14-3-3 regulates actin dynamics by stabilizing phosphorylated cofilin

The functionality of the actin cytoskeleton depends on a dynamic equilibrium between filamentous and monomeric actin. Proteins of the ADF/cofilin family are essential for the high rates of actin filament turnover observed in motile cells through regulation of actin polymerization/depolymerization cycles [1, 2]. Rho GTPases act through p21-activated kinase-1 (Pak-1) [3] and Rho kinase [4] to inhibit cofilin activity via the LIM kinase (LIMK)-mediated phosphorylation of cofilin on Sera [5, 6]. We report the identification of 14-3-3zeta as a novel phosphocofilin binding protein involved in the maintenance of the cellular phosphocofilin pool. A Sera phosphocofilin binding protein was purified from bovine brain and was identified as 14-3-3zeta by mass spectrometry. The phosphorylation-dependent interaction between cofilin and 14-3-3zeta was confirmed in pulldown and coimmunoprecipitation experiments. Both Sera phosphorylation and a 14-3-3 recognition motif in cofilin are necessary for 14-3-3 binding. The expression of 14-3-3 increases phosphocofilin levels, and the coexpression of 14-3-3zeta with LIMK further elevates phosphocofilin levels and potentiates LIMK-dependent effects on the actin cytoskeleton. This potentiation of cofilin action appears to be a result of the protection of phosphocofilin from phosphatase-mediated dephosphorylation at Sera by bound 14-3-3zeta. Taken together, these results suggest that 14-3-3zeta proteins may play a dynamic role in the regulation of cellular actin structures through the maintenance of phosphocofilin levels.