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

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 ( 348 LLKAKLTDPKED 359 ) 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 IKK␤. 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. 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 Xlinked 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)(52)(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 malig...

BRK Targets Dok1 for Ubiquitin-Mediated Proteasomal Degradation to Promote Cell Proliferation and Migration

“…A proportion of both proteins also localize in the nucleus [29], [44]. Others and we reported that BRK phosphorylates nuclear Sam68 and promotes its subcellular relocalization [29], [49].…”

Cutaneous Human Papillomavirus Type 38 E7 Regulates Actin Cytoskeleton Structure for Increasing Cell Proliferation through CK2 and the Eukaryotic Elongation Factor 1A

“…Immunoprecipitation and immunoblotting were performed as previously described (46). Briefly, cells were lysed in a lysis buffer containing 50 mM Tris-Cl (pH 7.4), 0.5% Nonidet P-40, 150 mM NaCl, 2 mM EDTA, 3% glycerol, and a Halt protease and phosphatase inhibitor cocktail (Pierce Biotechnology).…”

“…Immunofluorescence was performed as described previously, with minor modifications (46). Briefly, cells on cover slides were fixed in 4% formaldehyde at room temperature for 15 min and then permeabilized with 0.5% Triton X-100 in phosphate-buffered saline (PBS) for 20 min.…”

The Ras Inhibitors Caveolin-1 and Docking Protein 1 Activate Peroxisome Proliferator-Activated Receptor γ through Spatial Relocalization at Helix 7 of Its Ligand-Binding Domain

“…Dok1 shuttles between the plasma membrane and the nucleus in response to mitogens (43), similar to MEK1, by means of a functional NES. When Dok1 is present, it may facilitate release of PPAR␥ from its sequestering proteins MEK1 and Cav1 and, upon binding to ligand, promote its nuclear translocation.…”