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.
Szallasi
Z
Z
Laboratory of Cellular Carcinogenesis and Tumor Promotion, NCI, National Institutes of Health, Bethesda, Maryland 20892.
Smith
C B
CB
Blumberg
P M
PM
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