phosphorylation in response to CD3 stimulation (Fig. 3). However, when SLP-76 was cotransfected with Shb R522K (with a nonfunctional SH2 domain) or Shb DPTB-Tyr (with the four tyrosine phosphorylation sites and the PTB domain deleted), the cells exhibited a decreased response to CD3 stimulation (Fig. 3), which was verified using densitometric scannings. Particularly, decreased tyrosine phosphorylation of pro- teins corresponding to 160 (PLC-c1), 75 and 36 kDa (LAT) was observed and described in Fig. 3, with the relative phosphorylation of these proteins compared to the total amount of SLP-76 in the CD3 treated lanes. The 75-kDa protein comigrated exactly with SLP-76. These results indicate that both the SH2 domain and the PTB- Tyr region of Shb, are vital for the phosphorylation of several proteins upon TCR engagement, including SLP-76, PLC-c1 and LAT. The guanine nucleotide exchange factor Vav was also found to associate with Shb, utilizing fusion pro- teins (Fig. 4A). The PTB domain of Shb mediated this interaction and the binding could be blocked out with phosphotyrosine (Fig. 4A,B). To identify a putative binding site for Shb in the Vav protein, we searched the Vav sequence for the PTB domain consensus binding-site D-D- X-Y. Two such motifs were found in Vav and synthetic peptides corresponding to them were made. These peptides were used in binding experiments with a GST fusion protein containing only the Shb PTB domain. The PTB domain of Shb was found to associate with Vav and this binding is blocked by both phosphotyrosine and the more specific synthetic peptide corresponding to tyrosine 174 in Vav (Vav-Y174) (Fig. 4B), which is more known as the autoregulatory site of Vav [21]. We also tried to block this binding with the SLP-76 peptides, as Y113 and Y128 in SLP76 have been reported to be responsible for Vav binding to SLP76 [20], but observed no effect. Attempts to coimmunoprecipitate Vav with Shb in Jurkat cells were unsuccessful, due to an IgG-related band of the same size as Vav, which made detection of Vav impossible. An alternative approach was to express Vav and Shb in COS cells, and then perform a-Shb immunoprecipitations. Figure 4C shows how Vav coimmunoprecipitates with Shb upon pervanadate treatment of COS cells transfected with Shb and Vav. Our results suggest that although Vav and SLP-76 are known to interact with each other, they both bind directly and independently to Shb. Phosphorylation of SLP-76 and Vav is dependent on Shb with a functional SH2 domain To further study the effects of an R522K mutation in the SH2 domain of Shb on SLP-76 and Vav phosphorylation, we performed immunoprecipitations of SLP-76 and Vav in Fig. 3. Shb is vital for the phosphorylation of several proteins in Jurkat T cells. Jurkat cells, transfected with SLP-76 and different Shb con- structs, as indicated, were stimulated (+) or not (–) with CD3 antibody for2minin37°C, and lysed. The cell extracts were resolved on SDS/ PAGE and the blots were probed with anti-(SLP-76) Ig and 4G10 anti- phosphotyrosine Ig. Relative phosphorylation, as assessed by densio- metric scanning of the proteins indicated, is given in the table below the blot. The values are given relative the total amount of SLP-76. Ó FEBS 2002 Association of Shb, SLP-76 and Vav in Jurkat cells (Eur. J. Biochem. 