Phosphorylation of Protein Kinase C-α on Serine 657 Controls the Accumulation of Active Enzyme and Contributes to Its Phosphatase-resistant State

doi:10.1074/jbc.272.6.3544

Phosphorylation of Protein Kinase C-α on Serine 657 Controls the Accumulation of Active Enzyme and Contributes to Its Phosphatase-resistant State*

From the Imperial Cancer Research Fund, Lincoln's Inn Fields, London WC2A 3PX, United Kingdom

^§ To whom correspondence should be addressed:
The Imperial Cancer Research Fund, P. O. Box 123, 44 Lincoln's Inn Fields, London WC2A 3PX, United Kingdom
.

↵^‡ Current address: Synthelabo Recherche (L.E.R.S.), 31 Avenue Paul Vaillant-Couturier, 92220 Bagneux, France.

Abstract

Serine 657 in protein kinase C-α (PKCα) is a site of phosphorylation on expression of the recombinant protein in mammalian cells. To define the function of this phosphorylation, PKCα species with mutations of this site were investigated. The alanine mutant, S657A PKCα, displayed slow phosphate accumulation in pulse-chase experiments, indicating a rate-limiting role in the initial phase of phosphorylation. Consistent with this, the aspartic acid mutant, S657D PKCα, showed an increased rate of phosphate accumulation. Both the S657D and S657A PKCα mutants were slow to accumulate as fully phosphorylated forms during a second phase of phosphorylation. This latter property is shown to correlate with an increased phosphatase sensitivity and decreased protein kinase activity for these two PKCα mutants. It is further shown that once fully phosphorylated, the S657D PKCα mutant displays WT PKCα properties with respect to thermal stability and phosphatase sensitivity in vitro and in vivo; in contrast, the S657A PKCα mutant remains sensitive. The properties of the Ser-657 site PKCα mutants define functional roles for this phosphorylation in both the accumulation of phosphate on PKCα as well as in its agonist-induced dephosphorylation. These results are discussed in the context of a working model of PKCα behavior, providing insight into the workings of other kinases with equivalent sites of phosphorylation.

Footnotes

↵* This work was supported in part by a European Community fellowship (to F. B.) and Biomed Program Support (to P. J. P.). The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

PKC

protein kinase C

DMEM

Dulbecco's modified Eagle's medium

TPA

12-O-tetradecanoylphorbol-13-acetate

PAGE

polyacrylamide gel electrophoresis

PP1cγ, γ

isoform of human protein phosphatase 1

WT

wild-type.
↵² F. Bornancin, D. Rahman, D. J. C. Pappin, and P. J. Parker, unpublished data.
↵³ F. Bornancin, and P. J. Parker, unpublished data.
- Received October 22, 1996.
- Revision received November 19, 1996.