Protein kinase C, was used to phosphorylate the calmodulin-dependent enzyme, myosin light chain kinase. Phosphorylation of turkey gizzard myosin light chain kinase, which had calmodulin bound to it, by human platelet protein kinase C, resulted in the incorporation of 1 mole of phosphate per mole of myosin light chain kinase. This phosphorylation had no effect on the activity of myosin light chain kinase. An exhaustive tryptic digestion of the monophosphorylated myosin light chain kinase resulted in a single peptide that occupied a unique position following two-dimensional peptide mapping. This peptide did not co-chromatograph with the peptide that was phosphorylated by cyclic AMP-dependent protein kinase when calmoculin was bound to myosin light chain kinase. When calmodulin was no bound to myosin light chain kinase, protein kinase C incorporated two moles of phosphate per mole of myosin light chain kinase. Diphosphorylation of myosin light chain kinase decreased the activity of the enzyme by decreasing its ability to bind calmodulin. Two-dimensional peptide maps revealed that the additional site that was phosphorylated by protein kinase C in the absence of bound calmodulin was the same as the site that was phosphorylated by cyclic AMP-dependent protein kinase in the absence of bound calmodulin. Myosin and the enzyme, myosin light chain kinase, are being studied in extracts prepared from intact rat aorta as well as rat aortic cells which are being cultured in vitro. Preliminary experiments using nitrocellulose blots transferred from SDS-polyacrylamide gels indicate that antibodies raised to smooth muscle myosin appear to have a greater affinity for myosin extracted from the intact aorta than they do for myosin extracted from cultured smooth muscle cells.
