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Molecular and Cellular Biology, June 2006, p. 4288-4301, Vol. 26, No. 11
0270-7306/06/$08.00+0     doi:10.1128/MCB.01817-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

A Nuclear Export Signal and Phosphorylation Regulate Dok1 Subcellular Localization and Functions

Yamei Niu,1 François Roy ,1,{dagger},{ddagger} Frédéric Saltel,2,{dagger},§ Charlotte Andrieu-Soler,1, Wen Dong,1 Anne-Lise Chantegrel,1,|| Rosita Accardi,1 Amélie Thépot,1 Nadège Foiselle,1 Massimo Tommasino,1 Pierre Jurdic,2 and Bakary S. Sylla1*

International Agency for Research on Cancer, 69008 Lyon, France,1 LBMC, UMR 5161 CNRS/ENS, IFR 128 Biosciences, 69364 Lyon Cedex 07, France2

Received 15 September 2005/ Returned for modification 12 December 2005/ Accepted 9 March 2006


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ABSTRACT
 
Dok1 is believed to be a mainly cytoplasmic adaptor protein which down-regulates mitogen-activated protein kinase activation, inhibits cell proliferation and transformation, and promotes cell spreading and cell migration. Here we show that Dok1 shuttles between the nucleus and cytoplasm. Treatment of cells with leptomycin B (LMB), a specific inhibitor of the nuclear export signal (NES)-dependent receptor CRM1, causes nuclear accumulation of Dok1. We have identified a functional NES (348LLKAKLTDPKED359) that plays a major role in the cytoplasmic localization of Dok1. Src-induced tyrosine phosphorylation prevented the LMB-mediated nuclear accumulation of Dok1. Dok1 cytoplasmic localization is also dependent on IKKß. Serum starvation or maintaining cells in suspension favor Dok1 nuclear localization, while serum stimulation, exposure to growth factor, or cell adhesion to a substrate induce cytoplasmic localization. Functionally, nuclear NES-mutant Dok1 had impaired ability to inhibit cell proliferation and to promote cell spreading and cell motility. Taken together, our results provide the first evidence that Dok1 transits through the nucleus and is actively exported into the cytoplasm by the CRM1 nuclear export system. Nuclear export modulated by external stimuli and phosphorylation may be a mechanism by which Dok1 is maintained in the cytoplasm and membrane, thus regulating its signaling functions.


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INTRODUCTION
 
Dok1 belongs to a family of adaptor proteins that are heavily tyrosine phosphorylated after stimulation with epidermal growth factor receptor, insulin receptor, and antigen receptors. Tyrosine phosphorylation of Dok1 also occurs in several cell lines transformed by viral oncogenes including v-Src and v-Abl, and in chronic myelogenous leukemia cells, where it is a target of p210Bcr-Abl (2, 3, 29, 52). DOK family members (Dok1 to Dok6) and insulin receptor substrates are characterized by a pleckstrin homology domain (PH) that allows anchorage to the membrane, a phosphotyrosine binding domain that is involved in protein-protein interaction, and a C-terminal region rich in tyrosine and serine residues (3-6, 10, 23, 27, 30). Tyrosine phosphorylation modulates interactions with several SH2-containing signaling molecules such as RasGAP, Nck, and the X-linked lymphoproliferative syndrome gene product SH2D1A (3, 24, 26, 33, 43, 48, 49, 52).

Dok1 has emerged as a key negative regulator downstream of several receptor and nonreceptor tyrosine kinase cascades. Dok1 down-regulates cell proliferation and lymphocyte signaling, inhibits mitogen-activated protein (MAP) kinase activity, and mediates activin-induced apoptosis (18, 31, 45, 51-53). Dok1 is altered and down-regulated in chronic lymphocytic leukemia and can suppress cell transformation and leukemia (7, 22, 32, 41, 54). However, it appears not to play a major role in familial chronic lymphocytic leukemia cases (38). Knowing its tumor-suppressive activity, it is likely that genetic alterations or low expression of Dok1 and its related member Dok2 may be involved in a variety of malignancies affecting hematopoietic and probably nonhematopoietic cell populations.

In addition to its inhibitory effects on various cellular functions, Dok1 plays positive roles in cell adhesion, cell spreading, and cell migration (13, 21, 27, 33, 49). Tyrosine phosphorylation induced by Src tyrosine kinase family members or c-Abl and plasma membrane translocation mediated by its PH domain are reported to be required for Dok1 functions (22, 24, 29, 33, 49, 55). Phosphorylation of specific serine residues mediated by I{kappa}B kinase (IKK) signaling can also modulate Dok1 functions in inhibiting cell proliferation and Erk1/2 phosphorylation and in promotion of cell motility (21).

Nuclear-cytoplasmic shuttling plays an important role in regulating the activity of a number of proteins involved in cell proliferation, transformation, and tumorigenesis (12, 19). Their expulsion from the nucleus generally depends upon a nuclear export signal (NES) sequence, a short leucine-rich motif that is specifically recognized by the nuclear exporter protein known as chromosomal region maintenance 1 (CRM1) (8, 9, 12, 20). Such an NES motif has been identified in various regulatory shuttling proteins including cyclin D1, p53, c-Abl, and protein kinase A inhibitor (1, 35, 44, 47). Entry of a protein into the nucleus requires, in most cases, the presence of a distinct motif called a nuclear localization sequence (NLS) that is recognized by a family of importin {alpha}/ß heterodimers (for reviews, see references 14, 15, and 50). In many cases, posttranslational modifications of the NES or NLS, such as phosphorylation, affect the binding affinity with the transporter proteins and therefore regulate the intracellular movement and the functions of proteins (1, 14, 19, 34, 40, 46).

Although it is predominantly expressed as a cytoplasmic/membrane protein, a proportion of Dok1 has been found in the nuclear fraction (22, 33), suggesting a possible transit in the nucleus. The signaling events and mechanisms by which Dok1 activity is regulated in relation to its subcellular localization have not previously been determined. In the present study, we examined whether Dok1 can shuttle between the nucleus and cytoplasm. We found that Dok1 contains a functional NES and accumulates in the nucleus in the presence of leptomycin B (LMB), an inhibitor of the NES-dependent nuclear exporter CRM1. Src-induced tyrosine phosphorylation plays a critical role in the cytoplasmic localization of Dok1. IKKß is also involved in this process. Serum starvation or maintaining cells in suspension favor Dok1 nuclear localization, while serum stimulation, exposure to growth factor, or cell adhesion to a substrate induce cytoplasmic localization. Importantly, we found that, in contrast to wild-type Dok1, the Dok1 NES mutant retained in the nucleus has impaired ability to inhibit cell proliferation and to promote cell spreading and cell motility. Taken together, our data demonstrate for the first time an active nuclear-cytoplasmic shuttling of Dok1 controlled by external stimuli and phosphorylation, a possible mechanism for regulation of Dok1 functions.


