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.
Tsutakawa
S E
SE
Department of Biochemistry and Molecular Biology, University of California Berkeley 94720-3206, USA.
Medzihradszky
K F
KF
Flint
A J
AJ
Burlingame
A L
AL
Koshland
D E
DE
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D004789:Enzyme Activation; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D015394:Molecular Structure; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D011493:Protein Kinase C / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D051381:Rats; D017386:Sequence Homology, Amino Acid
D010446:Peptide Fragments; D011493:Protein Kinase C