Phosphoinositide 3-kinase-dependent and -independent activation of the small GTPase Rac2 in human neutrophils.
The small GTPase Rac participates in various cellular events such as cytoskeletal reorganization. It has remained, however, largely unknown about intracellular signaling pathways for Rac activation because of the lack of a simple and reliable assay to estimate the activation. Here we describe a novel method to detect the GTP-bound, active Rac in cells by pulling it down with the Rac-binding domain of the protein kinase PAK. Experiments using this method reveal that stimulation of human neutrophils with the Gi-coupled receptor agonists N-formyl-methionyl-leucyl-phenylalanine (fMLP) and leukotriene B4 (LTB4) leads to a rapid and transient increase in the GTP-bound state of Rac2, whereas phorbol myristate acetate (PMA) causes a slow but more sustained activation of Rac2. Pretreatment of cells with pertussis toxin results in defective activation of Rac2 in response to fMLP and LTB4, indicating that coupling of the receptors to Gi plays a crucial role in the activation. Furthermore, the phosphoinositide 3-kinase (PI3K) inhibitors wortmannin and LY294002 block Rac2 activation elicited by the receptor agonists, but not that by PMA. Thus the Gi-coupled receptors likely mediate Rac2 activation via PI3K, whereas PMA activates Rac2 in a PI3K-independent manner.
Akasaki
T
T
Department of Molecular and Structural Biology, Kyushu University Graduate School of Medical Science, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan.
Koga
H
H
Sumimoto
H
H
D000730:Androstadienes / Q000494:pharmacology; D002867:Chromones / Q000494:pharmacology; D004789:Enzyme Activation; D019204:GTP-Binding Proteins; D006160:Guanosine Triphosphate / Q000378:metabolism; D006801:Humans; D007211:Indoles / Q000494:pharmacology; D007975:Leukotriene B4 / Q000494:pharmacology; D008301:Maleimides / Q000494:pharmacology; D009025:Morpholines / Q000494:pharmacology; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D009504:Neutrophils / Q000201:enzymology*; D037342:Pertussis Toxin; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D015398:Signal Transduction; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D010566:Virulence Factors, Bordetella / Q000494:pharmacology; D000077191:Wortmannin; D020744:rac GTP-Binding Proteins
D000730:Androstadienes; D002867:Chromones; D019204:GTP-Binding Proteins; D006160:Guanosine Triphosphate; D007211:Indoles; D007975:Leukotriene B4; D008301:Maleimides; D009025:Morpholines; D009240:N-Formylmethionine Leucyl-Phenylalanine; D037342:Pertussis Toxin; D019869:Phosphatidylinositol 3-Kinases; D017346:Protein Serine-Threonine Kinases; D013755:Tetradecanoylphorbol Acetate; D010566:Virulence Factors, Bordetella; D000077191:Wortmannin; D020744:rac GTP-Binding Proteins; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; C070515:bisindolylmaleimide I