Protein kinase C alpha targeting is regulated by temporal and spatial changes in intracellular free calcium concentration [Ca2+](i)

Protein kinase C (PKC) isoforms exert specific intracellular functions, but the different isoforms display little substrate specificity in vitro. Sele ctive PKC isoform targeting may be a mechanism to achieve specificity. We u sed a green fluorescent fusion protein (GFP) to test the hypothesis that lo cal changes in [Ca2+](i); regulate translocation of PKC alpha and that diff erent modes of Ca2+ and Ca2+ release play a role in PKCa targeting. We cons tructed deletion mutants of PKC alpha. to analyze the Ca2+-sensitive domain s and their role in targeting. Confocal microscopy was used and [Ca2+](i) w as measured by fluo-3. The fusion protein PKC alpha-GFP was expressed in va scular smooth muscle cells and showed a cytosolic distribution similar to t he wildtype PKC alpha protein. The Ca2+ ionophore ionomycin induced a speck led cytosolic PKC alpha-GFP distribution, followed by membrane translocatio n, while depolarization by KCI induced primarily membrane translocation. Se lective voltage-operated Ca2+ channel opening led to a localized accumulati on of PKC alpha-GFP near the plasma membrane. Opening Ca2+ stores with InsP (3), thapsigargin, or ryanodine induced a specific PKC alpha-GFP targeting to distinct intracellular areas. The G-protein-coupled receptor agonist thr ombin induced a rapid translocation of the fusion protein to focal domains. The tyrosine kinase receptor agonist PDGF induced Ca2+ influx and led to a linear PKC alpha-GFP membrane association. PKC alpha-GFP deletion mutants demonstrated that the C2 domain, but not the catalytic subunit, is necessar y for Ca2+-induced PKC alpha targeting. Targeting was also abolished when t he ATP binding site was deleted. We conclude that PKC alpha can rapidly be translocated to distinct intracellular or membrane domains by local increas es in [Ca2+](i). The targeting mechanism is dependent on the C2 and ATP bin ding site of the enzyme. Localized [Ca2+](i) changes determine the spatial and temporal targeting of PKC alpha.