title: N-Ethylmaleimide inhibits platelet-derived growth factor BB-stimulated Akt phosphorylation via activation of protein phosphatase 2A.
abstract: The redox state plays an important role in gene regulation. Thiols maintain the intracellular redox homeostasis. To understand the role of thiols in redox signaling, we have studied the effect of thiol alkylation on platelet-derived growth factor-BB (PDGF-BB)-induced cell survival events in vascular smooth muscle cells. PDGF-BB stimulated Akt phosphorylation predominantly at Ser-473. N-Ethylmaleimide (NEM), a thiol alkylating agent, blocked PDGF-BB-induced Akt phosphorylation without affecting its upstream phosphatidylinositol 3-kinase (PI3K). On the other hand, LY294002 and wortmannin, specific inhibitors of PI3K, prevented PDGF-BB-induced phosphorylation of Akt and its downstream effector molecules, p70S6K, ribosomal protein S6, 4E-BP1, and eIF4E. NEM also abrogated the phosphorylation of p70S6K, ribosomal protein S6, 4E-BP1, and eIF4E induced by PDGF-BB, suggesting that thiol alkylation interferes with the PI3K/Akt pathway at the level of Akt. In addition, NEM blocked PDGF-BB-induced phosphorylation of BAD and forkhead transcription factor FKHR-L1, and these events correlated with increased apoptosis. NEM alone and in concert with PDGF-BB increased reactive oxygen species (ROS) production and protein phosphatase 2A (PP2A) activity in VSMC. The inhibition of PDGF-BB-induced Akt phosphorylation by NEM was completely reversed by PP2A inhibitors fostriecin and okadaic acid, ceramide synthase inhibitor fumonisin B1, and ROS scavenger N-acetylcysteine (NAC). NAC also attenuated the apoptosis induced by NEM, alone or in combination with PDGF-BB. Together, these findings demonstrate for the first time that PP2A mediates thiol alkylation-dependent redox regulation of Akt and cell survival.
authors.affiliation: Department of Physiology and Center for Vascular Biology, The University of Tennessee Health Science Center, Memphis, Tennessee 38163, USA.
authors.forename: Chandrahasa R
authors.initials: CR
authors.lastname: Yellaturu
authors.forename: Manjula
authors.initials: M
authors.lastname: Bhanoori
authors.forename: Indira
authors.initials: I
authors.lastname: Neeli
authors.forename: Gadiparthi N
authors.initials: GN
authors.lastname: Rao
mesh_terms: D000818:Animals; D001011:Aorta / Q000166:cytology; D017209:Apoptosis; D000077214:Becaplermin; D015153:Blotting, Western; D016923:Cell Death; D002455:Cell Division; D002470:Cell Survival; D004730:Endothelium, Vascular / Q000166:cytology; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005033:Ethylmaleimide / Q000494:pharmacology*; D008297:Male; D009130:Muscle, Smooth / Q000166:cytology*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010749:Phosphoprotein Phosphatases / Q000378:metabolism*; D010766:Phosphorylation; D010982:Platelet-Derived Growth Factor / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D054648:Protein Phosphatase 2; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt; D020574:Proto-Oncogene Proteins c-sis; D051381:Rats; D017207:Rats, Sprague-Dawley; D017382:Reactive Oxygen Species; D038601:Ribosomal Protein S6 / Q000378:metabolism; D013438:Sulfhydryl Compounds / Q000378:metabolism
chemical_list: D000077214:Becaplermin; D004791:Enzyme Inhibitors; D005033:Ethylmaleimide; D019869:Phosphatidylinositol 3-Kinases; D010749:Phosphoprotein Phosphatases; D010982:Platelet-Derived Growth Factor; D054648:Protein Phosphatase 2; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins; D051057:Proto-Oncogene Proteins c-akt; D020574:Proto-Oncogene Proteins c-sis; D017382:Reactive Oxygen Species; D038601:Ribosomal Protein S6; D013438:Sulfhydryl Compounds; C494920:Akt1 protein, rat