Phosphorylation of myosin light chain kinase by p21-activated kinase PAK2.
Phosphorylation of myosin II regulatory light chains (RLC) by Ca(2+)/calmodulin-dependent myosin light chain kinase (MLCK) is a critical step in the initiation of smooth muscle and non-muscle cell contraction. Post-translational modifications to MLCK down-regulate enzyme activity, suppressing RLC phosphorylation, myosin II activation, and tension development. Here we report that 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 placenta and recombinant, constitutively active PAK2 phosphorylate MLCK in vitro with a stoichiometry of 1.71 +/- 0. 21 mol of PO(4)/mol of MLCK. This phosphorylation inhibits MLCK phosphorylation of myosin II RLC. PAK2 catalyzes MLCK phosphorylation on serine residues 439 and 991. Binding calmodulin to MLCK blocks phosphorylation of Ser-991 by PAK2. These results demonstrate that PAK2 can directly phosphorylate MLCK, inhibiting its activity and limiting the development of isometric tension.
Goeckeler
Z M
ZM
Departments of Pathology and Anesthesiology, St. Louis University School of Medicine, St. Louis, Missouri 63104-1028, USA.
Masaracchia
R A
RA
Zeng
Q
Q
Chew
T L
TL
Gallagher
P
P
Wysolmerski
R B
RB
D002118:Calcium / Q000378:metabolism; D002147:Calmodulin / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004727:Endothelium / Q000201:enzymology; D006367:HeLa Cells; D006801:Humans; D016297:Mutagenesis, Site-Directed; D009219:Myosin-Light-Chain Kinase / Q000378:metabolism*; D010449:Peptide Mapping; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D054462:p21-Activated Kinases
D002147:Calmodulin; C515907:PAK2 protein, human; D017346:Protein Serine-Threonine Kinases; D054462:p21-Activated Kinases; D009219:Myosin-Light-Chain Kinase; D002118:Calcium