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* National Human Genome Research Institute, National Institutes of Health, Bethesda, MD 20892;
Department of Pathology, University of Chicago, Chicago, IL 60637;
National Institute of Aging, Gerontology Research Center, National Institutes of Health, Baltimore, MD 21224;
Department of Immunology and Division of Oncology Research, Mayo Clinic, Rochester, MN 55905; and
¶ Department of Pathology, Children’s Hospital of Philadelphia and University of Pennsylvania, Philadelphia, PA 19104
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The Tec family tyrosine kinases are key signaling intermediates that are required for full activation of phospholipase C-
(PLC-)6 and Ca2+ mobilization downstream from Ag receptors (9). Mutations affecting the Tec kinase Btk give rise to the human primary immunodeficiency X-linked agammaglobulinemia, which is associated with impaired responses to BCR engagement (10, 11). Similar defects have been observed in T cells from animals deficient in the Tec kinases Itk and Rlk and are associated with decreased ability to respond to infectious agents (12, 13). Notably, Itk is required for generating appropriate Th2 responses in vivo, a defect that may be linked to impaired activation of the Ca2+-sensitive NFAT transcription factors and/or altered T-bet expression (14, 15, 16, 17). More recently, Itk-deficient cells have been shown to have defects in actin polarization and the localized activation of the Rho family GTPase Cdc42 (18, 19). Overexpression of the Itk pleckstrin 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), suggesting that the Tec kinases may have important functions in the regulation of the actin cytoskeleton. However, Itk deficiency also causes altered thymic development and selection, and mature cells from Itk–/– animals can express decreased TCR levels and altered cell surface markers (21, 22), complicating interpretation of these results. Experiments in cell culture have to date relied on overexpression of mutant versions of Itk, which also may not accurately reflect protein function in vivo (18, 20, 23). Indeed, the first report to address the role of Itk in actin reorganization and cell polarization concluded that Itk was not involved in this process because a kinase-inactive version of Itk failed to disrupt cell contact and polarization to the APC (23).
Ideally, an Itk-deficient cell line would help address these concerns and aid the study of Itk function in T cell signaling. Indeed, analyses of mutant Jurkat cell lines that are deficient in signaling molecules have greatly advanced our understanding of TCR signal transduction (24). However, the majority of mutant Jurkat lines have been isolated by virtue of their inability to mobilize Ca2+ or induce expression of reporter genes upon TCR engagement, and mutants that permit partial T cell function, such as seen in Itk-deficient cells (13, 25), may not be isolated by such approaches. Although additional mutant T cell lines have been generated via somatic cell gene targeting, this process is highly labor-intensive, and few genes have been disrupted successfully (26, 27).
To evaluate functions of Itk, independent of altered T cell development, we have inhibited Itk expression in Jurkat cells and human peripheral blood T lymphocytes (PBT) using small interfering (si)RNA (28, 29). We demonstrate that reduction in endogenous Itk reproduces the functional deficit in actin organization seen in Itk–/– mouse cells, allowing us to re-express wild-type (WT) and mutant versions of murine Itk and identify domains of Itk required for TCR-stimulated actin cytoskeleton rearrangement. We confirm that Itk plays a critical role in TCR-induced actin cytoskeletal reorganization and demonstrates that the actin defects in Itk-deficient cells correlate with the ability of the Vav guanine nucleotide exchange factor to localize to the site of TCR stimulation both in Jurkat cells and in T cells derived from normal donors. Additionally, we show that Itk can directly interact with Vav and regulates Vav localization in a kinase-independent manner that requires function of the Itk SH2 domain. Finally, expression of a membrane-targeted mutant of Vav can rescue the defect in actin polarization in Itk-deficient cells. Our data extend recent observations from the Tsoukas laboratory (18) by demonstrating a kinase-independent role for Itk in regulating Vav localization, and suggest a mechanism for the actin defect in Itk-deficient cells.
