Kinase-Independent Functions for Itk in
TCR-Induced Regulation of Vav and the
Actin Cytoskeleton
This information is current as
of June 15, 2017.
Derek Dombroski, Richard A. Houghtling, Christine M.
Labno, Patricia Precht, Aya Takesono, Natasha J. Caplen,
Daniel D. Billadeau, Ronald L. Wange, Janis K. Burkhardt
and Pamela L. Schwartzberg
References
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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.
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J Immunol 2005; 174:1385-1392; ;
doi: 10.4049/jimmunol.174.3.1385
http://www.jimmunol.org/content/174/3/1385
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*
U
pon stimulation with Ag, T lymphocytes undergo a dramatic cellular reorganization leading to cell polarization,
which is accompanied by the accumulation of F-actin at
the site of contact with the APC (1). This regulation of the actin
cytoskeleton is required for full T cell activation. Accordingly,
mice deficient in the guanine nucleotide exchange factor Vav or
the Wiskott-Aldrich syndrome protein, two critical regulators of
the actin cytoskeleton, exhibit defective T cell responses and development (2–5). However, actin reorganization is not only required for proper T cell signaling, it also is initiated by and requires signals emanating from the TCR. A number of studies have
helped delineate the pathways between the TCR and the actin cytoskeleton and have revealed essential roles for proximal tyrosine
kinases and adaptor molecules in addition to Vav and WiskottAldrich syndrome protein (6 – 8). Recent data suggest that the Tec
kinase Itk is an important contributor to these pathways.
*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
Received for publication November 12, 2003. Accepted for publication November
10, 2004.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
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.
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 –
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).
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: [email protected]
Copyright © 2005 by The American Association of Immunologists, Inc.
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.
6
0022-1767/05/$02.00
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The Tec family kinase Itk is an important regulator of Ca2ⴙ 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 mutations 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.
1386
Electroporation
JTAg cells (107 cells/0.5 ml in RPMI 1640 plus 20% FBS and 10 mM
HEPES) were electroporated with siRNA (1–2 ␮g) and/or plasmids (3–5
␮g) 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 ␮g of Flagtagged Vav and/or 3–10 ␮g 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 ␮g/5 ⫻ 106 cells) and/or murine Myctagged Itk (3–10 ␮g) 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-␮m latex
beads (107 beads/ml (Interfacial Dynamics)) were coated with IgM, antiTCR C305 (1/130 dilution in PBS), or anti-CD3 OKT3 (for human PBT)
as described previously (13). For T cell-bead conjugations, 1.25 ⫻ 106
cells in serum-free media were mixed with 2 ⫻ 106 Ab-coated beads in 70
␮l, spun for 1 min at 100 ⫻ 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 ⫻40 and ⫻100 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
Materials and Methods
Cells and reagents
Jurkat-TAg (JTAg) cells were grown in RPMI 1640 containing 10% FBS
(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
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, 1⫻ nonessential amino acids, 10 ␮g/ml ciprofloxacin, and 20 U/ml human IL-2
(Roche)) containing 2 ␮g/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⬘-CAUCGAUGUGUGU
GAGAACUGC-3⬘; Lck-1 antisense, 5⬘-AGUUCUCACACACAUCGAU
GUU-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).
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-Llysine-coated (Sigma-Aldrich) slides for 1–5 min, fixed, and stained.
Immunoblotting
For verification of Itk levels, cells (4 ⫻ 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 ␮l) 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 SDSPAGE, transferred to nitrocellulose, immunoblotted with primary and HRPconjugated secondary Ab as per the manufacturer, and visualized by ECL
(Amersham Biosciences). In some experiments, FLAG peptide (SigmaAldrich) was included in the immunoprecipitation as a specificity control.
Results
Establishment of siRNA to reduce Itk expression in Jurkat cells
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 12fold; 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
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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 Itkdeficient 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.
Itk REGULATES Vav LOCALIZATION
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 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
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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 GFP⫹ 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,
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 ⬃1–2 ␮g of a 22-bp
siRNA electroporated per 107 cells (Fig. 1B). At doses of ⱖ5 ␮g
of siRNA, nonspecific effects were observed with decreased expression of multiple proteins, whereas at doses ⬍0.6 ␮g, less efficient 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 maximal suppression of Itk occurred in the first 2 days posttransfection
with normal protein levels restored by day 4 (Fig. 1E). Thus, for
functional studies of Itk, we used cells to which siItk had been
introduced 24 –36 h earlier.
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
FIGURE 2. Reduction of Itk expression impairs actin polarization. A,
JTAg cells were electroporated with siItk2 or siGFP. Twenty-four hours
later, cells were stimulated with anti-TCR-coated beads, fixed, and stained
with rhodamine-phalloidin. Representative polarized (positive) and nonpolarized (negative) cells are shown. B, JTAg cells were scored for actin
polarization in response to IgM- or C305-coated beads. C, WT murine Itk
rescues the actin polarization defect. JTAg cells were transfected with siItk
plus plasmid encoding murine WT Itk and scored for actin polarization to
C305-coated beads. Corresponding levels of endogenous Itk, murine myctagged Itk, and tubulin are shown below each lane.
