Autophosphorylation of Skeletal Muscle Myosin Light Chain Kinase

Ca²⁺/calmodulin-dependent myosin light chain kinase phosphorylates the regulatory light chain of myosin. Rabbit skeletal muscle myosin light chain kinase also catalyzes a Ca²⁺/calmodulin-dependent autophosphorylation with a rapid rate of incorporation of 1 mol of ³²P/mol of kinase and a slower rate of incorporation up to 1.52 mol of ³²P/mol. Autophosphorylation was inhibited by a peptide substrate that has a low K_m value for myosin light chain kinase. Autophosphorylation at both rates was concentrationindependent, indicating an intramolecular mechanism. There were no significant changes in catalytic properties toward light chain and MgATP substrates or in calmodulin activation properties upon autophosphorylation. After digestion with V8 protease, phosphopeptides were purified and sequenced. Two phosphorylation sites were identified, Ser 160 and Ser 234, with the former associated with the rapid rate of phosphorylation. Both sites are located amino terminal of the catalytic domain. These results indicate that the extended “tail” region of the enzyme can fold into the active site of the kinase.