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Materials and Methods |
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Jurkat-TAg (JTAg) cells were grown in RPMI 1640 containing 10% FBS (HyClone), 10 mM HEPES, 2 mM L-glutamine, 50 µM 2-ME, 100 U of penicillin/100 µg/ml streptomycin, and 1 µg/ml ciprofloxacin. In some experiments, antibiotics were removed to improve electroporation. Human PBT cells were obtained from donors at the National Institutes of Health Clinical Center and were purified by Ficoll separation. Cells were washed several times and cultured for 2 days in medium (RPMI 1640 supplemented with 10% FBS, 10 mM HEPES, 1 mM sodium pyruvate, 1x nonessential amino acids, 10 µg/ml ciprofloxacin, and 20 U/ml human IL-2 (Roche)) containing 2 µg/ml Con A. Con A was removed by 30 min of incubation with 10 mg/ml 
-methylpyrannoside at 37°C, followed by three washes in PBS; and the cells were rested for 5–9 days in IL-2 containing media before treatment. Reagents used included pEGFP-N1 (Clontech Laboratories), anti-Lck (BD Pharmingen), anti-Vav and anti-Itk 2F12 (Upstate Biotechnology), anti-tubulin (ICN Pharmaceuticals), anti-FLAG (M2; Sigma-Aldrich), and anti-SLP-76 (Antibody Solutions); anti-clonotypic TCR C305 and anti-CD3 OKT3 were concentrated from hybridoma supernatants. Myc-tagged Itk constructs were kind gifts from Drs. L. Berg (University of Massachusetts, Worcester, MA) and S. Bunnell (National Cancer Institute, Bethesda, MD).
siRNA
Synthetic siRNA oligonucleotides were purchased from Xeragon (Qiagen) and treated per the manufacturer’s recommendation. siRNA against human Lck or Itk included the following: Lck-1 sense, 5'-CAUCGAUGUGUGUGAGAACUGC-3'; Lck-1 antisense, 5'-AGUUCUCACACACAUCGAUGUU-3'; Itk-1 sense, 5'-CUGUUCUCAGCUGGAGAAGCUU-3'; Itk-1 antisense, 5'-GCUUCUCCAGCUGAGAACAGCA-3'; Itk-2 sense, 5'-GGAGCCUUCAUGGUAAGGGAUU-3'; and Itk-2 antisense, 5'-UCCCUUACCAUGAAGGCUCCUU-3'. Both sets of Itk oligos worked with similar efficiencies. siRNA against GFP and chloramphenicol acetyltransferase (CAT) have been described previously (30).
Electroporation
JTAg cells (107 cells/0.5 ml in RPMI 1640 plus 20% FBS and 10 mM HEPES) were electroporated with siRNA (1–2 µg) and/or plasmids (3–5 µg) in 0.4-mm cuvettes (300 V, 20 ms, BTX-850). To examine Vav localization, multiple cuvettes were electroporated with siRNA, combined, and 24 h later, live cells were purified over Ficoll and transfected with 10 µg of Flag-tagged Vav and/or 3–10 µg of Myc-tagged murine Itk constructs using the Amaxa nucleofector (program C16 in Solution T; Amaxa). Rested human PBT were transfected with siRNA (0.5 µg/5 x 106 cells) and/or murine Myc-tagged Itk (3–10 µg) using the Amaxa nucleofector (program T23 in solution 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.
T cell/bead conjugations
T cell/bead conjugates were prepared and evaluated in a manner similar to Lowin-Kropf et al. (8) and Labno et al. (19). In these studies, 5-µm latex beads (107 beads/ml (Interfacial Dynamics)) were coated with IgM, anti-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 x 106 cells in serum-free media were mixed with 2 x 106 Ab-coated beads in 70 µl, spun for 1 min at 100 x g, flicked to mix, and incubated at 37°C for 10 min. Conjugates were fixed with 4% paraformaldehyde at room temperature for 10 min, 0.7 ml of serum-free RPMI 1640 was added, and cells were stored rotating at 4°C until stained.