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MATERIALS AND METHODS
 
Cell culture conditions and transfection. Wild-type mouse embryonic fibroblasts (MEF) and IKK{alpha}, IKKß, and IKK{alpha}/ß-double-null MEF were obtained from I. Verma (Salk Institute). These cell lines, as well as HEK 293 (adenovirus E1a- and E1b-transformed human embryonic kidney) and HEK 293 cells expressing simian virus 40 large T antigen (HEK 293T), human osteosarcoma cell line U2OS cells, mouse fibroblast line NIH 3T3 cells, and mouse fibroblast line Swiss 3T3 cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (D10). Mouse fibroblast line SYF with inactivated Src, Yes, and Fyn was maintained in {alpha}-modified Eagle's medium supplemented with 10% FBS. The Burkitt lymphoma cell line BJAB was maintained in RPMI medium 1640 supplemented with 10% FBS. Transfection was performed using Superfect transfection reagent (QIAGEN) (21). For serum starvation, MEF were washed with DMEM without FBS (D0), and then incubated in D0 for 27 h. Effects of serum were evaluated by adding serum (20%) to the medium (16). For cell suspension and attachment, cells were trypsinized and washed with phosphate-buffered saline. Cells were then suspended in D10, half of which were kept in suspension for 3 h in a Falcon tube at 37°C, and then spread onto a slide glass for immunofluorescence. Remaining cells were seeded onto fibronectin (1 µg/ml)-coated coverslips in 12-well plates and incubated at 37°C for 3 h. Dok1 localization was then monitored by immunofluorescence. For platelet-derived growth factor (PDGF) stimulation, cells were starved in D0 for 72 h and then stimulated with human PDGF (40 ng/ml) (R&D) at different time points (37).

Plasmids, cloning, and mutagenesis. The Dok1 expression plasmids pEGFP-Dok1 and pcDNA3-Flag-Dok1 have been previously described (22, 43). Dok1 deletion variants were generated through standard cloning procedures. Specific mutants of Dok1 were obtained based on pEGFP-Dok1 using the QuikChange site-directed mutagenesis kit (Stratagene). The pcDNA3-Flag-Dok1-NES3 mutant was derived from the pEGFP-Dok1-NES3 mutant and pcDNA3-Flag-Dok1. All mutants were verified by checking the DNA sequence and protein expression. pRK5-Flag-IKKß was obtained from D. Goeddel (Tulirak). The expression plasmid for p210Bcr-Abl was obtained from A.-M. Pendergast (Duke University). The expression plasmids for c-Src and its kinase-dead (KD) form were obtained from A. August (Pennsylvania State University). pcI-HA-CRM1 was obtained from A. Diehl (Leonard and Madlyn Abramson Family Cancer Research Center).

Antibodies and reagents. The antibodies used were the following: rabbit anti-Dok1 (gifts of R. Kobayashi, University of Texas M. D. Anderson Cancer Center, and J. Cambier, St. Jude Children's Research Hospital); mouse anti-Flag (M5) monoclonal antibody, mouse anti-ß-tubulin (Sigma,); rabbit anti-green fluorescent protein (anti-GFP), mouse anti-pTyr, rabbit anti-Src, and mouse anti-hemagglutinin (anti-HA) (Santa Cruz); rabbit anti-human CRM1 (gift of G. Grosveld, St Jude Children's Research Hospital); mouse anti-poly(ADP-ribose) polymerase (anti-PARP, clone C2-10) (R&D); LMB was obtained from M. Yoshida (Chemical Genetics Laboratory, Japan) and Sigma; PP2 was from Calbiochem.

Immunofluorescence and microscopy. Cells were seeded on sterilized coverslips in 12-well plates at 1 x 105 cells per well. Twenty-four hours after transfection, cells were fixed with 4% paraformaldehyde for 10 min. Fixed cells were permeabilized with 0.1% Triton X-100 for 5 min and then blocked with 5% milk in 0.05% Tris-buffered saline-Tween at room temperature for 1 h, followed by incubation with the indicated primary antibodies and fluorescein isothiocyanate/tetramethyl rhodamine isothiocyanate-conjugated secondary antibodies (Interchim) as mentioned. Finally, the cellular localization of Dok1 was visualized using a confocal laser scanning microscope (LSM) 510 Pa or Axioplan2 microscope from Zeiss, and random fields were photographed.

Time-lapse confocal microscopy. Transfected cells seeded on coverslips were transferred to 35-mm glass-bottom petri dishes and then placed at 37°C in observation medium (DMEM without bicarbonate) (Life Technologies) containing 10% FBS in 20 mM HEPES. The dishes were placed on a 37°C heated stage (Zeiss), and cells were imaged with a Zeiss laser scanning microscope 510 (Axiovert 100 M) and a 40 x (numerical aperture 1.0) Zeiss Plan-Apochromat objective. Meta Imaging Series 4.5 (Universal Imaging Corporation) was used to mount AVI movies from image stacks. region of interest (ROI) analysis and three-dimensional representation of the fluorescence intensity were realized using the Zeiss software LSM 510. Images extracted from stacks were processed with Adobe Photoshop 6.0 (Adobe Systems).

Immunoprecipitation and immunoblotting. Immunoprecipitation and immunoblotting were performed as previously described (22, 42). Cells were lysed in cell lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 2 mM EDTA, 1% Nonidet P-40, 3% glycerol, 1 mM phenylmethylsulfonyl fluoride, 5 µg/ml leupeptin, 2 µg/ml aprotinin, 1 mM sodium fluoride, 1 mM sodium orthovanadate) for 30 min on ice. Insoluble material was removed by centrifugation at 14,000 x g for 15 min. Lysates were precleared with Sepharose CL-6B for 30 min and then immunoprecipitated with the anti-Flag M2 beads at 4°C for 2 h. Precipitates were washed five times with lysis buffer and analyzed by Western blotting. The nuclear and cytoplasmic fractions were prepared as previously described (22).

Cell spreading, cell proliferation, and cell motility assay. For the cell spreading assay, Swiss 3T3 cells were transfected with plasmids encoding GFP-Dok1 or GFP-Dok1-NES3. After 24 h, cells were trypsinized and reseeded onto collagen-coated coverslips. Four hours later, cells were fixed with 4% paraformaldehyde and observed for fluorescence and phase contrast. For the cell proliferation assay, HEK 293 cells stably expressing Dok1 were established by transfecting cells with pcDNA3, pcDNA3-Flag-Dok1, or pcDNA3-Flag-Dok1-NES3. G418 (1.0 mg/ml)-resistant clones were selected and expanded. To assess the growth curve, cells were seeded in six-well plates at a density of 5.0 x 104 cells per well and propagated in D10. The medium was changed every 48 h, and the number of cells was evaluated every 24 h. The cell motility was monitored by using the wound healing approach as described previously (21).