269) 3283 Jurkat-R522K-2 and Jurkat-neo cells (Fig. 5A,B). Western blot analyses of these immunoprecipitates revealed in the Shb SH2 defective clone, Jurkat-R522K-2, a failure of CD3 stimulation to increase the phosphorylation of either SLP-76 or Vav. The basal phosphorylation of Vav was also lower in the clone expressing the R522K mutant. The same experiment was performed on ZAP70 in these cells, with a minor difference in stimulation between mutant and control (Fig. 5C). The degree of phosphorylation, relative unstimulated Jurkat cells, are given below each blot. These findings are consistent with the view that ZAP70 operates upstream of Shb, whereas SLP-76 and Vav are effectors downstream of Shb in the signalling pathway following TCR engagement. Effects of expression of R522K Shb in Jurkat T cells on JNK and Rac1 activation The guanine nucleotide exchange factor Vav is known to activate Rac1, RhoA and Cdc42. Both Rac1 and Cdc42 are among other things known to cause activation of JNK. As Vav phosphorylation is decreased in the cells expressing Shb with a defective SH2 domain (Jurkat R522K-2), we decided to also examine Rac1 and JNK activation upon CD3 stimulation in these cells. As displayed in Fig. 6A, Rac1 activity (GTP-Rac1), as assessed by association with the PAK-CD fusion protein, is decreased in the Jurkat R522K- 2 cells upon CD3 stimulation, compared to normal Jurkat cells. Consequently, JNK activation is also abolished upon CD3 stimulation in the Jurkat R522K-2 cells compared to the Jurkat-neo cells (Fig. 6B). Shb is recruited to GEMs after TCR ligation It has recently been shown that several proteins involved in TCR signal transduction, including SLP-76, Vav, ZAP70 and Gads, localize to GEMs upon TCR stimulation [22–26]. To assess if this was also the case for Shb, Jurkat T cells were left unstimulated or stimulated with anti-CD3 Ig and then lysed in a Triton X-100-based buffer. Lysates were subjected to sucrose density gradient ultracentrifugation to separate the detergent resistant GEMs from the Triton-soluble Fig. 5. Phosphorylation of SLP-76 and Vav is dependent on the Shb SH2 domain. Jurkat-neo or Jurkat R522K-2 cells (10 7 ) were left unstimulated (–) or were stimulated with anti-CD3 Ig for 2 min in 37 °C (+). The cells were then lysed in triton lysis buffer and cell extracts were precleared with protein A sepharose and then subjected to immunoprecipitation with (A) anti-SLP-76 Ig (B) anti-Vav Ig or (C) anti-ZAP70 Ig. The precipitated proteins were resolved on SDS/PAGE and blotted for phosphotyrosine (4G10), SLP-76, Vav and ZAP70. The positions of SLP-76, Vav and ZAP-70 are indicated. The relative phosphorylation, after normalization for the total amount of protein, compared to unstimulated Jurkat cells, is also indicated under each blot. Fig. 4. Association of Shb and Vav in CD3 stimulated T cells and pervanadate stimulated COS cells. (A,B) Jurkat cells (10 7 )were unstimulated (–) or stimulated by CD3 cross-linking for 2 min in 37 °C (+) before lysis in triton lysis buffer. (A) Cell extracts were incubated for 30 min on ice with the indicated immobilized fusion proteins in the presence (+) or absence (–) of 10 m M phosphotyrosine (P-Y). (B) Jurkat cell extracts were incubated for 30 min on ice with immobilized Shb–PTB–GST fusion protein in the absence or presence of the indi- cated tyrosine-phosphorylated peptides or phosphotyrosine (P-Y). (C) COS cells, transfected with Shb and Vav, as indicated, were stimulated, or not, with pervanadate, for 15 min in 37 °C, before lysis. The lysates were subjected to immunoprecipitations using Shb antibody. The samplesin(A–C)werewashedandresolvedonSDS/PAGEand immunoblotted with the antibodies indicated. The positions of Vav and Shb are indicated with arrows. 3284 C. K. Lindholm et al. (Eur. J. Biochem. 269) Ó FEBS 2002 fractions. Both Shb isoforms (55 and 66 kDa) were found to localize to the GEM fraction (mainly fractions 3 and 4) after CD3 stimulation (Fig. 7A). The GEM fraction was defined by the presence of LAT, which is constitutively located to lipid rafts. LAT might therefore be responsible for the recruitment of Shb to the lipid rafts upon TCR engagment (Fig. 7B,C). SLP-76, Vav and Gads were also seen to localize to fraction 3, 4 and 5 upon CD3 stimulation (Fig. 7D–F), which is in agreement with previous studies [23–26]. DISCUSSION We have previously established a role for Shb in T cell signalling by describing the domain-specific