Immunofluorescence staining and microscopy
T cell/bead conjugates were stained with Alexa594-phalloidin (Molecular Bioprobes) as described previously (19). For Vav localization, cells were stained with anti-FLAG (1:500 in 1.25% BSA/0.1% saponin in PBS at room temperature for 40 min), followed by two washes and incubation with a solution of FITC-anti-mouse IgG (1:1000) and Alexa594-phalloidin (1:67) (19). Conjugates were examined on an inverted Zeiss Axiophot microscope with x40 and x100 oil immersion objectives. Cells were scored as having polarized actin if they bound a single bead and showed increased phalloidin staining at the T cell/bead interface. Thirty to 50 conjugates were scored per condition. Data are presented as the percent cells with polarized actin or Vav per conjugates scored ±SE for a minimum of three experiments. For human PBT cells, data were obtained using cells from two or more donors.
Immunofluorescence analysis of Jurkat-Nalm6 conjugates
Conjugations and microscopy were performed as previously described (31), except that CMAC stained Nalm 6 cells were allowed to interact with 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.
Immunoblotting
For verification of Itk levels, cells (4 x 107 cells/ml) were lysed in 1% Triton X-100 in PBS (pH 7.4) with protease inhibitors (Roche) for 20 min on ice and clarified (14,000 rpm, 10 min at 4°C). For biochemical assays, cells (107/100 µl) were stimulated with either soluble C305 or OKT3 for 2 min unless otherwise indicated, lysed in 1% Triton X-100, 150 mM NaCl, 20 mM HEPES, 50 mM 
-glycerolphosphate, 2 mM EGTA, 10% glycerol, 10 mM NaF, 1 mM Na3VO4, and protease inhibitors, and clarified as above. Proteins were immunoprecipitated with anti-FLAG, separated by SDS-PAGE, transferred to nitrocellulose, immunoblotted with primary and HRP-conjugated secondary Ab as per the manufacturer, and visualized by ECL (Amersham Biosciences). In some experiments, FLAG peptide (Sigma-Aldrich) was included in the immunoprecipitation as a specificity control.
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Results |
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To confirm the feasibility of using RNA interference (RNAi), JTAg cells were electroporated with a plasmid encoding enhanced GFP (EGFP), and siRNA against EGFP or a control siRNA (siCAT) (30) and GFP expression was monitored by flow cytometry at various times postelectroporation. Cotransfection of the siGFP oligonucleotide, but not the control siCAT nor the sense strand ribonucleotide, markedly reduced expression of GFP (5- to 12-fold; Fig. 1A). Reductions in GFP expression were observed both by percentage of cells positive for GFP and by mean fluorescence intensity of the GFP signal (Fig. 1B). Although optimal gene silencing required dsRNA, in some experiments, antisense RNA showed a modest reduction in expression of GFP. GFP silencing was dose dependent, reaching a plateau at 
1–2 µg of a 22-bp siRNA electroporated per 107 cells (Fig. 1B). At doses of 
5 µg of siRNA, nonspecific effects were observed with decreased expression of multiple proteins, whereas at doses <0.6 µg, less efficient silencing was seen (Fig. 1B and data not shown). On the basis of these results, we routinely used 1–2 µg of siRNA per 107 cells.
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Itk and actin polarization
Recent studies using both Itk-deficient mouse primary T cells and Jurkat cells expressing mutant versions of Itk suggest that Itk plays an important role in TCR-induced regulation of the actin cytoskeleton (18, 19, 20). To dissect this phenotype, JTAg cells were transiently 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 exhibited 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 cells treated with siGFP. Importantly, re-expression of WT murine Itk, which is not affected by the siRNA directed against human Itk, rescued actin polarization in siItk-treated cells, demonstrating the specificity of the defect for the loss of Itk expression (Figs. 2C and 6A).
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The ability of murine Itk to rescue the siItk effects on actin organization allowed us to address the requirements for Itk function. The Tec kinases share a common structure (Fig. 3A) with a tyrosine kinase catalytic domain preceded by Src homology protein-interaction domains, including a SH2 domain that interacts with tyrosine phosphorylated residues (32). Most Tec kinases also possess an N-terminal PH domain that interacts with products of PI3K and is required for inducible membrane localization, which is thought to be critical for protein function. Indeed, a mutant that disrupts the binding pocket of the PH domain and prevents Itk membrane localization failed to rescue actin polarization in siItk-treated cells (Fig. 3B).