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RESULTS
 
Dok1 shuttles between the nucleus and cytoplasm. The subcellular localization of Dok1 was evaluated by immunofluorescence using rabbit polyclonal antibody against Dok1. In agreement with previous reports (21, 22, 55), endogenous Dok1 was localized predominantly in the cytoplasm and plasma membrane region in four different cell lines (Fig. 1A, –LMB). No staining was detected when the secondary antibody alone was used as a control in NIH 3T3 cells (Fig. 1A, last panel at the bottom), demonstrating that the immunofluorescence staining of Dok1 was specific. A small proportion of Dok1 was also found in the nucleus in some untransfected cells, and in cells overexpressing Dok1 (Fig. 1A and B; see also Fig. 3F, Fig. 4C and D, and references 21 and 33). This pattern of distribution prompted us to investigate whether Dok1 can transit through the nucleus. Indeed, when cells were treated with LMB, an inhibitor of CRM1-dependent nuclear exporter (8, 12), over 90% of endogenous Dok1 accumulated in the nucleus in various Dok1-expressing cell lines (Fig. 1A, +LMB). The 4',6'-diamidino-2-phenylindole (DAPI) staining gives a clear compartmentalization of the nuclei of the cells used in this study. To confirm these findings, HEK 293T cells were transfected with an expression plasmid for the GFP fused to Dok1. The diffuse nuclear and cytoplasmic localization of the GFP protein did not change with the addition of LMB (Fig. 1C). However, similarly to the endogenous Dok1, ectopically expressed GFP-Dok1 had a prominently cytoplasmic localization in the absence of LMB treatment (Fig. 1C, –LMB) but showed strong nuclear accumulation in its presence (Fig. 1C, +LMB). Quantification of fluorescence in the nucleus by time-lapse video microscopy revealed that with LMB treatment GFP-Dok1 accumulated rapidly in the nucleus within 10 min and gradually increased after 1 h to reach a peak around 1 h 30 min (Fig. 1D). Thus, LMB induces nuclear accumulation of endogenous Dok1 from untransfected cells as well as from overexpressed Dok1 in transfected cells.


Figure 1
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  		FIG. 1. LMB sequesters Dok1 in the nucleus. (A) Subcellular localization of endogenous Dok1. Cells were seeded onto coverslips in 12-well plates. After 24 h, cells were left untreated or were treated with LMB (20 ng/ml) for 3 h at 37°C. Immunofluorescence (IF) was performed using rabbit anti-Dok1 antibody, and images were recorded using Axioplan2 microscope from Zeiss. The last panel is a negative control for IF, in which cells were incubated only with fluorescein isothiocyanate-conjugated anti-rabbit immunoglobulin G. Cells were stained with DAPI to visualize the nucleus. (B) Subcellular fractionation of NIH 3T3 cells. Cells (2 x 106) were harvested and fractionated into cytoplasmic (C) and nuclear (N) fractions. Total lysate (T) is half of the mixture of equal amounts of cytoplasmic and nuclear fractions. All of the fractions were applied to SDS-PAGE followed by immunoblotting with anti-Dok1, anti-ß-tubulin (cytoplasmic fraction marker), and anti-PARP antibodies (nuclear fraction marker). (C) Localization of overexpressed GFP-Dok1. HEK 293T cells were transfected with plasmids encoding GFP or GFP-Dok1. Twenty-four hours later, cells were treated with or without LMB (20 ng/ml) at 37°C for 3 h. (D) HEK 293T cells transfected with GFP-Dok1 and treated with LMB for 3 h were observed by confocal time-lapse microscopy. GFP fluorescence images were extracted at various times of observation. During the observation using the time lapse, the fluorescence of two ROI were quantified, one for the cytoplasm (ROI 1, in blue) and one for the nucleus (ROI 2, in red), and graphically represented using Zeiss LSM 510 software. Green fluorescence images and a three-dimensional representation of the intensity fluorescence using a color code (red for the maximum fluorescence intensity) are shown.


Figure 3
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  		FIG. 3. Identification of Dok1 nuclear export signal. (A) Three potential NESs in Dok1. Alignment of hDok1 and mDok1 NES sequences (NES1, 2, and 3) with other known NESs of protein kinase A inhibitor, p53, and c-Abl. Critical amino acids that are conserved or important for NES function are boxed. (B) Expression of GFP-Dok1-NES mutants. Amino acids underlined in panel A were mutated to alanine in Dok1. The Dok1 mutants were transfected into HEK 293T cells, and their expression levels were checked by immunoblotting with rabbit anti-Dok1 antibody. NES1 is the Dok1 mutant where L295, L300, L303, and I305 were converted to A; NES2 is the Dok1 mutant where L336, Y337, and L340 were changed to A. NES3 is the Dok1 mutant harboring mutations of L348, L349, and L353 to A. NES2, 3 includes the mutations from NES2 and NES3. (C) Subcellular localization of Dok1-NES mutants. GPF-Dok1-NES mutants were transfected into HEK 293T cells, and 24 h later, Dok1 localization was observed by confocal microscopy. (D) Interaction between overexpressed HA-CRM1 and Flag-Dok1. Plasmids encoding HA-CRM1 and Flag-Dok1, Flag-Dok1-NES3, or Flag-NES2,3 were transfected into HEK 293T cells. Flag-Dok1 was immunoprecipitated using anti-Flag M2 affinity gel, and coprecipitated proteins were immunoblotted using anti-HA antibody and anti-Dok1. Equal amounts of whole lysates were subjected to immunoblotting to determine protein expression levels. (E) Establishment of Dok1 stable transfectants. HEK 293 stable transfectants were established by transfection with empty pcDNA3 vector or Flag-Dok1-WT or Flag-Dok1-NES3 plasmid. Immunofluorescence using anti-Dok1 was performed to confirm its localization and cells homogenesis. (F) Subcellular fractionation of cells stably expressing Dok1-WT and Dok1-NES3. Equal numbers of cells were harvested and fractionated into cytoplasmic (C) and nuclear (N) extracts. Equal amounts of C and N, together with total lysates (T) (corresponding to half of C+N) were loaded to SDS-PAGE and probed with anti-Dok1, anti-ß-tubulin, and anti-PARP. (G) Dok1-CRM1 interactions in 293 stable transfectants. Cells (6 x 106) from each stable transfectant were lysed to confirm CRM1-Dok1 interaction by coimmunoprecipitation (IP). Anti-Flag was used to pull down Dok1, and coprecipitated protein was detected with anti-CRM1. Equal amounts of whole-cell lysates were also checked for similar protein expression levels.


Figure 4
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  		FIG. 4. Effects of tyrosine phosphorylation on subcellular localization of Dok1. (A) Effects of Src on Dok1 subcellular localization. Expression plasmids for wild-type GFP-Dok1 (GFP-Dok1-WT) and GFP-Dok1-NES3 were cotransfected with expression plasmids for c-Src or c-Src-KD into HEK 293T cells. Dok1 subcellular localization before and after LMB treatment was analyzed by confocal microscopy using fluorescence (flu). Src protein was visualized in red by staining with anti-Src antibody (Src). Arrows indicate cells coexpressing Dok1 and Src, while arrowheads indicate cells where only Dok1 is expressed. (B) c-Src induces tyrosine phosphorylation of Dok1 and Dok1 tyrosine mutants. Equal amounts of protein extracts from HEK 293T transfected with the indicated expression plasmids were analyzed by immunoblotting with anti-pTyr, anti-Dok1, and anti-c-Src antibodies. GFP-pY-Dok1 and pY-Src with respective molecular masses of 89 kDa and 60 kDa were clearly separated by PAGE. (C) PP2 enhances nuclear Dok1 accumulation in MEF. Cells on coverslips were treated with PP2 (10 µM) for 1 h at 37°C. Dok1 localization was visualized by immunofluorescence using anti-Dok1 antibody, and images were recorded using an Axioplan 2 microscope from Zeiss. (D) LMB induces faster migration of Dok1 in SYF cells. MEF and SYF cells were seeded onto coverslips in 12-well plates. Twenty-four hours later, cells were stimulated with LMB at the indicated time points and Dok1 localization was visualized by immunofluorescence as described above.

