Nuclear localization of the tyrosine kinase Itk and interaction of its SH3 domain with karyopherin
(Rch1
)
Juan J. Perez-Villar,
Kathleen O'Day,
Derek H. Hewgill,
Steven G. Nadler and
Steven B. Kanner
Immunology, Inflammation, Pulmonology and Dermatology Discovery, Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, NJ 08543, USA
Correspondence to:
Correspondence to J. J. Perez-Villar
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Abstract
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We report a physical and functional association between the Tec-family tyrosine kinase Itk (Emt/Tsk) and the nuclear import chaperone karyopherin
(Rch1
) in human T cells. The Itk-SH3 domain and the Rch1
proline-rich (PR) motif were crucial for the Itk/Rch1
constitutive interaction as demonstrated by directed mutagenesis of the Rch1
PR motif (proline 242 to alanine, P242A). TCRCD3 stimulation of Jurkat T cells resulted in increased Itk/Rch1
complex formation, recruitment of karyopherin ß to the protein complex and Rch1
tyrosine phosphorylation. Analysis of in vitro kinase reactions with a panel of recombinant glutathione-S-transferase (GST) fusion tyrosine kinases (Itk, Lck, ZAP-70 and Jak3) revealed that only GSTItk efficiently phosphorylated a recombinant GSTRch1
fusion. We observed constitutive nuclear localization of Itk that was up-regulated following either TCRCD3 stimulation or over-expression of wild-type Rch1
in T cells. Further, nuclear localization of Itk and TCRCD3-mediated IL-2 production were significantly down-regulated following expression of the Rch1
-P242A mutant, implicating a role for Rch1
in the nuclear translocation of Itk.
Keywords: Itk tyrosine kinase, karyopherin
/Rch1
, , TCR-CD3, T lymphocytes, nuclear import
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Introduction
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Itk (Emt, Tsk) is a member of the Tec-family of protein tyrosine kinases (PTK) expressed in T, NK and mast cells (1,2). Structurally, Itk comprises a pleckstrin-homology (PH) domain, a proline-rich (PR) sequence, a Src homology 3 (SH3) domain, a Src homology 2 (SH2) domain and a tyrosine kinase (TK) domain (1,2). Tec-family members lack an N-terminal myristylation site and the negative autoregulatory phosphorylation site present in members of Src-family PTK (3). Evidence suggests that the intramolecular association between the SH3 and PR domains regulates Tec-family kinase activity (4).
Several Itk associating proteins have been described, although the functional consequences of these interactions have not been elucidated. The PH domain interacts with different protein kinase C isoforms (5), inositol phospholipids (6) and activated TCRCD3 complexes (7). The PR motif associates with SH3 domains of Src-family PTK and Grb2 (3,4). The SH3 domain interacts with the WiscottAldrich syndrome protein, hnRNP-K, Fyn and c-Cbl (3,4). In addition, the SH2 domain associates with phosphatidylinositol 3-kinase (PI3-K) and phospholipase C-
1 (PLC-
1) in a tyrosine phosphorylation-dependent manner upon CD28- or TCRCD3-mediated stimulation respectively (8,9).
A model has emerged describing a protein complex involved in the active transport of cargo proteins from the cellular cytoplasm to the nucleus (10). The first transport receptor identified, importin ß/karyopherin ß, is essential for the nuclear import of many proteins (10,11). Karyopherin ß does not usually bind its cargo directly, but first bridges to importin
/karyopherin
(10,11). Three groups of karyopherin
molecules have been identified: the yeast protein SRP1 (12), and the human proteins hSRP1 and NPI-1 (13,14); importin 60 and the human proteins hSRP1
and Rch1
(15,16); and Qip-1 (10). Rch1
is required for the nuclear import of basic-type (classical) nuclear localization sequences (NLS), which comprise one or two short stretches of basic amino acids (i.e. lysine) (10), although non-classical NLS have been defined which lack basic residues (1721). The Rch1
/NLS-containing protein complex interacts in the cytosol with karyopherin
and hsp70, which together docks to the nuclear pore. The active transport of the NLS-containing cargo protein through the nuclear pore also involves the GTPase Ran and its interacting protein p10/nuclear transfer factor-2 (10,22).
