Myosin light-chain phosphorylation controls insulin secretion at a proximal step in the secretory cascade

1 Department of Pharmacology, 2 First Department of Surgery, and 3 Department of Anatomy, Nagoya University School of Medicine, Nagoya 466; 4 Department of Thoracic Surgery, Faculty of Medicine, Mie University, Tsu 514; and 5 Institute of Development, Aging and Cancer, Tohoku University, Sendai 980, Japan The aim of this study was to investigate how insulin secretion is controlled by phosphorylation of the myosin light chain (MLC). Ca 2+ -evoked insulin release from pancreatic islets permeabilized with streptolysin O was inhibited by different monoclonal antibodies against myosin light-chain kinase (MLCK) to an extent parallel to their inhibition of purified MLCK. Anti-MLCK antibody also inhibited insulin release caused by the stable GTP analog guanosine 5'- O -(3-thiodiphosphate), even at a substimulatory concentration (0.1 µM) of Ca 2+ . Free Ca 2+ increased MLC peptide phosphorylation by -cell extracts in vitro. In contrast to the phosphorylation by purified MLCK or by calmodulin (CaM) kinase II, the activity partially remained with the -cell under nonstimulatory Ca 2+ (0.1 µM) conditions. The MLCK inhibitor ML-9 inhibited the activity in the -cell with both substimulatory and stimulatory Ca 2+ , whereas KN-62, an inhibitor of CaM kinase II, only exerted an influence in the latter case. ML-9 decreased intracellular granule movement in MIN6 cells under basal and acetylcholine-stimulated conditions. We propose that MLC phosphorylation may modulate translocation of secretory granules, resulting in enhanced insulin secretion. calcium; calmodulin; myosin light-chain kinase; pancreatic -cell; protein kinase