Actin Cytoskeleton TCR-Induced Regulation of Vav and the Kinase

Kinase-Independent Functions for Itk in TCR-Induced Regulation of Vav and the Actin Cytoskeleton

This information is current as Derek Dombroski, Richard A. Houghtling, Christine M. of September 24, 2021. Labno, Patricia Precht, Aya Takesono, Natasha J. Caplen, Daniel D. Billadeau, Ronald L. Wange, Janis K. Burkhardt and Pamela L. Schwartzberg J Immunol 2005; 174:1385-1392; ;

doi: 10.4049/jimmunol.174.3.1385 Downloaded from http://www.jimmunol.org/content/174/3/1385

References This article cites 45 articles, 21 of which you can access for free at: http://www.jimmunol.org/content/174/3/1385.full#ref-list-1 http://www.jimmunol.org/

Why The JI? Submit online.

• Rapid Reviews! 30 days* from submission to initial decision

• No Triage! Every submission reviewed by practicing scientists by guest on September 24, 2021 • Fast Publication! 4 weeks from acceptance to publication

*average

Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts

The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2005 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Kinase-Independent Functions for Itk in TCR-Induced Regulation of Vav and the Actin Cytoskeleton1

Derek Dombroski,2* Richard A. Houghtling,2* Christine M. Labno,† Patricia Precht,‡ Aya Takesono,3* Natasha J. Caplen,4* Daniel D. Billadeau,§ Ronald L. Wange,‡ Janis K. Burkhardt,†¶ and Pamela L. Schwartzberg5* The Tec family kinase Itk is an important regulator of Ca2؉ mobilization and is required for in vivo responses to Th2-inducing agents. Recent data also implicate Itk in TCR-induced regulation of the actin cytoskeleton. We have evaluated the requirements for Itk function in TCR-induced actin polarization. Reduction of Itk expression via small interfering RNA treatment of the Jurkat human T lymphoma cell line or human peripheral blood T cells disrupted TCR-induced actin polarization, a defect that correlated with decreased recruitment of the Vav guanine nucleotide exchange factor to the site of Ag contact. Vav localization and actin polarization could be rescued by re-expression of either wild-type or kinase-inactive murine Itk but not by Itk containing mu- Downloaded from tations affecting the pleckstrin homology or Src homology 2 domains. Additionally, we ﬁnd that Itk is constitutively associated with Vav. Loss of Itk expression did not alter gross patterns of Vav tyrosine phosphorylation but appeared to disrupt the interactions of Vav with SLP-76. Expression of membrane-targeted Vav, Vav-CAAX, can rescue the small interfering RNA to Itk-induced phenotype, implicating the alteration in Vav localization as directly contributing to the actin polarization defect. These data suggest a kinase-independent scaffolding function for Itk in the regulation of Vav localization and TCR-induced actin

polarization. The Journal of Immunology, 2005, 174: 1385–1392. http://www.jimmunol.org/

pon stimulation with Ag, T lymphocytes undergo a dra- The Tec family tyrosine kinases are key signaling intermediates matic cellular reorganization leading to cell polarization, that are required for full activation of phospholipase C-␥ (PLC-␥)6 U which is accompanied by the accumulation of F-actin at and Ca2ϩ mobilization downstream from Ag receptors (9). Muta- the site of contact with the APC (1). This regulation of the actin tions affecting the Tec kinase Btk give rise to the human primary cytoskeleton is required for full T cell activation. Accordingly, immunodeficiency X-linked agammaglobulinemia, which is asso- mice deficient in the guanine nucleotide exchange factor Vav or ciated with impaired responses to BCR engagement (10, 11). Sim- the Wiskott-Aldrich syndrome protein, two critical regulators of ilar defects have been observed in T cells from animals deficient in the actin cytoskeleton, exhibit defective T cell responses and de- the Tec kinases Itk and Rlk and are associated with decreased by guest on September 24, 2021 velopment (2–5). However, actin reorganization is not only re- ability to respond to infectious agents (12, 13). Notably, Itk is quired for proper T cell signaling, it also is initiated by and re- required for generating appropriate Th2 responses in vivo, a defect quires signals emanating from the TCR. A number of studies have that may be linked to impaired activation of the Ca2ϩ-sensitive helped delineate the pathways between the TCR and the actin cy- NFAT transcription factors and/or altered T-bet expression (14– toskeleton and have revealed essential roles for proximal tyrosine 17). More recently, Itk-deficient cells have been shown to have kinases and adaptor molecules in addition to Vav and Wiskott- defects in actin polarization and the localized activation of the Rho Aldrich syndrome protein (6–8). Recent data suggest that the Tec family GTPase Cdc42 (18, 19). Overexpression of the Itk pleck- kinase Itk is an important contributor to these pathways. strin homology (PH) domain or Itk containing a mutation affecting the Src homology 2 (SH2) protein interaction domain in Jurkat cells also prevents TCR-induced actin polymerization (18, 20), *National Human Genome Research Institute, National Institutes of Health, Bethesda, MD 20892; †Department of Pathology, University of Chicago, Chicago, IL 60637; suggesting that the Tec kinases may have important functions in ‡National Institute of Aging, Gerontology Research Center, National Institutes of the regulation of the actin cytoskeleton. However, Itk deficiency § Health, Baltimore, MD 21224; Department of Immunology and Division of Oncol- also causes altered thymic development and selection, and mature ogy Research, Mayo Clinic, Rochester, MN 55905; and ¶Department of Pathology, Ϫ/Ϫ Children’s Hospital of Philadelphia and University of Pennsylvania, Philadelphia, cells from Itk animals can express decreased TCR levels and PA 19104 altered cell surface markers (21, 22), complicating interpretation of Received for publication November 12, 2003. Accepted for publication November these results. Experiments in cell culture have to date relied on 10, 2004. overexpression of mutant versions of Itk, which also may not ac- The costs of publication of this article were defrayed in part by the payment of page curately reflect protein function in vivo (18, 20, 23). Indeed, the charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. first report to address the role of Itk in actin reorganization and cell 1 This work was supported by Howard Hughes Medical Institute-National Institutes of polarization concluded that Itk was not involved in this process Health Research Scholar’s Program (to D.D.). because a kinase-inactive version of Itk failed to disrupt cell con- 2 D.D. and R.A.H. contributed equally to this work. tact and polarization to the APC (23). 3 Current address: Ludwig Institute for Cancer Research, Cell and Molecular Biology Branch, London, U.K. 4 Current address: Center for Cancer Research, National Cancer Institute, National 6 Abbreviations used in this paper: PLC-␥, phospholipase C-␥; PH, pleckstrin ho- Institutes of Health, Bethesda, MD 20892. mology; PBT, peripheral blood T lymphocyte; si, small interfering; WT, wild type; 5 Address correspondence and reprint requests to Dr. Pamela L. Schwartzberg, Na- JTAg, Jurkat-TAg; CAT, chloramphenicol acetyltransferase; EGFP, enhanced GFP; tional Human Genome Research Institute, National Institutes of Health, 4A38, 49 RNAi, RNA interference; LAT, linker for activation of T cells; SH2, Src homology Convent Drive, Bethesda, MD 20892. E-mail address: [email protected] 2; Itk-KD, Itk-kinase defective.

