Selective assay of protein kinase C with a specific peptide substrate.
Protein kinase C is a family of multifunctional protein serine/threonine kinase and generally accepted to be involved in a wide variety of cellular signal transduction. Biochemical and immunochemical studies as well as sequence analysis of its cDNA clones have revealed the existence of multiple subspecies of this enzyme with obvious tissue-specific expression. Enzymatic properties of type I, II, and III protein kinase C subspecies, which are encoded by gamma-, beta I- and beta II, and alpha-cDNA, respectively, are well characterized. Many proteins and peptides are reported as phosphate acceptors of these protein kinase C subspecies. In this study, it is shown that a synthetic peptide, Gln-Lys-Arg-Pro-Ser-Gln-Arg-Ser-Lys-Tyr-Leu, which corresponds to amino acid residues 4-14 of bovine myelin basic protein, is the most specific and convenient substrate for selective assay of protein kinase C among various phosphate acceptor proteins and peptides. This peptide is phosphorylated at Ser-8, but not Ser-11 by protein kinase C subspecies in a manner dependent on Ca2+, phosphatidylserine, and diacylglycerol. This peptide is not phosphorylated by other protein serine/threonine kinases such as cyclic AMP-dependent protein kinase. Thus, it is possible to assay protein kinase C activity in the crude tissue extracts selectively using this peptide as a phosphate acceptor.
Yasuda
I
I
Department of Biochemistry, Kobe University School of Medicine.
D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000201:enzymology; D006657:Histones / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D004676:Myelin Basic Protein / Q000737:chemistry / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000032:analysis* / Q000378:metabolism; D051381:Rats; D013379:Substrate Specificity
D006657:Histones; D004676:Myelin Basic Protein; D010446:Peptide Fragments; D011493:Protein Kinase C