Effect of phorbol esters on cytosolic protein kinase C content and activity in the human monoblastoid U937 cell.
12-O-Tetradecanoylphorbol-13-acetate (TPA) stimulates the human monoblastoid U937 cell to differentiate into a mature monocyte/macrophage-like cell. Since TPA may produce cellular responses by activating protein kinase C, the effects of TPA on kinase activity in the U937 cell were investigated. Brief exposures (less than or equal to 60 min) to TPA dramatically diminished protein kinase C-dependent phosphorylation of histone and endogenous substrates. However, using a peptide substrate corresponding to residues 720-737 of protein kinase C-epsilon, Ca2(+)-, phospholipid-, and diacylglycerol-dependent kinase activity was reduced only modestly after exposure to TPA. This phospholipid-dependent kinase activity coeluted on DEAE chromatography with protein kinase C. Examination of cytosolic protein kinase C content by Western blot analysis demonstrated a moderate decline in kinase content after TPA treatment. The decline was due primarily to loss of an 80-kDa species with preservation of a 76-kDa protein. The immunoreactive 76-kDa protein observed after TPA treatment comigrated on DEAE chromatography with the kinase activity phosphorylating the protein kinase C-epsilon peptide and had an elution profile similar to protein kinase C derived from untreated cells. Using antisera recognizing the catalytic and regulatory domains of the kinase, no evidence for proteolytic degradation of protein kinase C was observed. Although incubation of extracts from vehicle and TPA-treated cells inhibited the activity of partially purified protein kinase C, the degree of inhibition was similar in the two extracts. These findings suggest that TPA markedly diminishes protein kinase C-dependent phosphorylation of histone and endogenous substrates in part by altering kinase substrate specificity. These observations provide evidence for a novel post-translational process that can modulate protein kinase C-dependent phosphorylation.
Ways
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K
Department of Medicine, East Carolina University School of Medicine, Greenville, North Carolina 27858-4354.
Riddle
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R
Ways
M
M
Cook
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P
D001345:Autoradiography; D015153:Blotting, Western; D002458:Cell Fractionation; D002460:Cell Line; D002848:Chromatography, DEAE-Cellulose; D003600:Cytosol / Q000201:enzymology*; D006657:Histones / Q000378:metabolism; D006801:Humans; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D013379:Substrate Specificity; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*
D006657:Histones; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate