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, 26 (11), 4288-301

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

Affiliations

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

Yamei Niu et al. Mol Cell Biol.

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 IKKbeta. 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.

Figures

FIG. 1.
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 × 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-β-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.
FIG. 2.
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.
FIG. 3.
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-β-tubulin, and anti-PARP. (G) Dok1-CRM1 interactions in 293 stable transfectants. Cells (6 × 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.
FIG. 4.
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.
FIG. 5.
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.
FIG. 6.
FIG. 6.
IKKβ-dependent cytoplasmic localization of Dok1. (A) Subcellular localization of endogenous Dok1 in wild-type (WT), IKKα/β-null-, IKKα-null-, and IKKβ-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.
FIG. 7.
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.
FIG. 8.
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.

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