Ideally, an Itk-deficient cell line would help address these con- Electroporation cerns and aid the study of Itk function in T cell signaling. Indeed, JTAg cells (107 cells/0.5 ml in RPMI 1640 plus 20% FBS and 10 mM analyses of mutant Jurkat cell lines that are deficient in signaling HEPES) were electroporated with siRNA (1–2 ␮g) and/or plasmids (3–5 molecules have greatly advanced our understanding of TCR signal ␮g) in 0.4-mm cuvettes (300 V, 20 ms, BTX-850). To examine Vav local- transduction (24). However, the majority of mutant Jurkat lines ization, multiple cuvettes were electroporated with siRNA, combined, and 24 h ␮ have been isolated by virtue of their inability to mobilize Ca2ϩ or later, live cells were purified over Ficoll and transfected with 10 g of Flag- tagged Vav and/or 3–10 ␮g of Myc-tagged murine Itk constructs using the induce expression of reporter genes upon TCR engagement, and Amaxa nucleofector (program C16 in Solution T; Amaxa). Rested human PBT mutants that permit partial T cell function, such as seen in Itk- were transfected with siRNA (0.5 ␮g/5 ϫ 106 cells) and/or murine Myc- deficient cells (13, 25), may not be isolated by such approaches. tagged Itk (3–10 ␮g) using the Amaxa nucleofector (program T23 in solution Although additional mutant T cell lines have been generated via human T cells). The cells were placed into 12-well plates containing 2 ml of fresh media supplemented with 20% FBS and 20 U/ml IL-2. somatic cell gene targeting, this process is highly labor-intensive, and few genes have been disrupted successfully (26, 27). T cell/bead conjugations To evaluate functions of Itk, independent of altered T cell de- T cell/bead conjugates were prepared and evaluated in a manner similar to velopment, we have inhibited Itk expression in Jurkat cells and Lowin-Kropf et al. (8) and Labno et al. (19). In these studies, 5-␮m latex human peripheral blood T lymphocytes (PBT) using small inter- beads (107 beads/ml (Interfacial Dynamics)) were coated with IgM, anti- fering (si)RNA (28, 29). We demonstrate that reduction in endog- TCR C305 (1/130 dilution in PBS), or anti-CD3 OKT3 (for human PBT) as described previously (13). For T cell-bead conjugations, 1.25 ϫ 106 enous Itk reproduces the functional deficit in actin organization cells in serum-free media were mixed with 2 ϫ 106 Ab-coated beads in 70 Ϫ/Ϫ seen in Itk mouse cells, allowing us to re-express wild-type ␮l, spun for 1 min at 100 ϫ g, flicked to mix, and incubated at 37°C for

(WT) and mutant versions of murine Itk and identify domains of 10 min. Conjugates were fixed with 4% paraformaldehyde at room tem- Downloaded from Itk required for TCR-stimulated actin cytoskeleton rearrangement. perature for 10 min, 0.7 ml of serum-free RPMI 1640 was added, and cells We confirm that Itk plays a critical role in TCR-induced actin were stored rotating at 4°C until stained. cytoskeletal reorganization and demonstrates that the actin defects Immunofluorescence staining and microscopy in Itk-deficient cells correlate with the ability of the Vav guanine T cell/bead conjugates were stained with Alexa594-phalloidin (Molecular nucleotide exchange factor to localize to the site of TCR stimula- Bioprobes) as described previously (19). For Vav localization, cells were

