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-BBinduced 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 Nacetylcysteine (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.The cellular redox state plays an important role in the regulation of gene expression in prokaryotes and eukaryotes (1-4). The following observations support this notion: 1) Oxidants regulate the activities of several transcription factors, including activator protein-1, nuclear factor kappa B, and p53 (5-7); 2) Oxidants are capable of activating several early response events, including stimulation of protein tyrosine phosphorylation, activation of mitogen-activated protein kinases and induction of expression of proto-oncogenes (8 -11); 3) Oxidants are produced acutely in response to various agents, including growth factors and cytokines in several cell types (12, 13), and a requirement for their production in the mitogenic effects of receptor tyrosine kinase and G protein-coupled receptor agonists has been demonstrated (14, 15); and 4) In addition to producing oxidants, cells also possess enzymatic and non-enzymatic mechanisms for their removal (16 -18), and this feature attests to the role of oxidants as second messenger molecules (19). Despite the growing body of information on the role of oxidants in the regulation of gene expression, the mechanisms by which these molecules transmit the extracellular signals from the plasma membrane to the nucleus are less clear. Thiols play a critical role in the reduction/oxidation reactions as w...

The emerging role of the PI3-K-Akt pathway in prostate cancer progression

“…23 The activation of PP2A by N-ethylmaleimide, ceramide, palmitate or integrin a2b1 inhibits Akt phosphorylation. [23][24][25][26] Conversely, the inhibition of PP2A by okadaic acid increases Akt activities. 21,25,26 Previously, we and others found that expression of caveolin-1 (cav-1), the major component of caveolae, is elevated in prostate cancer and other malignancies.…”

“…98,107,108 In the past few years, the results of multiple studies have shown that PP2A or PP1 can participate in the regulation of PI3-K-Akt pathway through dephosphorylation of activated Akt and that these proteins can function as the major phosphatases for Akt in some systems. [21][22][23][24][25][26] Recent studies also show that alteration of PP2A and PP1 gene expression is associated with multiple human cancers. The PP2R1B gene that encodes the b isoform of the A subunit of PP2A is located in a region showing high-frequency LOH.…”

The FoxO code

“…Phosphorylation by Akt and SGK in response to insulin and growth factors The FoxO family is negatively regulated by the PI3K-Akt signaling pathway in response to insulin, insulinlike growth factors, growth factors and neurotrophic factors (Lin et al, 1997;Ogg et al, 1997;Brunet et al, 1999;Guo et al, 1999;Jackson et al, 2000;Medema et al, 2000;Yellaturu et al, 2002;Zheng et al, 2002). Phosphorylation of FoxO factors at three conserved sites by the protein kinases Akt and SGK (serum and glucocorticoid-induced kinase) causes the sequestration of FoxO factors in the cytoplasm, thereby preventing FoxO factors from transactivating their target genes (Figures 2 and 3) (Biggs et al, 1999;Brunet et al, 1999Brunet et al, , 2001Kops et al, 1999;Nakae et al, 1999).…”

Arachidonic Acid Mediates Apoptosis Induced by N-Ethylmaleimide in HepG2 Human Hepatoblastoma Cells

“…NEM appears to produce reactive oxygen species (ROS) in various cells (Leoncini and Signorello, 1999b;Meves et al, 2001;Yellaturu et al, 2002). Particularly, NEM has been reported to generate ROS by activation of membrane-bound NADPH oxidase which is known to exist in HepG2 cells (Ehleben et al, 1997;Cool et al, 1998).…”