Actin cytoskeleton remodeling is under the regulation of multiple proteins with various activities. Here, we demonstrate that the γ2 isoform of Casein Kinase I (CKIγ2) is part of a novel molecular path regulating the formation of actin stress fibers. We show that overexpression of CKIγ2 in fibroblasts alters cell morphology by impairing actin stress fibers formation. We demonstrate that this is concomitant with increased phosphorylation of the CDK inhibitor and lower levels of activated RhoA, and is dependent on CKIγ2 catalytic activity. Moreover, we report that roscovitine, a potent inhibitor of cyclin-dependent kinases, including Cdk5, decreases protein levels and restores actin stress fibers formation in CKIγ2 overexpressing cells, suggesting the existence of a CKIγ2-Cdk5- -RhoA pathway in regulating actin remodeling. On the other hand, we also show that in a manner independent of its catalytic activity, CKIγ2 delays cell cycle progression through G1. Collectively our findings reveal that CKIγ2 is a novel player in the control of actin cytoskeleton dynamics and cell proliferation. 1. Introduction The Rho family of GTPases comprising RhoA, Rac1, and Cdc42 regulates the organization of the cytoskeleton in eukaryotic cells [1]. These proteins cycle between an active GTP-bound and inactive GDP-bound state through the action of GTPase exchange factors (GEFs) and GTPase activating proteins (GAPs) [2]. Once activated, RhoA regulates actin stress fibers formation [3], while Rac1 triggers the assembly of actin in lamellipodia and membrane ruffles [4] and Cdc42 induces filopodial extensions [5] at the leading edge of the cell. Over the years, Rho GTPases were found to be crucial regulators of actin remodeling involved in a great deal of normal cellular functions, including cell migration and adhesion, cell cycle progression, and membrane trafficking [6]. In addition, Rho GTPases contribute to pathological conditions, particularly to cancer initiation and metastasis by controlling cell proliferation, migration, and adhesion during oncogenic transformation [7–9]. Accumulating evidence suggests that Rho GTPases are regulated at least in part by the cyclin-dependent kinase inhibitors (CDKIs) , , and through different mechanisms. As example, , which depends on its abundance and nuclear localization to inhibit the cyclin-dependent kinases (CDKs), inhibits RhoA activation in a cell-cycle independent manner, thereby modulates actin dynamics [10]. In fact, phosphorylation at Ser10 increases its stability and cytoplasmic localization [11, 12], where it binds to and
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