Membrane Targeting of cGMP-Dependent Protein Kinase is Required for Cystic Fibrosis Transmembrane Conductance Regulator Cl-Channel Activation

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 regu...

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Published in:	Proceedings of the National Academy of Sciences - PNAS 1998-02, Vol.95 (4), p.1466-1471
Main Authors:	Arie. B. Vaandrager, Smolenski, Albert, Tilly, Ben C., Houtsmuller, Adriaan B., Ehrich M. E. Ehlert, Alice G. M. Bot, Edixhoven, Marcel, Wendy E. M. Boomaars, Lohmann, Suzanne M., De Jonge, Hugo R.
Format:	Article
Language:	eng
Subjects:	Animals Biochemistry Biological Sciences Cell Compartmentation Cell lines Cell Membrane - enzymology Cell membranes Cellular immunity Chloride Channels - physiology COS cells Cyclic GMP-Dependent Protein Kinases - metabolism Cystic fibrosis Cystic Fibrosis Transmembrane Conductance Regulator - genetics Enzymes Epithelial cells Humans Infections Ion Channel Gating Membranes Myristates Phosphorylation Protein Processing, Post-Translational Proteins Rats Recombinant Proteins Transfection
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Summary:	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 1β , 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 of32P 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.
ISSN:	0027-8424 1091-6490