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.
We have used immunocytochemical analyses to characterize the subcellular distribution of protein kinase C (PKC)-alpha, -beta I, -beta II, -gamma, -delta, -epsilon, -zeta, and -eta 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-delta and -eta appears membrane-bound and concentrated in the Golgi apparatus. Activation of each isozyme's kinase activity (with the exception of PKC-zeta) 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-alpha and -epsilon concentrate at cell margins. In addition, PKC-alpha accumulates in the endoplasmic reticulum, PKC-beta II associates with actin-rich microfilaments of the cytoskeleton, PKC-gamma accumulates in Golgi organelles, and PKC-epsilon 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.
Goodnight
J A
JA
Molecular Genetics Section, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892-4255, USA.
Mischak
H
H
Kolch
W
W
Mushinski
J F
JF
D016475:3T3 Cells; D008841:Actin Cytoskeleton / Q000201:enzymology; D000199:Actins / Q000378:metabolism; D000818:Animals; D000918:Antibody Specificity; D001692:Biological Transport; D002462:Cell Membrane / Q000201:enzymology; D002467:Cell Nucleus / Q000201:enzymology; D003001:Cloning, Molecular; D004721:Endoplasmic Reticulum / Q000201:enzymology; D004789:Enzyme Activation; D005347:Fibroblasts / Q000201:enzymology; D006056:Golgi Apparatus / Q000201:enzymology; D007150:Immunohistochemistry; D007425:Intracellular Membranes / Q000201:enzymology; D007527:Isoenzymes / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D051379:Mice; D011493:Protein Kinase C / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D013347:Subcellular Fractions / Q000201:enzymology*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D000199:Actins; D007527:Isoenzymes; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate