Mechanism of recruitment of WASP to the immunological synapse and of its activation following TCR ligation

Molecular Cell, Vol. 10, 1269–1281, December, 2002, Copyright 2002 by Cell Press Mechanism of Recruitment of WASP to the Immunological Synapse and of Its Activation Following TCR Ligation Aldrich syndrome (WAS), is expressed only in hematopoietic cells and is the first identified member of an expanding family of proteins involved in signaling and cytoskeletal organization that includes N-WASP and Scar/WAVE (Derry et al., 1994; Machesky and Insall, Yoji Sasahara, 1,4 Rima Rachid, 1,4 Michael J. Byrne, 1 Miguel A. de la Fuente, 1 Robert T. Abraham, 2 Narayanaswamy Ramesh, 1,3 and Raif S. Geha 1,3 1 Division of Immunology Children’s Hospital and Department of Pediatrics 1998; Miki et al., 1996). WASP has an N-terminal Ena/ VASP homology domain 1 (EVH1) domain, a Cdc42/RacHarvard Medical School Boston, Massachusetts 02115 GTPase binding domain (GBD), a proline-rich domain, a G-actin binding verprolin homology (VH) domain, a 2 Department of Pharmacology Duke University Medical Center cofilin homology (CH) domain, and a C-terminal acidic (A) segment. WASP interacts with WIP via its N-terminalDurham, North Carolina 27710 EVH1 domain (Ramesh et al., 1997), with Cdc42-GTP via its GBD domain, with multiple SH3 domain-containing proteins that include Nck via its proline-rich region, and Summary with actin and the Arp2/3 complex via its VCA domain (Abo, 1998). WASP exists in cells in a closed inactive F-actin polymerization following engagement of the T conformation due to intramolecular interactions that incell receptor (TCR) is dependent on WASP and is critivolve the C-terminal acidic domain and a basic region cal for T cell activation. The link between TCR and that precedes the GBD domain. Binding of Cdc42-GTP WASP is not fully understood. In resting cells, WASP is thought to cause a conformational change in WASP, exists in a complex with WIP, which inhibits its activawhich allows the VCA domain to interact with and actition by Cdc42. We show that the adaptor protein CrkL vate the Arp2/3 complex (Higgs and Pollard, 2000; Kim binds directly to WIP. Further, TCR ligation results in et al., 2000; Rohatgi et al., 2000). WASP plays a critical the formation of a ZAP-70-CrkL-WIP-WASP complex, role in T cell activation and actin reorganization. T cells which is recruited to lipid rafts and the immunological from WAS patients and WASP⫺ / ⫺mice are severely defisynapse. TCR engagement also causes PKC␪-depencient in their ability to increase their F-actin content, dent phosphorylation of WIP, causing the disensecrete IL-2, and proliferate following TCR ligation (Galgagement of WASP from the WIP-WASP complex, lego et al., 1997; Snapper et al., 1998; Zhang et al., 1999). thereby releasing it from WIP inhibition. These results WIP is a 503 aa long proline-rich protein expressed suggest that the ZAP-70-CrkL-WIP pathway and PKC␪ at high levels in lymphoid tissues (Ramesh et al., 1997). link TCR to WASP activation. WIP binds actin via its VH domain (aa 1–151) and WASP via its carboxy-terminal end (aa 416–488). A WIP-WASP/ Introduction N-WASP complex is readily detected in resting cells (Martinez-Quiles et al., 2001). In lymphocytes, ⬎95% of Interaction between the T cell receptor (TCR) and pepWASP is complexed with WIP (our unpublished data). tide-loaded major histocompatibility complex (MHC) WIP inhibits Cdc42-mediated activation of N-WASP, molecules on the surface of antigen presenting cells suggesting that one function of WIP is to stabilize WASP/ (APCs) induces the formation of molecular clusters at N-WASP in their inactive closed conformation (Martinezthe contact site that are enriched in filamentous actin Quiles et al., 2001). Another function of WIP is to stabilize (F-actin) (Grakoui et al., 1999; Penninger and Crabtree, actin filaments (Martinez-Quiles et al., 2001). WIP, like 1999). These supramolecular activation clusters (SMACs), WASP, plays an important role in T cell activation. T also named immunological synapses (IS), contain sevcells from WIP⫺ / ⫺mice fail to proliferate, secrete IL-2, eral signaling components. They include src and Syk or increase their F-actin content after TCR ligation. Furtherfamily kinases, PKC␪and Cdc42-GTP (Bromley et al., more, WIP⫺ / ⫺T cells are deficient in conjugate formation 2001). The IS also contains adaptor proteins such as with superantigen-presenting B cells and anti-CD3/ICAMSLP-76, Fyb, and Nck that are linked directly or indirectly 1-containing lipid bilayers and have a disorganized actin to proteins such as WASP, Ena/VASP family members cytoskeleton (Anton et al., 2002). that are involved in actin polymerization (Krause et al., Recent data shows that WASP localizes with F-actin 2000; Monks et al., 1998). The accumulation of F-actin to the IS where it is thought to be activated by Cdc42at the T cell-APC interface is thought to stabilize a conGTP generated following activation of the exchange factinuous contact between T cells and APCs, which is tor Vav and its recruitment to lipid membranes (Arudrequired for optimal T cell activation. Inhibition of actin chandran et al., 2000). The mechanism(s) by which polymerization by cytochalasin blocks formation of the WASP is recruited to the IS and is released from WIP immunological synapse and T cell activation (Wulfing inhibition to initiate actin polymerization is not well unand Davis, 1998). derstood. We show here that WIP binds to the adaptor WASP, the product of the gene mutated in Wiskottprotein CrkL and that following TCR ligation, a CrkLWIP-WASP complex is recruited by ZAP-70 to lipid rafts 3 Correspondence: [email protected] (N.R.), and the IS. TCR ligation also causes PKC␪-dependent [email protected] (R.S.G.) 4 These authors contributed equally to this work. phosphorylation of WIP and disengagement of WASP Molecular Cell 1270 from the WIP-WASP complex, releasing it from WIP inhirecruit the WIP-WASP complex to ZAP-70 following TCR ligation. We first probed ZAP-70 immunoprecipitates bition. from Jurkat cells before and 3 min after anti-CD3 stimulation for CrkL, WIP, and WASP. As expected, anti-CD3 Results stimulation resulted in vigorous tyrosine phosphorylation of ZAP-70 (Figure 2A). CrkL and WIP became readily The Adaptor Protein CrkL Binds to WIP and detectable in ZAP-70 immunoprecipitates after anti-CD3 Associates with ZAP-70 after TCR Ligation stimulation. The weak association of these proteins with In a search for potential links between the activated ZAP-70 in unstimulated cells may be explained by the TCR-ZAP-70 complex and the WIP-WASP complex, we presence of small amounts of phosphorylated ZAP-70 considered the possibility that an SH2-SH3 domain-conin these cells. We were unable to detect WASP in ZAPtaining adaptor protein may link phosphorylated ZAP-70 70 immunoprecipitates. to the proline-rich WIP and WASP. Potential candidates In a further attempt to demonstrate association of included the CT10 regulator of kinase (Crk) family of ZAP-70 with WASP, we probed WASP immunoprecipiadaptor proteins. The prototype, CrkII, has one N-tertates for ZAP-70. Figure 2B shows that ZAP-70 copreminal SH2 domain and two SH3 domains. The second cipitated weakly with WASP in unstimulated cells. The SH3 domain (SH3.2) is alternatively spliced out to give association of