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 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 Ibeta, 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 Ibeta) 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.
|Animals, Cell Compartmentation, Cell Membrane/enzymology, Chloride Channels/*physiology, Cyclic GMP-Dependent Protein Kinases/*metabolism, Cystic Fibrosis Transmembrane Conductance Regulator/*genetics, Humans, Ion Channel Gating, Myristates, Phosphorylation, Protein Processing, Post-Translational, Rats, Recombinant Proteins, Transfection|
|Proceedings of the National Academy of Sciences of the United States of America|
|Organisation||Erasmus MC: University Medical Center Rotterdam|
Vaandrager, A.B, Smolenski, A, Tilly, B.C, Houtsmuller, A.B, Ehlert, E.M.E, Bot, A.G, … de Jonge, H.R. (1998). Membrane targeting of cGMP-dependent protein kinase is required for cystic fibrosis transmembrane conductance regulator Cl- channel activation. Proceedings of the National Academy of Sciences of the United States of America. doi:10.1073/pnas.95.4.1466