Protein phosphorylation by myosin light-chain kinase (MLCK), protein kinase A, and protein kinase C (PKC) plays a positive role in insulin secretion from the pancreatic beta-cell. To investigate the underlying mechanisms, we examined intracellular distribution of the insulin granules and MLCK by immunofluorescence and immunoelectron microscopies and also investigated intracellular traffic of the granules in cultured beta-cells (MIN6) by video microscopy. Considerable parts of MLCK immunoreactivity were colocalized with the insulin granules. Subcellular fractionation of MIN6 cell extracts revealed that myosin light chain (MLC) may be distributed with the insulin-rich fractions, and immunofluorescence staining using specific antibodies against mono- and diphosphorylated MLCs depicted presence of phosphorylated MLCs in the cytoplasm, in part, with colocalization with the insulin granules. Activation of PKC by 12-O-tetradecanoyl-phorbol 13-acetate (TPA) caused a shift of both insulin granules and MLCK to the cell periphery, which was not reproduced by the adenylate cyclase activator, forskolin. In contrast, forskolin, but not TPA, increased the granule movement. Costimulation of the beta-cell by TPA and forskolin induced drastic translocation of insulin granules and MLCK to the cell periphery, resulting in enormous potentiation of insulin release. These findings suggest that these protein kinases increase insulin granules in the ready-releasable pool by acting on different steps in the secretory cascade.
Skip Nav Destination
Article navigation
Abstract|
June 01 2000
Synergism of protein kinase A, protein kinase C, and myosin light-chain kinase in the secretory cascade of the pancreatic beta-cell.
W Yu;
W Yu
Department of Anatomy and Cell Biology, Nagoya University School of Medicine, Japan.
Search for other works by this author on:
T Niwa;
T Niwa
Department of Anatomy and Cell Biology, Nagoya University School of Medicine, Japan.
Search for other works by this author on:
T Fukasawa;
T Fukasawa
Department of Anatomy and Cell Biology, Nagoya University School of Medicine, Japan.
Search for other works by this author on:
H Hidaka;
H Hidaka
Department of Anatomy and Cell Biology, Nagoya University School of Medicine, Japan.
Search for other works by this author on:
T Senda;
T Senda
Department of Anatomy and Cell Biology, Nagoya University School of Medicine, Japan.
Search for other works by this author on:
Y Sasaki;
Y Sasaki
Department of Anatomy and Cell Biology, Nagoya University School of Medicine, Japan.
Search for other works by this author on:
I Niki
I Niki
Department of Anatomy and Cell Biology, Nagoya University School of Medicine, Japan.
Search for other works by this author on:
Citation
W Yu, T Niwa, T Fukasawa, H Hidaka, T Senda, Y Sasaki, I Niki; Synergism of protein kinase A, protein kinase C, and myosin light-chain kinase in the secretory cascade of the pancreatic beta-cell.. Diabetes 1 June 2000; 49 (6): 945–952. https://doi.org/10.2337/diabetes.49.6.945
Download citation file: