The Antibody Specific for Myristoylated Alanine-Rich C Kinase Substrate Phosphorylated by Protein Kinase C: Activation of Protein Kinase C in Smooth Muscle Cells in Human Coronary Arteries

Myristoylated alanine-rich C kinase substrate (MARCKS), a major substrate for protein kinase C, is distributed in a variety of cells. It has been reported that phosphorylation of MARCKS at serines 152 and 156 according to the numbering of rat brain MARCKS can be used as an indicator for protein kinase C activation in intact cells. To detect the activation of protein kinase Cin vivo,we produced a specific antibody against MARCKS phosphorylated at serines 152 and 156. We synthesized a phosphopeptide which contained phosphoserines 152 and 156 and prepared the antibody specific for this phosphopeptide. Immunoblot analysis with both purified MARCKS and the cytosol fraction from rat brain revealed that the antibody reacted only with MARCKS phosphorylated by protein kinase C. The antibody was suitable for immunoblot analysis and immunostaining with cultured human coronary artery smooth muscle cells. Phosphorylation of MARCKS was increased about eightfold by the treatment of the cells with phorbol 12-myristate 13-acetate, a protein kinase C activator. Furthermore, treatment of the cells with endothelin-1 and tumor necrosis factor α increased phosphorylation of MARCKS. Interestingly, phosphorylation of MARCKS was clearly observed in smooth muscle cells in atherosclerotic lesion of subjects at autopsy. These results suggest that the antibody is useful for examination of the activation of protein kinase C in vascular smooth muscle cellsin vivo.