Type 3 protein kinase C localization to the nuclear envelope of phorbol ester-treated NIH 3T3 cells.
We have examined the immunocytochemical localization of protein kinase C (PKC) in NIH 3T3 cells using mAbs that recognize Type 3 PKC. In control cells, the immunofluorescent staining was similar with mAbs directed to either the catalytic or the regulatory domain of PKC. Type 3 PKC localized in a diffuse cytoplasmic pattern, while the nuclei were apparently unstained. Cytoskeletal components also were Treatment of the cells with phorbol 12-myristate 13-acetate (PMA) resulted in a redistribution of PKC with a specific increase in nuclear PKC. Compared to control cells, the staining with the anticatalytic domain mAbs changed markedly, covering the entire cell surface. In contrast, the staining by the antiregulatory domain mAb did not cover the cell surface and the nuclei remained unstained; these results suggest that PKC activation leads to a conformational change of the regulatory domain such that the epitope recognized by the antiregulatory domain mAb is not readily accessible. We have demonstrated by three criteria that PMA treatment specifically increased PKC in the nucleus: (a) immunofluorescent staining in isolated nuclei increased; (b) Western blots showed that our mAbs detected only one protein, the 82-kD PKC, whose level increased in nuclear lysates from PMA-treated cells; and (c) PKC activity increased in nuclear lysates. In fractionation studies we demonstrated that PKC specifically localized to the nuclear envelope fraction. These results demonstrate that PMA activation leads to a rapid redistribution of Type 3 PKC to the nuclear envelope, and suggests that this isozyme may play a role in mediating PKC-induced changes in gene expression.
Leach
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KL
Department of Cell Biology, Upjohn Company, Kalamazoo, Michigan 49001.
Powers
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Ruff
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Jaken
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Kaufmann
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D000818:Animals; D000911:Antibodies, Monoclonal; D002458:Cell Fractionation; D002460:Cell Line; D003599:Cytoskeleton / Q000201:enzymology / Q000648:ultrastructure; D005347:Fibroblasts / Q000187:drug effects / Q000201:enzymology* / Q000648:ultrastructure; D005455:Fluorescent Antibody Technique; D007527:Isoenzymes / Q000276:immunology / Q000378:metabolism / Q000502:physiology; D051379:Mice; D009685:Nuclear Envelope / Q000187:drug effects / Q000201:enzymology* / Q000648:ultrastructure; D010703:Phorbol Esters / Q000494:pharmacology*; D011493:Protein Kinase C / Q000276:immunology / Q000378:metabolism* / Q000502:physiology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D000911:Antibodies, Monoclonal; D007527:Isoenzymes; D010703:Phorbol Esters; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate