NETWORK OF CALCIUM-BINDING PROTEINS IN THE SIGNAL CASCADE

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We have obtained new findings on the roles of calcium-binding proteins in the secretory events as described below.Secretion studies from permeabilized pancreatic B-cells suggested that calmodulin and calmodulin-dependent protein phosphorylation modulate Ca^<2+>-dependent insulin secretion via acting on a proximal step in the secretory cascade. Furthermore, observation of the intracellular movement of secretory granules in the living pancreatic B-cell under a phase-contrast microscope revealed that myosin light chain kinase and CaM kinase II,two calmodulin-dependent protein kinases, are involved in the control of the insulin granules via phosphorylating their common substrate, myosin regulatory light chain. Visualization of the granules in the living B-cell enabled us to obtain direct evidence that activation of the granule movement results in recruitment of the granules to a ready-release zone in the B-cell. We demonstrated that these two kinases are critical members which transduce ca … More lcium-signalling in the secretory events and that they play distinct roles in the control of the movement dependently on the concomitant Ca^<2+> concentrations. Calcyclin, was another calcium-binding proteins found to participate in the control of insulin secretion by ourselves. We obtained purified materials of rodent calcyclin by the expression system in E.Coli. We are now attempting to identify new calcyclin-binding proteins which react with calcyclin in the pancreatic B-cell line in a Ca^<2+>-dependent manner. We also selective antibodies against the expressed calcyclin and demonstrated that this protein is dominantly distributed in the cytoplasm by immunohistochemical and immunoblotting approaches. Since the roles of calcyclin will be better understood if we can visualize the localization of this calcyclin-binding protein in a living cell, we are now attempting to let insulinoma cells express the fusion protein between calcyclin and green fluorescent protein as a fluorescent marker. Less