Nuclear localization of the tyrosine kinase Itk and interaction of its SH3 domain with karyopherin {alpha} (Rch1{alpha})

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


    Abstract
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
We report a physical and functional association between the Tec-family tyrosine kinase Itk (Emt/Tsk) and the nuclear import chaperone karyopherin {alpha} (Rch1{alpha}) in human T cells. The Itk-SH3 domain and the Rch1{alpha} proline-rich (PR) motif were crucial for the Itk/Rch1{alpha} constitutive interaction as demonstrated by directed mutagenesis of the Rch1{alpha} PR motif (proline 242 to alanine, P242A). TCR–CD3 stimulation of Jurkat T cells resulted in increased Itk/Rch1{alpha} complex formation, recruitment of karyopherin ß to the protein complex and Rch1{alpha} 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 GST–Itk efficiently phosphorylated a recombinant GST–Rch1{alpha} fusion. We observed constitutive nuclear localization of Itk that was up-regulated following either TCR–CD3 stimulation or over-expression of wild-type Rch1{alpha} in T cells. Further, nuclear localization of Itk and TCR–CD3-mediated IL-2 production were significantly down-regulated following expression of the Rch1{alpha}-P242A mutant, implicating a role for Rch1{alpha} in the nuclear translocation of Itk.

Keywords: Itk tyrosine kinase, karyopherin {alpha}/Rch1{alpha}, , TCR-CD3, T lymphocytes, nuclear import


    Introduction
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
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 TCR–CD3 complexes (7). The PR motif associates with SH3 domains of Src-family PTK and Grb2 (3,4). The SH3 domain interacts with the Wiscott–Aldrich 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-{gamma}1 (PLC-{gamma}1) in a tyrosine phosphorylation-dependent manner upon CD28- or TCR–CD3-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 {alpha}/karyopherin {alpha} (10,11). Three groups of karyopherin {alpha} 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{alpha} and Rch1{alpha} (15,16); and Qip-1 (10). Rch1{alpha} 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{alpha}/NLS-containing protein complex interacts in the cytosol with karyopherin {alpha} 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{alpha}/karyopherin {alpha} in human T cells through the Itk-SH3 domain and the PR motif in Rch1{alpha}. We demonstrate a regulated nucleocytoplasmic transport of Itk, suggesting a novel role in T cell activation.


    Methods
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
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{alpha}-wt or mutated Rch1{alpha}-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 177–230 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-{gamma}1 mAb and anti-Oct-1 were purchased from Upstate Biotechnology (Lake Placid, NY). Anti-Rch1{alpha} and anti-hSRP1 rabbit sera were described (28), and anti-Rch1{alpha} 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 GST–Itk fusion proteins were produced as reported (9). GST–Rch1{alpha}, 6His–Rch1{alpha} and GST–CD28 fusion proteins were described previously (28,29). Full-length Rch1{alpha} 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{alpha}-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{alpha} 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 G–Sepharose 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 (polyGlu–Tyr, GST, GST–Rch1{alpha}, GST–LAT and GST–CD28) 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 450–650 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).


    Results
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Itk associates with Rch1{alpha} 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{alpha} (amino acids 1–253) (Fig. 1AGo). The isolated sequence contained the motif KNPAPP, a putative SH3-binding PR motif (31).



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Fig. 1. Itk/Rch1{alpha} association in yeast and human T cells. (A) Partial deduced amino acid sequence of human Rch1{alpha} in single-letter amino acid notation corresponding to the clones isolated in the yeast two-hybrid screen. A solid box indicates the PR motif. Residue numbers are on the left. (B) Jurkat T cells (left) or PHA-expanded T lymphoblasts (right) were lysed and subjected to immunoprecipitation with control IgG or mAb to Itk or Rch1{alpha}. Immunoprecipitates were immunoblotted with anti-Itk mAb (upper panels). The immunoblot was stripped and reprobed with anti-Rch1{alpha} (bottom panels). (C) Jurkat T cells were unstimulated or stimulated with anti-TCR–CD3 (5 min), lysed and subjected to immunoprecipitation with mAb to karyopherin {alpha}1/hSRP1, Rch1{alpha}, Itk or control IgG. Immunoprecipitates were immunoblotted with anti-Itk (upper panel) and then reprobed with anti-karyopherin {alpha}1 mAb (lower panel).

