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.