Complete activation of protein kinase C by an antipeptide antibody directed against the pseudosubstrate prototope.
It has been proposed that the regulatory domain of protein kinase C contains a pseudosubstrate site between amino acid residues 19 and 36 (House, C., and Kemp, B. E. (1987) Science 238, 1726-1728). Antiserum raised against this peptide sequence has now been shown to completely activate protein kinase C in the absence of calcium and phospholipids. Pre-clearing the antiserum with resin-immobilized pseudosubstrate peptide eliminates the ability of the serum to activate protein kinase C. Activation is not the result of degradation of the enzyme to a calcium- and phospholipid-independent fragment; the activated protein kinase remains intact. Although there are minor sequence differences in the pseudosubstrate region, the three principal protein kinase C isoforms (alpha, beta, and gamma) are recognized and apparently activated by the same pseudosubstrate antiserum. These results provide strong evidence that the pseudosubstrate region, presumably by interacting with the substrate binding site, is responsible for maintaining the catalytic domain in an inactive state. We propose that incubation of protein kinase C with the pseudosubstrate antiserum renders the catalytic domain accessible to protein substrates in a manner analogous to the conformational changes induced by physiological activators such as phospholipids.
Makowske
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Program in Molecular Biology, Memorial Sloan-Kettering Cancer Research Center, New York, New York 10021.
Rosen
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D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies; D000936:Antigen-Antibody Complex; D001921:Brain / Q000201:enzymology; D004789:Enzyme Activation; D015151:Immunoblotting; D007074:Immunoglobulin G; D007527:Isoenzymes / Q000276:immunology / Q000378:metabolism*; D008969:Molecular Sequence Data; D011493:Protein Kinase C / Q000276:immunology / Q000378:metabolism*; D051381:Rats; D013379:Substrate Specificity
D000906:Antibodies; D000936:Antigen-Antibody Complex; D007074:Immunoglobulin G; D007527:Isoenzymes; D011493:Protein Kinase C