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 γ-phosphate of [γ-32P]ATP to Hl 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.