Granulocyte-macrophage colony-stimulating factor-activated signaling pathways in human neutrophils. I. Tyrosine phosphorylation-dependent stimulation of phosphatidylinositol 3-kinase and inhibition by phorbol esters.
Phosphatidylinositol 3-kinase (PI3-kinase) is a cytosolic enzyme that plays key roles in mediating signaling through many receptors. The heterodimeric form of PI3-kinase is made up of a regulatory subunit, p85, and a catalytic subunit, p110. Although granulocyte-macrophage colony-stimulating factor (GM-CSF) has been shown to activate PI3-kinase, the mechanisms by which this activation is mediated and regulated are incompletely understood. Here we show that treatment of human neutrophils with GM-CSF induced both time- and concentration-dependent increases in the level of tyrosine phosphorylation of p85. The ability of GM-CSF to activate PI3-kinase was abolished by pretreating the cells with erbstatin, a tyrosine kinase inhibitor. The simultaneous treatment of the cells with GM-CSF and phorbol esters such as phorbol 12-myristate 13-acetate (PMA) and phorbol 12,13-dibutyrate (PDBu) significantly inhibited both the tyrosine phosphorylation of p85 and the activation of PI3-kinase. The inhibitory effects of phorbol esters were not induced by their inactive analogues and they were selective to the stimulation of tyrosine phosphorylation of p85 since phorbol esters did not alter the enhancement of the pattern of tyrosine phosphorylation of other cellular proteins, including that of Jak2 induced by GM-CSF. However, PMA significantly inhibited the in situ tyrosine phosphorylation and the activation of lyn observed in response to GM-CSF. The results suggest that the activation of PI3-kinase by GM-CSF is mediated by the tyrosine phosphorylation of p85 and that this activation is downregulated by PKC possibly via the inhibition of lyn.
al-Shami
A
A
Centre de Recherche en Rhumatologie et Immunologie, Faculty of Medicine, Laval University, Ste-Foy, Québec, Canada.
Bourgoin
S G
SG
Naccache
P H
PH
D000328:Adult; D004789:Enzyme Activation / Q000187:drug effects; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000494:pharmacology*; D006801:Humans; D053614:Janus Kinase 2; D018375:Neutrophil Activation / Q000187:drug effects*; D009504:Neutrophils / Q000187:drug effects / Q000201:enzymology* / Q000378:metabolism*; D010703:Phorbol Esters / Q000494:pharmacology*; D019869:Phosphatidylinositol 3-Kinases; D010766:Phosphorylation / Q000187:drug effects; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D019000:Phosphotyrosine / Q000502:physiology*; D011505:Protein-Tyrosine Kinases / Q000187:drug effects; D011518:Proto-Oncogene Proteins; D015398:Signal Transduction / Q000187:drug effects*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D019061:src-Family Kinases / Q000187:drug effects
D010703:Phorbol Esters; D011518:Proto-Oncogene Proteins; D019000:Phosphotyrosine; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D019869:Phosphatidylinositol 3-Kinases; D017853:Phosphotransferases (Alcohol Group Acceptor); D011505:Protein-Tyrosine Kinases; C507924:JAK2 protein, human; D053614:Janus Kinase 2; D019061:src-Family Kinases; D013755:Tetradecanoylphorbol Acetate