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.
Highlights
Exocytosis of insulin from pancreatic -cells has been suggested to be mediated by the same core fusion machinery that controls all membrane fusion events in organisms ranging from yeast to human [1,2,3]
cyclin-dependent kinase 5 (Cdk5) Activators Are Expressed in Insulin-secreting Primary -Cells—To examine if p35 and p39 mRNAs are transcribed in islets of Langerhans we performed reverse transcriptase polymerase chain reaction (RT-PCR) using p35- and p39-specific primers
The p35 protein was partially co-distributed with syntaxin 1, a protein that is used as a plasma membrane marker, whereas p39 in part was co-distributed both with syntaxin 1 and insulin-containing granules
Summary
Exocytosis of insulin from pancreatic -cells has been suggested to be mediated by the same core fusion machinery that controls all membrane fusion events in organisms ranging from yeast to human [1,2,3]. Several synaptic proteins regulating neuronal exocytosis including syntaxin, SNAP-25, VAMP, and Munc have been identified in pancreatic -cells, supporting the idea that the molecular machinery regulating insulin secretion is similar to that of neurotransmitter release from synaptic vesicles [7,8,9,10,11]. The molecular mechanisms regulating the transition of syntaxin 1 from a closed to an open conformation, releasing it from the donor Munc, prior SNARE complex formation with SNAP-25 and VAMP are not fully elucidated. We have demonstrated that Cdk in association with its regulatory protein p39 stimulates Ca2ϩ-dependent exocytosis in primary -cells via phosphorylation of Munc. We propose that Cdk5/p39 regulates insulin exocytosis at a biochemical step immediately after Ca2ϩ-influx but prior vesicle fusion with the plasma membrane
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