Nuclear translocation of the Tec-family member Btk was described previously (23). Btk is localized in the cytoplasm under steady-state conditions, yet upon cell activation it translocates to the plasma membrane via its PH domain and to the nucleus through an NLS-independent transport mechanism (23). In contrast, the Tec-family kinase Txk (Rlk) is transported to the nucleus through an NLS-specific translocation mechanism (24). Here we report the association between Itk and the nuclear importin Rch1
/karyopherin
in human T cells through the Itk-SH3 domain and the PR motif in Rch1
. We demonstrate a regulated nucleocytoplasmic transport of Itk, suggesting a novel role in T cell activation.
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Methods
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Cell lines and cell culture
The wild-type (wt) Jurkat T cell line was cultured as described (9). Jurkat T cells stably or transiently transfected with Rch1
-wt or mutated Rch1
-P242A cDNAs were cultured in medium supplemented with 1.5 mg/ml of G418 (Gibco/BRL, Gaithersburg, MD). Phytohemagglutinin (PHA) T lymphoblasts were generated as described previously (9).
Yeast two-hybrid screen
The protocol for the yeast two-hybrid screen has been described previously (25). The Itk-SH3 region defined by the amino acids at position 177230 was PCR amplified from a Jurkat cDNA library (37) using Expand polymerase (Boehringer Mannheim, Indianapolis, IN). Itk-SH3 `bait' expressing EGY48 cells were transformed with 30 µg of Jurkat cDNA library and screened for positive clones as described (26). Positive colonies were identified by blue color indication on plates containing 0.08% X-gal. Plasmids from positive colonies were transformed into AMA-1004 bacterial cells plated onto media lacking tryptophan to score for LexA-AD+ plasmids. DNA sequence analysis was performed using a forward primer for the LexA-AD region and sequences were analyzed by the Genetics Computer Group program (Madison, WI).
Antibodies and glutathione-S-transferase (GST) fusion proteins
Anti-CD3 mAb G19-4 and anti-mouse CD40 mAb 404.8E1 were described previously (27). Secondary antibodies [horseradish peroxidase (HRP)-conjugated anti-rabbit IgG and HRP-conjugated anti-mouse IgG] were purchased from Biosource International (Camarillo, CA)]. Rabbit anti-Itk, anti-Itk mAb, anti-PLC-
1 mAb and anti-Oct-1 were purchased from Upstate Biotechnology (Lake Placid, NY). Anti-Rch1
and anti-hSRP1 rabbit sera were described (28), and anti-Rch1
mAb and goat anti-karyopherin ß serum were purchased from Transduction Laboratories (Lexington, KY). Anti-GST mAb were generated using recombinant GST protein as the immunogen. The GSTItk fusion proteins were produced as reported (9). GSTRch1
, 6HisRch1
and GSTCD28 fusion proteins were described previously (28,29). Full-length Rch1
cDNA was cloned by PCR from the Jurkat cDNA library and mutation of the proline at position 242 was introduced by overlapping extension PCR using the pcDNA3-Rch1
-wt plasmid DNA as template.
Cell stimulation, immunoprecipitation and Western immunoblotting
Wild-type, stably or transiently transfected Jurkat T cells expressing the wild-type or P242A mutated Rch1
proteins, or PHA-expanded T lymphoblasts were stimulated and lysed as described (9). Immunoprecipitation and GST pull-down experiments were performed as reported (9). Cytosolic and nuclear extracts from Jurkat T cells were prepared by resuspending PBS washed cells 3 times in buffer `A' (20 mM HEPES, pH 7.2, 20mM NaCl, 2.5 mM MgCl2 and 0.1% Nonidet P-40), for 10 min (4°C). Nuclei were pelleted at 2000 g for 4 min (4°C), and cytosolic extracts removed and diluted 1:1 with PBS. Nuclear proteins were extracted with buffer `A' supplemented with 0.42 M NaCl. Samples were centrifuged at 14,000 r.p.m. for 2 min, lysates were precleared twice with 100 µl Protein GSepharose beads (Pharmacia, Uppsala, Sweden) for 60 min (4°C) and used for immunoprecipitation. Immunoprecipitates were analyzed by Western immunoblotting as described and blot stripping was carried out as reported (9).
