C2 Region-derived Peptides Inhibit Translocation and Function of Protein Kinase C in Vivo

doi:10.1074/jbc.270.41.24180

C2 Region-derived Peptides Inhibit Translocation and Function of Protein Kinase C in Vivo(*)

From the Department of Molecular Pharmacology, School of Medicine, Stanford University, Stanford, CA 94305-5332 and the
Columbia-Presbyterian Cancer Center, Columbia University, New York, New York 10032

^¶ To whom correspondence should be addressed. Tel.: 415-725-7720; Fax: 415-725-2952.

Abstract

RACK1 is a protein kinase C (PKC)-binding protein that fulfills the criteria previously established for a receptor for activated C-kinase (RACK). If binding of PKC to RACK anchors the activated enzyme near its protein substrates, then inhibition of this binding should inhibit translocation and function of the enzyme in vivo. Here, we have identified such inhibitors that mimic the RACK1-binding site on βPKC. We first found that a C2-containing fragment, but not a C1-containing fragment of βPKC, bound to RACK1 and inhibited subsequent βPKC binding. The RACK1-binding site was further mapped; peptides βC2-1 (βPKC(209-216)), βC2-2 (βPKC(186-198)), and βC2-4 (βPKC(218-226), but not a number of control peptides, bound to RACK1 and inhibited the C2 fragment binding to RACK1. Peptides βC2-1, βC2-2, and βC2-4 specifically inhibited phorbol ester-induced translocation of the C2-containing isozymes in cardiac myocytes and insulin-induced βPKC translocation and function in Xenopus oocytes. Therefore, peptides corresponding to amino acids 186-198, and 209-226 within the C2 region of the βPKC are specific inhibitors for functions mediated by βPKC.

Footnotes

↵* This research was supported by Grant HL43380 from the National Institutes of Health (to D. M. R.). 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:

PKC

protein kinase C

PS

phosphatidylserine

DG

diacylglycerol

RACK

receptors for activated protein kinase C

PMA

4β-phorbol 12-myristate 13-acetate

GST

glutathione S-transferase

PBS

phosphate-buffered saline

PAGE

polyacrylamide gel electrophoresis.
↵²J. A. Johnson, M. O. Gray, and D. Mochly-Rosen, manuscript in preparation.
↵³D. Ron and D. Mochly-Rosen, manuscript in preparation.
↵⁴B. L. Smith and D. Mochly-Rosen, unpublished result.
- Received July 9, 1995.