WASP and ZAP-70 increased after antirise to CrkI. A closely related member, CrkL (Crk like), CD3 stimulation, suggesting that WASP is recruited to resembles CrkII in that it also has two SH3 domains and ZAP-70 following TCR ligation. Probing of WASP immuis expressed at high levels in hematopoietic cells (Feller noprecipitates for CrkL and WIP revealed that CrkL, like et al., 1998). GST-CrkII and GST-CrkL have been reWIP, coprecipitated with WASP in unstimulated cells, ported to associate via their SH2 domains with phossuggesting that CrkL, WIP, and WASP exist as prephorylated ZAP-70 (Gelkop and Isakov, 1999). WIP, but formed complex, with WIP bridging WASP to CrkL. CrkI not WASP, contains two copies of the consensus motif and CrkII were not detected in WASP immunoprecipiPxLPxK/R, which binds to the N-terminal SH3 domain tates (data not shown). Anti-CD3 stimulation resulted in (SH3.1) of Crk proteins. decreased association of WASP with WIP and CrkL, To determine if WIP and WASP bind to Crk proteins, suggesting that TCR ligation may perturb the WIPwe expressed CrkII and CrkL as GST fusion proteins WASP complex. Similar results were obtained in normal and examined their ability to pull down Xpress-tagged T cell blasts (see Supplemental Figure S1 at http:// recombinant WIP and in vitro transcribed and translated www.molecule.org/cgi/content/full/10/6/1269/DC1). WASP. CrkI was not examined because it is an alternaTaken together, our results suggest that a CrkL-WIPtive splicing truncated product of CrkII. WIP bound to WASP complex is recruited to phosphorylated ZAP-70 CrkL, but not to CrkII. (Figure 1A). This indicates direct following TCR ligation. interaction between WIP and CrkL. WASP failed to bind to either CrkL or CrkII (Figure 1A), even in the presence CrkL, WIP, and WASP Translocate to GEMs of the constitutively active Cdc42 mutant Cdc42L61 after TCR Ligation (data not shown). Using the yeast two-hybrid system, Glycosphingolipids and cholesterol self-associate in we found that the SH3.1 domain of CrkL binds to a plasma membrane microdomains known as glycolipid region of WIP spanned by aa 321–415 (Figure 1B). This enriched microdomains (GEMs) or lipid rafts. GEMs loregion contains the two consensus binding motifs for calize at the IS in antigen-stimulated T cells (Bi et al., the Crk SH3.1 domain (aa 332–337 and 399–404) and is 2001) and are enriched in a number of molecules reledistinct from the WASP binding site (aa 416–488). vant for receptor signaling that leads to F-actin polymerWe next determined if CrkL associates with ZAP-70 ization and IL-2 gene expression. Signaling molecules and WIP in T cells. Western blotting of CrkL immunoprerecruited to lipid rafts after TCR ligation include phoscipitates from Jurkat cells revealed that CrkL associated phorylated CD3␨and its associated ZAP-70. Since ZAPwith ZAP-70 following anti-CD3 stimulation (Figure 1C). 70 associates with CrkL, WIP, and WASP in activated WIP coprecipitated with CrkL in unstimulated Jurkat T T cells, we examined whether these proteins translocate cells, and this association increased slightly after TCR to GEMs following TCR ligation. ligation (Figure 1C). Similar results were obtained in We used sucrose density gradient fractionation of TriPHA-derived T cell blasts derived from normal subjects ton X-100 lysates from Jurkat cells to analyze the trans- (see Supplemental Figure S1 at http://www.molecule. location of CrkL, WIP, and WASP to GEMs, which sediorg/cgi/content/full/10/6/1269/DC1). WIP coprecipiment in fractions 2–5 of the gradient, as evidenced by tated with CrkL from peripheral blood T cell blasts of a the presence of the glycosphingolipid GM1 in these