The activity of c-Src protein-tyrosine kinase is upregulated under a number of receptor signaling pathways. However, the activation mechanism of c-Src under physiological conditions has remained unclear. We show here that the Shc adaptor protein is a novel direct activator of c-Src in epidermal growth factor receptor signaling in A431 human epidermoid carcinoma cells. Among the three Shc isoforms, P66 and P52, but not P46, were found to interact with and activate c-Src in vitro and in vivo. Activation of c-Src accompanied autophosphorylation of c-Src in the activation segment, but the carboxyl-terminal dephosphorylation was not observed. We have identified the interaction sites between Shc and c-Src and constructed a point mutant of Shc that abolishes the c-Src activation. Using this mutant, we have confirmed that the Shc-mediated c-Src activation triggers Stat-p21/WAF1/Cip1 pathway that has been implicated in the cell cycle arrest and apoptosis of epidermal growth factor-stimulated A431 cells.

Background : Cell surface receptor for the epidermal growth factor (EGFR) and cytoplasmic tyrosine kinase c-Src co-operate in several cellular functions such as proliferation and apoptosis. Our previous studies have shown that ectopic expression of the adaptor protein p52 shc or p66 shc , but not p46 shc , and EGF stimulation lead to the activation of c-Src that is accompanied by phosphorylation of signal transducers and activators of transcription (Stat) in A431 cells.

The effects of β-adrenoceptor agonists (β-agonists) on the production of tumor necrosis factor-α (TNF-α), interleukin-1 β (IL-1β) and interleukin-8 (IL-8) by lipopolysaccharide (LPS)-stimulated human peripheral blood mononuclear cells (PBMCs) were investigated. The β-agonists, procaterol, clenbuterol, fenoterol and terbutaline, inhibited TNF-α and IL-1β production in a concentration-dependent manner, whereas they had no effect on IL-8 production. TNF-α production was inhibited more potently than IL-1β. Dibutyryl cyclic AMP (dbcAMP) also inhibited the production of TNF-α and IL-1β, but not IL-8. TNF-α production was almost completely inhibited by dbcAMP, whereas IL-1β production appeared to be partially refractory even at the highest concentration examined. Both procaterol and the-ophylline elevated cAMP levels in LPS-stimulated PBMCs, but the effect of procaterol was limited. The inhibition of TNF-α and IL-1β production by procaterol was additively potentiated with theophylline. dl-Propranolol, a β-adrenoceptor antagonist, abrogated the inhibition of TNF-α and IL-1β production by procaterol. These results indicate that β-agonists inhibit the production of proinflammatory cytokines, such as TNF-α and IL-1β, by elevating intracellular cAMP levels. These properties of β-agonists might be beneficial in the treatment of allergic inflammation.

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