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

Miho Oyasu ¹, Mineko Fujimiya ¹, Kaori Kashiwagi ¹, Shiho Ohmori ¹, Hirotsugu Imaeda ¹ and Naoaki Saito ^1*

¹ 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)

^* To whom correspondence should be addressed. E-mail: naosaito{at}kobe-u.ac.jp.

Submitted on June 11, 2007
Accepted on 5 November 2007

	Abstract

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, the treatment with 12-o-tetradecanoylphorbol 13-acetate (TPA) resulted in a slow and irreversible accumulation of GFP-tagged PKC (PKC-GFP) on the plasma membrane. In contrast, the 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 subtly but significantly different localization depending on stimulation. While 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 only for a moment then located this enzyme in a proper distance like touch-and-go, while 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.

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

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