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, Thr500-Lys520 and Glu490-Lys520, are phosphorylated at Thr500 greater than 90%. Peptide His636-Arg649 is phosphorylated about 75% at Thr641. 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 Asn650-Lys672 is phosphorylated at Thr660. A discussion of the potential implication of these results follows.