Dynamics of glucose-induced membrane recruitment of protein kinase C βII in living pancreatic islet β-cells

被引:76
作者
Pinton, P
Tsuboi, T
Ainscow, EK
Pozzan, T
Rizzuto, R
Rutter, GA [1 ]
机构
[1] Univ Bristol, Henry Wellcome Signalling Labs, Bristol BS8 1TD, Avon, England
[2] Univ Bristol, Dept Biochem, Bristol BS8 1TD, Avon, England
[3] Univ Padua, Dept Biomed Sci, I-35121 Padua, Italy
[4] Univ Padua, CNR, Ctr Study Biol Membranes, I-35121 Padua, Italy
[5] Univ Ferrara, Dept Expt & Diagnost Med, Sect Gen Pathol, I-44100 Ferrara, Italy
[6] Univ Ferrara, Interdisciplinary Ctr Study Inflammat, I-44100 Ferrara, Italy
关键词
D O I
10.1074/jbc.M204478200
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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 Ca2+ concentrations ([Ca2+](c)) were primarily responsible, prevention of [Ca2+](c) increases with EGTA or diazoxide completely eliminated membrane recruitment, whereas elevation of cytosolic [Ca2+](c) with KCl or tolbutamide was highly effective in redistributing PKC/betaII 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 Ca2+, was unaffected by glucose. Measurement of [Ca2+](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 [Ca2+](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 Ca2+ microdomains. Localized changes in PKCbetaII activity may thus have a role in the spatial control of insulin exocytosis.
引用
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页码:37702 / 37710
页数:9
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