title: Dynamics of glucose-induced membrane recruitment of protein kinase C beta II in living pancreatic islet beta-cells.
abstract: The mechanisms by which glucose may affect protein kinase C (PKC) activity in the pancreatic islet beta-cell are presently unclear. By developing adenovirally expressed chimeras encoding fusion proteins between green fluorescent protein and conventional (betaII), novel (delta), or atypical (zeta) PKCs, we show that glucose selectively alters the subcellular localization of these enzymes dynamically in primary islet and MIN6 beta-cells. Examined by laser scanning confocal or total internal reflection fluorescence microscopy, elevated glucose concentrations induced oscillatory translocations of PKCbetaII to spatially confined regions of the plasma membrane. Suggesting that increases in free cytosolic Ca(2+) concentrations ([Ca(2+)](c)) were primarily responsible, prevention of [Ca(2+)](c) increases with EGTA or diazoxide completely eliminated membrane recruitment, whereas elevation of cytosolic [Ca(2+)](c) with KCl or tolbutamide was highly effective in redistributing PKCbetaII both to the plasma membrane and to the surface of dense core secretory vesicles. By contrast, the distribution of PKCdelta.EGFP, which binds diacylglycerol but not Ca(2+), was unaffected by glucose. Measurement of [Ca(2+)](c) immediately beneath the plasma membrane with a ratiometric "pericam," fused to synaptic vesicle-associated protein-25, revealed that depolarization induced significantly larger increases in [Ca(2+)](c) in this domain. These data demonstrate that nutrient stimulation of beta-cells causes spatially and temporally complex changes in the subcellular localization of PKCbetaII, possibly resulting from the generation of Ca(2+) microdomains. Localized changes in PKCbetaII activity may thus have a role in the spatial control of insulin exocytosis.
authors.affiliation: Henry Wellcome Signalling Laboratories and the Department of Biochemistry, University of Bristol, Bristol BS8 1TD, United Kingdom.
authors.forename: Paolo
authors.initials: P
authors.lastname: Pinton
authors.forename: Takashi
authors.initials: T
authors.lastname: Tsuboi
authors.forename: Edward K
authors.initials: EK
authors.lastname: Ainscow
authors.forename: Tullio
authors.initials: T
authors.lastname: Pozzan
authors.forename: Rosario
authors.initials: R
authors.lastname: Rizzuto
authors.forename: Guy A
authors.initials: GA
authors.lastname: Rutter
mesh_terms: D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology / Q000502:physiology; D002999:Clone Cells; D003600:Cytosol / Q000201:enzymology; D003981:Diazoxide / Q000494:pharmacology; D004533:Egtazic Acid / Q000494:pharmacology; D005947:Glucose / Q000494:pharmacology*; D049452:Green Fluorescent Proteins; D007515:Islets of Langerhans / Q000187:drug effects / Q000201:enzymology / Q000502:physiology*; D007527:Isoenzymes / Q000378:metabolism*; D007700:Kinetics; D008164:Luminescent Proteins / Q000378:metabolism; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D051379:Mice; D011188:Potassium / Q000494:pharmacology; D011493:Protein Kinase C / Q000378:metabolism*; D064546:Protein Kinase C beta; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000378:metabolism
chemical_list: D007527:Isoenzymes; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D004533:Egtazic Acid; D011493:Protein Kinase C; D064546:Protein Kinase C beta; D005947:Glucose; D003981:Diazoxide; D011188:Potassium