Calmodulin inhibits the protein kinase C-catalysed phosphorylation of an endogenous protein in A10 smooth-muscle cells.
The protein kinase C (PKC) activator phorbol 12,13-dibutyrate stimulated the phosphorylation of a 75 kDa protein (p75) in intact cultured A10 smooth-muscle cells and sonicated cell preparations; p75 was the only major substrate for endogenous PKC in sonicated A10 cells. The Ca(2+)-dependent phosphorylation of p75 in vitro was dramatically decreased in PKC-down-regulated A10 cells; however, p75 from identical sonicated cell preparations was still phosphorylated by an exogenous aortic PKC preparation. Calmodulin inhibited the phosphorylation of p75 by PKC, but not the phosphorylation of other PKC substrates (platelet P47 protein and histone). The addition of calmodulin after the phosphorylation reaction was started prevented further phosphorylation, but did not decrease the extent of phosphorylation of p75 that was reached before the addition of calmodulin. The inhibition of p75 phosphorylation was concentration-dependent, with IC50 values (concn. giving 50% inhibition) ranging from less than 0.5 to 10 micrograms of calmodulin/ml, and was Ca(2+)-dependent, requiring a free Ca2+ concentration of 10 microM or greater. These results suggest that the inhibition of the PKC-catalysed phosphorylation of p75 by calmodulin may be due to its interaction with the substrate, rather than a direct inhibitory effect on the enzyme, and that this inhibition could be regulated by intracellular Ca2+ concentration. Therefore, p75 may be a physiological link between the PKC and Ca2+/calmodulin pathways.
Zhao
D Y
DY
Department of Pharmacology and Therapeutics, Faculty of Medicine, University of Calgary, Alberta, Canada.
Hollenberg
M D
MD
Severson
D L
DL
D000818:Animals; D001011:Aorta / Q000201:enzymology; D001921:Brain / Q000378:metabolism; D002147:Calmodulin / Q000494:pharmacology*; D002417:Cattle; D002460:Cell Line; D006657:Histones / Q000378:metabolism; D047908:Intracellular Signaling Peptides and Proteins; D007700:Kinetics; D008565:Membrane Proteins; D008970:Molecular Weight; D009124:Muscle Proteins / Q000378:metabolism*; D009130:Muscle, Smooth / Q000378:metabolism*; D009131:Muscle, Smooth, Vascular / Q000201:enzymology; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology*; D010750:Phosphoproteins / Q000302:isolation & purification; D010766:Phosphorylation; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism; D051381:Rats; D013379:Substrate Specificity
D002147:Calmodulin; D006657:Histones; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D009124:Muscle Proteins; D010750:Phosphoproteins; D011506:Proteins; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D015240:Phorbol 12,13-Dibutyrate; D011493:Protein Kinase C