Membrane targeting of cGMP-dependent protein kinase is required for cystic fibrosis transmembrane conductance regulator Cl− channel activation

Membrane targeting of cGMP-dependent protein kinase is required for cystic fibrosis transmembrane conductance regulator Cl⁻ channel activation

^*Departments of Biochemistry, Cardiovascular Research Institute COEUR, and ^§Pathology, Faculty of Medicine and Health Sciences, Erasmus University Rotterdam, P.O. Box 1738, 3000 DR Rotterdam, The Netherlands; and ^‡Laboratory of Clinical Biochemistry, Medical University Clinic Würzburg, Josef Schneider-Strasse 2, 97080 Würzburg, Germany

Edited by Ferid Murad, University of Texas Medical School at Houston, Houston TX, and approved December 12, 1997 (received for review July 18, 1997)

Abstract

A recently cloned isoform of cGMP-dependent protein kinase (cGK), designated type II, was implicated as the mediator of cGMP-provoked intestinal Cl⁻ secretion based on its localization in the apical membrane of enterocytes and on its capacity to activate cystic fibrosis transmembrane conductance regulator (CFTR) Cl⁻ channels. In contrast, the soluble type I cGK was unable to activate CFTR in intact cells, although both cGK I and cGK II could phosphorylate CFTR in vitro. To investigate the molecular basis for the cGK II isotype specificity of CFTR channel gating, we expressed cGK II or cGK I mutants possessing different membrane binding properties by using adenoviral vectors in a CFTR-transfected intestinal cell line, and we examined the ability of cGMP to phosphorylate and activate the Cl⁻ channel. Mutation of the cGK II N-terminal myristoylation site (Gly² → Ala) reduced cGK II membrane binding and severely impaired cGK II activation of CFTR. Conversely, a chimeric protein, in which the N-terminal membrane-anchoring domain of cGK II was fused to the N terminus of cGK Iβ, acquired the ability to associate with the membrane and activate the CFTR Cl⁻ channel. The potency order of cGK constructs for activation of CFTR (cGK II > membrane-bound cGK I chimer ≫ nonmyristoylated cGK II > cGK Iβ) correlated with the extent of ³²P incorporation into CFTR observed in parallel measurements. These results strongly support the concept that membrane targeting of cGK is a major determinant of CFTR Cl⁻ channel activation in intact cells.

Footnotes

↵ † To whom reprint requests should be addressed. e-mail: Vaandrager{at}bc1.fgg.eur.nl.
This paper was submitted directly (Track II) to the Proceedings Office.
Abbreviations: ANP, atrial natriuretic peptide; cAK, cAMP-dependent protein kinase; CFTR, cystic fibrosis transmembrane conductance regulator; cGK, cGMP-dependent protein kinase; 8-Br-PET-cGMP, β-phenyl-1,N²-etheno-8-bromo-cGMP; Ad, adenovirus; G2A, Gly at position 2 to an Ala.