Immunogold electron microscopic demonstration of distinct submembranous localization of the activated gammaPKC depending on the stimulation.
We examined the precise intracellular translocation of gamma subtype of protein kinase C (gammaPKC) after various extracellular stimuli using confocal laser-scanning fluorescent microscopy (CLSM) and immunogold electron microscopy. By CLSM, treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA) resulted in a slow and irreversible accumulation of green fluorescent protein (GFP)-tagged gammaPKC (gammaPKC-GFP) on the plasma membrane. In contrast, treatment with Ca(2+) ionophore and activation of purinergic or NMDA receptors induced a rapid and transient membrane translocation of gammaPKC-GFP. Although each stimulus resulted in PKC localization at the plasma membrane, electron microscopy revealed that gammaPKC showed a subtle but significantly different localization depending on stimulation. Whereas TPA and UTP induced a sustained localization of gammaPKC-GFP on the plasma membrane, Ca(2+) ionophore and NMDA rapidly translocated gammaPKC-GFP to the plasma membrane and then restricted gammaPKC-GFP in submembranous area (<500 nm from the plasma membrane). These results suggest that Ca(2+) influx alone induced the association of gammaPKC with the plasma membrane for only a moment and then located this enzyme at a proper distance in a touch-and-go manner, whereas diacylglycerol or TPA tightly anchored this enzyme on the plasma membrane. The distinct subcellular targeting of gammaPKC in response to various stimuli suggests a novel mechanism for PKC activation.
Oyasu
Miho
M
Laboratory of Molecular Pharmacology, Biosignal Research Center, Kobe University, Kobe, Japan.
Fujimiya
Mineko
M
Kashiwagi
Kaori
K
Ohmori
Shiho
S
Imaeda
Hirotsugu
H
Saito
Naoaki
N
D000818:Animals; D016466:CHO Cells; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D006224:Cricetinae; D003412:Cricetulus; D004789:Enzyme Activation; D020536:Enzyme Activators / Q000494:pharmacology*; D007476:Ionophores / Q000494:pharmacology; D007527:Isoenzymes / Q000378:metabolism; D018613:Microscopy, Confocal; D008854:Microscopy, Electron; D008856:Microscopy, Fluorescence; D016202:N-Methylaspartate / Q000494:pharmacology; D011493:Protein Kinase C / Q000378:metabolism*; D021381:Protein Transport; D016194:Receptors, N-Methyl-D-Aspartate / Q000819:agonists / Q000378:metabolism; D013347:Subcellular Fractions / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014544:Uridine Triphosphate / Q000494:pharmacology
D020536:Enzyme Activators; D007476:Ionophores; D007527:Isoenzymes; D016194:Receptors, N-Methyl-D-Aspartate; D000001:Calcimycin; D016202:N-Methylaspartate; C087857:protein kinase C gamma; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium; D014544:Uridine Triphosphate