The effects of LMB suggest that Dok1 is actively exported from the nucleus through a CRM1/exportin pathway and is likely to contain a functional NES.

The region between residues 250 and 430 in the C terminus of Dok1 is required for its cytoplasmic localization. To determine the region of Dok1 that is involved in its export from the nucleus, a number of deletion constructs of GFP-Dok1 were generated (Fig. 2A). The expression plasmids were then transfected into HEK 293T cells, and expression of the protein was evaluated by immunoblotting. All of the proteins were expressed at similar levels with the expected sizes (data not shown).


Figure 2
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  		FIG. 2. The region of aa 250 to 430 confers Dok1 cytoplasmic localization. (A) Schematic representation of GFP-Dok1 and its deletion mutants. A series of Dok1 deletion mutants were constructed based on pEGFP-Dok1. PH, pleckstrin homology domain; PTB, phosphotyrosine-binding domain. (B) Subcellular localization of GFP-Dok1 and its deletion mutants expressed in HEK 293T cells exposed to LMB or not, as determined by confocal microscopy.

We next examined the subcellular localization of these GFP-Dok1 deletion mutants in the presence or absence of LMB (Fig. 2B). As shown above, wild-type GFP-Dok1 accumulated in the nucleus after treatment with LMB. Consistent with previous reports from our laboratory and others (22, 55), GFP-Dok1 (1 to 150) expressing the PH domain showed diffuse staining with strong accumulation in the plasma membrane, where it associated with phospholipids, and this localization was not affected by LMB (Fig. 2B). GFP-Dok1 (150 to 250) and GFP-Dok1 (430 to 481) were distributed diffusely throughout the cytoplasm and the nucleus, and this pattern did not change significantly with LMB. However, as observed for full-length GFP-Dok1, GFP-Dok1 (250 to 430) had a cytoplasmic localization in the absence of LMB and prominent nuclear staining in its presence (Fig. 2B). Thus, the region between amino acids (aa) 250 and 430 plays a major role in the cytoplasmic localization of Dok1. Further studies restricted the minimal responsible region for the nuclear exclusion of Dok1 to aa 240 to 354 (data not shown).

NES at residues 348 to 359 plays a major role in nuclear export of Dok1. The results described above suggest that the region between amino acids 240 and 354 of human Dok1 (hDok1) is likely to contain an NES motif required for its export from the nucleus. Examination of the sequence of this region revealed the presence of three leucine-rich motifs (NES1, NES2, and NES3) that display significant similarities to previously identified NESs. These NES-like elements are also present in mouse Dok1 (mDok1) (Fig. 3A).

To determine the relative contributions of NES1, NES2, and NES3 to the cytoplasmic localization of Dok1, GFP-Dok1 constructs mutated in the conserved hydrophobic residues of these NESs were generated. All of the GFP-Dok1 NES mutants were expressed efficiently with the expected sizes (Fig. 3B). Converting the leucine (L) or isoleucine (I) residues within NES1 to alanine (A) (L295A, L300A, L303A, I305A), either singly or all together, did not significantly affect the cytoplasmic localization of Dok1 (Fig. 3C and data not shown). Some presence of Dok1 in the nucleus was observed when L336, Y337, and L340 of NES2 were changed to alanine, suggesting that NES2 plays a marginal role in the nuclear export of Dok1. Further mutation of H343 and Q345 had no additional effect (data not shown). In contrast, inactivation of NES3 located at aa 348 to 359, by replacing L348 or L353 (but not L349) with alanine, rendered the resulting Dok1 mutants constitutively nuclear in transfected HEK 293T cells (Fig. 3C). Similar results were obtained with the U2OS, NIH 3T3, and Swiss 3T3 cell lines (data not shown). Mutation of L348, 353, and 349 all together to alanine did not further enhance the nuclear accumulation (Fig. 3C, compare mutants NES3 [L348] and NES3 [L353] to NES3). Likewise, no further increase in nuclear localization of GFP-Dok1 was seen when both NES2 and NES3 were mutated (Fig. 3C, compare mutants NES2 and NES3 to mutant NES2,3), indicating the major contribution of NES3 to the overall process of nuclear export of Dok1.

Abrogation of the nuclear export of Dok1 by LMB is probably mediated by its interaction with CRM1. To investigate this, HEK 293T cells were transiently transfected with expression plasmids for Dok1 and human CRM1, and their interaction was evaluated by immunoprecipitation and immunoblotting. As shown in Fig. 3D, ectopically expressed CRM1 interacts with Dok1. Further experiments showed that NES2,3 binds slightly less to CRM1 than to NES3 or wild-type Dok1 (Fig. 3D and G). However, this slight difference in binding to CRM1 was not sufficient to increase significantly the level of Dok1-NES2,3 in the nucleus (Fig. 3C, compare NES3 and NES2,3).

The contribution of NES3 to the cytoplasmic localization of Dok1 was further assessed in cells stably expressing wild-type Dok1 and the Dok1-NES3 mutant. As shown in Fig. 3E, wild-type Dok1 is homogeneously expressed in these stable HEK 293 cells as a cytoplasmic protein, while Dok1-NES3 is predominantly a nuclear protein. Cell fractionation showed increased Dok1 in the nuclear fraction of cells expressing the Dok1-NES mutant compared to wild-type Dok1 (Fig. 3F). Interaction of endogenous CRM1 with Dok1 was also assessed in these stable HEK 293 cells expressing low levels of endogenous Dok1 (Fig. 3G and data not shown). In these cells, endogenous CRM1 also interacted with wild-type Dok1, but this interaction occurred to a lesser extent in cells expressing Dok1-NES3 (Fig. 3G, compare lanes 2 and 3). The expression of Dok1 and CRM1 was similar in both types of expressing cell lines (Fig. 3D and G, whole lysate). Taken together, these data further support the role of NES3 in the cytoplasmic localization of Dok1.

Thus, the NES3 located at aa 348 to 359 of hDok1 mediates the CRM1-dependent nuclear export of Dok1 and plays a major role in its cytoplasmic localization. In view of this result, the Dok1 NES3 mutant (L348A, L349A, L353A) was used in subsequent studies.