ELISA kinase assays
Substrate proteins (polyGluTyr, GST, GSTRch1
, GSTLAT and GSTCD28) at 4 µg/ml were loaded onto 96-well ELISA plates in 50 mM NaHCO3, pH 9, overnight (4°C). Wells were blocked for 1 h with 5% non-fat milk in PBS + 0.05% Tween 20 (PBS-T). Wells were washed 3 times with PBS-T, once with PBS and once with 25 mM HEPES, pH 7. Kinase reactions were performed by adding the indicated amount of purified enzyme in kinase buffer (25 mM HEPES, pH 7, 0.1 mg/ml BSA, 100 µM ATP, 5 mM MgCl2, 1 mM DTT and 1 mM Na3VO4) for 45 min. After washing, HRP-conjugated anti-phosphotyrosine antibodies PY99 (Transduction Laboratories) and 5H1 (30) diluted 1:1000 in PBS-T + 1% BSA were added to the wells for 1h, followed by extensive washing. Reactions were developed using a Kirkegaard & Perry (Gaithersburg, MD) TMB ELISA kit (100 µl/well of 50:50 TMB substrate and H2O2 solutions). Color reactions were stopped with 1N H2SO4 and the absorbency at 450650 nm was measured.
IL-2 assay
To induce IL-2 production, transiently transfected Jurkat T cells were stimulated with solid-bound anti-CD3 mAb (2 µg/ml) or PHA (10 µg/ml) for 18 h at 37°C. IL-2 levels in culture supernatants were measured using an ELISA kit (PharMingen, San Diego, CA).
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Results
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Itk associates with Rch1
in human T lymphocytes
To further understand the role of Itk, we sought to identify Itk interacting proteins using the yeast two-hybrid technology. The region of Itk defined as the SH3 domain was fused to the LexA binding domain to generate a `bait' construct which was co-transformed into reporter yeast cells with a Jurkat T cell cDNA library fused to the LexA activation domain. Two of the 115 ß-galactosidase-positive clones encoded an identical portion of the gene product Rch1
(amino acids 1253) (Fig. 1A
). The isolated sequence contained the motif KNPAPP, a putative SH3-binding PR motif (31).
To confirm and characterize the Itk/Rch1
association in vivo, Jurkat T cells or normal PHA-expanded T lymphoblasts were lysed and Itk and Rch1
were immunoprecipitated with specific antibodies (Fig. 1B
). Constitutive association between Itk and Rch1
was detected in both T cell sources using either combination of specific antibodies. Upon examining a possible association between Itk and karyopherin
1 (hSRP1), another member of the karyopherin
family (10), we did not detect association between Itk and karyopherin
1 in Jurkat T cells (Fig. 1C
).
We investigated the effect of TCRCD3 stimulation in regulating the Itk/Rch1
association. Jurkat T cells were stimulated with an anti-TCRCD3 mAb and immunoprecipitates of Itk from varying time points were immunoblotted with anti-Rch1
mAb. As indicated in Fig. 2
(upper panel), there was a quantitative enhancement of protein complex formation between Itk and Rch1
that peaked at 5 min of TCRCD3 stimulation. Densitometric analysis showed that under steady-state conditions ~4% of total cellular Rch1
associated with Itk; however, upon TCRCD3 activation the pool of Rch1
in complex with Itk increased to 14%. We investigated whether Itk/Rch1
associated with karyopherin ß, another protein involved in nuclear transport (19,21). The Itk immunoprecipitates were reprobed with anti-karyopherin ß. Karyopherin ß was detected in the complex (Fig. 2
, middle panel), associating after the Rch1
/Itk interaction suggesting a sequential binding mechanism. This association was up-regulated upon TCRCD3 stimulation. The level of Itk immunoprecipitated during the time course was unchanged (Fig. 2
, bottom panel).