binding of Shb to the TCR f-chain,LAT,PLC-c1 and Grb2 [14] [16]. We have also demonstrated that a mutation in the SH2 domain of Shb affects TCR signalling through MAPK and Ca 2+ causing abolished activation of the NFAT element in the IL-2 promoter [16]. In this report, we show interactions between Shb and SLP-76, Gads, Vav and ZAP70 and that Shb is recruited to GEMs upon TCR ligation. We also examine how some of these interactions are affected by a mutation in the SH2 domain of Shb, and consequently how the activation of JNK is affected. To further study the effects of the mutation in the Shb SH2 domain, we examined the proteins found to coprecipi- tate with Shb in Jurkat cells. Except for the previously studied LAT, PLC-c1andtheTCRf-chain, we also noted the presence of other tyrosine phosphorylated proteins possibly corresponding to SLP-76, Vav and ZAP70. In the cells expressing a nonfunctional SH2 domain, the phos- phorylation of SLP-76 and Vav is decreased. This indicates that Shb forms a signalling complex with LAT, SLP-76, Vav and PLC-c1, and that this complex is linked to the TCR via the f-chain, through association of the Shb SH2 domain. Phosphorylation of ZAP70 is not affected, or affected very little, by the R522K mutation in the Shb SH2 domain. This is consistent with our model (Fig. 8), where ZAP70 is bound to the ITAMs of the f-chain, to which Shb with its associated proteins also are attached, and thus brought in proximity to the tyrosine kinase ZAP70. Introduction of the Shb SH2 point mutation decreases the ability of the Shb–LAT–SLP76–Vav complex to interact with ZAP70. In addition, expression of Shb with a deletion of the PTB domain and the tyrosine phosphorylation sites, also reduces the phosphorylation of phospho-proteins corresponding to PLC-c1, SLP-76 and LAT, implicating the involvement of these regions of Shb in TCR signaling. We have previously investigated the effects of overex- pression of wild-type Shb in Jurkat cells, and seen no differences in the CD3-mediated tyrosine phosphorylation, when Shb is overexpressed compared to mock transfected Jurkat cells [14]. This is further confirmed in Fig. 3, where cotransfection of Shb and SLP-76 produces Jurkat cells with a normal CD3-induced tyrosine phosphorylation pattern. To elucidate how SLP-76, Vav and ZAP70 associate with Shb, we utilized fusion proteins comprising different parts of Shb. An association of the adapter protein SLP-76 to the p55 Shb–pET and the Shb PTB-Proline-rich fusion protein could be seen both in unstimulated cells and after CD3 crosslinking. As the phosphopeptides did not efficiently displace this association and as SLP-76 did not bind directly to the Shb PTB-domain fusion protein only, we considered it plausible that the Shb proline-rich motifs associated with SLP-76 via some adapter protein, as SLP-76 lacks an SH3 domain. However, this interaction does not explain the CD3-dependency of the SLP-76–Shb interaction; we there- fore performed binding experiments using fusion proteins corresponding to different domains of SLP-76. We also cotransfected Shb together with different SLP-76 mutants in COS cells. Both these approaches showed that the SLP-76 SH2 domain associated with Shb after CD3 stimulation in Jurkat cells, or pervanadate treatment in COS cells. We have previously reported tyrosine phosphorylation of Shb after CD3 stimulation [16], and therefore suggest that phosphorylated tyrosines in Shb are binding sites for the SLP-76 SH2 domain. Gads is a potential adapter that links the Shb proline-rich motifs with SLP-76 as the latter associates with Gads via an SH3-domain/proline–rich motif interaction. The strong association of Gads to the Shb fusion protein, and the previously reported association between Shb and other proteins of the Gads family, Grb2 and Grap, all suggest that an association between Shb and Gads exists. Accordingly, Gads