Src-mediated tyrosine phosphorylation prevents entry of Dok1 into the nucleus. Dok1 is a substrate of a variety of receptor and nonreceptor tyrosine kinases including the Src tyrosine kinase family members Tec and p210Brc-Abl, and tyrosine phosphorylation is important for Dok1 cellular functions (10, 22, 24, 33, 49). To study the importance of tyrosine phosphorylation of Dok1 in its nuclear-cytoplasmic shuttling, GFP-Dok1 was coexpressed in HEK 293T cells with active c-Src tyrosine kinase or kinase-dead c-Src (c-Src-KD). As shown in Fig. 4A, the cytoplasmic localization of GFP-Dok1 was not affected by the presence of Src before LMB treatment, indicating that tyrosine phosphorylation did not induce detectable nuclear localization of Dok1. Surprisingly, when coexpressed with Src kinase, most GFP-Dok1 remained in the cytoplasm despite LMB exposure (Fig. 4A, c-Src, +LMB). No change in the cytoplasmic localization of GFP-Dok1 occurred even after 12 h of incubation with LMB (data not shown). In addition, GFP-Dok1 that was insensitive to LMB treatment colocalized in the cytoplasm with Src kinase (Fig. 4A), indicating a direct effect of Src on the subcellular distribution of Dok1. In addition, cytoplasmic retention of Dok1 with LMB treatment was not observed in the absence of Src or with Src-KD (Fig. 4A, c-Src-KD, +LMB, right), suggesting a direct role of Src kinase activity in Dok1 localization. Indeed, and as expected, Src, but not the KD form, induced a robust Dok1 tyrosine phosphorylation (Fig. 4B). GFP-pY-Dok1, of about 89 kDa in size, was clearly distinct in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) from the 60-kDa pY-Src. Taken together, these results indicate that Src-mediated tyrosine phosphorylation of Dok1 interferes with its nuclear accumulation induced by the CRM1 inhibitor LMB.

Next, we coexpressed the constitutive nuclear GFP-Dok1-NES3 mutant with Src kinase or Src-KD and examined its subcellular localization following LMB treatment. As shown in Fig. 4B, active Src kinase, but not Src-KD, induced tyrosine phosphorylation of GFP-Dok1-NES3 at a level similar to that of GFP-Dok1. Surprisingly, GFP-Dok1-NES3 was no longer found in the nucleus when it was coexpressed with active Src but was distributed in the cytoplasm and colocalized with Src (Fig. 4A, bottom, c-Src). In contrast, GFP-Dok1-NES3 expressed in cells without Src or coexpressed with Src-KD remained in the nucleus following LMB treatment (Fig. 4A, bottom). Thus, tyrosine phosphorylation of GFP-Dok1-NES may interfere with its entry into the nucleus or, alternatively, induce its translocation from the nucleus to the cytoplasm.

The abolition by Src of the nuclear shuttling of GFP-Dok1 in the presence of LMB may result from the lack of affinity of nuclear import transporters for tyrosine-phosphorylated Dok1, leading to the accumulation of tyrosine-phosphorylated GFP-Dok1 (GFP-pY-Dok1) in the cytoplasm. Alternatively, pY-Dok1 may effectively enter the nucleus, and Src-mediated tyrosine phosphorylation may activate a CRM1-independent nuclear exporter pathway leading to cytoplasmic localization of Dok1. In this case, the reentry of pY-Dok1 into the nucleus would become insensitive to LMB treatment. Since Src has never been detected in the nucleus in these assays, we believe that Src-mediated cytoplasmic localization of Dok1 or Dok1-NES3 resulted from its inability to enter the nucleus rather than a CRM1-independent nuclear exclusion pathway.

The role of tyrosine phosphorylation in the cytoplasmic localization of Dok1 was further investigated by using PP2, a specific inhibitor of Src tyrosine kinase family members (11). Dok1 was found as a diffuse cytoplasmic protein with increased staining in the nucleus in PP2-treated MEF compared to untreated control cells (Fig. 4C). Similar observations were made in SYF cells harboring inactivated Src, and its family members Yes and Fyn (Fig. 4D, 0 min). As in wild-type MEF, LMB blocked Dok1 in the nucleus of PP2-treated and SYF-treated cells (Fig. 4C and D). However, in SYF-treated cells, Dok1 moved faster into the nucleus following LMB exposure in comparison to wild-type MEF. While Dok1 is a still diffuse cytoplasmic protein in wild-type MEF 15 min after LMB treatment, most Dok1 became localized in the nucleus in treated SYF cells at the same time of exposure (Fig. 4D). Complete translocation into the nucleus occurred after 30 and 60 min in both cases, although a faint cytoplasmic staining was observed in MEF at 30 min (Fig. 4D).

It is worth noting that LMB, a strong inhibitor of CRM1-mediated nuclear export, induced a stronger and sustained nuclear localization of Dok1 compared to the effects of PP2 or in SYF cells (compare Fig. 1A and C, Fig. 4A, and Fig. 4C –LMB and +LMB). This suggests the involvement of additional kinases or cellular factors in preventing Dok1 nuclear localization.

Taken together, these data strongly support the notion that entry of Dok1 into the nucleus is negatively regulated by tyrosine phosphorylation mediated in part by Src tyrosine kinase. Other tyrosine kinases may contribute to the cytoplasmic/membrane retention of Dok1, since p210Bcr-Abl had similar effects to Src, albeit to a lesser extent (N. Niu, N. Foisella, W. Dong, and B. S. Sylla, unpublished data).

To map the tyrosine residues whose phosphorylation by Src is important for keeping Dok1 in the cytoplasm, we took advantage of the finding by Shah and Shokat (39) that Y295, Y361, and Y450 of mDok1 (equivalent to Y296, Y362, and Y449 of hDok1) are direct phosphorylation sites of v-Src. We generated a human GFP-Dok1 mutant (Y296F, Y362F, and Y449F; GFP-Dok1-YF; equivalent to mDok1 Y295F, Y361F, and Y450F) and examined the effects of Src on its subcellular localization in the presence or absence of LMB (Fig. 5A). As expected, active Src, but not Src-KD, induced a robust tyrosine phosphorylation of the wild type. However, phosphorylation of Dok1-YF occurred relatively less, confirming that Y296, Y362, and Y449 are phosphorylation sites for Src (Fig. 5B). Without LMB stimulation, Dok1-YF alone or coexpressed with Src or Src-KD remained in the cytoplasm, indicating that converting the indicated Y residues to F did not significantly affect the cytoplasmic localization of Dok1 (Fig. 5, –LMB). This suggests that phosphorylation of other tyrosine residues plays a role in maintaining Dok1 in the cytoplasm. Interestingly, following exposure to LMB, in about 60% of cells coexpressing GFP-Dok1-YF Src and Src, GFP-Dok1-YF translocated to the nucleus (like wild-type Dok1 exposed to LMB in the absence of Src, or coexpressed with Src-KD) (Fig. 5 and data not shown). Knowing that Src prevented the entry of wild-type Dok1 into the nucleus in the presence of LMB in all of the transfected cells and that all Dok1-YF accumulated in the nucleus of cells cotransfected with Src-KD, these data strongly indicate that phosphorylation of Y296, Y362, and Y449 of human Dok1 contributes to keeping Dok1 in the cytoplasm. However, not all cells coexpressing Dok1-YF and Src showed nuclear staining of Dok1 in the presence of LMB, suggesting the potential contribution of additional tyrosine residues. The possibility that the GFP-Dok1-YF mutant is leaky cannot be excluded.