We analyzed the contribution of individual Itk domains to the Itk/Rch1
complex. Three GSTItk fusion proteins including single domains or domain combinations (9) were used in GST pull-down assays in Jurkat T cells. As shown in Fig. 3
(A, upper panel), the GSTPTR32 (Itk lacking the kinase domain) and GSTSH3, but not the GSTSH2 fusion protein, isolated Rch1
from lysates of unstimulated T cells. The constitutive association was moderately augmented upon TCRCD3 engagement when using the GSTPTR32 fusion protein. The GSTSH2 fusion isolated Rch1
from T cells only following TCRCD3 stimulation. As a loading control, the immunoblot was stripped and reprobed with anti-GST (Fig. 3A
, bottom panel).
Proline-to-alanine point mutation within the Rch1
PR motif disrupts Itk/Rch1
association
We generated myc-tagged fusions of wild-type Rch1
(Rch1
-wt) and a proline-to-alanine point mutant form (proline 242 to alanine, Rch1
-P242A), and studied their ability to associate with endogenous Itk. Rch1
-wt or Rch1
-P242A myc-tagged proteins were immunoprecipitated from stably transfected Rch1
-wt or Rch1
-P242A Jurkat T cells with an anti-myc mAb. Immunoblotting with anti-Itk showed that Rch1
-P242A did not associate with Itk (Fig. 3B
, upper panel), while the Rch1
-wt protein interacted with Itk. For protein loading control, the immunoprecipitates were immunoblotted with anti-Rch1
(Fig. 3B
, bottom panel). In both Rch1
-wt- and Rch1
-P242A-expressing cells, endogenous Rch1
co-immunoprecipitated endogenous Itk (data not shown).
Tyrosine phosphorylation of Rch1
We evaluated whether the Itk/Rch1
association resulted in tyrosine phosphorylation of Rch1
. Immunoprecipitates of Rch1
from either unstimulated or TCRCD3-activated Jurkat T cells were immunoblotted with anti-phosphotyrosine. Tyrosine phosphorylation of Rch1
peaked at 5 min after TCRCD3 stimulation (Fig. 4A
, upper panel) and was undetectable after 30 min of T cell stimulation (data not shown). Additional tyrosine phosphorylated proteins were associated with Rch1
(115, 80 and 72 kDa). Reprobing of the immunoblot with anti-Itk revealed its presence in the complex following TCRCD3 stimulation (Fig. 4A
, middle panel), while no change in Rch1
levels were observed (Fig. 4A
, bottom panel).
We evaluated in vitro tyrosine phosphorylation of Rch1
with purified recombinant GSTRch1
and GSTItk-TK (a fusion including only the Itk tyrosine kinase domain, residues 341621). Recombinant GSTRch1
was efficiently tyrosine phosphorylated by GSTItk-TK (Fig. 4B
). The cytoplasmic tail of CD28 (GSTCD28) was used as a positive control for Itk-mediated tyrosine phosphorylation (32). We investigated whether kinases from different PTK families including Syk (ZAP-70), Src (Lck) and Janus (Jak3) were effective in tyrosine phosphorylation of Rch1
in vitro. None of these tyrosine kinases mediated detectable Rch1
tyrosine phosphorylation (Fig. 4C
), yet they actively phosphorylated control GSTLAT protein (Fig. 4D
). Since Rch1
becomes tyrosine phosphorylated following T cell stimulation, we addressed whether the Itk-SH2 domain was involved in the enhanced Itk/Rch1
association upon TCRCD3 stimulation. Purified recombinant 6HisRch1
was phosphorylated in vitro by GSTItk-TK or left unphosphorylated and equal amounts of protein were incubated with GST, GSTItk-SH3 or GSTItk-SH2. Isolation of GST-containing fusion proteins was followed by immunoblotting with anti-Rch1
. Specific binding of the GSTItk-SH3 domain to 6HisRch1
was observed (Fig. 4E
), while no interaction between GSTItk-SH2 and Rch1
was detected even following in vitro Itk tyrosine phosphorylation. For GST loading control, the immunoblot was stripped and reprobed with anti-GST (Fig. 4E
).
