Endothelial cell retraction is induced by PAK2 monophosphorylation of myosin II

The p21-activated kinase (PAK) family includes several enzyme isoforms regu lated by the GTPases Rac1 and Cdc42, PAK1, found in brain, muscle and splee n, has been implicated in triggering cytoskeletal rearrangements such as th e dissolution of stress fibers and reorganization of focal complexes. The r ole of the more widely distributed PAK2 in controlling the cytoskeleton has been less well studied. Previous work has demonstrated that PAK2 can monop hosphorylate the myosin II regulatory light chain and induce retraction of permeabilized endothelial cells. In this report we characterize PAK2's morp hological and biochemical effect on intact endothelial cells utilizing micr oinjection of constitutively active PAK2 Under these conditions we observed a modification of the actin cytoskeleton with retraction of endothelial ce ll margins accompanied by an increase in monophosphorylation of myosin II. Selective inhibitors were used to analyze the mechanism of action of PAK2, Staurosporine, a direct inhibitor of PAK2, largely prevented the action of microinjected PAK2 in endothelial cells. Butanedione monoxime, a non-specif ic myosin ATPase inhibitor, also inhibited the effects of PAK2 implicating myosin in the changes in cytoskeletal reorganization. In contrast, KT5926, a specific inhibitor of myosin light chain kinase was ineffective in preven ting the changes in morphology and the actin cytoskeleton, The additional f inding that endogenous PAK2 associates with myosin II is consistent with th e proposal that cell retraction and cytoskeletal rearrangements induced by microinjected PAK2 depend on the direct activation of myosin II by PAK2 mon ophosphorylation of the regulatory light chain.