Thrombin stimulates wortmannin-inhibitable phosphoinositide 3-kinase and membrane blebbing in CHRF-288 cells.

Abstract

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.

• Authors: G S Vemuri, J Zhang, R Huang, J H Keen, S E Rittenhouse
• Journal: The Biochemical journal (Biochem J) Vol. 314 ( Pt 3) Pg. 805-10 (Mar 15 1996) ISSN: 0264-6021 [Print] England
• PMID: 8615773 (Publication Type: Comparative Study, Journal Article, Research Support, U.S. Gov't, P.H.S.)

Chemical References

• Androstadienes
• Enzyme Inhibitors
• Isoenzymes
• Peptide Fragments
• Phosphatidylinositols
• Phosphorus Radioisotopes
• Receptors, Thrombin
• thrombin receptor peptide (42-47)
• Phosphatidylinositol 3-Kinases
• Phosphotransferases (Alcohol Group Acceptor)
• Thrombin
• Wortmannin

Topics

• Androstadienes (pharmacology)
• Blood Platelets (enzymology)
• Blotting, Western
• Cell Membrane (drug effects, physiology, ultrastructure)
• Cell Membrane Permeability
• Enzyme Inhibitors (pharmacology)
• Humans
• Isoenzymes (analysis, antagonists & inhibitors, metabolism)
• Kinetics
• Leukemia
• Peptide Fragments (pharmacology)
• Phosphatidylinositol 3-Kinases
• Phosphatidylinositols (metabolism)
• Phosphorus Radioisotopes
• Phosphotransferases (Alcohol Group Acceptor) (analysis, antagonists & inhibitors, metabolism)
• Receptors, Thrombin (physiology)
• Thrombin (pharmacology)
• Tumor Cells, Cultured
• Wortmannin