THROMBIN STIMULATES WORTMANNIN-INHIBITABLE PHOSPHOINOSITIDE 3-KINASE AND MEMBRANE BLEBBING IN CHRF-288 CELLS

We have investigated thrombin-stimulated morphological changes and the activation of phosphoinositide 3-kinase (PI 3-K), as manifested by th e accumulation of PtdIns(3,4)P-2 and PtdIns(3,4,5)P-3 (labelled with P -32 or myo-[H-3]inositol), in CHRF-288 cells, a leukaemic cell line de rived from a platelet progenitor cell. We report that these cells, whe n exposed to thrombin or SFLLRN (the peptide Ser-Phe-Leu-Leu-Arg-Asn, a thrombin-receptor ligand) rapidly change shape, forming membrane 'bl ebs', detectable by differential interference contrast or confocal mic roscopy, as well as labelled 3-phosphorylated phosphoinositides. The ' blebs' are distinguishable from 'ruffles' or lamellae, since they do n ot contain phalloidin-detectable actin. Studies with permeabilized cel ls indicate that PI 3-K is activated synergistically by thrombin + gua nosine 5'-[gamma-thio]triphosphate. Two forms of PI 3-K, i.e. PI 3-K(g amma) and p85/PI 3-K, regulated by G beta gamma subunits of heterotrim eric G-protein and the small G-protein Rho, respectively, are present in these cells, as is true for platelets. Wortmannin, a known potent a nd specific inhibitor of PI 3-K activities, inhibits thrombin-stimulat ed accumulation of 3-phosphorylated phosphoinositides in a dose-depend ent manner (IC50 similar to 10nM), without affecting phospholipase C a ctivation. Pretreatment of CHRF-288 cells with either wortmannin (100 nM) or an unrelated synthetic PI 3-K inhibitor, LY294002 (50 mu M), ab olishes thrombin-receptor-stimulated blebbing. These results suggest t hat thrombin-stimulated accumulation of 3-phosphorylated phosphoinosit ide(s) is required for the shape-change response in CHRF-288 cells.