Figure 5
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  		FIG. 5. Effects of Src on subcellular localization of Dok1 tyrosine mutant. (A) Subcellular localization of Dok1 tyrosine mutant with (+) and without (–) LMB exposure. GFP-Dok1-WT and GFP-Dok1-YF (Y296F, Y362F, and Y449F) were transfected into 293T cells, and immunofluorescence was performed as described in the legend to Fig. 4A. Arrows and arrowheads indicate cells coexpressing Dok1 and Src, respectively. Dok1-WT and Dok1-YF remained in the cytoplasm in the absence of LMB (see arrows, –LMB). In LMB-stimulated cells cotransfected with Dok1-YF and Src, 60% of cells show nuclear localization of Dok1-YF with LMB treatment (arrows, +LMB), while 40% of them show cytoplasmic staining (arrowheads, +LMB). In contrast, Dok1-YF and Dok1-WT are nuclear in all cells treated with LMB when cotransfected with Src-KD (see arrows, +LMB). (B) Src induces tyrosine phosphorylation of Dok1 and Dok1-YF. Equal amounts of protein extracts from HEK 293T transfected with the indicated expression plasmids were analyzed by immunoblotting using anti-pTyr, anti-Dok1, and anti-c-Src antibodies. Dok1-YF is less tyrosine phosphorylated than wild-type (WT) Dok1.

Thus, Src negatively regulates the entry of Dok1 into the nucleus by tyrosine phosphorylation which is partially mediated by phosphorylation of Y296, Y362, and Y449.

IKKß is required for nuclear export of Dok1. We showed recently that IKK-mediated serine phosphorylation of Dok1 is important in modulating Dok1 activities downstream of tyrosine kinase (21). Since both IKK{alpha} and IKKß induce Dok1 serine phosphorylation, we examined the direct effect of IKK on the subcellular localization of endogenous Dok1 in IKK{alpha}- and IKKß-null MEF. While in the absence of LMB Dok1 is mainly a cytoplasmic protein in wild-type MEF, a substantial amount of Dok1 was concentrated in the nucleus of IKK{alpha}/ß-double null MEF (Fig. 6A), suggesting a role of the IKK complex in the cytoplasmic localization of Dok1. However, not all Dok1 accumulated in the nucleus of IKK{alpha}/ß-double null cells, suggesting that kinases or cellular factors other than IKK may also contribute to the cytoplasmic localization of Dok1. Indeed, LMB treatment induced the translocation of most, if not all, of the Dok1 protein in the nucleus in both wild-type and IKK-null MEF (Fig. 6A). To determine the specific contributions of IKK{alpha} and IKKß, the subcellular distribution of Dok1 was examined in IKK{alpha}- or IKKß-single-null MEF. Although LMB induced nuclear concentration of Dok1 in all of these cell types with similar efficiency, different patterns of distribution were observed in untreated cells (Fig. 6A). Indeed, the cytoplasmic localization of Dok1 in IKK{alpha}-single-null cells was similar to that of Dok1 in wild-type MEF (Fig. 6A). However, as in IKK{alpha}/ß-double-null cells, Dok1 was found predominantly in the nucleus of IKKß-single-null cells (Fig. 6A), suggesting a role of IKKß in the nuclear export of Dok1. Immunoblotting analyses confirmed the lack of IKK{alpha} in IKK{alpha}-null cells, IKKß in IKKß-null cells and both IKK{alpha} and IKKß in IK{alpha}/ß-null cells (Fig. 6B). In contrast to the effect of Src tyrosine kinase, the LMB-mediated nuclear translocation was not lost when Dok1 was coexpressed with IKKß (data not shown), suggesting that IKK, particularly IKKß, may be involved in the nuclear export of Dok1 rather than in its nuclear entry.


Figure 6
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  		FIG. 6. IKKß-dependent cytoplasmic localization of Dok1. (A) Subcellular localization of endogenous Dok1 in wild-type (WT), IKK{alpha}/ß-null-, IKK{alpha}-null-, and IKKß-null MEF. Cells were treated (+) or not treated (–) with LMB for 3 h, and Dok1 was visualized using rabbit anti-Dok1 antibody. (B) Protein extracts from indicated MEF lines were analyzed by immunoblotting.

In a recent study, we reported that S439, S443, S446, and S450 of human Dok1 are targets of phosphorylation by IKKß and that this phosphorylation can modulate Dok1 functions (21). We therefore evaluated the potential contribution of these IKKß phosphorylation sites to Dok1 nuclear export. We found that the Dok1 mutant (S439A, S443A, S446A, and S450A) had a cytoplasmic localization similarly to wild-type Dok1 and also translocated to the nucleus following exposure to LMB (data not shown). Therefore, phosphorylation of S439, S443, S446, and S450 by IKKß appears not to be important for cytoplasmic localization of Dok1. IKKß-mediated phosphorylation of additional serine residues may be required for nuclear export of Dok1.

Nuclear-cytoplasmic shuttling of Dok1 in response to serum, growth factor, and cell adhesion. To obtain insight into the physiological relevance of nuclear-cytoplasmic shuttling of Dok1, we analyzed the effects of serum, PDGF, and cell adhesion to a substrate on the subcellular localization of Dok1 in murine fibroblast lines. Under basal cell culture conditions in D10 medium containing 10% of serum, Dok1 is predominantly present in the cytoplasm (Fig. 7A, control panel and previous data). However, when cells were maintained under conditions of serum starvation for 27 h, Dok1 was less compartmentalized in the cytoplasm, becoming more diffuse with a significant nuclear localization in a proportion of the cells (Fig. 7A, middle panel). Remarkably, addition of serum to the medium for 2 h led to relocalization of most, if not all, Dok1 in the cytoplasm (Fig. 7A, right panel). Taken together, these data strongly indicate that critical components of serum (such as growth factors) regulate the subcellular localization of Dok1. To explore these effects further, we examined the effect of PDGF on Dok1 subcellular localization. PDGF has been reported to induce tyrosine phosphorylation of Dok1 and triggers translocation of Dok1 to the plasma membrane (55). Stimulation of serum-starved cells (where Dok1 has prominent nuclear staining) with PDGF at various time intervals resulted in cytoplasmic localization 15 min after growth factor exposure (Fig. 7B). Dok1 reverted to the normal diffuse staining 30 and 60 min after stimulation, probably due to the transient action of PDGF (Fig. 7B). Thus, growth factors such as PDGF play a role in regulating the shuttling of Dok1 between the nucleus and cytoplasm.