1388
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).
Requirements for Itk function in actin regulation
The ability of murine Itk to rescue the siItk effects on actin organization allowed us to address the requirements for Itk function.
Itk REGULATES Vav LOCALIZATION
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 antiCD3 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
FIGURE 3. Requirements for Itk function in TCR-mediated actin reorganization. A, Domain structure of Itk. Mutations affecting the PH (R29C),
SH2 (R265A), and kinase domains (Itk-KD, K390R) are indicated. B, PH
mutant fails to rescue actin polarization. C, Itk-KD rescues actin polarization defect. D, SH2 mutant fails to rescue and blocks actin polarization.
Levels of endogenous Itk, murine myc-tagged Itk, and tubulin are shown
below each lane.
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 TCRinduced Vav phosphorylation (19, 21). To determine whether Vav
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The Tec kinases share a common structure (Fig. 3A) with a tyrosine kinase catalytic domain preceded by Src homology proteininteraction 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 siItktreated 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 addressed 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
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
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.
The Journal of Immunology
1389
FIGURE 4. Reduction of Itk expression impairs actin polarization in
human PBT cells. A, Human PBT cells were treated with siItk2 or no
siRNA and, 24 h later, stimulated with OKT3-coated beads and fixed and
stained with rhodamine-phalloidin. Representative polarized and nonpolarized cells are shown. B, Human PBT cells were stimulated with either
control (IgM) or OKT3-coated beads, and actin polarization was scored. C,
Human PBT cells were transfected with siItk in the presence or absence of
plasmids encoding WT and mutant murine Itk, stimulated with OKT3coated beads and scored for actin polarization. Si0 cells received no
siRNA. Data represent the average ⫾ SD of results from two to three
donors. D, Itk-SH2 mutant blocks actin polarization in human PBT cells.
Data represent the average ⫾ SD of results from two donors.
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 siItktreated 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).
We then examined how re-expression of WT or mutant Itk affected
Vav localization in cells transfected with siItk. Expression of either
WT or KD murine Itk rescued both actin polarization and Vav localization (Fig. 6, A and B). In contrast, cells expressing the SH2 mutant
of Itk showed abnormal Vav localization, which paralleled their
defect in actin polarization, and the SH2 mutant failed to rescue Vav
localization in siItk-treated cells (Fig. 6C). Thus, the ability to polarize
actin correlated with proper Vav localization, and both regulation of
Vav localization and actin polarization appeared to be independent of
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
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
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 membranetargeted construct of Vav, Vav-CAAX (36). Expression of VavCAAX restored actin polarization to the site of TCR stimulation
in siItk-treated cells (Fig. 7), suggesting that the actin defect in
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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 antiTCR-coated beads (A; ⴱ, marks beads) or Staph enterotoxin E-pulsed
Nalm-6 cells (B; labeled with 4⬘,6⬘-diamidino-2-phenylindole and an arrowhead) and stained with rhodamine-phalloidin and anti-FLAG. T cells in
B are marked with a star.
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.
Itk expression affects protein interactions with Vav
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). Further-
FIGURE 7. Vav-CAAX rescues actin polarization. Cells were electroporated with siItk and, 24 h later, transfected with FLAG-Vav-CAAX and
evaluated for actin polarization. Levels of endogenous Itk, murine myctagged Itk, Vav, and tubulin are shown below each lane.
more, 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.
The constitutive association of Itk with Vav suggested that the
presence of Itk may influence the ability of Vav to interact with
other proteins in TCR-mediated signaling complexes. Therefore, we
examined proteins that coimmunoprecipitated with Vav in the presence or absence of Itk. Immunoprecipitation of Vav with an antiFLAG monoclonal revealed the presence of a 76-kDa tyrosine phosphorylated protein that comigrated with the adaptor molecule SLP-76,
as determined by immunoblotting with anti-SLP-76 and Itk Abs (Fig.
8C and data not shown). However, depletion of Itk, via siRNA,
reduced the amount of this protein that was observed in the Vav immunoprecipitations. Thus, depletion of Itk appears to impair the ability of Vav to interact with other proteins in the signaling complex.
Discussion
Regulation of the actin cytoskeleton is critical for T cell signal
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 WiskottAldrich 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
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Itk-deficient cells results at least in part from improper Vav
localization.
The Journal of Immunology
1391
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 kinaseindependent 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 –
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-76mediated 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
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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.
1392
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.
Acknowledgments
We thank S. Bunnell and L. Berg for the murine Itk constructs, B. Hill and
members of the Schwartzberg and Burkhardt labs for helpful discussions
and reagents, and M. Cichanowski for assistance with graphics.
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References
Itk REGULATES Vav LOCALIZATION