Dissociation of protein kinase C redistribution from the phosphorylation of its substrates.
Increases in cytoplasmic [Ca2+] caused by receptor activation are thought to stimulate the redistribution of loosely associated protein kinase C (PKC) to a tightly membrane-bound form that is activated by diacylglycerol. The precise role of Ca2(+)-dependent redistribution of PKC in the activation of this enzyme has not been critically assessed. We examined the relationship between PKC redistribution and substrate phosphorylation by comparing the kinetics and the Ca2+ dependence of the two events. Using immunoblotting with specific PKC antibodies, we find that 1321N1 cells express the alpha form of PKC, approximately 10-20% of which is membrane-associated in unstimulated cells. This fraction is increased to 60% in response to muscarinic receptor stimulation. Agonist-induced redistribution of PKC is rapid and transient, peaking at 30 s and returning to control levels by 2-5 min. Stimulation of muscarinic receptors also rapidly increases phosphorylation of both an endogenous 80-kDa protein and the peptide substrate, VRKRTLRRL. However, unlike the time course of PKC redistribution, PKC-mediated phosphorylation of these substrates is sustained for up to 30 min. To compare the Ca2+ dependence of PKC redistribution and substrate phosphorylation, we buffered muscarinic receptor-induced increases in cytoplasmic [Ca2+] with the divalent cation chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid. Under these conditions, redistribution of PKC and phosphorylation of the exogenous peptide substrate are inhibited by about 80%. In contrast, muscarinic receptor-stimulated phosphorylation of the 80-kDa protein occurs even when increases in cytoplasmic [Ca2+] are prevented. Taken together, these data demonstrate that the redistribution of PKC does not correlate in extent or duration with phosphorylation of PKC substrates.
Trilivas
I
I
Department of Pharmacology, University of California at San Diego, La Jolla 92093-0636.
McDonough
P M
PM
Brown
J H
JH
D000595:Amino Acid Sequence; D002118:Calcium / Q000378:metabolism; D002217:Carbachol / Q000494:pharmacology; D002462:Cell Membrane / Q000378:metabolism; D002614:Chelating Agents / Q000494:pharmacology; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D004789:Enzyme Activation; D015151:Immunoblotting; D047908:Intracellular Signaling Peptides and Proteins; D007527:Isoenzymes / Q000378:metabolism; D007700:Kinetics; D008565:Membrane Proteins; D008969:Molecular Sequence Data; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D009842:Oligopeptides / Q000378:metabolism; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism; D011976:Receptors, Muscarinic / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014407:Tumor Cells, Cultured
D002614:Chelating Agents; D047908:Intracellular Signaling Peptides and Proteins; D007527:Isoenzymes; D008565:Membrane Proteins; D009842:Oligopeptides; D011506:Proteins; D011976:Receptors, Muscarinic; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D004533:Egtazic Acid; D002217:Carbachol; D011493:Protein Kinase C; C025603:1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium