EFFECTS OF GRANULOCYTE-MACROPHAGE COLONY-STIMULATING FACTOR AND TUMOR-NECROSIS-FACTOR-ALPHA ON TYROSINE PHOSPHORYLATION AND ACTIVATION OF MITOGEN-ACTIVATED PROTEIN-KINASES IN HUMAN NEUTROPHILS

The present study was undertaken to determine the identities and chara cteristics of proteins with molecular masses between 40 and 44 kDa who se tyrosine phosphorylation increases in human neutrophils following s timulation of these cells with tumour necrosis factor alpha: (TNF-alph a) and granulocyte-macrophage colony-stimulating factor (GM-CSF), Immu noblotting results demonstrate that addition of GM-CSF to human neutro phils increases the tyrosine phosphorylation of two proteins with mole cular masses of 42 and 44 kDa, However, the addition of TNF-alpha to n eutrophils induces a time- and dose-dependent increase in tyrosine pho sphorylation of a 40 kDa protein. Immunoprecipitation using specific m itogen-activated protein kinase (MAPK) isoform antibodies and an antib ody which recognizes phosphotyrosine-containing proteins demonstrated that the 42 and 44 kDa proteins are isoforms of MAPKs. Utilizing an in situ gel kinase activity assay, GM-CSF increases the kinase activity of the 42 and 44 kDa proteins. Moreover, using immunoprecipitated p42 and p44 MAPK isoforms in this gel assay revealed activity associated w ith the p42 and p44 MAPK isoforms. Using the same in situ assay, TNF-a lpha induces an increase in kinase activity of a 40-42 kDa protein. Ho wever, the 40 kDa protein whose phosphorylation on tyrosine residues i ncreased in human neutrophils following stimulation with TNF-alpha is not a member of the known MAPK family, demonstrating the divergences i n pathways utilized by GM-CSF and TNF-alpha. This 40 kDa protein may b e related to the recently identified protein that becomes phosphorylat ed on tyrosine residues upon stimulation of the human epidermal carcin oma cell line KB by interleukin-1. In these cells the p40 protein is p art of a protein kinase cascade which results in the phosphorylation o f the small heat shock protein, hsp27.