Involvement of tyrosine kinases, Ca2+ and PKC in activation of mitogen-activated protein (MAP) kinase in human polymorphonuclear neutrophils.

1. Activation of mitogen-activated protein (MAP) kinase is an early response to a wide variety of stimuli and plays an important role in the regulation of cellular functions. In the present study we investigated the activation of MAP kinase in human polymorphonuclear neutrophils (PMNs). 2. Activity of MAP kinase and protein kinase C (PKC) was measured radiometrically from the rate of phosphorylation of specific peptide substrates. Protein phosphorylation was measured by immunoprecipitation and Western blot analysis. 3. N-Formyl-Met-Leu-Phe (fMLP), phorbol 12-myristate, 13-acetate (PMA) and the Ca2+-ATPase inhibitors thapsigargin (Tg) and cyclopiazonic acid (CPA) increased MAP kinase activity significantly. The tyrosine kinase inhibitors erbstatin and herbimycin A partially inhibited the effects of fMLP and PMA, and completely abolished the effects of both Tg and CPA. The specific PKC inhibitor calphostin C suppressed activation of MAP kinase produced by fMLP and PMA, but had no effect on that produced by Tg and CPA. Tg and CPA were without effect on PKC activity. 4. Immunoprecipitation and Western blot analysis indicated that the 42 and 44 kDa tyrosine-phosphorylated proteins found after stimulation of PMNs were both members of the MAP kinase family. Pretreatment of PMNs with staurosporine, EGTA or erbstatin significantly reduced the tyrosine phosphorylation of MAP kinase(s). 5. These results suggest that in human PMNs, MAP kinase can be stimulated in both a PKC-dependent and a PKC-independent manner. The Ca2+ signal leads to activation of tyrosine kinases, which contribute to the activation of MAP kinase. However, a PMA-sensitive Ca2+-independent pathway also exists. Mobilization of Ca2+ and activation of PKC synergistically induce maximal MAP kinase activation and tyrosine phosphorylation.