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Direct stimulation by tyrosine phosphorylation of microtubule-associated protein (MAP) kinase activity by
J Gomez-Cambronero1, J M Colasanto, C K Huang
1Department of Physiology, University of Connecticut Health Center, Farmington 06030.
Abstract:
Human polymorphonuclear neutrophils exhibit a low level of the microtubule-associated protein kinase (MAPK) activity. This enzymic activity is enhanced up to 3-fold upon cell stimulation with the human haematopoietic hormone granulocyte-macrophage colony-stimulating factor (GM-CSF). This is demonstrated both in whole-cell lysates and in DEAE-anion-exchange semi-purified fractions prepared from GM-CSF-stimulated neutrophils, by assaying the kinase activity against either myelin basic protein or a phosphoacceptor peptide that bears the specific phosphorylation site of the MAPK natural substrate. Similarly, phosphorylation of MAPK in tyrosine residues, as found in immunoblots using anti-phosphotyrosine antibodies, follows similar time- and dose-response curves as the kinase activation. Pretreatment of the cells with the tyrosine kinase inhibitor genistein abrogates the above-mentioned effect, whereas the phosphatase inhibitor okadaic acid enhances both the basal and the GM-CSF-stimulated kinase activities. Likewise, MAPK tyrosine phosphorylation is diminished in genistein-treated neutrophils, and enhanced in okadaic acid-treated cells. We conclude that MAPK activity is present in human neutrophils, and that it is stimulated by GM-CSF. This stimulation of the activity is most likely due to the phosphorylation of MAPK in tyrosine residues triggered upon binding of GM-CSF to its receptors.
Insights
Human neutrophils possess microtubule-associated protein kinase (MAPK) activity, which is significantly boosted by granulocyte-macrophage colony-stimulating factor (GM-CSF). This GM-CSF-induced MAPK activation involves tyrosine phosphorylation.
Area of Science:
- Immunology
- Cell Biology
- Biochemistry
Background:
- Human polymorphonuclear neutrophils exhibit low basal microtubule-associated protein kinase (MAPK) activity.
- Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a key hematopoietic hormone involved in neutrophil function.
Purpose of the Study:
- To investigate the effect of GM-CSF on MAPK activity in human neutrophils.
- To elucidate the molecular mechanisms underlying GM-CSF-mediated MAPK activation.
Main Methods:
- Assessing MAPK kinase activity in whole-cell lysates and semi-purified fractions using myelin basic protein and a specific phosphoacceptor peptide.
- Utilizing immunoblotting with anti-phosphotyrosine antibodies to detect MAPK tyrosine phosphorylation.
- Employing tyrosine kinase inhibitor (genistein) and phosphatase inhibitor (okadaic acid) to probe signaling pathways.
Main Results:
- GM-CSF stimulation enhanced MAPK activity up to 3-fold in human neutrophils.
- MAPK tyrosine phosphorylation levels correlated with kinase activity, increasing upon GM-CSF stimulation.
- Genistein inhibited GM-CSF-induced MAPK activation and tyrosine phosphorylation, while okadaic acid enhanced both.
Conclusions:
- MAPK is enzymatically active in human neutrophils and its activity is stimulated by GM-CSF.
- GM-CSF-induced MAPK activation is mediated by tyrosine phosphorylation, likely triggered by GM-CSF receptor binding.