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

Abstract

A BSTR ACTA recently cloned isoform of cGMPdependent 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 (Gly 2 3 Ala) reduced cGK II membrane binding and severely impaired cGK II activation of CFTR. Conversely, a chimeric protein, in which the N-terminal membraneanchoring domain of cGK II was fused to the N terminus of cGK I␤, 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 > membranebound cGK I chimer > > nonmyristoylated cGK II > cGK I␤) correlated with the extent of 32 P 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.

The Large Conductance, Voltage-dependent, and Calcium-sensitive K+ Channel, Hslo, Is a Target of cGMP-dependent Protein Kinase Phosphorylation in Vivo

“…However, they stress the point that maintenance of a native intracellular architecture may be important for specific channel responses. In fact, other kinases like cAMP-dependent protein kinase and PKG-II are targeted to ion channels via anchoring proteins or anchoring domain(s) enhancing channel regulation (47,48). In line with this view, PKG-I␣ has been found to interact with the intermediate filament protein vimentin (49).…”

ICln159 Folds into a Pleckstrin Homology Domain-like Structure

“…It is noteworthy that in contrast to other substrates of PKGII in vivo, i.e. cystic fibrosis transmembrane conductance regulator (CFTR) (48,49), ICln is able to discriminate between PKGI and PKGII in vitro (Fig. 4a).…”

Status of Fluid and Electrolyte Absorption in Cystic Fibrosis

“…CFTR is also unique among ion channels with respect to (1) complex molecular structure (Riordan et al 1989;Welsh et al 1992); (2) multiple regulatory controls involving PKC (Berger et al 1993;Jia et al 1997), PKA (Cheng et al 1991;Berger et al 1993;Dulhanty and Riordan 1994), PKG (Picciotto et al 1992;French et al 1995;Vaandrager et al 1996Vaandrager et al , 1998, Ca 2þ calmodulin-dependent protein kinases (Picciotto et al 1992), protein tyrosine kinases (Gadsby and Nairn 1999), and different protein phosphatases (Berger et al 1993;Fischer et al 1998;Luo et al 1998;Gadsby and Nairn 1999); and (3) multifunctional behavior involving several transport functions (Schwiebert et al 1999). Structurally, it is distinguished from most ion channels by the presence of a regulatory domain (R-domain) with multiple consensus phosphorylation sites and two nucleotide-binding domains (NBDs) (Riordan et al 1989;Welsh et al 1992).…”