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Immunogold Electron Microscopic Demonstration of Distinct Submembranous Localization of the Activated PKC Depending on the Stimulation

Miho Oyasu, Mineko Fujimiya, Kaori Kashiwagi, Shiho Ohmori, Hirotsugu Imaeda and Naoaki Saito

Laboratory of Molecular Pharmacology, Biosignal Research Center, Kobe University, Kobe, Japan (MO,KK,SO,NS), and Department of Anatomy, Shiga University of Medical Science, Shiga, Japan (MF,HI)

Correspondence to: Naoaki Saito, Laboratory of Molecular Pharmacology, Biosignal Research Center, Kobe University, Rokkodai-cho 1-1, Nada-ku, Kobe 657-8501, Japan. E-mail: naosaito{at}kobe-u.ac.jp

We examined the precise intracellular translocation of subtype of protein kinase C (PKC) 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 PKC (PKC–GFP) on the plasma membrane. In contrast, treatment with Ca²⁺ ionophore and activation of purinergic or NMDA receptors induced a rapid and transient membrane translocation of PKC–GFP. Although each stimulus resulted in PKC localization at the plasma membrane, electron microscopy revealed that PKC showed a subtle but significantly different localization depending on stimulation. Whereas TPA and UTP induced a sustained localization of PKC–GFP on the plasma membrane, Ca²⁺ ionophore and NMDA rapidly translocated PKC–GFP to the plasma membrane and then restricted PKC–GFP in submembranous area (<500 nm from the plasma membrane). These results suggest that Ca²⁺ influx alone induced the association of PKC 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 PKC in response to various stimuli suggests a novel mechanism for PKC activation. (J Histochem Cytochem 56:253–265, 2008)

Key Words: protein kinase C • translocation • Ca²⁺ ionophore • phorbol ester • electron microscopy • green fluorescent protein • immunogold

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