Phosphorylation of an acidic mol. wt. 80 000 cellular protein in a cell-free system and intact Swiss 3T3 cells: a specific marker of protein kinase C activity.
Activation of the endogenous Ca2+-activated phospholipid-dependent protein kinase (protein kinase C) by Ca2+, phosphatidylserine (PS) and phorbol dibutyrate (PBt2) in detergent-solubilized extracts of Swiss 3T3 cells resulted in a very rapid increase (detectable within seconds) in the phosphorylation of an 80 000 mol. wt. protein (termed 80 K). Neither cyclic AMP nor Ca2+ had any effect on 80 K phosphorylation. The 80 K phosphoproteins generated after activation of protein kinase C, both in cell-free conditions and in intact fibroblasts, are identical as judged by one and two-dimensional polyacrylamide slab gel electrophoresis and peptide mapping. Prolonged treatment of cells with phorbol esters causes a selective decrease in protein kinase C activity and prevents the stimulation of 80 K phosphorylation in intact fibroblasts. We now show that extracts from PBt2-treated cultures fail to stimulate 80 K phosphorylation after the addition of the protein kinase C activators. This effect was due to the lack of protein kinase C activity since the addition of exogenous protein kinase C from mouse brain stimulated 80 K phosphorylation in both control and PBt2-treated preparations. The 80 K phosphoprotein generated by activation of endogenous and exogenous protein kinase C yielded similar phosphopeptide fragments after peptide mapping by limited proteolysis. We conclude that the detection of changes in the phosphorylation of 80 K provides a useful approach to ascertain which extracellular ligands activate protein kinase C in intact cells.
D000818:Animals; D002474:Cell-Free System; D002478:Cells, Cultured; D004789:Enzyme Activation; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D051379:Mice; D008970:Molecular Weight; D010750:Phosphoproteins / Q000096:biosynthesis* / Q000302:isolation & purification; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*
D010750:Phosphoproteins; D011493:Protein Kinase C