 
To confirm and characterize the Itk/Rch1{alpha} association in vivo, Jurkat T cells or normal PHA-expanded T lymphoblasts were lysed and Itk and Rch1{alpha} were immunoprecipitated with specific antibodies (Fig. 1BGo). Constitutive association between Itk and Rch1{alpha} was detected in both T cell sources using either combination of specific antibodies. Upon examining a possible association between Itk and karyopherin {alpha}1 (hSRP1), another member of the karyopherin {alpha} family (10), we did not detect association between Itk and karyopherin {alpha}1 in Jurkat T cells (Fig. 1CGo).

We investigated the effect of TCR–CD3 stimulation in regulating the Itk/Rch1{alpha} association. Jurkat T cells were stimulated with an anti-TCR–CD3 mAb and immunoprecipitates of Itk from varying time points were immunoblotted with anti-Rch1{alpha} mAb. As indicated in Fig. 2Go (upper panel), there was a quantitative enhancement of protein complex formation between Itk and Rch1{alpha} that peaked at 5 min of TCR–CD3 stimulation. Densitometric analysis showed that under steady-state conditions ~4% of total cellular Rch1{alpha} associated with Itk; however, upon TCR–CD3 activation the pool of Rch1{alpha} in complex with Itk increased to 14%. We investigated whether Itk/Rch1{alpha} 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. 2Go, middle panel), associating after the Rch1{alpha}/Itk interaction suggesting a sequential binding mechanism. This association was up-regulated upon TCR–CD3 stimulation. The level of Itk immunoprecipitated during the time course was unchanged (Fig. 2Go, bottom panel).



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Fig. 2. TCR–CD3 stimulation enhances the Itk/Rch1{alpha} association. Jurkat T cells were unstimulated (–) or stimulated with anti-TCR–CD3, lysed and subjected to immunoprecipitation with mAb to Itk or control IgG at the indicated time points. Immunoprecipitates were immunoblotted with anti-Rch1{alpha} (upper panel). The immunoblot was stripped and reprobed with anti-karyopherin ß mAb (middle panel), followed by stripping and reprobing with anti-Itk (bottom panel). Mol. wt markers in kDa are shown (right).

 
We analyzed the contribution of individual Itk domains to the Itk/Rch1{alpha} complex. Three GST–Itk fusion proteins including single domains or domain combinations (9) were used in GST pull-down assays in Jurkat T cells. As shown in Fig. 3Go(A, upper panel), the GST–PTR32 (Itk lacking the kinase domain) and GST–SH3, but not the GST–SH2 fusion protein, isolated Rch1{alpha} from lysates of unstimulated T cells. The constitutive association was moderately augmented upon TCR–CD3 engagement when using the GST–PTR32 fusion protein. The GST–SH2 fusion isolated Rch1{alpha} from T cells only following TCR–CD3 stimulation. As a loading control, the immunoblot was stripped and reprobed with anti-GST (Fig. 3AGo, bottom panel).



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Fig. 3. Specificity of Itk/Rch1{alpha} interaction. (A) Jurkat T cells were unstimulated or stimulated with anti-TCR–CD3 (5 min), lysed and incubated with equivalent amounts of GST or the indicated GST–Itk fusion proteins. Reactants were immunoblotted with anti-Rch1{alpha} (upper panel). The membrane was stripped and reprobed with anti-GST mAb (lower panel). (B) Jurkat T cells stably transfected with the empty vector pcDNA3 (mock), the expression vector containing wild-type Rch1{alpha} (Rch1{alpha}-wt) or the point mutation P242A (Rch1{alpha}-P242A) were lysed and immunoprecipitated with an anti-myc mAb. Immunoprecipitates were immunoblotted with anti-Itk (upper panel). The immunoblot was stripped and reprobed with anti-Rch1{alpha} mAb (lower panel).

