Localizing a control region in the pathway to leukotriene C₄ secretion following stimulation of human basophils with anti-IgE antibody

Mediator release from human basophils is a self-limited process, but down-regulation of the signaling cascades leading to secretion of leukotriene C₄ (LTC₄) is controlled independently of the pathway leading to IL-4 secretion. In the current studies, we have explored the regulation of upstream signaling events leading to activation of extracellular signal-related kinases (ERKs; previously shown to be required for LTC₄ generation) in human basophils. IgE-, but not FMLP-mediated activation, induced sustained tyrosine phosphorylation of syk, of shc, and an association of shc to the Grb2/son of sevenless 2 complex. In contrast, IgE-mediated activation resulted in transient activation of p21^ras and mitogen-activated protein/ERK kinase 1, which were kinetically associated with phosphorylation of ERKs. The canonical Shc/Grb2/son of sevenless pathway to activation of p21^ras is therefore sustained, while p21^ras activity is not. We have previously shown that phosphatidylinositol 3 kinase activity is required for p21^ras activity and, in the current studies, we show that of the p85-sensitive forms of p110 possible, basophils express only p110 δ and that there are no changes in association between p21^ras and p110 δ in stimulated basophils. We used the generation of phospho-Akt as a marker of the presence of phosphatidylinositol-3,4,5-trisphosphate and found that phospho-Akt is transient on a time scale consistent with p21^ras activity. On the basis of information obtained in these and other studies, we localize down-regulation of IgE-mediated LTC4 secretion to a region of the signaling cascade antecedent to p21^ras activation, downstream of phosphatidylinositol 3 kinase activity and probably involving regulation of phosphatidylinositol-3,4,5-trisphosphate levels.