Figure 7
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  		FIG. 7. Normal physiological conditions modulate Dok1 subcellular localization. MEF were submitted to various treatments, followed by immunofluorescence using anti-Dok1 antibody to detect endogenous Dok1 localization. MEF in routine culture conditions with 10% of serum are shown as a control on the far left in panels A, B, and C. (A) Middle panel, cells were seeded onto coverslips for 24 h and then serum starved in D0 for 27 h; right panel, serum-starved cells were stimulated with 20% serum for 2 h. (B) Cells seeded onto coverslips were starved in D0 for 72 h and then stimulated by PDGF (40 ng/ml) at 37°C at the indicated time points. (C) Cells were trypsinized, washed, and maintained in suspension in D10 for 3 h at 37°C or seeded onto fibronectin (1 µg/ml)-coated coverslips and incubated at 37°C for 3 h.

Another factor which has been reported to play a role in Dok1 signaling is cell adhesion. Cell adhesion to extracellular matrix (ECM) proteins induces tyrosine phosphorylation of a number of proteins including focal adhesion kinase, paxillin, and p130 Cas (36). Concentration of such proteins at focal contacts is known to play an important role in modulating cell attachment, cell spreading, and cell motility (28). Noguchi et al. have reported that cell adhesion to ECM induces Src-mediated tyrosine phosphorylation of Dok1 (33). We therefore examined the effects of cell adhesion on the subcellular localization of Dok1. When detached cells were maintained in suspension, they had a round shape and Dok1 was present throughout the cell, with a substantial staining in the nucleus (Fig. 7C, middle panel). When cells were replated on fibronectin-coated dishes, they adhered and spread within 20 min and became fully spread in about 1 h. In these attached cells, Dok1 shifted almost entirely into the cytoplasm (Fig. 7C, right panel), indicating that external stimuli such as adhesion to ECM can induce movement of Dok1 into the cytoplasm and probably to the plasma membrane as well.

Thus, the overall results demonstrate that cytoplasmic/nuclear shuttling of Dok1 occurs under physiological conditions, with extracellular factors such as serum, growth factors, or attachment to a substrate playing an important modulating role. As PDGF or cell adhesion induce tyrosine phosphorylation of Dok1, these observations are also consistent with our findings on the role of Src in keeping Dok1 in the cytoplasm.

The nuclear Dok1 NES mutant has impaired ability to inhibit cell proliferation and to promote cell spreading and cell motility. Dok1 inhibits cell proliferation and induces cell spreading and cell migration (21, 27, 32, 33, 53). To examine the effect of the mislocalization of Dok1 on cell proliferation, we stably expressed Dok1 and Dok1-NES3 in HEK 293 cells (Fig. 3E). As previously reported, overexpressed wild-type Dok1 inhibited cell proliferation in the presence of serum which contains growth factors (Fig. 8A). In contrast to wild-type Dok1, no inhibition of cell proliferation was observed in cells expressing a comparable level of Dok1-NES3 (Fig. 8A). However, it has been reported that effects of Dok1 in promoting cell migration can occur without serum or external stimulation, but addition of insulin increases the migration effects of Dok1 (33).


Figure 8
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  		FIG. 8. Impaired ability of Dok1-NES3 mutant to inhibit cell proliferation and to promote cell spreading and cell motility. (A) HEK 293 stable transfectant cells containing the plasmid vector (V) or constitutively expressing wild-type Dok1 (WT) and Dok1-NES3 (NES3) were monitored for cell proliferation. Data are representative of results from three independent experiments performed in duplicate. Similar results were also obtained from another independent set of HEK 293 stable transfectant clones (data not shown). Levels of Dok1 expression were determined by immunoblotting. (B) Impaired ability of Dok1-NES3 to induce cell spreading. Swiss 3T3 cells were transfected with plasmids encoding GFP-Dok1 or GFP-Dok1-NES3. Twenty-four hours later, cells were trypsinized and reseeded onto collagen-coated coverslips. Four hours later, cells were observed for fluorescence and phase contrast. (C) Quantification of spread cells expressing wild-type and NES mutant Dok1. Percentages of spread cells were determined by counting a total of 600 cells per sample, performed by two independent investigators. *, P < 0.05. (D) Cell migration based on the cell filling of the wound area by using METAMORPH for HEK 293 stable transfectant cells containing the plasmid vector (Vector) or constitutively expressing wild-type Dok1 (Dok1-WT) and Dok1-NES3 (Dok1-NES3) was evaluated. (E) The percentage of wound filling at 6 h is plotted. Data are means ± standard errors of results from three independent experiments carried out in triplicate. *, P < 0.05.

We next examined the effects of Dok1 mislocalization in the nucleus on cell spreading and cell migration. While the presence of Dok1 in the cytoplasmic and membrane compartments promoted cell spreading as expected, this effect was significantly restricted with Dok1-NES3 (Fig. 8B and C). In agreement with these observations, HEK 293 cells stably expressing wild-type Dok1 were flat and spread out, whereas Dok1-NES3-expressing cells exhibited a rounded shape (Fig. 3E and 8B). In addition, Dok1-NES3 is altered in Dok1 functions to promote cell migration (Fig. 8D and E). Although we cannot exclude a possible inhibitory effect per se of NES3 mutation on Dok1, the results confirm that the cytoplasmic localization of Dok1 is important for its capacity to down-regulate cell proliferation and in promoting cell spreading and cell motility.

During this study, we noticed consistently that IKK{alpha}/ß-double-null and IKKß-single-null MEF showed a larger size than wild-type or IKK{alpha}-null cells (Fig. 6), implying a potential role of IKKß in the control of cellular shape.


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DISCUSSION
 
Our data demonstrate for the first time that Dok1, a cytoplasmic/membrane adaptor protein with tumor-suppressive activity, shuttles between the cytoplasm and nucleus. LMB treatment of cells expressing endogenous or exogenous forms of Dok1 induced accumulation of Dok1 in the nucleus, suggesting that a CRM1-dependent pathway mediated Dok1 export from the nucleus. It is established that nuclear export can occur via recognition of an NES motif by the exporter protein CRM1. Mutagenesis studies indicated that the cytoplasmic localization of Dok1 depends mainly on the NES motif, located at aa 348 to 359. Mutating two leucine residues in the NES3 motif of Dok1 resulted in a constitutive nuclear localization of Dok1. In addition, CRM1 bound to wild-type Dok1 but to a lesser extent to Dok1-NES mutants. Thus, the cytoplasmic localization of Dok1 is highly dependent on the presence of a strongly functional NES.

The cellular functions of Dok1 are mainly regulated by tyrosine phosphorylation (24, 33, 49, 55). However, the link between Dok1 tyrosine phosphorylation and its subcellular localization has not been previously addressed. We have shown that expression of Src kinase with Dok1 completely prevents the LMB-mediated accumulation of Dok1 in the nucleus. In addition, constitutive nuclear Dok1-NES3 showed cytoplasmic localization in the presence of Src. This process is directly mediated by the tyrosine kinase activity of Src, since inactive Src had no effect on this localization. Others kinases such as p210Bcr-Abl may also be involved in this process. Thus, the Src kinase activity interferes with entry of Dok1 into the nucleus. This conclusion is supported by our observation that a significant proportion of Dok1 is found in the nucleus of cells treated with Src kinase inhibitor PP2 or cells lacking Src kinase. As Src appears to be a major tyrosine kinase for Dok1 (21, 33) and is a regulatory kinase which modulates the nuclear-cytoplasmic localization of a number of proteins (25), we believe that Src is the tyrosine kinase that contributes most to maintaining Dok1 in the cytoplasm.

