Determination of in Vivo Phosphorylation Sites in Protein Kinase C

doi:10.1074/jbc.270.45.26807

Determination of in Vivo Phosphorylation Sites in Protein Kinase C (*)

From the (1) Department of Biochemistry and Molecular Biology, University of California Berkeley, California 94720-3206, the
(2) Department of Pharmaceutical Chemistry, School of Pharmacy, University of California, San Francisco, California 94143-0446, and the
(3) Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724

^§ To whom correspondence should be addressed:
229 Stanley Hall, University of California, Berkeley, CA 94720-3206
. Tel.: 510-642-0416; Fax: 510-643-6386.

Abstract

The primary structure of rat protein kinase C βII 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.

Footnotes

↵* This work was supported by Grant DK09765 from NIDDK, National Institutes of Health (to D. E. K.), Grant RR01614 from the National Institutes of Health National Center for Research Resources (to A. L. B.), Grant DIR8700766 from the National Science Foundation (to A. L. B.), and Grant ES04705 from NIEHS, National Institutes of Health (to A. L. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

HPLC

high pressure liquid chromatography

CID

collision-induced dissociation

ESIMS

electrospray ionization mass spectrometry

LC/ESIMS

HPLC directly coupled with electrospray ionization mass spectrometry

LSIMS

liquid secondary ionization mass spectrometry

rt

retention time.
↵² L. M. Keranen, E. M. Dutil, and A. C. Newton, personal communication.
- Received May 3, 1995.
- Revision received July 10, 1995.