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Actin Cytoskeleton TCR-Induced Regulation of Vav and the Kinase

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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. Copyright © 2005 by The American Association of Immunologists, Inc. 0022-1767/05/$02.00 1386 Itk REGULATES Vav LOCALIZATION 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.
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