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Granulocyte-macrophage colony-stimulating factor-induced protein tyrosine phosphorylation of microtubule-associated
J Gomez-Cambronero1, C K Huang, T M Gomez-Cambronero
1Department of Physiology, University of Connecticut Health Center, Farmington 06030.
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.
Insights
Granulocyte-macrophage colony-stimulating factor (GM-CSF) and other stimuli increase tyrosine phosphorylation of a 41 kDa protein (p41) in neutrophils. This protein is identified as microtubule-associated protein kinase (MAPK), crucial for GM-CSF signaling.
Area of Science:
- Cellular signaling
- Molecular biology
- Immunology
Background:
- Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a key cytokine in neutrophil activation.
- Tyrosine phosphorylation plays a critical role in cellular signal transduction pathways.
- Identifying specific signaling molecules involved in GM-CSF responses is essential for understanding neutrophil function.
Purpose of the Study:
- To identify the 41 kDa protein (p41) that undergoes tyrosine phosphorylation in response to GM-CSF and other stimuli.
- To investigate the role of this p41 protein in the GM-CSF neutrophil stimulation cascade.
Main Methods:
- Western blotting using anti-phosphotyrosine (anti-PY) and anti-MAPK antibodies.
- Analysis of protein phosphorylation in response to various agonists including GM-CSF.
- Immunoprecipitation of MAPK followed by Western blotting with anti-PY.
Main Results:
- A 41 kDa protein (p41) consistently showed increased tyrosine phosphorylation upon stimulation with GM-CSF, formylmethionylleucylphenylalanine, TNF-alpha, PAF, PMA, and A23187.
- Western blotting confirmed that p41 is microtubule-associated protein kinase (MAPK, p42MAPK).
- MAPK phosphorylation was time- and dose-dependent on GM-CSF stimulation and localized in the cytosol.
Conclusions:
- The 41 kDa tyrosine-phosphorylated protein in neutrophils stimulated by GM-CSF is MAPK.
- MAPK phosphorylation is an early and crucial signaling event in the GM-CSF neutrophil activation pathway.