fracWAS patient with a point mutation in the WASP gene tions. Small amounts of 21 kDa phosphorylated CD3␨, (C73Y), who does not express WASP protein (Figure ZAP-70, CrkL, WIP, and WASP were present in GEMs 1D). These results suggest that WIP associates with before stimulation. As expected, phosphorylated CD3␨ ZAP-70 after TCR ligation and associates constitutively and ZAP-70 translocated to GEMs after anti-CD3 stimuwith CrkL independently of WASP. lation. TCR ligation caused translocation of CrkL, WIP, and WASP to GEMs (Figure 3C). WIP and WASP Are Recruited to ZAP-70 after TCR Ligation Translocation of WIP and WASP to GEMs Since WIP exists as a preformed complex with WASP Is Dependent on ZAP-70 and CrkL and since the WIP binding sites for CrkL and WASP We used ZAP-70-deficient P116 Jurkat cells to examine the role of ZAP-70 in the translocation of CrkL, WIP, andare distinct, we examined the possibility that CrkL may Mechanism of WASP Activation Following TCR Ligation 1271 Figure 1. Interaction between CrkL and WIP (A) Pull-down assay using GST-CrkL, GST-CrkII fusion proteins and GST with purified Xpress-tagged WIP or in vitro translated WASP. Bound proteins were probed with anti-Xpress or anti-WASP mAbs. The left lane in each panel was loaded with WIP or WASP protein (“Input”). (B) Mapping by yeast two-hybrid assay of the WIP binding site in CrkL (left panel) and of the CrkL binding site in WIP (right panel). (C) Coprecipitation of WIP and ZAP-70 with CrkL in unstimulated and anti-CD3-stimulated Jurkat cells. CrkL and control (“Ctrl. Ig.”) immunoprecipitates were probed for ZAP-70, WIP, and CrkL. The right lane in each panel was loaded with cell lysates (“Lys.”). (D) WIP association with CrkL is independent of WASP. Left: expression levels of WIP and WASP in lysates from T cells of a WAS patient (“pt.”) with C73Y WASP mutation and a normal control (“Ctrl.”). Right: CrkL and control immunoprecipitates from T cells of the patient and normal control were probed for WIP and CrkL. WASP to GEMs. The parent P116 cell line was stably the transfected P116 clones was confirmed by Western blotting (Figure 3A). In contrast to wild-type Jurkat cells,transfected with either Myc-tagged wt (wild-type) ZAP70 or Myc-tagged kinase-dead (KD) ZAP-70. At least there was no translocation of CrkL, WIP, or WASP to GEMs in ZAP-70-deficient P116 cells (Figure 3B). Trans-two clones that showed equivalent surface CD3 expression to wt Jurkat cells were studied for each construct, location of all three proteins to GEMs was restored in P116 cells reconstituted with wt ZAP-70, but not in P116with similar results. Expression of ZAP-70 proteins in Molecular Cell 1272 Figure 2. ZAP-70, CrkL, WIP, and WASP Form a Complex following TCR Ligation and Are Recruited to Lipid Rafts (A) ZAP-70 immunoprecipitates from unstimulated and anti-CD3 stimulated Jurkat T cells and lysates (“Lys.”) were probed with Abs to phosphotyrosine (“PY”), CrkL, WIP, and ZAP70 as a loading control. (B) WASP immunoprecipitates and lysates from the same cells were probed for ZAP-70, CrkL, WIP, and WASP as a loading control. (C) Sucrose density gradient fractions of Triton X-100 lysates from unstimulated and antiCD3 stimulated Jurkat cells were probed for CD3␨, ZAP-70, CrkL, WIP, and WASP. The visualized 21 kDa band corresponds to phosphorylated CD3␨. Cholera toxin B subunit was used to probe for GM1 that constitutively resides in GEMs as a loading control. cells reconstituted with KD ZAP-70. These results sugall translocated to the T cell-APC interface (Figure 4A and Table 1). As expected, F-actin also accumulated atgest that ZAP-70 and its kinase activity are essential for the translocation