tion both in Jurkat cells and in T cells derived from normal donors. stained with anti-FLAG (1:500 in 1.25% BSA/0.1% saponin in PBS at http://www.jimmunol.org/ Additionally, we show that Itk can directly interact with Vav and room temperature for 40 min), followed by two washes and incubation with regulates Vav localization in a kinase-independent manner that re- a solution of FITC-anti-mouse IgG (1:1000) and Alexa594-phalloidin (1: 67) (19). Conjugates were examined on an inverted Zeiss Axiophot mi- quires function of the Itk SH2 domain. Finally, expression of a mem- croscope with ϫ40 and ϫ100 oil immersion objectives. Cells were scored brane-targeted mutant of Vav can rescue the defect in actin polariza- as having polarized actin if they bound a single bead and showed increased tion in Itk-deficient cells. Our data extend recent observations from phalloidin staining at the T cell/bead interface. Thirty to 50 conjugates the Tsoukas laboratory (18) by demonstrating a kinase-independent were scored per condition. Data are presented as the percent cells with polarized actin or Vav per conjugates scored ϮSE for a minimum of three role for Itk in regulating Vav localization, and suggest a mechanism experiments. For human PBT cells, data were obtained using cells from for the actin defect in Itk-deficient cells. two or more donors. by guest on September 24, 2021 Immunofluorescence analysis of Jurkat-Nalm6 conjugates Materials and Methods Conjugations and microscopy were performed as previously described Cells and reagents (31), except that CMAC stained Nalm 6 cells were allowed to interact with Jurkat-TAg (JTAg) cells were grown in RPMI 1640 containing 10% FBS JTAg cells for only 5 min at room temperature, then plated onto poly-L- lysine-coated (Sigma-Aldrich) slides for 1–5 min, fixed, and stained. (HyClone), 10 mM HEPES, 2 mM L-glutamine, 50 ␮M 2-ME, 100 U of penicillin/100 ␮g/ml streptomycin, and 1 ␮g/ml ciprofloxacin. In some Immunoblotting experiments, antibiotics were removed to improve electroporation. Human PBT cells were obtained from donors at the National Institutes of Health For verification of Itk levels, cells (4 ϫ 107 cells/ml) were lysed in 1% Clinical Center and were purified by Ficoll separation. Cells were washed Triton X-100 in PBS (pH 7.4) with protease inhibitors (Roche) for 20 min several times and cultured for 2 days in medium (RPMI 1640 supple- on ice and clarified (14,000 rpm, 10 min at 4°C). For biochemical assays, mented with 10% FBS, 10 mM HEPES, 1 mM sodium pyruvate, 1ϫ non- cells (107/100 ␮l) were stimulated with either soluble C305 or OKT3 for essential amino acids, 10 ␮g/ml ciprofloxacin, and 20 U/ml human IL-2 2 min unless otherwise indicated, lysed in 1% Triton X-100, 150 mM (Roche)) containing 2 ␮g/ml Con A. Con A was removed by 30 min of NaCl, 20 mM HEPES, 50 mM ␤-glycerolphosphate, 2 mM EGTA, 10% incubation with 10 mg/ml ␣-methylpyrannoside at 37°C, followed by three glycerol, 10 mM NaF, 1 mM Na3VO4, and protease inhibitors, and clarified as washes in PBS; and the cells were rested for 5–9 days in IL-2 containing above. Proteins were immunoprecipitated with anti-FLAG, separated by SDS- media before treatment. Reagents used included pEGFP-N1 (Clontech PAGE, transferred to nitrocellulose, immunoblotted with primary and HRP- Laboratories), anti-Lck (BD Pharmingen), anti-Vav and anti-Itk 2F12 (Up- conjugated secondary Ab as per the manufacturer, and visualized by ECL state Biotechnology), anti-tubulin (ICN Pharmaceuticals), anti-FLAG (M2; (Amersham Biosciences). In some experiments, FLAG peptide (Sigma- Sigma-Aldrich), and anti-SLP-76 (Antibody Solutions); anti-clonotypic Aldrich) was included in the immunoprecipitation as a specificity control. TCR C305 and anti-CD3 OKT3 were concentrated from hybridoma su- pernatants. Myc-tagged Itk constructs were kind gifts from Drs. L. Berg Results (University of Massachusetts, Worcester, MA) and S. Bunnell (National Establishment of siRNA to reduce Itk expression in Jurkat cells Cancer Institute, Bethesda, MD). To confirm the feasibility of using RNA interference (RNAi), siRNA JTAg cells were electroporated with a plasmid encoding enhanced GFP (EGFP), and siRNA against EGFP or a control siRNA (si- Synthetic siRNA oligonucleotides were purchased from Xeragon (Qiagen) CAT) (30) and GFP expression was monitored by flow cytometry and treated per the manufacturer’s recommendation. siRNA against human Lck or Itk included the following: Lck-1 sense, 5Ј-CAUCGAUGUGUGU at various times postelectroporation. Cotransfection of the siGFP GAGAACUGC-3Ј; Lck-1 antisense, 5Ј-AGUUCUCACACACAUCGAU oligonucleotide, but not the control siCAT nor the sense strand GUU-3Ј; Itk-1 sense, 5Ј-CUGUUCUCAGCUGGAGAAGCUU-3Ј; ribonucleotide, markedly reduced expression of GFP (5- to 12- Itk-1 antisense, 5Ј-GCUUCUCCAGCUGAGAACAGCA-3Ј; Itk-2 fold; Fig. 1A). Reductions in GFP expression were observed both sense, 5Ј-GGAGCCUUCAUGGUAAGGGAUU-3Ј; and Itk-2 antisense, 5Ј-UCCCUUACCAUGAAGGCUCCUU-3Ј. Both sets of Itk oligos by percentage of cells positive for GFP and by mean fluorescence worked with similar efficiencies. siRNA against GFP and chloramphenicol intensity of the GFP signal (Fig. 1B). Although optimal gene si- acetyltransferase (CAT) have been described previously (30). lencing required dsRNA, in some experiments, antisense RNA The Journal of Immunology 1387

an important role in TCR-induced regulation of the actin cytoskeleton (18–20). To dissect this phenotype, JTAg cells were tran- siently transfected with siItk, and 24 h later, the cells were stimulated with either mouse IgM-coated beads as a negative control or anti-TCR-coated beads, stained with rhodamine-phalloidin, and examined by fluorescence microscopy. Conjugates between a single cell and bead were counted and scored positive if the conjugate showed increased actin accumulation at the site of bead contact (Fig. 2, A and B). Conjugates of T cells to IgM-coated beads ex- hibited a basal level of polarized actin of 25–30%, which was unaffected by treatment with siItk (Fig. 2B). However, similar to Itk-deficient cells, JTAg cells treated with siItk showed reduced TCR-induced actin polarization (Fig. 2B). This effect appeared to be specific for siItk because actin polarization remained normal in Downloaded from http://www.jimmunol.org/

