Protein kinase C activation in human monocytes: regulation of PKC isoforms.
The distribution of protein kinase C (PKC) isoforms and phorbol 12-myristate 13-acetate (PMA)-induced activation of PKC in human monocytes was investigated. Using Western blot analysis, PKC beta was found to be the most abundant isoform in monocytes. PKC beta was equally distributed in the cytosol and membrane. PKC-alpha was readily detectable and found predominantly in the cytosol. Little to no PKC-epsilon, gamma, delta, and zeta were observed. Following the treatment of monocytes with PMA, the physical translocation of PKC alpha from the cytosol to the membrane occurred over 60 min. PMA-induced translocation of PKC-beta was difficult to detect by Western blot. Fura-2 analysis demonstrated that PMA-induced PKC translocation was not accompanied by a net change in cytosolic calcium levels. Using histone as a substrate for PKC activity, an extremely rapid translocation of PKC-dependent histone phosphorylation (PKC-DHP) was induced by PMA. Cytosolic PKC-DHP activity decreased to undetectable levels within 8 min. In contrast, analysis of PKC-dependent endogenous substrate phosphorylation (PKC-DESP) showed a pattern with a time-course similar to that observed with Western blot. Thus, translocation of PKC-DESP but not PKC-DHP activity correlated with PKC-alpha as determined by Western blot. The data support the concept that PKC activity is substrate dependent and suggest that using one assay for the measurement of PKC activity may lead to erroneous conclusions.
Chang
Z L
ZL
Laboratory of Macrophage Biology, Guthrie Research Institute, Sayre, Pennsylvania 18840.
Beezhold
D H
DH
D000818:Animals; D015153:Blotting, Western; D002462:Cell Membrane / Q000201:enzymology; D002478:Cells, Cultured; D003600:Cytosol / Q000201:enzymology; D006657:Histones; D006801:Humans; D007527:Isoenzymes / Q000097:blood* / Q000187:drug effects; D051379:Mice; D008807:Mice, Inbred BALB C; D009000:Monocytes / Q000201:enzymology*; D010766:Phosphorylation / Q000187:drug effects; D011493:Protein Kinase C / Q000097:blood* / Q000187:drug effects; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D006657:Histones; D007527:Isoenzymes; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate