H2O2-induced phosphorylation of ERK1/2 and PKB requires tyrosine kinase activity of insulin receptor and c-Src.
Hydrogen peroxide (H2O2) mimics many physiological responses of insulin, and increased H2O2 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 H2O2 has also been shown to activate several key components of the insulin signaling cascade. H2O2-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 H2O2-induced signaling events. Treatment of CHO-IR cells with H2O2 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 H2O2 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 H2O2-stimulated phosphorylation of the p85 subunit of PI3K, ERK1/2, and PKB. Moreover, H2O2, 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 H2O2-induced signaling in CHO-IR cells albeit IR-PTK has a less dominant role in this process.
Laboratory of Cell Signaling, Research Centre, Centre hospitalier de l'Université de Montréal-Hôtel-Dieu and Department of Medicine, Université de Montréal, Montréal, Quebec, Canada.
Mohamad Z
MZ
Mehdi
Nihar R
NR
Pandey
Sanjay K
SK
Pandey
Ashok K
AK
Srivastava
D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism*; D038362:Glycogen Synthase Kinase 3 / Q000378:metabolism; D000071679:Glycogen Synthase Kinase 3 beta; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010766:Phosphorylation / Q000187:drug effects; D019000:Phosphotyrosine / Q000378:metabolism; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D021122:Protein Subunits / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000378:metabolism*; D011972:Receptor, Insulin / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D000077191:Wortmannin
D000730:Androstadienes; D048049:Extracellular Signal-Regulated MAP Kinases; D038362:Glycogen Synthase Kinase 3; D000071679:Glycogen Synthase Kinase 3 beta; D006861:Hydrogen Peroxide; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D019000:Phosphotyrosine; D047428:Protein Kinase Inhibitors; D017346:Protein Serine-Threonine Kinases; D021122:Protein Subunits; D011518:Proto-Oncogene Proteins; D051057:Proto-Oncogene Proteins c-akt; D016392:Proto-Oncogene Proteins pp60(c-src); D011972:Receptor, Insulin; D000077191:Wortmannin