The role of phosphorylation in the regulatory mechanism of the Diaphanous‐related formins

Abstract

Diaphanous‐related formins (DRFs) are a conserved family of Rho GTPase effector proteins that coordinate the actin and microtubule cytoskeletal networks. This situates the DRFs in a key position to influence cellular shape, motility, and cytokinesis; therefore, it is critical that these proteins are tightly regulated. Normally, DRFs are maintained in an inactive state via intramolecular interactions and can be activated by the binding of Rho GTPases. However, recent studies of mammalian DRFs have demonstrated that Rho GTPase binding is necessary, but not sufficient for DRF activation. Based upon the hypothesis that a second activation ‘signal’ might be phosphorylation, we show here that the mouse DRF, mDia2, is phosphorylated by p21‐activated kinase 1 (PAK1). In vitro kinase assays using purified PAK1 and mDia2 mutants have identified a serine residue in mDia2 as the site of phosphorylation. The main activity of PAK1 is to coordinate extracellular signals with the reorganization of the actin cytoskeleton, and mDia2 phosphorylation may be one way this is accomplished. Currently, we are probing the role of PAK1 phosphorylation in mDia2 regulation by using fluorescence anisotropy to monitor mDia2 intramolecular binding. The cytoskeletal effects of PAK1‐mDia2 regulation, as well as the effects of PAK1 and Rho GTPases on mDia2‐mediated actin nucleation will provide insight into the mechanism of mDia2 regulation.