Identification of a Phosphorylation Site on Skeletal Muscle Myosin Light Chain Kinase That Becomes Phosphorylated during Muscle Contraction

A protein phosphorylated efficiently in vitro by MAP kinase-activated protein kinase-2 (MAPKAP-K2) was purified from skeletal muscle extracts and identified as the calcium/calmodulin-dependent myosin light chain kinase (MLCK). The phosphorylation site was mapped to Ser161, a residue shown previously to be autophosphorylated by MLCK. The residue equivalent to Ser161 became phosphorylated in vivo when rat hindlimbs were stimulated electrically. However, phosphorylation was triggered within seconds, whereas activation of MAPKAP-K2 required several minutes. Moreover, contraction-induced Ser161 phosphorylation was similar in wild-type or MAPKAP-K2−/− mice. These results indicate that contraction-induced phosphorylation is probably catalyzed by MLCK and not MAPKAP-K2. Ser161 phosphorylation induced the binding of MLCK to 14-3-3 proteins, but did not detectably affect the kinetic properties of MLCK. The sequence surrounding Ser161 is unusual in that residue 158 is histidine. Previously, an arginine located three residues N-terminal to the site of phosphorylation was thought to be critical for the specificity of MAPKAP-K2.