of the CrkL-WIP-WASP complex to the interface. Translocation of CrkL, WIP and WASP to the T cell-lipid rafts. To examine the role of CrkL in the translocation of APC interface was dependent on ZAP-70, because it was markedly diminished in ZAP-70-deficient P116WIP and WASP to GEMs, Jurkat T cells were stably transfected with a CrkL deletion mutant that lacked the cells. Both the percentages of conjugates that contained these proteins at the interface and the amount of accu-SH3.1 domain that is essential for WIP binding (CrkL⌬SH3.1), or vector alone. The mutant would commulated proteins were decreased (Figure 4A and Table 1). Conjugate formation by these cells was normal (datapete with native CrkL for ZAP-70 binding. Two clones that showed equivalent surface CD3 expression to wt not shown). Consistent with previous data (Morgan et al., 2001), there was a much weaker accumulation ofJurkat cells were studied with similar results. Western blotting revealed that the mutant was expressed 2.5F-actin at the interface in P116 cells. Translocation of CrkL, WIP, and WASP and F-actin accumulation at thefold higher than endogeneous protein (Figure 3C). Mutant CrkL, like native CrkL, translocated to GEMs after interface was completely corrected in P116 cells reconstituted with wt ZAP-70, but remained deficient in P116TCR ligation, consistent with the notion that the mutant can be recruited by ZAP-70 via its SH2 domain. In concells reconstituted with KD ZAP-70. Translocation of WIP and WASP to the T cell-APC interface was alsotrast, WIP and WASP translocated poorly to GEMs in CrkL⌬SH3.1-transfected cells (Figure 3D). dependent on CrkL, because it was markedly diminished in CrkL⌬SH3.1-transfected Jurkat cells (Figure 4A and Table 1). Conjugate formation by these cells was normal WIP and WASP Translocate to the T Cell-APC (data not shown). Interface in a ZAP-70and CrkL-Dependent Manner Since CrkL, WIP, and WASP translocate to GEMs, we examined whether they also localize to the T cell-APC ZAP-70 and CrkL Play an Important Role in Cellular F-Actin Increase and IL-2 Synthesis following TCRinterface. Jurkat T cells were incubated with Raji B cells in the presence of the superantigen SEE, and T:B cell Ligation by Superantigen Presented by APCs We next investigated the role of ZAP-70 and CrkL inconjugates were examined for localization of CrkL, WIP, WASP, and F-actin after 10 min. CrkL, WIP, and WASP the increase of total cellular F-actin content and in IL-2 Mechanism of WASP Activation Following TCR Ligation 1273 Figure 3. Recruitment of WIP-WASP Complex to Lipid Rafts Is Dependent on ZAP-70 and CrkL (A) Expression of ZAP-70 protein in wt Jurkat cells and in P116 clones stably transfected with vector alone, wild-type (“WT”) ZAP-70, and a kinase-dead (“KD”) ZAP-70 mutant. Lysates were probed with anti-ZAP-70 mAb. (B) Recruitment of CrkL, WIP, and WASP to lipid rafts in Jurkat cells, P116 cells, and P116 reconstituted cells. Fractions 2–5 of the sucrose gradient were pooled and probed for ZAP-70, CrkL, WIP, and WASP and for GM1 as control. (C) Expression of CrkL protein in Jurkat clones stably transfected with vector alone or with a CrkL mutant lacking the SH3.1 domain (“⌬SH3.1”). Lysates were probed with antiCrkL mAb. (D) Recruitment of ZAP-70, CrkL, WIP, and WASP to lipid rafts in CrkL⌬SH3.1-transfected Jurkat cells and controls. synthesis following TCR ligation. P116 cells failed to single consensus PKC phosphorylation motif (RxxS/ increase their F-actin content after anti-CD3 stimulation TxR) that surrounds S488. PKC␪, the major PKC in T and to secrete IL-2 following stimulation with APCs and cells, is activated and translocates to GEMs and