title: Ca2+-dependent dephosphorylation of kinesin heavy chain on beta-granules in pancreatic beta-cells. Implications for regulated beta-granule transport and insulin exocytosis.
abstract: The specific biochemical steps required for glucose-regulated insulin exocytosis from beta-cells are not well defined. Elevation of glucose leads to increases in cytosolic [Ca2+]i and biphasic release of insulin from both a readily releasable and a storage pool of beta-granules. The effect of elevated [Ca2+]i on phosphorylation of isolated beta-granule membrane proteins was evaluated, and the phosphorylation of four proteins was found to be altered by [Ca2+]i. One (a 18/20-kDa doublet) was a Ca2+-dependent increase in phosphorylation, and, surprisingly, three others (138, 42, and 36 kDa) were Ca2+-dependent dephosphorylations. The 138-kDa beta-granule phosphoprotein was found to be kinesin heavy chain (KHC). At low levels of [Ca2+]i KHC was phosphorylated by casein kinase 2, but KHC was rapidly dephosphorylated by protein phosphatase 2B beta (PP2Bbeta) as [Ca2+]i increased. Inhibitors of PP2B specifically reduced the second, microtubule-dependent, phase of insulin secretion, suggesting that dephosphorylation of KHC was required for transport of beta-granules from the storage pool to replenish the readily releasable pool of beta-granules. This is distinct from synaptic vesicle exocytosis, because neurotransmitter release from synaptosomes did not require a Ca2+-dependent KHC dephosphorylation. These results suggest a novel mechanism for regulating KHC function and beta-granule transport in beta-cells that is mediated by casein kinase 2 and PP2B. They also implicate a novel regulatory role for PP2B/calcineurin in the control of insulin secretion downstream of a rise in [Ca2+]i.
authors.affiliation: Pacific Northwest Research Institute and Department of Pharmacology, University of Washington, Seattle, Washington 98112, USA.
authors.forename: Matthew J
authors.initials: MJ
authors.lastname: Donelan
authors.forename: Gerardo
authors.initials: G
authors.lastname: Morfini
authors.forename: Richard
authors.initials: R
authors.lastname: Julyan
authors.forename: Scott
authors.initials: S
authors.lastname: Sommers
authors.forename: Lori
authors.initials: L
authors.lastname: Hays
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Kajio
authors.forename: Isabelle
authors.initials: I
authors.lastname: Briaud
authors.forename: Richard A
authors.initials: RA
authors.lastname: Easom
authors.forename: Jeffery D
authors.initials: JD
authors.lastname: Molkentin
authors.forename: Scott T
authors.initials: ST
authors.lastname: Brady
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Rhodes
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000494:pharmacology* / Q000502:physiology; D047390:Casein Kinase II; D002460:Cell Line; D003594:Cytoplasmic Granules / Q000378:metabolism*; D004533:Egtazic Acid / Q000494:pharmacology; D005089:Exocytosis; D007328:Insulin / Q000378:metabolism*; D000078790:Insulin Secretion; D007340:Insulinoma; D007515:Islets of Langerhans / Q000502:physiology*; D016547:Kinesins / Q000378:metabolism*; D007700:Kinetics; D010190:Pancreatic Neoplasms; D010710:Phosphates / Q000378:metabolism; D010749:Phosphoprotein Phosphatases / Q000378:metabolism; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D021122:Protein Subunits; D021381:Protein Transport; D051381:Rats; D014407:Tumor Cells, Cultured
chemical_list: D007328:Insulin; D010710:Phosphates; D021122:Protein Subunits; D004533:Egtazic Acid; D000255:Adenosine Triphosphate; D011494:Protein Kinases; D047390:Casein Kinase II; D017346:Protein Serine-Threonine Kinases; D010749:Phosphoprotein Phosphatases; D016547:Kinesins; D002118:Calcium