 
Proline-to-alanine point mutation within the Rch1{alpha} PR motif disrupts Itk/Rch1{alpha} association
We generated myc-tagged fusions of wild-type Rch1{alpha} (Rch1{alpha}-wt) and a proline-to-alanine point mutant form (proline 242 to alanine, Rch1{alpha}-P242A), and studied their ability to associate with endogenous Itk. Rch1{alpha}-wt or Rch1{alpha}-P242A myc-tagged proteins were immunoprecipitated from stably transfected Rch1{alpha}-wt or Rch1{alpha}-P242A Jurkat T cells with an anti-myc mAb. Immunoblotting with anti-Itk showed that Rch1{alpha}-P242A did not associate with Itk (Fig. 3BGo, upper panel), while the Rch1{alpha}-wt protein interacted with Itk. For protein loading control, the immunoprecipitates were immunoblotted with anti-Rch1{alpha} (Fig. 3BGo, bottom panel). In both Rch1{alpha}-wt- and Rch1{alpha}-P242A-expressing cells, endogenous Rch1{alpha} co-immunoprecipitated endogenous Itk (data not shown).

Tyrosine phosphorylation of Rch1{alpha}
We evaluated whether the Itk/Rch1{alpha} association resulted in tyrosine phosphorylation of Rch1{alpha}. Immunoprecipitates of Rch1{alpha} from either unstimulated or TCR–CD3-activated Jurkat T cells were immunoblotted with anti-phosphotyrosine. Tyrosine phosphorylation of Rch1{alpha} peaked at 5 min after TCR–CD3 stimulation (Fig. 4AGo, upper panel) and was undetectable after 30 min of T cell stimulation (data not shown). Additional tyrosine phosphorylated proteins were associated with Rch1{alpha} (115, 80 and 72 kDa). Reprobing of the immunoblot with anti-Itk revealed its presence in the complex following TCR–CD3 stimulation (Fig. 4AGo, middle panel), while no change in Rch1{alpha} levels were observed (Fig. 4AGo, bottom panel).



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Fig. 4. Tyrosine phosphorylation of Rch1{alpha} in vitro and in vivo. (A) Jurkat T cells were unstimulated (–) or stimulated with anti-TCR–CD3, lysed and subjected to immunoprecipitation with control IgG or mAb to Rch1{alpha}. Immunoprecipitates were immunoblotted with anti-phosphotyrosine (upper panel). The immunoblot was stripped and reprobed with anti-Itk (middle panel), followed by stripping and reprobing with anti-Rch1{alpha} mAb (bottom panel). Mol. wt markers in kDa are shown (right). (B) Substrate proteins poly-Glu–Tyr (poly-GY), GST, GST–Rch1{alpha} and GST–CD28, (C) GST–Rch1{alpha} or (D) GST–LAT were loaded onto 96-well plates. Kinase reactions were performed by adding the indicated amounts of the different enzymes as GST fusions with TK domains only (GST–Itk, GST–ZAP-70, GST–Lck or GST–Jak3) in kinase buffer. The data are representative means of quadruplicate samples from two independent experiments. (E) Phosphorylation of recombinant 6His–Rch1{alpha} protein with GST–Itk-TK (+) or not (–) was followed by incubation with equivalent amounts of GST or the indicated GST–Itk fusion proteins. Reactants were immunoblotted with anti-Rch1{alpha} (upper panel). The membrane was stripped and immunoblotted with anti-GST mAb (lower panel).

