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 (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 IKKβ. 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.

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.).