PALMITOYLATION OF G-PROTEIN-COUPLED RECEPTOR KINASE, GRK6 - LIPID MODIFICATION DIVERSITY IN THE GRK FAMILY

GRK6, a 66-kDa serine/threonine protein kinase, is a recently identifi ed member of the G protein-coupled receptor kinase (GRK) family. GRKs are involved in the phosphorylation of seven-transmembrane receptors, a process mediating desensitization of signal transduction. An importa nt feature of these enzymes is their membrane-associated nature, which for some members is stimulus dependent. The structural basis for this membrane association previously has been shown in different members o f the GRK family to include isoprenylation, G protein beta gamma-bindi ng domains, and basic regions to provide electrostatic interactions wi th phospholipids. We provide evidence that another mechanism includes fatty acid acylation. GRK6, but not other GRKs tested, incorporated tr itium after incubation with [H-3]palmitate in Sf9 and in COS-7 cells o verexpressing the kinase. The incorporated radioactivity was released from the protein by neutral hydroxylamine, indicating the presence of a thioester bond, and was confirmed as palmitic acid by high performan ce liquid chromatography analysis. Site-directed mutagenesis defined t he region of palmitate attachment as a cluster of 3 cysteines (Cys(561 ), Cys(562), and Cys(565)) the carboxyl-terminal domain of the kinase, consistent with the location of the membrane targeting domains of GRK s 1, 2, 3, and 5. Palmitoylation of GRK6 appears essential for membran e association, since palmitoylated kinase was found only in the membra ne fraction. This lipid modification provides a structural basis for p otential regulation of the subcellular distribution of GRK6 through ac ylation/deacylation cycles.