FIGURE 1. RNAi is functional in Jurkat cells. A, JTAg cells were electroporated with pEGFP-N1 with or without siRNA against EGFP or CAT. Percentages of GFPϩ cells 24 h posttransfection are shown. Data represent one of three independent experiments. B, Cells were transfected with pEGFP-N1 plus varying amounts of siGFP. Data are presented as the mean fluorescence intensity (MFI). C, Reduction of endogenous gene expression using siRNA. JTAg cells were electroporated with siRNA against GFP, by guest on September 24, 2021 Lck, or Itk, and 24 h later, cell lysates were immunoblotted for Itk, Lck, and Tubulin. D, PBT cells from a normal donor were treated with siRNA against Itk, and 24 h later, cell lysates were immunoblotted for Itk and Tubulin. E, JTAg cells were electroporated with siItk, lysed at the indicated times after electroporation, and immunoblotted for Itk and Tubulin. showed a modest reduction in expression of GFP. GFP silencing was dose dependent, reaching a plateau at ϳ1–2 ␮g of a 22-bp siRNA electroporated per 107 cells (Fig. 1B). At doses of Ն5 ␮g of siRNA, nonspecific effects were observed with decreased expression of multiple proteins, whereas at doses Ͻ0.6 ␮g, less ef- ficient silencing was seen (Fig. 1B and data not shown). On the basis of these results, we routinely used 1–2 ␮g of siRNA per 107 cells. To test whether RNAi could reduce expression of endogenous Itk, we used siRNA against the genes encoding Itk or the Src family kinase Lck (siItk or siLck). Transfection of siItk or siLck led to specific reductions in expression of Itk or Lck, respectively (Fig. 1C). Similar results were found using siItk in human PBT (Fig. 1D). TCR and Tubulin levels were also unaffected by siRNA treatment (Fig. 1, C and D, and data not shown), confirming the specificity of the effects. Time course studies revealed that maxi- FIGURE 2. Reduction of Itk expression impairs actin polarization. A, mal suppression of Itk occurred in the first 2 days posttransfection JTAg cells were electroporated with siItk2 or siGFP. Twenty-four hours with normal protein levels restored by day 4 (Fig. 1E). Thus, for later, cells were stimulated with anti-TCR-coated beads, fixed, and stained functional studies of Itk, we used cells to which siItk had been with rhodamine-phalloidin. Representative polarized (positive) and nonpo- larized (negative) cells are shown. B, JTAg cells were scored for actin introduced 24–36 h earlier. polarization in response to IgM- or C305-coated beads. C, WT murine Itk Itk and actin polarization rescues the actin polarization defect. JTAg cells were transfected with siItk plus plasmid encoding murine WT Itk and scored for actin polarization to Recent studies using both Itk-deficient mouse primary T cells and C305-coated beads. Corresponding levels of endogenous Itk, murine myc- Jurkat cells expressing mutant versions of Itk suggest that Itk plays tagged Itk, and tubulin are shown below each lane. 1388 Itk REGULATES Vav LOCALIZATION cells treated with siGFP. Importantly, re-expression of WT murine The Tec kinases share a common structure (Fig. 3A) with a ty- Itk, which is not affected by the siRNA directed against human Itk, rosine kinase catalytic domain preceded by Src homology protein- rescued actin polarization in siItk-treated cells, demonstrating the interaction domains, including a SH2 domain that interacts with specificity of the defect for the loss of Itk expression (Figs. 2C tyrosine phosphorylated residues (32). Most Tec kinases also pos- and 6A). sess an N-terminal PH domain that interacts with products of PI3K and is required for inducible membrane localization, which is Requirements for Itk function in actin regulation thought to be critical for protein function. Indeed, a mutant that The ability of murine Itk to rescue the siItk effects on actin orga- disrupts the binding pocket of the PH domain and prevents Itk nization allowed us to address the requirements for Itk function. membrane localization failed to rescue actin polarization in siItk- treated cells (Fig. 3B). Although kinase-inactive mutants are often used to block functions of tyrosine kinases, data suggest that overexpression of kinase-inactive Itk may not affect actin cytoskeletal reorganization (18, 23). However, such experiments only address the ability of mutants to act as dominant-negative proteins but do not necessarily address requirements for protein function, which are better ad- dressed by genetic complementation. To definitively address whether actin cytoskeleton regulation required Itk kinase activity,

we re-expressed a point mutant of murine Itk that impaired its Downloaded from kinase activity (Itk-kinase defective (Itk-KD)) in cells transfected with siItk. Although this mutant has previously been shown to fail to rescue IL-2 transcription in Itk-deﬁcient mouse cells, Itk-KD was able to rescue actin polarization to a similar extent as WT Itk in JTAg cells treated with siItk (Fig. 3C). In contrast, Itk-KD that

also carried a mutation of the PH domain failed to rescue actin http://www.jimmunol.org/ polarization (data not shown). Thus, the PH domain, but not kinase activity of Itk, is required to rescue the TCR-induced actin polarization defect in siItk-treated cells. Finally, similar to findings from Tsoukas et al. (18), we observed that expression of a SH2 mutant of Itk dominantly inhibited actin polarization in JTAg cells (Fig. 3D). This effect occurred to a similar extent as that caused by transfection of siItk. Consistent with these observations, the SH2 mutant of Itk also failed to rescue actin polarization in siItk-treated cells. by guest on September 24, 2021 Kinase-independent functions of Itk in primary human T cells Jurkat T cells have been shown to be deficient in the phosphatase and tensin homologue deleted on chromosome 10 (PTEN), an ino- sitol phosphatase that acts on the products of PI3K (33, 34). De- ficiency of this phosphatase has been found to alter Itk localization (33). To confirm that our findings are not an artifact of the use of a Jurkat subline, we examined actin polarization in human PBT cells from normal donors that were treated with siItk. Because our studies with JTAg cells used the C305 anti-TCR Ab that is specific for the clonotypic TCR on Jurkat cells, we first confirmed that we could visualize actin polarization using PBT lymphocytes or JTAg cells that were stimulated with beads coated with OKT3, an anti- CD3 Ab (Fig. 4, A and B, and data not shown). We then demonstrated that siItk could also reduce actin polarization in PBT cells and that both WT and the kinase-defective mutant of Itk could rescue the actin defect in PBT treated with siItk (Fig. 4, A and C). Finally, we found that expression of the SH2 mutant of Itk also blocked anti-CD3-induced actin polarization in human PBT cells (Fig. 4D). Thus, a kinase-independent function for Itk in actin polarization is also seen in primary human T cells. Regulation of Vav localization One of the key regulators of the actin cytoskeleton in T cells is the guanine nucleotide exchange factor Vav, which is activated by FIGURE 3. Requirements for Itk function in TCR-mediated actin reor- tyrosine phosphorylation upon TCR stimulation and helps regulate ganization. A, Domain structure of Itk. Mutations affecting the PH (R29C), the Rho family GTPases, Rac, Rho, and Cdc42 (35). We have SH2 (R265A), and kinase domains (Itk-KD, K390R) are indicated. B,PH Ϫ/Ϫ mutant fails to rescue actin polarization. C, Itk-KD rescues actin polariza- recently found that the altered actin polarization in Itk T cells tion defect. D, SH2 mutant fails to rescue and blocks actin polarization. correlates with defective Cdc42 activation and a failure to localize Levels of endogenous Itk, murine myc-tagged Itk, and tubulin are shown Vav to the site of TCR stimulation, despite relatively normal TCR- below each lane. induced Vav phosphorylation (19, 21). To determine whether Vav The Journal of Immunology 1389 Downloaded from http://www.jimmunol.org/

