H(2)O(2)-Induced Phosphorylation of ERK1/2 and PKB Requires Tyrosine Kinase Activity of Insulin Receptor and c-Src.

摘要

Hydrogen peroxide (H(2)O(2)) mimics many physiological responses of insulin, and increased H(2)O(2) generation via the Nox-4 subunit of NAD(P)H oxidase was recently demonstrated to serve as a critical early step in the insulin signaling pathway. Exogenously added H(2)O(2) has also been shown to activate several key components of the insulin signaling cascade. H(2)O(2)-induced signaling responses have been found to be associated with the activation of receptor and nonreceptor protein tyrosine kinases (PTK), including the insulin receptor (IR)-beta subunit. Therefore, in the present studies on Chinese hamster ovary cells overexpressing wild-type IR-PTK (CHO-IR) or a PTK-inactive form of IR (CHO-1018), we investigated whether IR-PTK plays a role in H(2)O(2)- induced signaling events. Treatment of CHO-IR cells with H(2)O(2) increased the phosphorylation of extracellular signal-regulated kinases 1 and 2 (ERK1/2), protein kinase B (PKB), and glycogen synthase kinase-3beta while enhancing tyrosine phosphorylation of the IR-beta subunit and the p85 subunit of phosphatidylinositol 3-kinase (PI3K). Compared with CHO-IR cells, the stimulatory effect of H(2)O(2) on ERK1/2 and PKB was partially reduced in CHO-1018 cells. However, pharmacological inhibition of Src family PTK by 4-amino-5-(4- chlorophenyl)-7-(tert-butyl)pyrazolo[3,4-d]pyrimidine (PP-2) almost completely blocked H(2)O(2)-stimulated phosphorylation of the p85 subunit of PI3K, ERK1/2, and PKB. Moreover, H(2)O(2), but not insulin, induced Tyr- 418 phosphorylation of Src, which was also suppressed by PP-2. Taken together, these data suggest that both IR-PTK and Src family PTKs contribute to H(2)O(2)-induced signaling in CHO-IR cells albeit IR-PTK has a less dominant role in this process. Antioxid. Redox Signal. 7, 1014-1020.