Nuclear localization of Itk
Given the function of the karyopherin complex in the nucleocytoplasmic transport of protein cargo, we investigated the intracellular localization of Itk. A cell fractionation procedure was used to yield highly purified cytosol/membrane and nuclear extracts from Jurkat T cells (28). As shown by immunoprecipitation and immunoblotting of Itk from cytosol/membrane or nuclear lysate preparations from three different T cell clones (I, II and III), Itk was observed in both the cytosol/membrane fraction and in the nucleus (Fig. 5A
, upper panels). Densitometric analysis showed 15% of total cellular Itk in the nuclear fraction using this technique. PLC-
1 was detected in the cytosol/membrane extracts, but was undetectable in the nuclear preparations (Fig. 5A
, bottom panels), confirming undetectable contamination of nuclear extracts. We performed direct immunoblotting of the cell fractions with anti-Itk, confirming that the levels of Itk in the different fractions correlated with that using immunoprecipitation techniques (Fig. 5B
).
We also analyzed the subcellular localization of Itk in Jurkat T cells by confocal microscopy. Cells were co-stained with propidium iodide to label DNA and anti-Itk-specific mAb plus anti-mouseFITC. As a control, the antigen CD5 was analyzed for cell surface staining as a comparison. In proliferating Jurkat T cells, Itk was found in both the nucleus and the cytoplasm; however, staining for Itk was more intense in the nuclear compartment in all experimental conditions and may have reflected non-specific binding (data not shown).
To analyze the function of the Itk/Rch1
association, we tested whether transient transfection of Rch1
-wt regulated import of Itk into the nucleus. Jurkat T cells were transiently transfected with either an empty expression vector or vector containing the Rch1
-wt construct. Transfectants were unstimulated or were stimulated with anti-TCRCD3, and Itk was immunoprecipitated from cytosol/membrane and nuclear extracts. An enhancement of nuclear Itk was observed after transient transfection of Rch1
-wt (relative to mock transfected) in both unstimulated and TCRCD3-stimulated cells (Fig. 5C
). PLC-
1 was observed only in the cytosolic fraction and was unaffected by cell transfection with the Rch1
constructs. Rch1
-wt myc-tagged proteins immunoprecipitated with anti-myc mAb from aliquots of the same cytosolic and nuclear extracts confirmed equivalent amounts of Rch1
-wt proteins in the transfectants (data not shown).
We investigated TCRCD3-induced regulation of endogenous Itk nuclear translocation. Jurkat T cells were stimulated with anti-TCRCD3 and immunoprecipitates of Itk were immunoblotted with anti-Itk. An enhancement of Itk translocation from the cytoplasm to the nucleus was observed upon TCRCD3 stimulation (Fig. 5D
). For loading control, whole-cell lysates (nuclear and cytosolic) corresponding to each condition were immunoblotted for PLC-
1 (cytosolic extracts) or the nuclear transcription factor Oct-1 (nuclear extracts) (Fig. 5D
).
To compare the activation status of cytosolic versus nuclear Itk, immunoprecipitates of Itk from each fraction were analyzed for phosphotyrosine content. An increase in tyrosine phosphorylation of both cytosolic and nuclear Itk was observed after TCRCD3 stimulation, with similar kinetics (Fig. 5E
). The relative tyrosine phosphorylation levels of cytoplasmic and nuclear Itk 3 min after TCRCD3 stimulation were equivalent when normalized to the total amount of Itk protein in each fraction.
An analysis of normal human T cells was carried out to confirm the observation that Itk was observed in the nuclear compartment. Fresh peripheral blood-derived T cells were expanded in PHA for 5 days and then subjected to TCRCD3 stimulation over the course of 30 min prior to subcellular fractionation as performed above. We observed nuclear localization of Itk in normal T cells (Fig. 6
), with an increase following TCRCD3 stimulation as observed in Jurkat T cells. Further, there was no translocation of PLC-
1 into the nucleus, no translocation of Oct-1 into the cytoplasm and no cross-contamination of the cell fractions (Fig. 6
).