the IS SEE. The latter result is consistent with results obtained following TCR ligation (Bi and Altman, 2001). This raised with anti-CD3 stimulation (Williams et al., 1998). These the possibility that PKC␪phosphorylation of WIP may deficits were completely corrected by reconstitution perturb the WIP-WASP complex. In addition to the C45 with wt ZAP-70, but not with KD ZAP-70 (Figures 4B antiserum which recognizes the carboxy-terminal 45 aa and 4C). CrkL⌬SH3.1-transfected Jurkat cells were also of WIP (aa 459–503), we have raised another antiserum, markedly impaired in their capacity to increase their C14, against a 14 aa peptide of WIP, 483 ESRSGSNRRER F-actin content (Figure 4B and Supplemental Figure S2 GGAP 496 , which contains S488 (Figure 5A). C14 Ab reacat http://www.molecule.org/cgi/content/full/10/6/1269/ tivity was markedly decreased 5 min after treatment of DC1) and secrete IL-2 following TCR ligation (Figure 4C), Jurkat cells with anti-CD3 and was restored 30 min later but had normal calcium fluxes (data not shown). (Figure 5B). In contrast, C45 Ab reactivity remained unchanged. Reactivity with C14 Ab was fully restored by treatment of lysates with alkaline phosphatase (Figure WIP Is Phosphorylated by PKC␪ 5C). Similar results were obtained in peripheral blood T following TCR Ligation cells (data not shown). These findings indicate that WIP Translocation of the WIP-WASP complex to the IS brings is phosphorylated after TCR ligation and that C14 Ab it into proximity with Cdc42-GTP. However, since almost recognizes a nonphosphorylated WIP epitope, whereas all of the WASP (⬎95%) in resting T cells is complexed C45 Ab is insensitive to phosphorylation. with WIP, which inhibits its activation by Cdc42, we To explore the identity of the enzyme that results in investigated potential mechanisms of WASP activation following TCR ligation. WIP, but not WASP, contains a WIP phosphorylation, we first examined the effect of Molecular Cell 1274 Figure 4. Recruitment of CrkL, WIP, WASP, and F-Actin at the T Cell-APC Interface, F-Actin Content, and IL-2 Secretion Are Dependent on ZAP-70 and CrkL (A) Intracellular immunofluorescence staining for CrkL, WIP, WASP, and F-actin in T cells stimulated by SEE (5 ␮g/ml), and Raji B cells preloaded with CMAC (blue). Cells were stained simultaneously for CrkL and WIP. In separate experiments, cells were stained for WASP and F-actin. Magnification is 600⫻. (B) FACS analysis of intracellular F-actin content of cells following CD3 crosslinking. Representative results of three independent experiments are shown. (C) IL-2 secretion by T cells stimulated with SEE and Raji B cells. Supernatants collected at 24 hr were assayed for IL-2 by ELISA. Means ⫾ SD of three independent experiments are shown. selective PKC inhibitors. Pretreatment of Jurkat cells To ascertain the role of PKC␪in WIP phosphorylation, we examined splenic T cells from PKC␪⫺ / ⫺mice. Anti-with rottlerin, an inhibitor of the nonclassical PKCs ␦and ␪(Villalba et al., 1999), inhibited WIP phosphorylation CD3 stimulation resulted in loss of C14 Ab reactivity in T cells from wt mice. In contrast, there was no loss offollowing TCR ligation, as evidenced by retention of C14 Ab reactivity (Figure 5D). In contrast, G06976, an inhibiC14 Ab reactivity in T cells of PKC␪⫺ / ⫺mice (Figure 5E). ZAP-70, SLP-76, and Vav-1 all play an important role intor of the classical calcium-dependent PKCs ␣,␤, and ␥(Martiny-Baron et al., 1993), and the PKA inhibitor 14the activation of PKC␪(Herndon et al., 2001; Villalba et al., 2000). Following TCR ligation, WIP phosphorylation22 amide had no effect (Figure 5D and data not shown). Mechanism of WASP Activation