 
We evaluated in vitro tyrosine phosphorylation of Rch1{alpha} with purified recombinant GST–Rch1{alpha} and GST–Itk-TK (a fusion including only the Itk tyrosine kinase domain, residues 341–621). Recombinant GST–Rch1{alpha} was efficiently tyrosine phosphorylated by GST–Itk-TK (Fig. 4BGo). The cytoplasmic tail of CD28 (GST–CD28) 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{alpha} in vitro. None of these tyrosine kinases mediated detectable Rch1{alpha} tyrosine phosphorylation (Fig. 4CGo), yet they actively phosphorylated control GST–LAT protein (Fig. 4DGo). Since Rch1{alpha} becomes tyrosine phosphorylated following T cell stimulation, we addressed whether the Itk-SH2 domain was involved in the enhanced Itk/Rch1{alpha} association upon TCR–CD3 stimulation. Purified recombinant 6His–Rch1{alpha} was phosphorylated in vitro by GST–Itk-TK or left unphosphorylated and equal amounts of protein were incubated with GST, GST–Itk-SH3 or GST–Itk-SH2. Isolation of GST-containing fusion proteins was followed by immunoblotting with anti-Rch1{alpha}. Specific binding of the GST–Itk-SH3 domain to 6His–Rch1{alpha} was observed (Fig. 4EGo), while no interaction between GST–Itk-SH2 and Rch1{alpha} was detected even following in vitro Itk tyrosine phosphorylation. For GST loading control, the immunoblot was stripped and reprobed with anti-GST (Fig. 4EGo).

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. 5AGo, upper panels). Densitometric analysis showed 15% of total cellular Itk in the nuclear fraction using this technique. PLC-{gamma}1 was detected in the cytosol/membrane extracts, but was undetectable in the nuclear preparations (Fig. 5AGo, 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. 5BGo).



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Fig. 5. Nuclear localization of Itk in Jurkat T cells. (A) Nuclear or cytosolic extracts were prepared from three Jurkat T cell clones (I, II and III) and immunoprecipitated with control IgG (Ig), anti-Itk (upper panels) or anti-PLC-{gamma}1 (lower panels). Immunoprecipitates were immunoblotted with anti-Itk (upper panels) or anti-PLC-{gamma}1 (lower panels). (B) Equal amounts of nuclear or cytosolic extracts as in (A) were directly immunoblotted with anti-Itk. (C) Jurkat T cells were transfected with pcDNA3 empty vector (mock) or the expression vector containing Rch1{alpha}-wt. Transfectants were unstimulated or stimulated with anti-TCR–CD3 and lysed to harvest nuclear and cytosolic fractions. Immunoprecipitates of Itk and PLC-{gamma}1 were prepared and immunoblotted with their respective antibodies. (D) Jurkat T cells were unstimulated or stimulated with anti-TCR–CD3 over the course of 1 h. Nuclear and cytosolic extracts were prepared at the indicated time points, and Itk was immunoprecipitated from each fraction and immunoblotted with anti-Itk. For loading control, equivalent amounts of the cell fractions were immunoblotted for PLC-{gamma}1 (cytosolic extracts) or the nuclear transcription factor Oct-1 (nuclear extracts). (E) Jurkat T cells were stimulated (up to 5 min) and fractionated as above. Immunoprecipitates of Itk were immunoblotted with anti-phosphotyrosine (upper panel). The blot was stripped and reprobed with anti-Itk (lower panel).

 
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-mouse–FITC. 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{alpha} association, we tested whether transient transfection of Rch1{alpha}-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{alpha}-wt construct. Transfectants were unstimulated or were stimulated with anti-TCR–CD3, and Itk was immunoprecipitated from cytosol/membrane and nuclear extracts. An enhancement of nuclear Itk was observed after transient transfection of Rch1{alpha}-wt (relative to mock transfected) in both unstimulated and TCR–CD3-stimulated cells (Fig. 5CGo). PLC-{gamma}1 was observed only in the cytosolic fraction and was unaffected by cell transfection with the Rch1{alpha} constructs. Rch1{alpha}-wt myc-tagged proteins immunoprecipitated with anti-myc mAb from aliquots of the same cytosolic and nuclear extracts confirmed equivalent amounts of Rch1{alpha}-wt proteins in the transfectants (data not shown).

