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 microtu bule-associated protein kinase (MAPK) activity. This enzymic activity is enhanced up to 3-fold upon cell stimulation with the human haematop oietic hormone granulocyte-macrophage colony-stimulating factor (GM-CS F). This is demonstrated both in whole-cell lysates and in DEAE-anion- exchange semi-purified fractions prepared from GM-CSF-stimulated neutr ophils, by assaying the kinase activity against either myelin basic pr otein or a phosphoacceptor peptide that bears the specific phosphoryla tion site of the MAPK natural substrate. Similarly, phosphorylation of MAPK in tyrosine residues, as found in immunoblots using anti-phospho tyrosine antibodies, follows similar time- and dose-response curves as the kinase activation. Pretreatment of the cells with the tyrosine ki nase inhibitor genistein abrogates the above-mentioned effect, whereas the phosphatase inhibitor okadaic acid enhances both the basal and th e GM-CSF-stimulated kinase activities. Likewise, MAPK tyrosine phospho rylation is diminished in genistein-treated neutrophils, and enhanced in okadaic acid-treated cells. We conclude that MAPK activity is prese nt in human neutrophils, and that it is stimulated by GM-CSF. This sti mulation of the activity is most likely due to the phosphorylation of MAPK in tyrosine residues triggered upon binding of GM-CSF to its rece ptors.