Effect of phorbol esters on protein kinase C-zeta.
Protein kinase C-zeta (PKC-zeta) is a member of the protein kinase C gene family which using in vitro preparations has been described as being resistant to activation by phorbol esters. PKC-zeta was found to be expressed in several cell types as an 80-kDa protein. In vitro translation of a full-length PKC-zeta construct also yielded as a primary translation product an 80-kDa protein. In the U937 cell, PKC-zeta was slightly more abundant in the cytosol than in the particulate fraction. Acute exposure of U937 cells to tetradecanoyl-phorbol-13-acetate (TPA), phorbol dibutyrate, mezerin, or diacylglycerol derivatives did not induce translocation of this isoform to the particulate fraction. Chronic exposure to 1 microM TPA failed to translocate or down-regulate PKC-zeta in U937, HL-60, COS, or HeLa-fibroblast fusion cells. To examine whether PKC-zeta was activated by TPA, PKC activity was evaluated in COS cells transiently over-expressing this isoform. In non-transfected cells, two peaks of phospholipid- and TPA-dependent kinase activity were observed. Eluting at a lower salt concentration was a peak of activity associated with PKC-alpha. PKC-zeta eluted with the second peak of activity and at a higher salt concentration. In transfected cells which expressed PKC-zeta at 4-10-fold over endogenous levels, there was only a slight increase in activity associated with the second peak. The activity and quantity of PKC-zeta did not strictly correlate. Treatment with TPA under conditions that did not alter PKC-zeta content abolished detection of the second peak of PKC activity eluting from the Mono Q column. Thus, PKC-zeta does not translocate or down-regulate in response to phorbol esters or diacylglycerol derivatives. However, for reasons discussed these studies do not resolve the issue of whether this isoform is activated by TPA.
Ways
D K
DK
Department of Medicine and Microbiology/Immunology, East Carolina University School of Medicine, Greenville, North Carolina 27858-4354.
Cook
P P
PP
Webster
C
C
Parker
P J
PJ
D000595:Amino Acid Sequence; D015153:Blotting, Western; D002460:Cell Line; D004075:Diglycerides / Q000494:pharmacology; D015536:Down-Regulation; D004591:Electrophoresis, Polyacrylamide Gel; D006367:HeLa Cells; D006801:Humans; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D005810:Multigene Family; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology; D011493:Protein Kinase C / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D014162:Transfection
D004075:Diglycerides; D007527:Isoenzymes; D015240:Phorbol 12,13-Dibutyrate; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate