Phosphorylation of non-muscle myosin II regulatory light chain by p21-activated kinase (gamma-PAK)

Myosin regulatory light chain (RLC) phosphorylation has been implicated in Rho-mediated stress fibre formation. The recent observation that Rho kinase phosphorylates RLC in vitro suggests that serine/threonine kinases other t han those in the myosin light chain kinase (MLCK) family have the potential to activate myosin II. In this study we report that gamma-PAK, which is ac tivated by the GTP-binding proteins Cdc42 and Rac, catalyses phosphorylatio n of intact non-muscle myosin II and isolated recombinant RLC. gamma-PAK ph osphorylated endothelial cell myosin II to 0.85 +/- 0.02 mol PO4 per mol RL C. Phosphorylation is Ca2+/calmodulin-independent and the enzyme has a K-m and V-max for myosin II regulatory light chain of 12 mu M and 180 nmol/min/ mg respectively. No myosin II heavy chain phosphorylation was detected. Pho sphopeptide maps and phosphoamino acid analysis revealed that gamma-PAK pho sphorylates Ser-19 but does not phosphorylate Thr-18. A panel of recombinan t RLC mutants was used to confirm that Ser-19 is the only phosphorylation s ite modified by gamma-PAK. On substitution of both Ser-19 and Thr-18 with A la or Glu, no phosphorylation of other Ser/Thr residues in the RLC was dete cted. Similar to MLCK, Arg-16 is required for interaction of gamma-PAK with the substrate, since converting Arg-16 to Ala significantly reduced RLC ph osphorylation. Endothelial cell monolayers permeabilized with saponin retra ct upon exposure to either Cdc42 or trypsin-activated gamma-PAK and ATP. Ac tivation of gamma-PAK is required to initiate Ca2+/calmodulin-independent c ell retraction and actin rearrangement. Taken together, these data suggest that myosin II activation by the p21-activated family of kinases may be phy siologically important in regulating cytoskeletal organization. (C) Kluwer Academic Publishers.