We investigated TCR–CD3-induced regulation of endogenous Itk nuclear translocation. Jurkat T cells were stimulated with anti-TCR–CD3 and immunoprecipitates of Itk were immunoblotted with anti-Itk. An enhancement of Itk translocation from the cytoplasm to the nucleus was observed upon TCR–CD3 stimulation (Fig. 5DGo). For loading control, whole-cell lysates (nuclear and cytosolic) corresponding to each condition were immunoblotted for PLC-{gamma}1 (cytosolic extracts) or the nuclear transcription factor Oct-1 (nuclear extracts) (Fig. 5DGo).

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 TCR–CD3 stimulation, with similar kinetics (Fig. 5EGo). The relative tyrosine phosphorylation levels of cytoplasmic and nuclear Itk 3 min after TCR–CD3 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 TCR–CD3 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. 6Go), with an increase following TCR–CD3 stimulation as observed in Jurkat T cells. Further, there was no translocation of PLC-{gamma}1 into the nucleus, no translocation of Oct-1 into the cytoplasm and no cross-contamination of the cell fractions (Fig. 6Go).



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Fig. 6. Nuclear translocation of Itk in normal human T cells. Peripheral blood-derived human T cells were expanded in PHA for 5 days. The T cells were stimulated with anti-TCR–CD3 over the course of 30 min and were lysed by the subcellular fractionation method. For control analysis, immunoprecipitation using a control Ig was included. Immunoprecipitates of Itk, PLC-{gamma}1 or Oct-1 were prepared from the T cell fractions from each time point and were immunoblotted with their respective antibodies. No contamination of either the cytolosolic or nuclear compartments was observed by cross-comparison with PLC-{gamma}1 or Oct-1.

 
To further analyze the function of the Itk/Rch1{alpha} association, we tested whether the transiently transfected Rch1{alpha}-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{alpha}-wt or Rch1{alpha}-P242A DNA constructs. After 48 h in culture, the cells were left unstimulated or were stimulated through TCR–CD3, and Itk was immunoprecipitated from cytosolic/membrane and nuclear extracts. As indicated in Fig. 7AGo (upper panels), no nuclear Itk was detected upon Rch1{alpha}-P242A transient transfection in either resting or TCR–CD3-activated cells, in contrast to mock or Rch1{alpha}-wt-transfected cells. Densitometric analysis showed that 5% of total cellular Itk was nuclear (15% upon TCR–CD3 activation) and that this amount was augmented upon transient expression of Rch1{alpha}-wt (12 and 23% respectively) or inhibited (<1% present) following Rch1{alpha}-P242A transient expression. As a control for cytosolic contamination in the nuclear extracts, PLC-{gamma}1 was immunoprecipitated from cytosolic and nuclear extracts from a fraction of the same transiently transfected Jurkat cells. Clearly, PLC-{gamma}1 was only observed in the cytosolic fraction and was unaffected by transfection of the cells with the Rch1{alpha} constructs. In addition, Rch1{alpha}-wt and Rch1{alpha}-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{alpha}-wt and Rch1{alpha}-P242A proteins in the transient transfections (Fig. 7BGo).



