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 than 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 activated by the GTP-binding proteins Cdc42 and Rac, catalyses phosphorylation of intact non-muscle myosin II and isolated recombinant RLC. gamma-PAK phosphorylated endothelial cell myosin II to 0.85 +/- 0.02 mol PO4 per mol RLC. Phosphorylation is Ca2+/calmodulin-independent and the enzyme has a K(m) and Vmax for myosin II regulatory light chain of 12 microM and 180 nmol/min/mg respectively. No myosin II heavy chain phosphorylation was detected. Phosphopeptide maps and phosphoamino acid analysis revealed that gamma-PAK phosphorylates Ser-19 but does not phosphorylate Thr-18. A panel of recombinant RLC mutants was used to confirm that Ser-19 is the only phosphorylation site modified by gamma-PAK. On substitution of both Ser-19 and Thr-18 with Ala or Glu, no phosphorylation of other Ser/Thr residues in the RLC was detected. Similar to MLCK, Arg-16 is required for interaction of gamma-PAK with the substrate, since converting Arg-16 to Ala significantly reduced RLC phosphorylation. Endothelial cell monolayers permeabilized with saponin retract upon exposure to either Cdc42 or trypsin-activated gamma-PAK and ATP. Activation of gamma-PAK is required to initiate Ca2+/calmodulin-independent cell retraction and actin rearrangement. Taken together, these data suggest that myosin II activation by the p21-activated family of kinases may be physiologically important in regulating cytoskeletal organization.
SciCrunch is a data sharing and display platform. Anyone can create a custom portal where they can select searchable subsets of hundreds of data sources, brand their web pages and create their community. SciCrunch will push data updates automatically to all portals on a weekly basis. User communities can also add their own data to scicrunch, however this is not currently a free service.