Granulocyte-macrophage colony-stimulating factor-induced protein tyrosine phosphorylation of microtubule-associated protein kinase in human neutrophils.

Abstract

Granulocyte-macrophage colony-stimulating factor (GM-CSF), formylmethionylleucylphenylalanine, tumor necrosis factor a, platelet-activating factor, phorbol ester (phorbol 12-myristate 13-acetate), and calcium ionophore A23187 are able to increase the level of tyrosine phosphorylation of different protein substrates, as demonstrated by Western blotting with anti-phosphotyrosine antibody (anti-PY). A protein of 41 kDa (p41) consistently showed more intense reactivity to anti-PY than controls. Blots treated with anti-PY, stripped of the antibody, and reblotted with microtubuleassociated protein kinase (MAPK, p42m"K) antibody show only one band. The molecular mass of that band exactly matches that of p41. MAPK-reactive protein is present in control and stimulated cells, although the intensity of the band is greater in the latter. GM-CSF-stimulated phosphorylation of p41 is time-and dose-dependent. Anti-MAPK antibody detects a single band of 41 kDa, whose intensity increases with time of incubation and concentration of the agonist. Thus, the anti-MAPK antibody appears to react better to the phosphorylated form of p41 from GM-CSF-stimulated cells than to the dephosphorylated form. The p41 and MAPK proteins are localized in the cytosol. Finally, MAPK immunoprecipitates were probed with anti-PY in Western blots and a band of 41 kDa was found. In summary, these results suggest that this 41-kDa protein in neutrophils that is tyrosine phosphorylated in response to GM-CSF and other stimuli is MAPK. Its phosphorylation may represent an early and crucial signal associated with the GM-CSF neutrophil stimulation cascade.

Regulation of Proliferation, Differentiation and Survival by the IL-3/IL-5/GM-CSF Receptor Family

“…SHP2 in IL-3/IL-5/GM-CSF signalling remains to be determined, because it also seems to play a positive role in IL-3/ A role for ERK activation by IL-3/IL-5/GM-CSF in neutrophil effector functions was suggested [151,152]. However, IL-5/GM-CSF signalling, coupling the receptor to Grb2 and PI3K [44,164].…”

Activation of mitogen-activated protein kinase cascades during priming of human neutrophils by TNF-α and GM-CSF

“…As GM-CSF primes PMN functional responses [7,12] and stimulates ERK activity in PMNs [24,29,30], ERK activation by GM-CSF was compared between GM-CSF and TNF-␣. Using the in vitro kinase assay, GM-CSF stimulated maximal ERK activity at concentrations from 300 to 500 pM, and maximal stimulation occurred at 5 min and fell gradually though 30 min (data not shown).…”

“…Although three priming agents, TNF-␣, GM-CSF, and LPS, stimulate p38 MAPK activity in human PMNs [24][25][26][27][28], they differ in their ability to stimulate ERK activation. GM-CSF stimulates a robust increase in ERK activity in human PMNs [24,29,30]. Several studies report that TNF-␣ fails to stimulate ERK activation in PMNs in suspension [24,26,31], whereas TNF-␣ increases ERK activity in adherent PMNs [26].…”

Bacterial phagocytosis activates extracellular signal-regulated kinase and p38 mitogen-activated protein kinase cascades in human neutrophils

“…Inflammatory stimuli, including LPS, tumor necrosis factor ␣ (TNF-␣), granulocyte-macrophage colony-stimulating factor (GM-CSF), and chemoattractants, have been shown to activate ERKs and/or p38 kinases, but not JNK, in human PMNs [41][42][43][44][45][46][47][48][49][50][51]. Pharmacological inhibition of ERK and p38 kinase cascades attenuates N-formyl-methionyl-leucyl-phenylalanine (fMLP)-stimulated respiratory burst activity, adherence, and/or chemotaxis [42,48,52,53].…”