Chemotactic peptide-induced activation of Ras in human neutrophils is associated with inhibition of p120-GAP activity.
The monomeric G-protein Ras is now considered to function as an initial regulator of multiple signaling pathways in both normal and transformed cell types. Adhesion and chemoattractant receptors are known to trigger activation of Ras in human neutrophils, but the signaling mechanism that activates Ras has only been partially elucidated. The present results show that in neutrophils, a time- and dose-dependent f-Met-Leu-Phe (FMLP)-induced activation of Ras is mediated by Gi2-proteins, because such activation is inhibited by pertussis toxin and because direct stimulation of heterotrimeric G-proteins with AlF4- is sufficient to activate Ras. Pretreatment of neutrophils with tyrosine kinase inhibitors, i.e. genistein or erbstatin that completely block FMLP-stimulated protein tyrosine phosphorylations, did not affect the FMLP-induced activation of Ras. Moreover, FMLP did not induce any detectable translocation of Grb2 and Sos to the plasma membrane of neutrophils. Other signaling molecules, such as protein kinase C, phosphatidylinositol 3-kinase and Ca2+, do not appear to be involved in the FMLP-induced Ras activation. Instead, stimulation of neutrophils with FMLP or C5a, the latter of which also activates Gi2-proteins, resulted in transient inhibition of the activity of Ras GTPase-activating proteins (GAP) with kinetics that correlated well with the kinetics of Ras activation. Moreover, decreased Ras-GAP activity was found in p120-GAP but not in neurofibromin immunoprecipitates of FMLP-stimulated cells. These results suggest that tyrosine kinase-dependent Ras exchange factors do not contribute to the FMLP-induced activation of Ras but that such activation is mediated via inhibition of p120-GAP in neutrophils.
Zheng
L
L
Division of Experimental Pathology, Department of Laboratory Medicine, Wallenberg Laboratory, Lund University, U-MAS, S-205 02 Malmö, Sweden. limin.zheng@medforsk.mas.lu.se
Eckerdal
J
J
Dimitrijevic
I
I
Andersson
T
T
D048868:Adaptor Proteins, Signal Transducing; D002118:Calcium / Q000378:metabolism; D005459:Fluorides / Q000494:pharmacology; D051380:GRB2 Adaptor Protein; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000378:metabolism; D020690:GTPase-Activating Proteins; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology*; D025542:Neurofibromin 1; D009504:Neutrophils / Q000187:drug effects / Q000378:metabolism*; D037342:Pertussis Toxin; D019869:Phosphatidylinositol 3-Kinases; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000378:metabolism; D011233:Precipitin Tests; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011506:Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism; D015398:Signal Transduction; D020664:Son of Sevenless Proteins; D010738:Type C Phospholipases / Q000378:metabolism; D010566:Virulence Factors, Bordetella / Q000494:pharmacology; D020703:ras GTPase-Activating Proteins; D018631:ras Proteins / Q000378:metabolism*
D048868:Adaptor Proteins, Signal Transducing; D051380:GRB2 Adaptor Protein; C495948:GRB2 protein, human; D020690:GTPase-Activating Proteins; D008565:Membrane Proteins; D025542:Neurofibromin 1; D011506:Proteins; D020664:Son of Sevenless Proteins; D010566:Virulence Factors, Bordetella; D020703:ras GTPase-Activating Proteins; D006153:Guanosine Diphosphate; D009240:N-Formylmethionine Leucyl-Phenylalanine; D006160:Guanosine Triphosphate; D037342:Pertussis Toxin; D019869:Phosphatidylinositol 3-Kinases; D017853:Phosphotransferases (Alcohol Group Acceptor); D011505:Protein-Tyrosine Kinases; D010738:Type C Phospholipases; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D018631:ras Proteins; D005459:Fluorides; D002118:Calcium