Endogenous reactive oxygen intermediates activate tyrosine kinases in human neutrophils

In response to invading microorganisms, neutrophils produce large amounts of superoxide and other reactive oxygen intermediates (ROI) by assembly and activation of a multicomponent enzyme complex, the NADPH oxi dase. While fulfilling a microbicidal role, ROI have also been postulated to serve as signaling molecules, because activation of the NADPH oxidase was found to be associated with increased tyrosine phosphorylation (Fialkow, L., Chan, C, K., Grinstein, S., and Downey, G, P. (1993) J. Biol. Chem. 268, 17131-17137), The mechanism whereby ROI induces phosphotyrosine accumulation was investigated using electroporated neutrophils stimulated with guanosine 5'-O-3-thiotriphosphate in order to bypass membrane receptors. In vitro immune complex assays and immunoblotting were used to identify five tyrosine kinases present in human neutrophils. Of these, p56/59(hck), p72(syk), and p77(bth) were activated during production of ROI. Interestingly, the in vitro autophosphorylation activities of p53/56(lyn) and p5(fgr) were found to decline with ROI production. The mode of regulation of p56/59(hch) was explored in detail. Oxidizing agents were unable to activate p56/59(hck) in vitro and, once activated in situ, reducing agents failed to inactivate it, suggesting that the effects of ROI are indirect. Tyrosine phosphorylation of p56/59(hck) paralleled its activation, and dephosphorylation in vitro reversed the stimulation, We therefore conclude that tyrosine phosphorylation is central to the regulation of p56/59(hck) and likely also of p72(syk), which is similarly phosphorylated upon activation of the oxidase, Because ROI have been shown to reduce the activity of tyrosine phosphatases, we suggest that this inhibition allows constitutively active kinases to auto/transphosphorylate on stimulatory tyrosine residues, leading to an increase in their catalytic activity, Enhanced phosphotyrosine accumulation would then result from the combined effects of increased phosphorylation with decreased dephosphorylation.