The primary structure of rat protein kinase C 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-Lys and Glu-Lys, are phosphorylated at Thr greater than 90%. Peptide His-Arg is phosphorylated about 75% at Thr. 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-Lys is phosphorylated at Thr. A discussion of the potential implication of these results follows.

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