bridges SLP-76 and Shb, Fig. 6. Activation of Rac1 and JNK is dependent on a functional Shb SH2 domain. Jurkat-neo or Jurkat R522K-2 cells (10 7 )were unstimulated (–) or stimulated by CD3 crosslinking for 2 min (+) before lysis in triton lysis buffer. (A) Jurkat cell extracts were incubated for 30 min on ice with immobilized PAK–CD–GST fusion protein. Fusion-protein complexes and cell extracts were resolved on SDS/ PAGE, and the blot was probed with Rac1 antisera. (B) Cell extracts were resolved on SDS/PAGE and the blot was probed with anti- (phospho-JNK) Ig, and after stripping, reprobed with anti-JNK Ig. The positions of Rac1, phospho-JNK and total JNK are indicated in the figures. The relative amount of active Rac1, after normalization for the total amount of Rac1, is also indicated under the blot in (A). Ó FEBS 2002 Association of Shb, SLP-76 and Vav in Jurkat cells (Eur. J. Biochem. 269) 3285 and the SLP-76 SH2 domain binds to tyrosine phosphor- ylated Shb. The guanine nucleotide exchange factor Vav associates directly with the PTB domain of Shb through tyrosine-174. Tyrosine 174 has previously been shown to be of import- ance for Vav GEF function [9] and is a possible site for negative regulation of Vav. The binding of Vav to the Shb PTB domain might stabilize its association to SLP-76, and thus aids in the activation process of Vav. Our data also show abolished CD3-mediated Rac1 activation and JNK phosphorylation, as a consequence of the mutation in the Shb SH2 domain (R522K). This is probably due to the abolished activation of Vav in these cells, as several reports have shown that activated Vav is required for Rac1-mediated JNK activation in lymphoid cells [27,28]. Recent attention has focused on the concentration of effector molecules into subdomains, so-called GEMs or lipid rafts, upon receptor stimulation. These membrane subdomains are characterized by their detergent insolubility. Some proteins involved in T cell signalling are always present in GEMs, for example LAT, which is anchored to the membrane via its palmitoylated tail. We presently show that Shb is recruited to the GEMs upon TCR stimulation, where SLP-76, Vav, ZAP70, Gads and Grb2 have all previously been shown to target after CD3 ligation [23,24,26,29]. In conclusion, our results suggest that Shb is important for the early events of T cell signalling. Shb is recruited to GEMs, possibly by LAT, after antigen stimulation, and can also associate with several important signalling proteins such as PLC-c1, Vav, Gads, Grb2, GRAP and SLP-76. This generates a signalling complex that is brought in proximity with the ZAP70 tyrosine kinase, the main phosphate-donor and thus activator, of these signaling proteins. These interactions are then of importance for the activation of both the MAP kinase and the c-Jun kinase pathways. Fig. 7. Recruitment of Shb to GEMs after TCR ligation. Jurkat T cells were not treated (NT) or stimulated with CD3 antibody (CD3) for 3 min, followed by lysis in Mes lysis buffer. Lysates were subjected to sucrose gradient ultracentrifugation. Sequential fractions were removed from the top of the gradient and indicated as fraction number. The fractions were precipitated using TCA, and after washing with acetone, dissolved in 40 lLof SDS sample buffer. The samples were resolved on SDS/PAGE, followed by immunoblot analysis using anti-Shb Ig (A), anti-phosphotyrosine Ig (4G10) (B), anti-LAT Ig (C), anti-Gads Ig (D), anti-Vav Ig (E) and anti-(SLP-76) Ig (F). 3286 C. K. Lindholm et al. (Eur. J. Biochem. 269) Ó FEBS 2002 ACKNOWLEDGEMENTS We gratefully acknowledge the skillful technical assistance of Ing-Britt Hallgren and Ing-Marie Mo ¨ rsare and peptide synthesis by Dr A ˚ ke Engstro ¨ m. We thank Dr Jane McGlade for providing the anti-Gads Ig and Dr Arthur Weiss for providing the Vav expression plasmid. 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