Following TCR Ligation 1275 Table 1. Translocation of CrkL, WIP, and WASP to the T-APC Interface Cells % of Conjugates with Accumulation at the T Cell-APC Interface CrkL WIP WASP SEE: ⫺⫹ ⫺ ⫹ ⫺⫹ Wild-type Jurkat 8.5 ⫾5.7 26.5 ⫾5.0 15.0 ⫾1.2 36.0 ⫾2.3 8.0 ⫾2.2 34.0 ⫾2.4 P116/vector 7.5 ⫾3.4 10.5 ⫾5.7* 13.0 ⫾1.2 13.5 ⫾1.9* 4.0 ⫾1.5 6.0 ⫾2.3* P116/ZAP-70 wt 8.5 ⫾4.1 26.0 ⫾2.3 14.0 ⫾1.6 38.5 ⫾1.9 6.0 ⫾1.6 36.0 ⫾2.0 P116/ZAP-70 KD 9.0 ⫾6.0 13.0 ⫾6.0* 15.5 ⫾2.5 17.0 ⫾4.2* 5.0 ⫾2.4 9.0 ⫾3.6* CrkL/⌬SH3.1 9.5 ⫾5.2 34.0 ⫾5.0 12.0 ⫾2.0 26.0 ⫾2.5* 6.0 ⫾2.4 22.0 ⫾2.2* Results represent the mean ⫾SD of four experiments (fifty conjugates were examined in each). *p ⬍0.01 compared to wt Jurkat cells. resulting in loss of Ab C14 reactivity was not detectable in WASP immunoprecipitates from anti-CD3-stimulated Jurkat cells (Figure 2B). To confirm this result, WIP im-in Jurkat T cells that lacked ZAP-70 (P116 cells) or SLP76 (J14 cells) nor in T cells from Vav-1⫺ / ⫺mice. These munoprecipitates were prepared from anti-CD3-stimulated Jurkat cells using C45 Ab and were probed forresults suggest that PKC␪plays an important role in the phosphorylation of WIP. WASP using a polyclonal anti-WASP antibody that recognizes the C-terminal 18 aa of WASP. Anti-CD3 stimulation resulted in a marked decrease in the associationThe WASP-WIP Complex Is Disrupted following TCR Ligation of WASP with WIP (Figure 6A). Similar results were obtained using a mAb that recognizes the WASP GBDThe WASP binding region of WIP (aa 416–488) overlaps with the region recognized by C14 Ab (aa 483–496). domain and with peripheral blood T cells (data not shown). It is unlikely that the decreased WASP signal isWe considered the possibility that WIP phosphorylation after TCR ligation may result in the dissociation of WASP due to failure of the antibodies used to recognize WASP, which may have been phosphorylated following TCRfrom WIP, which would allow its activation by Cdc42. In fact, we had observed decreased amounts of WIP ligation, because treatment of WIP immunoprecipitates Figure 5. WIP Is Phosphorylated after TCR Ligation by PKC␪ (A) Map showing WIP peptides used to raise C14 and C45 Abs. (B) Reactivity of lysates from Jurkat cells before and after anti-CD3 stimulation (5 and 30 min) with C14 and C45 Abs. (C) Reversal of loss of C14 Ab reactivity after anti-CD3 stimulation (5 min) by treatment of lysates with alkaline phosphatase. (D) Effect of the PKC inhibitors on loss of C14 Ab reactivity after anti-CD3 stimulation (5 min). Cells were pretreated with 20 ␮M rottlerin or 62 nM G06976 for 30 min at 37⬚C. (E) Role of PKC␪, Vav-1, ZAP-70, and SLP76 in WIP phosphorylation after TCR ligation. Splenic T cells from PKC␪ ⫺ / ⫺ and Vav-1 ⫺ / ⫺ mice and from wt Jurkat T cells, P116 cells, and J14 cells were stimulated with anti-CD3 for 5 min, lysed, and probed with C14 and C45 Abs. Molecular Cell 1276 Figure 6. WIP Dissociation from WASP following TCR Ligation (A) Dissociation of WASP from WIP following anti-CD3 stimulation (5 and 30 min) of Jurkat cells. WIP C45 Ab immunoprecipitates were probed for WASP and WIP as loading controls. (B) Reversal of the dissociation of WIP and WASP by treatment of cell lysates with alkaline phosphatase, 1 U/10 ␮lat25⬚C for 3 hr. (C) Effect of pretreatment with rottlerin (left panel) and G06976 (right panel) on the dissociation of WIP and WASP after TCR ligation (5 min). In all cases, WIP C45 Ab immunoprecipitates from Jurkat cells were probed with WASP polyclonal Ab and WIP C45 Ab as loading control. (D) Pull-down assay using wt GST-WIP 401-503 fusion protein and its mutants S405D, S486D, and S488D with in vitro transcribed and translated WASP. Bound