DETERMINATION OF IN-VIVO PHOSPHORYLATION SITES IN PROTEIN-KINASE-C

The primary structure of rat protein kinase C beta II was probed by high pressure liquid chromatography directly coupled to an electrospray ionization mass spectrometer and by high energy collision-induced dissociation analysis to identify in vivo phosphorylation sites, The N-terminal methionine was found to be cleaved post-translationally and replaced with an acetyl group, Four phosphopeptides were identified, Two peptides, Thr(500)-Lys(520) and Glu(490)-Lys(520), are phosphorylated at Thr(500) greater than 90%. Peptide His(636)-Arg(649) is phosphorylated about 75% at Thr(641). It is the only site that was previously identified during the in vitro autophosphorylation studies (Flint, A. J., Paladini, R. D., and Koshland, D. E., Jr. (1990) Science 249, 408-411). The fourth peptide Asn(650)-Lys(672) is phosphorylated at Thr(660). A discussion of the potential implication of these results follows.