FIGURE 5. Reduction of Itk expression impairs Vav polarization. Cells were electroporated with siItk and, 24 h later, transfected with FLAG-Vav and murine Itk constructs. After 4–6 h, cells were stimulated with anti- marks beads) or Staph enterotoxin E-pulsed ,ء ;TCR-coated beads (A Nalm-6 cells (B; labeled with 4Ј,6Ј-diamidino-2-phenylindole and an ar- rowhead) and stained with rhodamine-phalloidin and anti-FLAG. T cells in B are marked with a star. by guest on September 24, 2021

showed a reduction in Vav localization that correlated with the extent of actin polarization (Figs. 5A and 6A). This defect in Vav localization was also observed in human PBT cells from normal donors treated with siRNA to Itk (data not shown). Moreover, defective actin polarization and Vav localization were observed both in siItk- treated cells stimulated with Ab-coated beads, as well as those stimulated with Staph enterotoxin E-treated Nalm6 cells, a system which FIGURE 4. Reduction of Itk expression impairs actin polarization in more closely resembles Ag stimulation (Fig. 5B; Ref. 8). human PBT cells. A, Human PBT cells were treated with siItk2 or no We then examined how re-expression of WT or mutant Itk affected siRNA and, 24 h later, stimulated with OKT3-coated beads and ﬁxed and Vav localization in cells transfected with siItk. Expression of either stained with rhodamine-phalloidin. Representative polarized and nonpo- WT or KD murine Itk rescued both actin polarization and Vav local- larized cells are shown. B, Human PBT cells were stimulated with either control (IgM) or OKT3-coated beads, and actin polarization was scored. C, ization (Fig. 6, A and B). In contrast, cells expressing the SH2 mutant Human PBT cells were transfected with siItk in the presence or absence of of Itk showed abnormal Vav localization, which paralleled their plasmids encoding WT and mutant murine Itk, stimulated with OKT3- defect in actin polarization, and the SH2 mutant failed to rescue Vav coated beads and scored for actin polarization. Si0 cells received no localization in siItk-treated cells (Fig. 6C). Thus, the ability to polarize siRNA. Data represent the average Ϯ SD of results from two to three actin correlated with proper Vav localization, and both regulation of donors. D, Itk-SH2 mutant blocks actin polarization in human PBT cells. Vav localization and actin polarization appeared to be independent of Data represent the average Ϯ SD of results from two donors. Itk kinase activity but dependent on SH2 domain interactions.

Membrane-targeted Vav rescues actin polarization localization correlated with the ability of Itk mutants to rescue Although the defect in Vav localization could lead to an actin actin polarization, we expressed a FLAG-tagged version of Vav, defect in siItk-treated cells, it is also possible that the defective which facilitated subcellular localization studies. Although we in- Vav localization was actually secondary to the actin defect. To troduced siItk for 24–36 h to allow maximal gene silencing, ex- determine whether the altered Vav localization directly contributes pression of Vav-FLAG was performed for short periods of time to the actin defect in siItk-treated cells, we expressed a membrane- (4–8 h), which did not alter cellular viability, adhesion of cells to targeted construct of Vav, Vav-CAAX (36). Expression of Vav- anti-TCR-coated beads, nor polarization of actin (Fig. 5A and data CAAX restored actin polarization to the site of TCR stimulation not shown). Similar to primary cells, siItk-treated Jurkat cells in siItk-treated cells (Fig. 7), suggesting that the actin defect in 1390 Itk REGULATES Vav LOCALIZATION

FIGURE 6. Kinase-inactive Itk rescues Vav localization. A and B, WT and KD murine Itk rescues Vav localization and actin polarization in JTAg cells treated with siItk. C, SH2 mutant blocks both actin polarization and Vav localization. Levels of endogenous Itk, murine myc-tagged Itk, Vav, and Tubulin are shown below each lane. Downloaded from

Itk-deficient cells results at least in part from improper Vav more, coimmunoprecipitation of Itk with Vav was not affected by localization. mutation of the SH2 domain of Itk (Fig. 8B). Thus, the Vav-Itk interaction appears to be independent of Itk SH2 function. Itk expression affects protein interactions with Vav The constitutive association of Itk with Vav suggested that the To additionally investigate the mechanism by which Itk affects presence of Itk may influence the ability of Vav to interact with http://www.jimmunol.org/ Vav, we examined Vav phosphorylation and protein interactions in other proteins in TCR-mediated signaling complexes. Therefore, we siItk-treated cells. Unlike Vav localization, which appeared to be examined proteins that coimmunoprecipitated with Vav in the pres- dependent on the presence of Itk, patterns of Vav tyrosine phos- ence or absence of Itk. Immunoprecipitation of Vav with an anti- phorylation were not grossly affected by the loss of Itk (Fig. 8A), FLAG monoclonal revealed the presence of a 76-kDa tyrosine phos- consistent with what we have observed in Itk and Rlk/Itk-deficient phorylated protein that comigrated with the adaptor molecule SLP-76, thymocytes. Thus, tyrosine phosphorylation of Vav appears to oc- as determined by immunoblotting with anti-SLP-76 and Itk Abs (Fig. cur before or independent of stable recruitment of Vav to the site 8C and data not shown). However, depletion of Itk, via siRNA, of TCR contact. To better understand how Itk can affect Vav lo- reduced the amount of this protein that was observed in the Vav im- by guest on September 24, 2021 calization, we performed coimmunoprecipitation experiments to munoprecipitations. Thus, depletion of Itk appears to impair the abil- examine whether Itk directly interacts with Vav using cells coex- ity of Vav to interact with other proteins in the signaling complex. pressing Vav-FLAG and myc-tagged Itk. Immunoprecipitation of FLAG-tagged Vav revealed Itk-myc in the immunoprecipitates (Fig. 8B). The association of Vav and Itk appeared to be consti- Discussion tutive and did not depend on TCR stimulation. The same was also Regulation of the actin cytoskeleton is critical for T cell signal observed for endogenous Itk and Vav (data not shown). Further- transduction and normal immune cell interactions (1, 37). We have used RNAi to dissect the requirements for Itk in TCR-induced actin polarization. Although certain data suggest that siRNA can give rise to nonspecific effects in mammalian cells, we find that the actin defect in siItk-treated cells is rescued by WT murine Itk, supporting the specificity of this phenotype for the loss of expression of Itk. Our results suggest that Itk has a potential scaffolding function that is independent of its tyrosine kinase activity, which helps regulate Vav localization and cytoskeletal reorganization in response to TCR stimulation. We have previously reported that Itk-deficient cells show defective TCR-induced actin polarization that is associated with decreased activation of Cdc42, a key regulator of the Wiskott- Aldrich syndrome protein (19). We additionally found that Vav was not properly targeted to the site of TCR contact. The observation that Vav regulates Cdc42 (7) suggested that altered Vav localization contributed to the actin defect in ItkϪ/Ϫ cells. Our demonstration in this study that a membrane-targeted version of Vav can rescue actin polarization in siItk-treated cells directly im- plicates Vav in the altered actin regulation in these cells. The mechanism(s) by which Itk affects Vav localization remains FIGURE 7. Vav-CAAX rescues actin polarization. Cells were electro- unclear. Itk has been shown to phosphorylate tyrosine 171 of linker porated with siItk and, 24 h later, transfected with FLAG-Vav-CAAX and for activation of T cells (LAT), which can bind Vav (38). How- evaluated for actin polarization. Levels of endogenous Itk, murine myc- ever, our results argue that proper Vav localization does not re- tagged Itk, Vav, and tubulin are shown below each lane. quire Itk kinase activity. Indeed, the role of Itk kinase activity in The Journal of Immunology 1391 Downloaded from http://www.jimmunol.org/