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Fig. 7. Disruption of Itk nuclear translocation by expression of Rch1{alpha}-P242A down-regulates TCR–CD3-mediated IL-2 production in Jurkat T cells. (A) The pcDNA3 empty vector (mock) and the expression vector containing Rch1{alpha}-wt or Rch1{alpha}-P242A were introduced into Jurkat T cells. After 48 h of culture at 37°C, cells were left unstimulated or were stimulated with anti-TCR–CD3 mAb. Nuclear and cytosolic extracts were then prepared as described in the Methods and subjected to immunoprecipitation with anti-Itk mAb (upper panels) or anti-PLC-{gamma}1 (bottom panels). Immunoprecipitated proteins were analyzed by SDS–PAGE on 8% gels and immunoblotted with anti-Itk or anti-PLC-{gamma}1 mAb respectively. (B) Equivalent aliquots of cytosolic or nuclear extracts from (A) were subjected to immunoprecipitation with anti-myc mAb. Immunoprecipitated proteins were analyzed by SDS–PAGE on 8% gels and immunoblotted with anti-Rch1{alpha} mAb. (C) Jurkat T cells were transiently transfected with equal amounts of the pcDNA3 empty vector (mock) or the expression vector containing Rch1{alpha}-wt or Rch1{alpha} P242A. After 24 h of culture at 37°C, cells were left unstimulated, or were stimulated with either anti-TCR–CD3 or PHA for 18 h at 37°C. Supernatants were harvested and the production of IL-2 was analyzed by ELISA. The mean measurements from quadruplicate cultures with error bars is shown and the results are representative of three independent experiments.

 
We also studied the effect of Rch1{alpha}-P242A over-expression on the nuclear import of other unrelated proteins also crucial for cytokine production (i.e. NF-{kappa}B and NFATc1). Overexpression of Rch1{alpha}-P242A did not interfere with the nuclear translocation of NF-{kappa}B or NFATc1 transcription factors upon cellular activation (data not shown). Taken together, the above results demonstrate that Rch1{alpha} is involved in the nuclear translocation of Itk.

Expression of Rch1{alpha}-P242A down-regulates TCR–CD3-mediated IL-2 production by Jurkat T cells
We assessed whether blocking Itk nuclear import was able to impact the downstream outcome of TCR–CD3-mediated intracellular signaling. For this purpose, the effect of Rch1{alpha}-wt or Rch1{alpha}-P242A expression on TCR–CD3-mediated cytokine production was evaluated. Jurkat T cells were transiently transfected with equal amounts of the empty expression vector or ones containing Rch1{alpha}-wt or Rch1{alpha}-P242A (5 µg). After 24 h in culture, the transfected cells were left unstimulated or were activated with anti-TCR–CD3 or PHA for 18 h and the levels of IL-2 production were measured. The experiments showed that expression of the mutant Rch1{alpha}-P242A, but not Rch1{alpha}-wt, markedly down-regulated (50–70%) IL-2 production in response to TCR–CD3- or PHA-mediated activation (Fig. 7CGo). This observation suggests that the TCR–CD3-induced nuclear import of Itk accounts in part for the IL-2 production mediated by TCR–CD3 activation.


    Discussion
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
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 1–253 of the importin Rch1{alpha} 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{alpha} 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 TCR–CD3 stimulation and its role in IFN-{gamma} 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 57–71). 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{alpha} association following TCR–CD3 engagement coincided with Rch1{alpha} tyrosine phosphorylation. Our in vitro analysis suggested that Itk may account for Rch1{alpha} tyrosine phosphorylation in vivo; however, the Itk-SH2 domain association with Rch1{alpha} after TCR–CD3 stimulation is indirect, not directly due to Rch1{alpha} phosphorylation.

Of particular interest is the observation of inhibiting both Itk nuclear translocation and concomitant TCR–CD3- or PHA-mediated IL-2 production by expression of the Rch1{alpha}-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{alpha} and the Rch1{alpha}-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 TCR–CD3 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{alpha} tyrosine phosphorylation and that of the nuclear localization of Itk will be paramount to understanding their roles in TCR–CD3-directed effector functions. Disruption of the Itk/Rch1{alpha} complex by genetic and/or chemical approaches may have value in the treatment of immunological disorders.


    Acknowledgments
 
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
 
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
 
Transmitting editor: L. H. Glimcher

Received 16 March 2001, accepted 2 July 2001.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
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