Phosphorylation of myosin light chain kinase by p21-activated kinase PAK2

Phosphorylation of myosin II regulatory light chains (RLC) by Ca2+/calmodul in-dependent myosin light chain kinase (MLCK) is a critical step in the ini tiation of smooth muscle and non-muscle cell contraction. Posttranslational modifications to MLCK down-regulate enzyme activity suppressing RLC phosph orylation, myosin II activation, and tension development. Here we report th at PAK2, a member of the Rho family of GTPase-dependent kinases, regulates isometric tension development and myosin II RLC phosphorylation in saponin permeabilized endothelial monolayers. PAK2 blunts tension development by 75 % while inhibiting diphosphorylation of myosin II RLC. Cdc42-activated plac enta and recombinant, constitutively active PAK2 phosphorylate MLCK in vitr o with a stoichiometry of 1.71 +/- 0.21 mol of PO4/mol of MLCK. This phosph orylation inhibits MLCK phosphorylation of myosin II RLC. PAK2 catalyzes ML CK phosphorylation on serine residues 439 and 991. Binding calmodulin to ML CK blocks phosphorylation of Ser-991 by PAK2. These results demonstrate tha t PAK2 can directly phosphorylate MLCK; inhibiting its activity and limitin g the development of isometric tension.