FIGURE 8. Interaction of Itk and Vav stabilizes Vav protein interactions. A, Reduction of Itk expression does not alter Vav tyrosine phosphorylation. JTAg cells were transfected with siItk and, 24 h later, stimulated with soluble C305, lysed, immunoprecipitated with anti-Vav, and immunoblotted with anti-phosphotyrosine. Right panel, Itk levels. B, Itk and Vav are constitutively associated, independent of Itk-SH2 domain function. JTAg cells were transfected with either WT or the SH2 mutant of murine myc-tagged Itk and FLAG-tagged Vav, then 24 h later, stimulated with OKT3, lysed, and immunoprecipitated with anti-FLAG and immunoblotted with anti-myc or anti-Vav1. For speciﬁcity controls, immunoprecipitations were performed in the presence of the cognate FLAG peptide. C, Reduction of Itk expression impairs the ability of Vav to associate with SLP-76. JTAg cells were transfected with FLAG-tagged Vav with or without siItk, then stimulated with C305 and immunoprecipitated with anti-FLAG and sequentially immunoblotted for

phosphotyrosine, SLP-76, and Vav. Si0 cells received no siRNA. by guest on September 24, 2021

regulation of the actin cytoskeleton has been somewhat controver- function of the Itk SH2 domain, suggesting that the SH2 domain of sial. Work from Donnadieu et al. (23) originally argued that Itk did Itk is interacting with another protein. Direct binding of Tec ki- not participate in actin cytoskeleton regulation because kinase-in- nases and Vav have also been previously reported both in vitro and by active Itk failed to block T cell polarization. Interpretations of this coimmunoprecipitation (39, 43). However, it is also possible that a work have been challenged by two recent studies demonstrating an separate function of Itk may also contribute to Vav localization. Of actin polarization defect in ItkϪ/Ϫ cells, despite the inability of interest are findings that Btk can act in a kinase-independent fashion Itk-KD to inhibit this process (18, 19). Nonetheless, it should be to increase phosphatidylinositol 3,4,5-phosphate levels (44). Because noted that data from Shimizu and colleagues (20) argue that a Vav contains a PH domain, it is possible that Itk contributes to Vav kinase-inactive Itk mutant can block TCR-induced actin polymer- localization through this type of secondary influence. ization. Such discrepancies may be the result of differences in as- Nonetheless, the cross-regulation of and interactions between say systems, expression levels, or the use of different mutants of Itk and Vav are complex. Tybulewicz and colleagues (45) have Itk. By demonstrating that a mutant affecting the kinase activity of also demonstrated impaired Itk activation in Vav-deficient cells. Itk can indeed rescue the actin defect in siItk-treated Jurkat or Although this observation may be secondary to defective PI3K human PBT cells, our work provides strong evidence for a kinase- activation in VavϪ/Ϫ cells, rather than a direct effect on Itk, these independent function of Itk. data underscore the complex regulatory interactions among com- Our findings suggest that Itk has an adaptor function that re- ponents of the TCR signaling complex and argue that linear rela- quires its PH and SH2 domains, but not kinase activity, for the tionships cannot necessarily be used to describe the biochemical regulation of Vav recruitment and actin reorganization. Therefore, pathways regulating T cell activation. Therefore, it is of interest it is relevant that both Itk and Vav bind via their SH2 domains to that Tybulewicz and colleagues (45) also noted disruption of the the adaptor SLP-76 as part of the LAT-SLP-76 mediated complex interactions of SLP-76 with PLC-␥ in Vav1-deficient cells, sug- that helps orchestrate downstream readouts in TCR signaling (39– gesting there may be defects in the integrity of the LAT-SLP-76- 42). Moreover, depletion of Itk appears to reduce the amount of mediated complex in the absence of either Vav1 or Itk. It should SLP-76 found in Vav immunoprecipitates. Because the SH2 do- also be noted that our data does not rule out the possibility that main of Itk is required for actin polarization, it is possible that an Vav1 associates with the LAT-SLP-76 complex in Itk-deficient interaction between the Itk-SH2 domain and SLP-76 (or LAT) cells, but does suggest that Vav1 phosphorylation can occur before may be required to stabilize the interactions of Vav with other or independent of stable SLP-76 association. proteins in this signaling complex. Indeed, we observed that Vav The Tec kinases have been previously implicated in the regu- and Itk can coimmunoprecipitate and that this does not require that lation of PLC-␥ and Ca2ϩ mobilization (9). Nonetheless, we see 1392 Itk REGULATES Vav LOCALIZATION effects on actin under conditions where we see minimal changes in 18. Grasis, J. A., C. D. Browne, and C. D. Tsoukas. 2003. Inducible T cell tyrosine Ca2ϩ and NFAT activation (data not shown). It is possible that kinase regulates actin-dependent cytoskeletal events induced by the T cell antigen receptor. J. Immunol. 