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. 1992 Aug 15;89(16):7551–7555. doi: 10.1073/pnas.89.16.7551

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

J Gomez-Cambronero 1, C K Huang 1, T M Gomez-Cambronero 1, W H Waterman 1, E L Becker 1, R I Sha'afi 1
PMCID: PMC49748  PMID: 1323842

Abstract

Granulocyte-macrophage colony-stimulating factor (GM-CSF), formylmethionylleucylphenylalanine, tumor necrosis factor alpha, 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 microtubule-associated protein kinase (MAPK, p42MAPK) 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.

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