Phosphorylation of the Inositol 1,4,5-Trisphosphate Receptor

doi:10.1074/jbc.271.36.21933

Phosphorylation of the Inositol 1,4,5-Trisphosphate Receptor

CYCLIC GMP-DEPENDENT PROTEIN KINASE MEDIATES cAMP AND cGMP DEPENDENT PHOSPHORYLATION IN THE INTACT RAT AORTA*

From the Department of Pathology, Division of Molecular and Cellular Pathology, The University of Alabama at Birmingham, Birmingham, Alabama 35294-0019

^‡ To whom correspondence should be addressed:
Pathology Dept., University of Alabama at Birmingham, Volker Hall Rm. G038, 1670 University Blvd., Birmingham, AL 35294-0019.
Tel.: 205-975-9569; Fax: 205-934-1775.

Abstract

The effects of cyclic GMP (cGMP) and activation of cGMP-dependent protein kinase (PKG) on the phosphorylation of the inositol 1,4,5-trisphosphate (IP₃) receptor were examined in intact rat aorta using the technique of back phosphorylation. Aorta treated with the nitric oxide donors, S-nitroso-N-acetylpenicillamine and sodium nitroprusside, or the selective PKG activator, 8-(4-para-chlorophenylthio)-cGMP (8-CPT-cGMP), demonstrated increased IP₃ receptor phosphorylation in situ, which was both time- and concentration-dependent with a stoichiometry of 0.5 mol of phosphate/mol of receptor above control. Treatment of aorta with the adenyl cyclase activator, forskolin, also demonstrated increased phosphorylation of the IP₃ receptor on the PKG site, although the selective cAMP-dependent protein kinase activator, 8-(4-para-chlorophenylthio)-cAMP (8-CPT-cAMP), did not increase the phosphorylation of the IP₃ receptor. Moreover, the PKG selective inhibitor, KT 5823, inhibited both sodium nitroprusside and forskolin-induced IP₃ receptor phosphorylation more potently than the selective cAMP-dependent protein kinase inhibitor, KT 5720, suggesting that PKG mediates the increase in IP₃ receptor phosphorylation by both cyclic nucleotides in intact aorta. These results provide further support for the notion that PKG is activated by both cAMP and cGMP in intact vascular smooth muscle and that PKG performs a critical role in cyclic nucleotide-dependent relaxation of blood vessels.

Footnotes

↵* This work was supported by National Heart, Lung, and Blood Institute Grant HL-34646 (to T. M. L.) and American Heart Association, Alabama Affiliate, Fellowship ALF 930038 (to P. K.). 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:

NO

nitric oxide

PKG

cGMP-dependent protein kinase

PKA

cAMP-dependent protein kinase

IP₃

inositol 1,4,5-trisphosphate

[Ca²⁺]_i

cytosolic calcium

PAGE

polyacrylamide gel electrophoresis

SNP

sodium nitroprusside

SNAP

S-nitroso-N-acetylpenicillamine

SR

sarcoplasmic reticulum

8-CPT-cAMP

8-(4-para-chlorophenylthio)-cAMP

8-CPT-cGMP

8-(4-para-chloro phenylthio)-cGMP.
- Received March 22, 1996.
- Revision received May 23, 1996.