The apical membrane of intestinal epithelial cells harbors a unique
isozyme of cGMP-dependent protein kinase (cGK type II) which acts as a key
regulator of ion transport systems, including the cystic fibrosis
transmembrane conductance regulator (CFTR)-chloride channel. To explore
the mechanism of cGK II membrane-anchoring, recombinant cGK II was
expressed stably in HEK 293 cells or transiently in COS-1 cells. In both
cell lines, cGK II was found predominantly in the particulate fraction.
Immunoprecipitation of solubilized cGK II did not reveal any other tightly
associated proteins, suggesting a membrane binding motif within cGK II
itself. The primary structure of cGK II is devoid of hydrophobic
transmembrane domains; cGK II does, however, contain a penultimate
glycine, a potential acceptor for a myristoyl moiety. Metabolic labeling
showed that cGK II was indeed able to incorporate [3H]myristate. Moreover,
incubation of cGK II-expressing 293 cells with the myristoylation
inhibitor 2-hydroxymyristic acid (1 mM) significantly increased the
proportion of cGK II in the cytosol from 10 +/- 5 to 35 +/- 4%.
Furthermore, a nonmyristoylated cGK II Gly2 --> Ala mutant was localized
predominantly in the cytosol after transient expression in COS-1 cells.
The absence of the myristoyl group did not affect the specific enzyme
activity or the Ka for cGMP and only slightly enhanced the thermal
stability of cGK II. These results indicate that N-terminal myristoylation
fulfills a crucial role in directing cGK II to the membrane