To further analyze the function of the Itk/Rch1
association, we tested whether the transiently transfected Rch1
-P242A mutant regulated import of Itk into the nucleus. Wild-type Jurkat T cells were transiently transfected with equal amounts of the empty expression vector or ones containing Rch1
-wt or Rch1
-P242A DNA constructs. After 48 h in culture, the cells were left unstimulated or were stimulated through TCRCD3, and Itk was immunoprecipitated from cytosolic/membrane and nuclear extracts. As indicated in Fig. 7A
(upper panels), no nuclear Itk was detected upon Rch1
-P242A transient transfection in either resting or TCRCD3-activated cells, in contrast to mock or Rch1
-wt-transfected cells. Densitometric analysis showed that 5% of total cellular Itk was nuclear (15% upon TCRCD3 activation) and that this amount was augmented upon transient expression of Rch1
-wt (12 and 23% respectively) or inhibited (<1% present) following Rch1
-P242A transient expression. As a control for cytosolic contamination in the nuclear extracts, PLC-
1 was immunoprecipitated from cytosolic and nuclear extracts from a fraction of the same transiently transfected Jurkat cells. Clearly, PLC-
1 was only observed in the cytosolic fraction and was unaffected by transfection of the cells with the Rch1
constructs. In addition, Rch1
-wt and Rch1
-P242A myc-tagged proteins were immunoprecipitated with anti-myc mAb from aliquots of the same cytosolic and nuclear extracts, showing equivalent amounts of the Rch1
-wt and Rch1
-P242A proteins in the transient transfections (Fig. 7B
).
We also studied the effect of Rch1
-P242A over-expression on the nuclear import of other unrelated proteins also crucial for cytokine production (i.e. NF-
B and NFATc1). Overexpression of Rch1
-P242A did not interfere with the nuclear translocation of NF-
B or NFATc1 transcription factors upon cellular activation (data not shown). Taken together, the above results demonstrate that Rch1
is involved in the nuclear translocation of Itk.
Expression of Rch1
-P242A down-regulates TCRCD3-mediated IL-2 production by Jurkat T cells
We assessed whether blocking Itk nuclear import was able to impact the downstream outcome of TCRCD3-mediated intracellular signaling. For this purpose, the effect of Rch1
-wt or Rch1
-P242A expression on TCRCD3-mediated cytokine production was evaluated. Jurkat T cells were transiently transfected with equal amounts of the empty expression vector or ones containing Rch1
-wt or Rch1
-P242A (5 µg). After 24 h in culture, the transfected cells were left unstimulated or were activated with anti-TCRCD3 or PHA for 18 h and the levels of IL-2 production were measured. The experiments showed that expression of the mutant Rch1
-P242A, but not Rch1
-wt, markedly down-regulated (5070%) IL-2 production in response to TCRCD3- or PHA-mediated activation (Fig. 7C
). This observation suggests that the TCRCD3-induced nuclear import of Itk accounts in part for the IL-2 production mediated by TCRCD3 activation.
 |
Discussion
|
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Upon T cell activation, Itk is tyrosine phosphorylated and transiently associates with phospholipids and different cell surface antigens (19). Although Itk remains largely in the cytosolic compartment of T cells, we provide evidence for the nuclear localization of Itk and propose a mechanism for this observation. We isolated a sequence corresponding to residues 1253 of the importin Rch1
including the motif KNP242APP, which matches the consensus SH3-binding PR motif (R/KXPXPP). Most SH3 domains bind preferentially to arginine-containing PR motifs; however, screening of phage display peptide libraries using the murine Itk-SH3 domain revealed a preference for lysine as the critical upstream basic residue (3), suggesting that the Rch1
PR motif is a binding sequence for the Itk SH3 domain.
