Cyclin-dependent kinase 5 associated with p39 promotes Munc18-1 phosphorylation and Ca(2+)-dependent exocytosis.
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.
Lilja
Lena
L
Department of Molecular Medicine, Karolinska Institutet, Karolinska University Hospital, SE-171 76 Stockholm, Sweden.
Johansson
Jenny Ulrika
JU
Gromada
Jesper
J
Mandic
Slavena Andrea
SA
Fried
Gabriel
G
Berggren
Per-Olof
PO
Bark
Christina
C
D000818:Animals; D001921:Brain / Q000378:metabolism; D002118:Calcium / Q000378:metabolism*; D002478:Cells, Cultured; D051360:Cyclin-Dependent Kinase 5; D018844:Cyclin-Dependent Kinases / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation; D005089:Exocytosis / Q000502:physiology*; D005260:Female; D019382:Human Growth Hormone / Q000378:metabolism; D006801:Humans; D007328:Insulin / Q000378:metabolism; D007515:Islets of Langerhans / Q000166:cytology / Q000378:metabolism; D008564:Membrane Potentials / Q000502:physiology; D051379:Mice; D008810:Mice, Inbred C57BL; D008820:Mice, Obese; D051938:Munc18 Proteins; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism*; D016376:Oligonucleotides, Antisense / Q000378:metabolism; D018408:Patch-Clamp Techniques; D010766:Phosphorylation; D013347:Subcellular Fractions / Q000378:metabolism; D033921:Vesicular Transport Proteins / Q000235:genetics / Q000378:metabolism*
C108298:Cdk5 activator p39; D007328:Insulin; D051938:Munc18 Proteins; D009419:Nerve Tissue Proteins; D016376:Oligonucleotides, Antisense; C497937:STXBP1 protein, human; C497938:Stxbp1 protein, mouse; D033921:Vesicular Transport Proteins; C089495:neuronal Cdk5 activator (p25-p35); D019382:Human Growth Hormone; D051360:Cyclin-Dependent Kinase 5; C495905:CDK5 protein, human; C495906:Cdk5 protein, mouse; D018844:Cyclin-Dependent Kinases; D002118:Calcium