Determination of in Vivo Phosphorylation Sites in Protein Kinase C

作者：Susan E. Tsutakawa, Katalin F. Medzihradszky, Andrew Flint, Alma L. Burlingame, Daniel E. Koshland · 发表于：Journal of Biological Chemistry · 年份：1995 · DOI：10.1074/jbc.270.45.26807 · 被引用次数：100 · 研究领域：Protein Kinase Regulation and GTPase Signaling、Metabolism, Diabetes, and Cancer、Enzyme Structure and Function

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.