Dissociation of phorbol esters leads to immediate redistribution to the cytosol of protein kinases C alpha and C delta in mouse keratinocytes.
We have measured the dissociation rate of phorbol 12-myristate 13-acetate (PMA), a potent tumor promoter, phorbol 12,13-dibutyrate (PDBu), a weak tumor promoter, and 12-deoxyphorbol 13-phenylacetate (dPP), an antitumor promoter, from intact mouse keratinocytes. PDBu and dPP showed a very rapid release from the cells (t1/2 = 1 min), whereas PMA showed a slower release (t1/2 = 9 min). Western blot analysis of the amounts of protein kinase C alpha (PKC alpha) and PKC delta in the soluble fraction and the Triton X-100-soluble particulate fraction revealed that translocation of both isozymes from the soluble to the particulate fraction was reversible when the phorbol esters were washed off. Washes of 5-15 min resulted in complete redistribution of the PKC isozymes when the cells were previously treated with 1 microM dPP or 1 microM PDBu for 5 min. In the case of treatment with 100 or 10 nM PMA, the redistribution required a longer time; nevertheless, the PKC isozymes returned to the soluble fraction within 60 min. Longer initial treatments with PMA, dPP, and PDBu (up to 60 min) translocated PKC in a very similar, completely reversible fashion. We conclude that in this cell line phorbol esters do not induce the conversion of PKC isozymes to an integral membrane state.
Laboratory of Cellular Carcinogenesis and Tumor Promotion, NCI, National Institutes of Health, Bethesda, Maryland 20892.
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Szallasi
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Smith
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Blumberg
D000818:Animals; D029742:Caenorhabditis elegans Proteins; D002352:Carrier Proteins; D002451:Cell Compartmentation; D002478:Cells, Cultured; D003600:Cytosol / Q000201:enzymology; D066298:In Vitro Techniques; D007527:Isoenzymes / Q000378:metabolism*; D015603:Keratinocytes / Q000201:enzymology* / Q000648:ultrastructure; D051379:Mice; D010703:Phorbol Esters / Q000378:metabolism*; D011493:Protein Kinase C / Q000378:metabolism*; D051571:Protein Kinase C-alpha; D051745:Protein Kinase C-delta; D011955:Receptors, Drug / Q000378:metabolism*
D029742:Caenorhabditis elegans Proteins; D002352:Carrier Proteins; D007527:Isoenzymes; D010703:Phorbol Esters; D011955:Receptors, Drug; C083141:phorbol ester binding protein; C027776:phorbol ester receptor; C497259:Prkcd protein, mouse; C496630:Prkca protein, mouse; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha; D051745:Protein Kinase C-delta