The major target of the endogenously generated reactive oxygen species in response to insulin stimulation is phosphatase and tensin homolog and not phosphoinositide-3 kinase (PI-3 kinase) in the PI-3 kinase/Akt pathway.
Phosphoinositide-3 kinase (PI-3 kinase) and its downstream signaling molecules PDK-1 and Akt were analyzed in SK-N-SH and SK-N-BE(2) human neuroblastoma cell lines. When cells were stimulated with insulin, PI-3 kinase was activated in both cell lines, whereas the translocation of PDK-1 to the membrane fraction and phosphorylated Akt were observed only in SK-N-SH cells. Analyses of the insulin-mediated reactive oxygen species (ROS) generation and Phosphatase and Tensin homolog (PTEN) oxidation indicate that PTEN oxidation occurred in SK-N-SH cells, which can produce ROS, but not in SK-N-BE(2) cells, which cannot increase ROS in response to insulin stimulation. When SK-N-SH cells were pretreated with the NADPH oxidase inhibitor diphenyleneiodonium chloride before insulin stimulation, insulin-mediated translocation of PDK-1 to the membrane fraction and phosphorylation of Akt were remarkably reduced, whereas PI-3 kinase activity was not changed significantly. These results indicate that not only PI-3 kinase activation but also inhibition of PTEN by ROS is needed to increase cellular level of phosphatidylinositol 3,4,5-trisphosphate for recruiting downstream signaling molecules such as PDK-1 and Akt in insulin-mediated signaling. Moreover, the ROS generated by insulin stimulation mainly contributes to the inactivation of PTEN and not to the activation of PI-3 kinase in the PI-3 kinase/Akt pathway.
Seo
Ji Hae
JH
Department of Biology, Ewha Women's University, Seoul 120-750, Korea.
Ahn
Younghee
Y
Lee
Seung-Rock
SR
Yeol Yeo
Chang
C
Chung Hur
Kyu
K
D064413:3-Phosphoinositide-Dependent Protein Kinases; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D015151:Immunoblotting; D047468:Immunoprecipitation; D007328:Insulin / Q000378:metabolism*; D019255:NADPH Oxidases / Q000378:metabolism; D009861:Onium Compounds / Q000494:pharmacology; D010100:Oxygen / Q000378:metabolism; D051059:PTEN Phosphohydrolase; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism*; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D021381:Protein Transport; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt; D017382:Reactive Oxygen Species; D013997:Time Factors; D025521:Tumor Suppressor Proteins / Q000378:metabolism*
D064413:3-Phosphoinositide-Dependent Protein Kinases; D004791:Enzyme Inhibitors; D006861:Hydrogen Peroxide; D007328:Insulin; D019255:NADPH Oxidases; D009861:Onium Compounds; D010100:Oxygen; D051059:PTEN Phosphohydrolase; D019869:Phosphatidylinositol 3-Kinases; D010744:Phosphoric Monoester Hydrolases; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins; D051057:Proto-Oncogene Proteins c-akt; D017382:Reactive Oxygen Species; D025521:Tumor Suppressor Proteins; C007517:diphenyleneiodonium; C494918:AKT1 protein, human; C494929:PTEN protein, human