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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 inositol phosphatase that acts on the products of PI3K (33, 34). Deficiency 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.
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One of the key regulators of the actin cytoskeleton in T cells is the guanine nucleotide exchange factor Vav, which is activated by tyrosine phosphorylation upon TCR stimulation and helps regulate the Rho family GTPases, Rac, Rho, and Cdc42 (35). We have recently found that the altered actin polarization in Itk–/– T cells correlates with defective Cdc42 activation and a failure to localize Vav to the site of TCR stimulation, despite relatively normal TCR-induced Vav phosphorylation (19, 21). To determine whether Vav localization correlated with the ability of Itk mutants to rescue actin polarization, we expressed a FLAG-tagged version of Vav, which facilitated subcellular localization studies. Although we introduced siItk for 24–36 h to allow maximal gene silencing, expression of Vav-FLAG was performed for short periods of time (4–8 h), which did not alter cellular viability, adhesion of cells to anti-TCR-coated beads, nor polarization of actin (Fig. 5A and data not shown). Similar to primary cells, siItk-treated Jurkat cells 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 more closely resembles Ag stimulation (Fig. 5B; Ref.8).
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Membrane-targeted Vav rescues actin polarization
Although the defect in Vav localization could lead to an actin defect in siItk-treated cells, it is also possible that the defective Vav localization was actually secondary to the actin defect. To determine whether the altered Vav localization directly contributes to the actin defect in siItk-treated cells, we expressed a membrane-targeted construct of Vav, Vav-CAAX (36). Expression of Vav-CAAX restored actin polarization to the site of TCR stimulation in siItk-treated cells (Fig. 7), suggesting that the actin defect in Itk-deficient cells results at least in part from improper Vav localization.
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To additionally investigate the mechanism by which Itk affects Vav, we examined Vav phosphorylation and protein interactions in siItk-treated cells. Unlike Vav localization, which appeared to be dependent on the presence of Itk, patterns of Vav tyrosine phosphorylation were not grossly affected by the loss of Itk (Fig. 8A), consistent with what we have observed in Itk and Rlk/Itk-deficient thymocytes. Thus, tyrosine phosphorylation of Vav appears to occur before or independent of stable recruitment of Vav to the site of TCR contact. To better understand how Itk can affect Vav localization, we performed coimmunoprecipitation experiments to examine whether Itk directly interacts with Vav using cells coexpressing 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 constitutive and did not depend on TCR stimulation. The same was also observed for endogenous Itk and Vav (data not shown). Furthermore, coimmunoprecipitation of Itk with Vav was not affected by mutation of the SH2 domain of Itk (Fig. 8B). Thus, the Vav-Itk interaction appears to be independent of Itk SH2 function.
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Discussion |
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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 Itk–/– cells. Our demonstration in this study that a membrane-targeted version of Vav can rescue actin polarization in siItk-treated cells directly implicates Vav in the altered actin regulation in these cells.
The mechanism(s) by which Itk affects Vav localization remains unclear. Itk has been shown to phosphorylate tyrosine 171 of linker for activation of T cells (LAT), which can bind Vav (38). However, our results argue that proper Vav localization does not require Itk kinase activity. Indeed, the role of Itk kinase activity in regulation of the actin cytoskeleton has been somewhat controversial. Work from Donnadieu et al. (23) originally argued that Itk did not participate in actin cytoskeleton regulation because kinase-inactive Itk failed to block T cell polarization. Interpretations of this work have been challenged by two recent studies demonstrating an actin polarization defect in Itk–/– cells, despite the inability of Itk-KD to inhibit this process (18, 19). Nonetheless, it should be noted that data from Shimizu and colleagues (20) argue that a kinase-inactive Itk mutant can block TCR-induced actin polymerization. Such discrepancies may be the result of differences in assay systems, expression levels, or the use of different mutants of Itk. By demonstrating that a mutant affecting the kinase activity of Itk can indeed rescue the actin defect in siItk-treated Jurkat or human PBT cells, our work provides strong evidence for a kinase-independent function of Itk.
