Immunocytochemical Localization of Eight Protein Kinase C Isozymes Overexpressed in NIH 3T3 Fibroblasts

doi:10.1074/jbc.270.17.9991

Immunocytochemical Localization of Eight Protein Kinase C Isozymes Overexpressed in NIH 3T3 Fibroblasts

ISOFORM-SPECIFIC ASSOCIATION WITH MICROFILAMENTS, GOLGI, ENDOPLASMIC RETICULUM, AND NUCLEAR AND CELL MEMBRANES (*)

From the (1) Molecular Genetics Section, Laboratory of Genetics, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892-4255 and the
(2) Institute for Clinical Molecular Biology and Tumor Genetics, GSF-Forschungszentrum Hämatologikum Marchioninistrasse 25, D-81377 Munich, Federal Republic of Germany

^§ To whom correspondence should be addressed:
Bldg. 37, Rm. 2B04, National Institutes of Health, 37 CONVENT DR. MSC 4255, BETHESDA, MD 20892-4255.

Abstract

We have used immunocytochemical analyses to characterize the subcellular distribution of protein kinase C (PKC)-α, -βI, -βII, -, -, -, -, and - in NIH 3T3 fibroblasts that overexpress these different PKC isozymes. Immunofluorescence studies and Western blotting with antibodies specific for individual isoforms revealed that before activation the majority of the PKCs are not membrane-bound and are diffusely distributed throughout the cytoplasm. In addition, a fraction of PKC- and - appears membrane-bound and concentrated in the Golgi apparatus. Activation of each isozyme's kinase activity (with the exception of PKC-) by treatment of these cells with the phorbol ester 12- O-tetradecanoylphorbol-13-acetate results in isozyme-specific alterations of cell morphology, as well as in a rapid, selective redistribution of the different PKC isozymes to distinct subcellular structures. Within minutes after 12- O-tetradecanoylphorbol-13-acetate treatment, PKC-α and - concentrate at cell margins. In addition, PKC-α accumulates in the endoplasmic reticulum, PKC-βII associates with actin-rich microfilaments of the cytoskeleton, PKC- accumulates in Golgi organelles, and PKC- associates with nuclear membranes. Our results demonstrate that each activated PKC isozyme specifically associates with a particular cellular structure, presumably containing the substrate for that isozyme. These findings support the hypothesis that PKC substrate specificity in vivo is mediated, at least in part, by the restricted subcellular locale for each PKC isozyme and its target protein.

Footnotes

↵* This work was supported in part by Grant 10318 from the Deutsche Krebshilfe Doktor Mildred Scheel Stiftung. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

PKC

protein kinase C

DiC₈

1,2-dioctanoylglycerol

MARCKS

myristoylated alanine-rich protein kinase C substrate

TPA

12- O-tetradecanoylphorbol-13-acetate

DMEM

Dulbecco's modified Eagle's medium

FCS

fetal calf serum

TBS

Tris-buffered saline

FITC

fluorescein isothiocyanate

ER

endoplasmic reticulum

PBS

phosphate-buffered saline.