Direct stimulation by tyrosine phosphorylation of microtubule-associated protein (MAP) kinase activity by granulocyte-macrophage colony-stimulating factor in human neutrophils.
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.
Gomez-Cambronero
J
J
Department of Physiology, University of Connecticut Health Center, Farmington 06030.
Colasanto
J M
JM
Huang
C K
CK
Sha'afi
R I
RI
D017871:Calcium-Calmodulin-Dependent Protein Kinases; D002852:Chromatography, Ion Exchange; D004988:Ethers, Cyclic / Q000494:pharmacology; D019833:Genistein; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000494:pharmacology*; D006801:Humans; D015151:Immunoblotting; D007529:Isoflavones / Q000494:pharmacology; D009504:Neutrophils / Q000201:enzymology*; D019319:Okadaic Acid; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors; D010766:Phosphorylation; D019000:Phosphotyrosine; D011494:Protein Kinases / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000378:metabolism
D004988:Ethers, Cyclic; D007529:Isoflavones; D019319:Okadaic Acid; D019000:Phosphotyrosine; D014443:Tyrosine; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D019833:Genistein; D011494:Protein Kinases; D011505:Protein-Tyrosine Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D010749:Phosphoprotein Phosphatases