A phosphatase resistant substrate for the assay of protein kinase C in crude tissue extracts.
Protein kinase C (PKC) is routinely assayed, after it is partially purified over DEAE-cellulose chromatography to eliminate any interfering protein kinases and phosphatases, by measuring the transfer of gamma-phosphate of [gamma-32P]ATP to H1 histone. Recently, it has been shown that a synthetic peptide, comprising residues 4-14 of myelin basic protein (MBP4-14), is a very selective PKC substrate which is not phosphorylated effectively by cyclic AMP-dependent protein kinase, casein kinase I and II, Ca2+/calmodulin dependent protein kinase II or phosphorylase kinase [Yasuda, I., Kishimoto, A., Tanaka, S-I., Tominaga, M., Sakurai, A. and Nishizuka, Y. (1990) BBRC 166, 1220-1227]. We report here that once MBP4-14 is phosphorylated, it is not dephosphorylated by okadaic acid-sensitive phosphatases (protein phosphatases 1, 2A and 3) or other protein phosphatases such as calcineurin and/or PP 2C present in hippocampal homogenates. Therefore, MBP4-14 can be used for PKC assay in crude extracts of neural tissue.
Farrar
Y J
YJ
Veterans Administration Medical Center, Lexington, KY 40511.
Vanaman
T C
TC
Slevin
J T
JT
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D002848:Chromatography, DEAE-Cellulose; D004988:Ethers, Cyclic / Q000494:pharmacology; D006624:Hippocampus / Q000201:enzymology*; D007700:Kinetics; D008297:Male; D008969:Molecular Sequence Data; D004676:Myelin Basic Protein / Q000138:chemical synthesis / Q000378:metabolism; D019319:Okadaic Acid; D009842:Oligopeptides / Q000138:chemical synthesis; D010749:Phosphoprotein Phosphatases / Q000378:metabolism*; D011493:Protein Kinase C / Q000302:isolation & purification / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D013379:Substrate Specificity
D004988:Ethers, Cyclic; D004676:Myelin Basic Protein; D009842:Oligopeptides; D019319:Okadaic Acid; D000255:Adenosine Triphosphate; D011493:Protein Kinase C; D010749:Phosphoprotein Phosphatases