Abstract
Cyclin-dependent kinase 5 (Cdk5) is a proline-directed serine/threonine protein kinase that requires association with a regulatory protein, p35 or p39, to form an active enzyme. Munc18-1 plays an essential role in membrane fusion, and its function is regulated by phosphorylation. We report here that both p35 and p39 were expressed in insulin-secreting beta-cells, where they exhibited individual subcellular distributions and associated with membranous organelles of different densities. Overexpression of Cdk5, p35, or p39 showed that Cdk5 and p39 augmented Ca(2+)-induced insulin exocytosis. Suppression of p39 and Cdk5, but not of p35, by antisense oligonucleotides selectively inhibited insulin exocytosis. Transient transfection of primary beta-cells with Munc18-1 templates mutated in potential Cdk5 or PKC phosphorylation sites, in combination with Cdk5 and the different Cdk5 activators, suggested that Cdk5/p39-promoted Ca(2+)-dependent insulin secretion from primary beta-cells by phosphorylating Munc18-1 at a biochemical step immediately prior to vesicle fusion.
Publication types
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Research Support, Non-U.S. Gov't
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Research Support, U.S. Gov't, P.H.S.
MeSH terms
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Animals
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Brain / metabolism
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Calcium / metabolism*
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Cells, Cultured
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Cyclin-Dependent Kinase 5
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Cyclin-Dependent Kinases / genetics
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Cyclin-Dependent Kinases / metabolism*
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Enzyme Activation
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Exocytosis / physiology*
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Female
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Human Growth Hormone / metabolism
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Humans
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Insulin / metabolism
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Islets of Langerhans / cytology
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Islets of Langerhans / metabolism
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Membrane Potentials / physiology
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Mice
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Mice, Inbred C57BL
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Mice, Obese
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Munc18 Proteins
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Nerve Tissue Proteins / genetics
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Nerve Tissue Proteins / metabolism*
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Oligonucleotides, Antisense / metabolism
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Patch-Clamp Techniques
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Phosphorylation
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Subcellular Fractions / metabolism
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Vesicular Transport Proteins / genetics
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Vesicular Transport Proteins / metabolism*
Substances
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Cdk5 activator p39
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Insulin
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Munc18 Proteins
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Nerve Tissue Proteins
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Oligonucleotides, Antisense
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STXBP1 protein, human
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Stxbp1 protein, mouse
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Vesicular Transport Proteins
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neuronal Cdk5 activator (p25-p35)
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Human Growth Hormone
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Cyclin-Dependent Kinase 5
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CDK5 protein, human
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Cdk5 protein, mouse
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Cyclin-Dependent Kinases
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Calcium