proteins were eluted, run on SDS-PAGE, and Western blotted for WASP and GST as loading controls. with alkaline phosphatase did not increase the intensity binding site (aa 416–488) as well as the point mutants S488D, S405D, and S486D. Affinity precipitation usingof the WASP signal (data not shown). It is also unlikely that the decreased association of WASP with WIP was recombinant WASP showed that wt WIP 401-503 bound to WASP. The S488D mutant, but not the S405D and S486Ddue to degradation of WASP, because the intensity of the WASP band in cell lysates remained unchanged after mutants, showed markedly decreased WASP binding (Figure 6D). These results suggest that phosphorylationTCR ligation, and no smaller molecular weight bands that react with anti-WASP were detected (data not at S488 disrupts WIP binding to WASP. shown). We next investigated the role of phosphorylation in the dissociation of the WIP-WASP complex following Activation of PKC Is Essential for F-Actin Accumulation and IL-2 Synthesis, but Not for WASPTCR ligation. Treatment of cell lysates with alkaline phosphatase prior to immunoprecipitation restored the Translocation to the T Cell-APC Interface To test the hypothesis that WASP-mediated F-actinassociation of WASP with WIP (Figure 6B). Since WIP is a target for PKC␪phosphorylation, we examined the polymerization may be dependent on PKC phosphorylation of WIP and the subsequent release of WASP fromeffect of PKC inhibitors on the dissociation of the WIPWASP complex. Figure 6C shows that pretreatment of WIP inhibition, we examined F-actin accumulation at the T cell-APC interface in Jurkat cells pretreated withJurkat cells with rottlerin partially inhibited this dissociation, whereas G06976 had no effect. These results sugPKC inhibitors. F-actin accumulation at the interface was diminished in cells pretreated with rottlerin, but notgest that WIP phosphorylation by PKC␪results in dissociation of the WIP-WASP complex. in cells pretreated with G06976 (Figure 7A). In contrast, accumulation of CrkL and WIP and WASP was not af-S488 is the only residue in WIP that falls within a consensus motif for PKC phosphorylation. We therefore fected. Since SLP-76 is essential for PKC␪activation, we also examined SLP-76-deficient J14 cells. CrkL andexamined the effect of an S488→D substitution, which mimics the negative charge of phosphoserine, on the WIP accumulated normally at the interface in these cells. However, the amounts of WASP and F-actin that accu-ability of WIP to bind WASP. Since full-length WIP is poorly expressed in bacterial systems, we constructed mulated at the interface were diminished, although the percentage of conjugates with accumulation of WASPa GST-WIP 401-503 fusion protein that contains the WASP Mechanism of WASP Activation Following TCR Ligation 1277 Figure 7. Effects of PKC Inhibitors and SLP-76 on the T Cell-APC interface, F-Actin Content, and IL-2 Secretion (A) Effect of PKC inhibitors and of SLP-76 deficiency (J14 cells) on the accumulation of CrkL, WIP, WASP, and F-actin at the IS. Magnification is 600⫻. (B) F-actin content following TCR ligation in wt Jurkat cells untreated or pretreated with rottlerin or G06976, splenic T cells from wt and PKC␪ ⫺ / ⫺ mice, and J14 cells were analyzed by FACS. Representative results of three independent experiments are shown. (C) Effect of PKC inhibitors (upper panel) and SLP-76 (lower panel) on IL-2 secretion by Jurkat T cells or J14 cells stimulated with SEE and Raji B cells. Means ⫾SD of three independent experiments are shown. (D) Model for the recruitment of WASP to GEMs and the IS and for its activation that results in actin polymerization following TCR ligation.