Recruitment of pleckstrin and phosphoinositide 3-kinase gamma into the cell membranes, and their association with G beta gamma after activation of NK cells with chemokines

Abstract

The role of phosphoinositide 3 kinases (PI 3-K) in chemokine-induced NK cell chemotaxis was investigated. Pretreatment of NK cells with wortmannin inhibits the in vitro chemotaxis of NK cells induced by lymphotactin, monocyte-chemoattractant protein-1, RANTES, IFN-inducible protein-10, or stromal-derived factor-1 alpha. Introduction of inhibitory Abs to PI 3-K gamma but not to PI 3-K alpha into streptolysin O-permeabilized NK cells also inhibits chemokine-induced NK cell chemotaxis. Biochemical analysis showed that within 2-3 min of activating NK cells, pleckstrin is recruited into NK cell membranes, whereas PI 3-K gamma associates with these membranes 5 min after stimulation with RANTES. Recruited PI 3-K gamma generates phosphatidylinositol 3,4,5 trisphosphate, an activity that is inhibited upon pretreatment of NK cells with wortmannin. Further analysis showed that a ternary complex containing the beta gamma dimer of G protein, pleckstrin, and PI 3-K gamma is formed in NK cell membranes after activation with RANTES. The recruitment of pleckstrin and PI 3-K gamma into NK cell membranes is only partially inhibited by pertussis toxin, suggesting that the majority of these molecules form a complex with pertussis toxin-insensitive G proteins. Our results may have application for the migration of NK cells toward the sites of inflammation.