170:3971. PLC-␥ activation may be compensated for by expression of other 19. Labno, C. M., C. M. Lewis, D. You, D. W. Leung, A. Takesono, N. Kamberos, Tec kinases, as in mice, where T cells lacking two Tec kinases, Rlk A. Seth, L. D. Finkelstein, M. K. Rosen, P. L. Schwartzberg, et al. 2003. Itk 2ϩ Ϫ/Ϫ functions to control actin polymerization at the immune synapse through local- and Itk, have more impaired Ca mobilization than Itk cells ized activation of Cdc42 and WASP. Curr. Biol. 13:1619. 2ϩ (13). Alternatively, it is possible that the defects in prolonged Ca 20. Woods, M. L., W. J. Kivens, M. A. Adelsman, Y. Qiu, A. August, and Ϫ/Ϫ Y. Shimizu. 2001. A novel function for the Tec family tyrosine kinase Itk in mobilization and immune responses in Itk mice may actually ␤ activation of 1 integrins by the T-cell receptor. EMBO J. 20:1232. result in part from the altered Vav localization and cytoskeletal 21. Schaeffer, E. M., C. Broussard, J. Debnath, S. Anderson, D. W. McVicar, and organization, which may prevent formation of a stable TCR signaling P. L. Schwartzberg. 2000. Tec family kinases modulate thresholds for thymocyte complex. We have recently found that T cells expressing mutant ver- development and selection. J. Exp. Med. 192:987. 22. Lucas, J. A., L. O. Atherly, and L. J. Berg. 2002. The absence of Itk inhibits sions of Tec kinases also show decreased cell polarization and acti- positive selection without changing lineage commitment. J. Immunol. 168:6142. vation of Cdc42 and Rac in response to chemokines (46). It will be of 23. Donnadieu, E., V. Lang, G. Bismuth, W. Ellmeier, O. Acuto, F. Michel, and A. Trautmann. 2001. Differential roles of Lck and Itk in T cell response to antigen interest to see whether Vav plays a role in these processes and whether recognition revealed by calcium imaging and electron microscopy. J. Immunol. Btk-deficient B cells also exhibit similar actin cytoskeleton defects. 166:5540. 24. Abraham, R. T. 2000. Mutant T cell lines as model systems for the dissection of T cell antigen receptor signaling pathways. Immunol. Res. 22:95. Acknowledgments 25. Miller, A. T., and L. J. Berg. 2002. Defective Fas ligand expression and activa- We thank S. Bunnell and L. Berg for the murine Itk constructs, B. Hill and tion-induced cell death in the absence of IL-2-inducible T cell kinase. J. Immunol. members of the Schwartzberg and Burkhardt labs for helpful discussions 168:2163. 26. Braaten, D., and J. Luban. 2001. Cyclophilin A regulates HIV-1 infectivity, as and reagents, and M. Cichanowski for assistance with graphics. Downloaded from demonstrated by gene targeting in human T cells. EMBO J. 20:1300. 27. Cao, Y., E. M. Janssen, A. W. Duncan, A. Altman, D. D. Billadeau, and References R. T. Abraham. 2002. Pleiotropic defects in TCR signaling in a Vav-1-null Jurkat 1. Cannon, J. L., and J. K. Burkhardt. 2002. The regulation of actin remodeling T-cell line. EMBO J. 21:4809. during T-cell-APC conjugate formation. Immunol. Rev. 186:90. 28. Elbashir, S. M., J. Harborth, K. Weber, and T. Tuschl. 2002. Analysis of gene 2. Costello, P. S., A. E. Walters, P. J. Mee, M. Turner, L. F. Reynolds, A. Prisco, function in somatic mammalian cells using small interfering RNAs. Methods N. Sarner, R. Zamoyska, and V. L. Tybulewicz. 1999. The Rho-family GTP 26:199. exchange factor Vav is a critical transducer of T cell receptor signals to the 29. Caplen, N. J. 2002. A new approach to the inhibition of gene expression. Trends calcium, ERK, and NF-␬B pathways. Proc. Natl. Acad. Sci. USA 96:3035. Biotechnol. 20:49. http://www.jimmunol.org/ 3. Fischer, K. D., Y. Y. Kong, H. Nishina, K. Tedford, L. E. Marengere, 30. Caplen, N. J., S. Parrish, F. Imani, A. Fire, and R. A. Morgan. 2001. Specific I. Kozieradzki, T. Sasaki, M. Starr, G. Chan, S. Gardener, et al. 1998. Vav is a inhibition of gene expression by small double-stranded RNAs in invertebrate and regulator of cytoskeletal reorganization mediated by the T-cell receptor. Curr. vertebrate systems. Proc. Natl. Acad. Sci. USA 98:9742. Biol. 8:554. 31. Cannon, J. L., C. M. Labno, G. Bosco, A. Seth, M. H. McGavin, 4. Snapper, S. B., F. S. Rosen, E. Mizoguchi, P. Cohen, W. Khan, C. H. Liu, K. A. Siminovitch, M. K. Rosen, and J. K. Burkhardt. 2001. Wasp recruitment to T. L. Hagemann, S. P. Kwan, R. Ferrini, L. Davidson, et al. 1998. Wiskott- the T cell:APC contact site occurs independently of Cdc42 activation. Immunity Aldrich syndrome protein-deficient mice reveal a role for WASP in T but not B 15:249. cell activation. Immunity 9:81. 32. Smith, C. I., T. C. Islam, P. T. Mattsson, A. J. Mohamed, B. F. Nore, and 5. Zhang, J., A. Shehabeldin, L. A. da Cruz, J. Butler, A. K. Somani, M. McGavin, M. Vihinen. 2001. The Tec family of cytoplasmic tyrosine kinases: mammalian I. Kozieradzki, A. O. dos Santos, A. Nagy, S. Grinstein, et al. 1999. Antigen Btk, Bmx, Itk, Tec, Txk and homologs in other species. Bioessays 23:436. receptor-induced activation and cytoskeletal rearrangement are impaired in Wis- 33. Shan, X., M. J. Czar, S. C. Bunnell, P. Liu, Y. Liu, P. L. Schwartzberg, and kott-Aldrich syndrome protein-deficient lymphocytes. J. Exp. Med. 190:1329. R. L. Wange. 2000. Deficiency of PTEN in Jurkat T cells causes constitutive by guest on September 24, 2021 6. Bunnell, S. C., V. Kapoor, R. P. Trible, W. Zhang, and L. E. Samelson. 