Our findings suggest that Itk has an adaptor function that requires its PH and SH2 domains, but not kinase activity, for the regulation of Vav recruitment and actin reorganization. Therefore, it is relevant that both Itk and Vav bind via their SH2 domains to the adaptor SLP-76 as part of the LAT-SLP-76 mediated complex that helps orchestrate downstream readouts in TCR signaling (39, 40, 41, 42). Moreover, depletion of Itk appears to reduce the amount of SLP-76 found in Vav immunoprecipitates. Because the SH2 domain of Itk is required for actin polarization, it is possible that an interaction between the Itk-SH2 domain and SLP-76 (or LAT) may be required to stabilize the interactions of Vav with other proteins in this signaling complex. Indeed, we observed that Vav and Itk can coimmunoprecipitate and that this does not require that function of the Itk SH2 domain, suggesting that the SH2 domain of Itk is interacting with another protein. Direct binding of Tec kinases and Vav have also been previously reported both in vitro and by coimmunoprecipitation (39, 43). However, it is also possible that a separate function of Itk may also contribute to Vav localization. Of interest are findings that Btk can act in a kinase-independent fashion to increase phosphatidylinositol 3,4,5-phosphate levels (44). Because Vav contains a PH domain, it is possible that Itk contributes to Vav localization through this type of secondary influence.
Nonetheless, the cross-regulation of and interactions between Itk and Vav are complex. Tybulewicz and colleagues (45) have also demonstrated impaired Itk activation in Vav-deficient cells. Although this observation may be secondary to defective PI3K activation in Vav–/– cells, rather than a direct effect on Itk, these data underscore the complex regulatory interactions among components of the TCR signaling complex and argue that linear relationships cannot necessarily be used to describe the biochemical pathways regulating T cell activation. Therefore, it is of interest that Tybulewicz and colleagues (45) also noted disruption of the interactions of SLP-76 with PLC- in Vav1-deficient cells, suggesting there may be defects in the integrity of the LAT-SLP-76-mediated complex in the absence of either Vav1 or Itk. It should also be noted that our data does not rule out the possibility that Vav1 associates with the LAT-SLP-76 complex in Itk-deficient cells, but does suggest that Vav1 phosphorylation can occur before or independent of stable SLP-76 association.
The Tec kinases have been previously implicated in the regulation of PLC- and Ca2+ mobilization (9). Nonetheless, we see effects on actin under conditions where we see minimal changes in Ca2+ and NFAT activation (data not shown). It is possible that PLC- activation may be compensated for by expression of other Tec kinases, as in mice, where T cells lacking two Tec kinases, Rlk and Itk, have more impaired Ca2+ mobilization than Itk–/– cells (13). Alternatively, it is possible that the defects in prolonged Ca2+ mobilization and immune responses in Itk–/– mice may actually result in part from the altered Vav localization and cytoskeletal organization, which may prevent formation of a stable TCR signaling complex. We have recently found that T cells expressing mutant versions of Tec kinases also show decreased cell polarization and activation of Cdc42 and Rac in response to chemokines (46). It will be of interest to see whether Vav plays a role in these processes and whether Btk-deficient B cells also exhibit similar actin cytoskeleton defects.
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Acknowledgments |
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Footnotes |
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1 This work was supported by Howard Hughes Medical Institute-National Institutes of Health Research Scholar’s Program (to D.D.). 
2 D.D. and R.A.H. contributed equally to this work.
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 Institutes of Health, Bethesda, MD 20892.
5 Address correspondence and reprint requests to Dr. Pamela L. Schwartzberg, National Human Genome Research Institute, National Institutes of Health, 4A38, 49 Convent Drive, Bethesda, MD 20892. E-mail address: pams{at}nhgri.nih.gov
6 Abbreviations used in this paper: PLC-, phospholipase C-; PH, pleckstrin homology; PBT, peripheral blood T lymphocyte; si, small interfering; WT, wild type; JTAg, Jurkat-TAg; CAT, chloramphenicol acetyltransferase; EGFP, enhanced GFP; RNAi, RNA interference; LAT, linker for activation of T cells; SH2, Src homology 2; Itk-KD, Itk-kinase defective.
Received for publication November 12, 2003. Accepted for publication November 10, 2004.
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