TYROSINE PHOSPHORYLATION AND ACTIVATION OF A NEW MITOGEN-ACTIVATED PROTEIN (MAP)-KINASE CASCADE IN HUMAN NEUTROPHILS STIMULATED WITH VARIOUS AGONISTS

The presence of a novel 38 kDa protein that is tyrosine phosphorylated in human neutrophils, a terminally differentiated cell, upon stimulat ion of these cells with low concentrations of lipopolysaccharide (LPS) in combination with serum has been demonstrated. This 38 kDa protein was identified as the mammalian homologue of HOG1 in yeast, the p38 mi togen-activated protein (MAP) kinase. This conclusion is based on the experimental findings that anti-phosphotyrosine (anti-PY) antibody imm unoprecipitates a 38 kDa protein that is recognized by anti-p38 MAP ki nase antibody, and conversely, anti-p38 MAP kinase antibody immunoprec ipitates a 38 kDa protein that can be recognized by anti-PY antibody. Moreover, this tyrosine phosphorylated protein is found associated ent irely with the cytosol. It was also found that this p38 MAP kinase is activated following stimulation of these cells with low concentrations of LPS in combination with serum. This conclusion is based on three e xperimental findings. First, soluble fractions isolated from LPS-stimu lated cells phosphorylate heat shock protein 27 (hsp27) in an in vitro assay, and this effect is not inhibited by protein kinase C and prote in kinase A inhibitor peptides. This effect is similar to the effect p roduced by the commercially available phosphorylated and activated MAP KAP kinase-2 (MAP kinase activated protein kinase-2). Secondly, a 27 k Da protein that aligns with a protein recognized by anti-hsp27 antibod y is phosphorylated upon LPS stimulation of intact human neutrophils p relabelled with radioactive phosphate, Lastly, immune complex protein kinase assays, using [gamma-P-32]ATP and activating transcription fact or 2 (ATF2) as substrates, showed increased p38 MAP kinase activity fr om LPS-stimulated human neutrophils. The phosphorylation and activatio n of this p38 MAP kinase can be affected by both G-protein-coupled rec eptors such as platelet-activating factor (PAF) and non-G-protein-coup led receptors such as the cytokine-coupled receptors for granulocyte-m acrophage colony-stimulating factor (GM-CSF) and tumour necrosis facto r alpha (TNF-alpha). The effect of low concentrations of PAF is greatl y increased in cells pretreated with LPS. The tyrosine phosphorylation of the p38 MAP kinase is not restricted to stimuli that mediate their actions through membrane-associated receptors, but it can be affected by agents that bypass membrane-associated receptors such as the prote in translation blocker anisomycin. While anisomycin is known to increa se the tyrosine phosphorylation of the 54 kDa SAPK (stress-activated p rotein kinase), this is the first report that shows that anisomycin al so tyrosine phosphorylates the p38 MAP kinase. Cytokine receptors that increase the tyrosine phosphorylation and activation of the erk1 and erk2 MAP kinases have less effect on this p38 MAP kinase than those th at do not affect the erkl and erk2 MAP kinases. The possible role of t he p38 MAP kinase in the phosphorylation of cytosolic phospholipase A( 2) is discussed.