2001. localization of Itk to the plasma membrane and hyperresponsiveness to CD3 Dynamic actin polymerization drives T cell receptor-induced spreading: a role for stimulation. Mol. Cell. Biol. 20:6945. the signal transduction adaptor LAT. Immunity 14:315. 34. Xu, Z., D. Stokoe, L. P. Kane, and A. Weiss. 2002. The inducible expression of 7. Zeng, R., J. L. Cannon, R. T. Abraham, M. Way, D. D. Billadeau, the tumor suppressor gene PTEN promotes apoptosis and decreases cell size by J. Bubeck-Wardenberg, and J. K. Burkhardt. 2003. SLP-76 coordinates Nck- inhibiting the PI3K/Akt pathway in Jurkat T cells. Cell Growth Differ. 13:285. dependent Wiskott-Aldrich syndrome protein recruitment with Vav-1/Cdc42-de- 35. Bustelo, X. R. 2001. Vav proteins, adaptors and cell signaling. Oncogene pendent Wiskott-Aldrich syndrome protein activation at the T cell-APC contact 20:6372. site. J. Immunol. 171:1360. 36. Billadeau, D. D., S. M. Mackie, R. A. Schoon, and P. J. Leibson. 2000. Specific 8. Lowin-Kropf, B., V. S. Shapiro, and A. Weiss. 1998. Cytoskeletal polarization of subdomains of Vav differentially affect T cell and NK cell activation. J. Immunol. T cells is regulated by an immunoreceptor tyrosine-based activation motif-de- 164:3971. pendent mechanism. J. Cell Biol. 140:861. 37. Fuller, C. L., V. L. Braciale, and L. E. Samelson. 2003. All roads lead to actin: 9. Lewis, C. M., C. Broussard, M. J. Czar, and P. L. Schwartzberg. 2001. Tec the intimate relationship between TCR signaling and the cytoskeleton. Immunol. kinases: modulators of lymphocyte signaling and development. Curr. Opin. Im- Rev. 191:220. munol. 13:317. 38. Perez-Villar, J. J., G. S. Whitney, M. T. Sitnick, R. J. Dunn, S. Venkatesan, K. 10. Tsukada, S., D. C. Saffran, D. J. Rawlings, O. Parolini, R. C. Allen, I. Klisak, O’Day, G. L. Schieven, T. A. Lin, and S. B. Kanner. 2002. Phosphorylation of the R. S. Sparkes, H. Kubagawa, T. Mohandas, S. Quan, et al. 1993. Deficient ex- linker for activation of T-cells by Itk promotes recruitment of Vav. Biochemistry pression of a B cell cytoplasmic tyrosine kinase in human X-linked agamma- 41:10732. globulinemia. Cell 72:279. 39. Bunnell, S., M. Diehn, M. Yaffe, P. Findell, L. Cantley, and L. Berg. 2000. 11. Vetrie, D., I. Vorechovsky, P. Sideras, J. Holland, A. Davies, F. Flinter, Biochemical interactions integrating Itk with the T cell receptor-initiated signal- L. Hammarstrom, C. Kinnon, R. Levinsky, M. Bobrow, et al. 1993. The gene ing cascade. J. Biol. Chem. 276:2219. involved in X-linked agammaglobulinaemia is a member of the src family of 40. Su, Y. W., Y. Zhang, J. Schweikert, G. A. Koretzky, M. Reth, and J. Wienands. protein-tyrosine kinases [Published erratum appears in 1993 Nature 364:362.] 1999. Interaction of SLP adaptors with the SH2 domain of tec family kinases. Nature 361:226. Eur. J. Immunol. 29:3702. 12. Liu, K. Q., S. C. Bunnell, C. B. Gurniak, and L. J. Berg. 1998. T cell receptor- 41. Tuosto, L., F. Michel, and O. Acuto. 1996. p95vav associates with tyrosine-phos- initiated calcium release is uncoupled from capacitative calcium entry in Itk- phorylated SLP-76 in antigen-stimulated T cells. J. Exp. Med. 184:1161. deficient T cells. J. Exp. Med. 187:1721. 42. Wu, J., D. G. Motto, G. A. Koretzky, and A. Weiss. 1996. Vav and SLP-76 13. Schaeffer, E. M., J. Debnath, G. Yap, D. McVicar, X. C. Liao, D. R. Littman, interact and functionally cooperate in IL-2 gene activation. Immunity 4:593. A. Sher, H. E. Varmus, M. J. Lenardo, and P. L. Schwartzberg. 1999. Require- 43. Kline, J. B., D. J. Moore, and C. V. Clevenger. 2001. Activation and association ment for Tec kinases Rlk and Itk in T cell receptor signaling and immunity. of the Tec tyrosine kinase with the human prolactin receptor: mapping of a Tec/ Science 284:638. Vav1-receptor binding site. Mol. Endocrinol. 15:832. 14. Fowell, D. J., K. Shinkai, X. C. Liao, A. M. Beebe, R. L. Coffman, D. R. Littman, 44. Saito, K., K. F. Tolias, A. Saci, H. B. Koo, L. Humphries, A. M. Scharenberg, and R. M. Locksley. 1999. Impaired NFATc translocation and failure of Th2 D. J. Rawlings, J. P. Kinet, and C. L. Carpenter. 2003. BTK regulates PtdIns-4,5-P2 development in Itk-deficient CD4ϩ T cells. Immunity 11:399. synthesis important for calcium signaling and PI3K activity. Immunity 19:669. 15. Mueller, C., and A. August. 2003. Attenuation of immunological symptoms of 45. Reynolds, L. F., L. A. Smyth, T. Norton, N. Freshney, J. Downward, D. Kioussis, allergic asthma in mice lacking the tyrosine kinase ITK. J. Immunol. 170:5056. and V. L. Tybulewicz. 2002. Vav1 transduces T cell receptor signals to the 16. Schaeffer, E. M., G. S. Yap, C. M. Lewis, M. J. Czar, D. W. McVicar, activation of phospholipase C-␥1 via phosphoinositide 3-kinase-dependent and A. W. Cheever, A. Sher, and P. L. Schwartzberg. 2001. Mutation of Tec family -independent pathways. J. Exp. Med. 195:1103. kinases alters T helper cell differentiation. Nat. Immunol. 2:1183. 46. Takesono, A., R. Horai, M. Mandai, D. Dombroski, and P. L. Schwartzberg. 17. Miller, A. T., H. M. Wilcox, Z. Lai, and L. J. Berg. Signaling through Itk pro- 2004. Requirement for tec kinases in chemokine-induced migration and activa- motes T helper 2 differentiation via negative regulation of T-bet. Immunity 21:67. tion of cdc42 and rac. Curr. Biol. 14:917.