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 the accumulation of PtdIns(3,4)P2 and PtdIns(3,4,5)P3 (labelled with 32P or myo-[3H]inositol), in CHRF-288 cells, a leukaemic cell line derived from a platelet progenitor cell. We report that these cells, when exposed to thrombin or SFLLRN (the peptide Ser-Phe-Leu-Leu-Arg-Asn, a thrombin-receptor ligand) rapidly change shape, forming membrane 'blebs', detectable by differential interference contrast or confocal microscopy, as well as labelled 3-phosphorylated phosphoinositides. The 'blebs' are distinguishable from 'ruffles' or lamellae, since they do not contain phalloidin-detectable actin. Studies with permeabilized cells indicate that PI 3-K is activated synergistically by thrombin+guanosine 5'[gamma-thio]triphosphate. Two forms of PI 3-K, i.e. PI 3-K(gamma) and p85/PI 3-K, regulated by G beta gamma subunits of heterotrimeric G-protein and the small G-protein Rho, respectively, are present in these cells, as is true for platelets. Wortmannin, a known potent and specific inhibitor of PI 3-K activities, inhibits thrombin-stiumlated accumulation of 3-phosphorylated phosphoinositides in a dose-dependent manner (IC50 approximately 10nM), without affecting phospholipase C activation. Pretreatment of CHRF-288 cells with either wortmannin (100 nM) or an unrelated synthetic PI 3-K inhibitor, LY294002 (50 microM), abolishes thrombin-receptor-stimulated blebbing. These results suggest that thrombin-stimulated accumulation of 3-phosphorylated phosphoinositide(s) is required for the shape-change response in CHRF-288 cells.
Vemuri
G S
GS
Department of Pharmacology, Thomas Jefferson University, Philadelphia, PA 19107, USA.
Zhang
J
J
Huang
R
R
Keen
J H
JH
Rittenhouse
S E
SE
D000730:Androstadienes / Q000494:pharmacology*; D001792:Blood Platelets / Q000201:enzymology*; D015153:Blotting, Western; D002462:Cell Membrane / Q000187:drug effects / Q000502:physiology* / Q000648:ultrastructure; D002463:Cell Membrane Permeability; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006801:Humans; D007527:Isoenzymes / Q000032:analysis / Q000037:antagonists & inhibitors / Q000378:metabolism; D007700:Kinetics; D007938:Leukemia; D010446:Peptide Fragments / Q000494:pharmacology*; D019869:Phosphatidylinositol 3-Kinases; D010716:Phosphatidylinositols / Q000378:metabolism*; D010761:Phosphorus Radioisotopes; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000032:analysis / Q000037:antagonists & inhibitors / Q000378:metabolism*; D018179:Receptors, Thrombin / Q000502:physiology; D013917:Thrombin / Q000494:pharmacology*; D014407:Tumor Cells, Cultured; D000077191:Wortmannin
D000730:Androstadienes; D004791:Enzyme Inhibitors; D007527:Isoenzymes; D010446:Peptide Fragments; D010716:Phosphatidylinositols; D010761:Phosphorus Radioisotopes; D018179:Receptors, Thrombin; C082835:thrombin receptor peptide (42-47); D019869:Phosphatidylinositol 3-Kinases; D017853:Phosphotransferases (Alcohol Group Acceptor); D013917:Thrombin; D000077191:Wortmannin