Dynamics of Glucose-induced Membrane Recruitment of Protein Kinase C βII in Living Pancreatic Islet β-Cells

Paolo Pinton, Takashi Tsuboi, Edward K. Ainscow, Tullio Pozzan, Rosario Rizzuto, Guy A. Rutter
2002 Journal of Biological Chemistry  
The mechanisms by which glucose may affect protein kinase C (PKC) activity in the pancreatic islet ␤-cell are presently unclear. By developing adenovirally expressed chimeras encoding fusion proteins between green fluorescent protein and conventional (␤II), novel (␦), or atypical () PKCs, we show that glucose selectively alters the subcellular localization of these enzymes dynamically in primary islet and MIN6 ␤-cells. Examined by laser scanning confocal or total internal reflection
more » ... lection fluorescence microscopy, elevated glucose concentrations induced oscillatory translocations of PKC␤II 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 PKC␤II both to the plasma membrane and to the surface of dense core secretory vesicles. By contrast, the distribution of PKC␦⅐EGFP, 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 ␤-cells causes spatially and temporally complex changes in the subcellular localization of PKC␤II, possibly resulting from the generation of Ca 2؉ microdomains. Localized changes in PKC␤II activity may thus have a role in the spatial control of insulin exocytosis. Ca 2ϩ and phospholipid-dependent protein kinases (PKC) 1 are important mediators of intracellular signals (1). PKC iso-
doi:10.1074/jbc.m204478200 pmid:12149258 fatcat:f7slfls6jrctpp6duvtmjhdoge