Non-receptor PTK generally observed in the cytosol or plasma membrane are found in the nucleus: c-Abl, Fer, Wee1, ZAP-70, Btk, Src and Fgr (3336). Some tyrosine kinases include a classical NLS in their structure (c-Abl and Fgr), while no evident NLS has been found in ZAP-70, Src or Btk (23). Although the nuclear substrates for these PTK are largely unknown, the presence of tyrosine phosphorylated proteins in the nucleus of different cell types is well established and increases following cell activation.
Recent reports describe changes in subcellular localization of the Tec-family PTK Txk (Rlk) in response to TCRCD3 stimulation and its role in IFN-
production by T cells (24,37). The Txk gene encodes proteins of 58 and 52 kDa that have distinct properties and subcellular localization depending upon the presence of a cysteine-string motif essential for palmitoylation. The larger 58-kDa Txk is cytosolic, while the smaller isoform (52 kDa) lacks the cysteine-string motif and localizes mainly in the nucleus. Both proteins contain a cryptic bipartite NLS (residues 5771). Itk lacks both the cysteine-rich motif and an evident NLS. Thus, different members in the same PTK family may be utilizing distinct nuclear import mechanisms.
Enhancement of the Itk/Rch1
association following TCRCD3 engagement coincided with Rch1
tyrosine phosphorylation. Our in vitro analysis suggested that Itk may account for Rch1
tyrosine phosphorylation in vivo; however, the Itk-SH2 domain association with Rch1
after TCRCD3 stimulation is indirect, not directly due to Rch1
phosphorylation.
Of particular interest is the observation of inhibiting both Itk nuclear translocation and concomitant TCRCD3- or PHA-mediated IL-2 production by expression of the Rch1
-P242A mutant. Our finding that IL-2 production was not completely inhibited upon blocking Itk nuclear import suggests that there are likely pathways leading to IL-2 production independent of nuclear Itk. We would suggest that interference of Itk nuclear translocation may be due to competition between endogenous Rch1
and the Rch1
-P242A mutant for nuclear pore binding sites. Overall, the data suggest that the fraction of Itk that is translocated to the nuclear compartment has a role in downstream T cell activation events.
The increase in nuclear Itk phosphotyrosine content after TCRCD3 stimulation suggests nuclear enzymatic regulation. Itk may be phosphorylated or autophosphorylate in the cytosol and subsequently get transported into the nucleus or it may be activated in the nuclear compartment. A model for cytoplasmic Itk activation has been proposed following T cell stimulation where cytosolic PI3-K generate phospholipid-based PH domain docking sites in the plasma membrane for Itk, where Src-family PTK then activate Itk by a transphosphorylation mechanism. PI3-K activity also occurs in the nucleus leading to nuclear accumulation of 3-phosphorylated phosphoinositides (38,39). Nuclear phospholipids may recruit Itk to the nuclear membrane leading to its activation by nuclear-localized kinases (i.e. Src and Fgr). Understanding the role of Rch1
tyrosine phosphorylation and that of the nuclear localization of Itk will be paramount to understanding their roles in TCRCD3-directed effector functions. Disruption of the Itk/Rch1
complex by genetic and/or chemical approaches may have value in the treatment of immunological disorders.
 |
Acknowledgments
|
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We thank Dr Deryk Loo and Glen Mikesell for critical reading of the manuscript, and Gena Whitney for her valuable technical advice and helpful discussions. We also thank Dr Alejandro Aruffo and members of the IIPD Department at Bristol-Myers Squibb for their continuous support during this project.
 |
Abbreviations
|
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GST glutathione-S-transferase |
HRP horseradish peroxidase |
NLS nuclear localization sequence |
PH pleckstrin homology |
PI3-K phosphoinositide 3-kinases |
PHA phytohemagglutinin |
PR proline rich |
PTK protein tyrosine kinase |
SH2 Src homology 2 |
SH3 Src homology 3 |
TK tyrosine kinase |
wt wild-type |
 |
Notes
|
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Transmitting editor: L. H. Glimcher
Received 16 March 2001,
accepted 2 July 2001.
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