In nonmuscle cells, myosin mediates several functions including cytokinesis, granular secretion and shape change. Previous studies have shown that protein kinase C (PKC) can phosphorylate the 20 Kd myosin light chain as well as a single serine residue (serine-1915) in the rod portion of the MHC-A isoform found in human platelets, rat basophil leukemic cells and chicken intestinal epithelial cells. It is also reported that PKC phosphorylates brain myosin in the globular head region. The goal of this project is to examine whether 1) myosin heavy chain B (MHC-B) isoform(s) found in vertebrate brain can serve as substrates for PKC; 2) to analyze the site(s) of phosphorylation to see whether any of the site(s) are located in the head region of the MHC-B; 3) to compare these results with those from human lymphocytes, i.e., Jurkat T-cell and CD16 lymphocytes (CD16 is a marker for natural killer cells which, following stimulation with IL-2, become lymphokine- activated); 4) and to determine the functional significance of this phosphorylation. We found that 1) PKC catalyzes the phosphorylation of purified bovine brain myosin heavy chains and light chains. Similar results were also obtained in both 32-P-labeled Jurkat T-cells and CD16 cells following phorbol ester and IL-2 treatment, respectively; 2) isoelectric focusing of the tryptic phosphopeptides generated from the brain myosin heavy chain revealed multiple sites of phosphorylation. This is in contrast to the myosin heavy chain-A isoform isolated from human lymphocytes, which showed only a single phosphorylated peptide following PKC activation; 3) when the MHC-B isoforms present in bovine brain myosin are separated by SDS-PAGE following phosphorylation in vitro by protein kinase C, two distinct phosphopeptide maps are obtained, suggesting that additional sites are phosphorylated in the slower migrating MHC-B.
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