Our findings on the role of Src in the cytoplasmic localization of Dok1 are physiologically relevant and important. Indeed, normal cellular growth conditions in the serum, exposure to growth factors, or adhesion to ECM proteins induce Dok1 to localize predominantly in the cytoplasm (Fig. 7). Interestingly, these external stimuli induce tyrosine phosphorylation of Dok1, mediated mainly by Src tyrosine kinase family members (33). Others tyrosine kinases might also be required. Thus, exposure of cells to growth factors or cell adhesion leads to Src-mediated tyrosine phosphorylation of Dok1, with subsequent inhibition of its entry into the nucleus and, thus, accumulation of Dok1 in the cytoplasm and membrane. Phosphorylation of Dok1 Y296, Y362, and Y449 by Src partially contributes to this process.

In addition to its regulation by tyrosine phosphorylation, Dok1 is also a substrate of IKK (21), a master kinase complex required for NF-{kappa}B activation (17). While Dok1 is mainly cytoplasmic in wild-type MEF and in IKK{alpha}-null cells, it was found mostly in the nucleus of IKK{alpha}/ß-double-null and IKKß-single-null cells, indicating the direct role of IKK ß in the cytoplasmic localization of Dok1. In contrast to c-Src effects, coexpression of Dok1 with IKKß did not prevent nuclear accumulation of Dok1 after LMB treatment, suggesting that IKKß plays a major role in the nuclear export of Dok1. Phosphorylation of S439, S443, S446, and S450 of Dok1, previously reported as IKKß phosphorylation sites (21), appears not to play a direct role in the cytoplasmic localization of Dok1, indicating a potential contribution of other unidentified serine residues. The incomplete localization of Dok1 in the nucleus of IKK-null cells points, however, to the possible existence of an IKK-independent pathway contributing to its cytoplasmic localization.

Taken together, our data reveal a mechanism by which subcellular localization and functions of Dok1 are regulated in the cell. Following its synthesis, Dok1 moves into the nucleus but is rapidly exported to the cytoplasm by the nuclear exporter protein CRM1. Src-mediated tyrosine phosphorylation, induced by growth factors or adhesion to a substrate, interferes with entry of Dok1 into the nucleus, while IKKß is involved in its export. It is not yet clear whether the kinase activity of IKKß is required for its effect on the nuclear export of Dok1. Phosphorylation of Dok1 may reduce its interaction with nuclear importin factors and/or facilitate its interaction with other cytoplasmic and membrane proteins. Thus, tyrosine kinases such as Src and serine kinases such as IKKß are required for efficient maintenance of Dok1 in the cytoplasm/membrane compartments. The domain of Dok1 responsible for its entry into the nucleus has yet to be determined. A small stretch of basic residue (RKK) at position 332 of hDok1 that might function as an NLS has been identified. Mutagenesis studies indicated, however, that this putative NLS probably does not contribute to the process, suggesting that other nonclassical NLSs may be involved (data not shown).

Our findings from functional studies of the nuclear Dok1-NES3 mutant support the hypothesis that cytoplasmic/membrane localization of Dok1 is important for functions attributed to Dok1 downstream of tyrosine kinase signaling. Constitutive nuclear Dok1-NES3 had impaired capacity to inhibit cell proliferation and to promote cell spreading and cell motility. Thus, Dok1 needs to be exported from the nucleus into the cytoplasm and membrane compartments, where it participates in signaling that regulates cell proliferation, cell spreading, and cell motility. In addition, Dok1 may have suppressive activity when present in the nucleus. As Dok1 (like Dok2) acts as a tumor suppressor (32, 54), it is tempting to speculate from our present and previous results (22) that mislocalization of Dok1 may be a mechanism leading to inactivation of its tumor suppressor activity. Thus, the possibility cannot be excluded that Dok1 might be found constitutively present in the nucleus in some tumor cells (either by mutation or misregulation). This scenario is not in complete contradiction with the oncogenic properties of v-Src or BCR-ABL. In fact, it is also possible that in selected human tumor samples Dok1 is inactivated by mutation and subsequent nuclear localization, which may contribute to tumor formation independent of activated Src or BCR. Due to conformational change or mutations, this nuclear Dok1 may not be a good substrate for Src or BCR. Indeed, we have recently reported a truncated Dok1 mutant in chronic lymphocytic leukemia, that was found exclusively in the nucleus. Interestingly, this Dok1 mutant was not phosphorylable by either Src or BCR-ABL (22). Thus, inactivation of Dok1 in some tumor samples can contribute to the process of oncogenesis by a pathway that does not necessarily require Src or BCR-ABL.

In conclusion, the CRM1 pathway is an important regulator of the subcellular localization of Dok1 mediated by tyrosine phosphorylation that is modulated by external stimuli. Further research is needed to identify additional factors involved in this regulation and to determine whether Dok1 has a direct role, if any, in the nucleus.


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ACKNOWLEDGMENTS
 
We thank J. Hall, E. van Dyck, Z.-Q. Wang, P. Hainaut, and V. Krutovskikh for advice and critical reading of the manuscript. We thank J. A. Diehl, R. Kobayashi, J. Cambier, D. Goeddel, M. Yoshida, I. Verma, G. Grosveld, A.-M. Pendergast, and A. August for reagents and J. Michelon and A. Burzynska for technical assistance. We are also grateful to J. Cheney for editing and G. Mollon for illustrations.

This work was partially supported by the Association pour la Recherche sur le Cancer and La Ligue Contre le Cancer, Comité du Rhône, Lyon, and Comité de la Drôme, Valence (to B.S.S.), and by a fellowship from the Ministère de l'Education Nationale de la Recherche et de la Technologie (to F.S.).


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FOOTNOTES
 
* Corresponding author. Mailing address: Infections and Cancer Biology Group, International Agency for Research on Cancer, 150 cours Albert-Thomas, 69008 Lyon, France. Phone: 33 4 72 73 80 96. Fax: 33 4 72 73 84 42. E-mail: sylla{at}iarc.fr. Back

{dagger} These authors made equal contributions to the work. Back

{ddagger} Present address: NUCLEIS, 69008 Lyon, France. Back

§ Present address: CMU-Centre Médicale Universitaire, 1211 Geneva 4, Switzerland. Back

Present address: INSERM U450, Institut Biomédical des Cordeliers, 75006 Paris, France. Back

|| Present address: BioMérieux, 69280 Marcy l'Etoile, France. Back


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Molecular and Cellular Biology, June 2006, p. 4288-4301, Vol. 26, No. 11
0270-7306/06/$08.00+0     doi:10.1128/MCB.01817-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.




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