N-Ethylmaleimide inhibits platelet-derived growth factor BB-stimulated Akt phosphorylation via activation of protein phosphatase 2A

Chandrahasa R. Yellaturu, Manjula Bhanoori, Indira Neeli, Rao Gadiparthi

Research output: Contribution to journalArticle

102 Citations (Scopus)

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.

Original languageEnglish (US)
Pages (from-to)40148-40155
Number of pages8
JournalJournal of Biological Chemistry
Volume277
Issue number42
DOIs
StatePublished - Oct 18 2002

Fingerprint

Protein Phosphatase 2
Phosphorylation
Ethylmaleimide
Chemical activation
Sulfhydryl Compounds
Phosphatidylinositol 3-Kinase
Oxidation-Reduction
Alkylation
Ribosomal Protein S6
70-kDa Ribosomal Protein S6 Kinases
Cells
Acetylcysteine
Reactive Oxygen Species
Cell Survival
Apoptosis
Forkhead Transcription Factors
platelet-derived growth factor BB
Okadaic Acid
2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one
Alkylating Agents

All Science Journal Classification (ASJC) codes

  • Biochemistry

Cite this

N-Ethylmaleimide inhibits platelet-derived growth factor BB-stimulated Akt phosphorylation via activation of protein phosphatase 2A. / Yellaturu, Chandrahasa R.; Bhanoori, Manjula; Neeli, Indira; Gadiparthi, Rao.

In: Journal of Biological Chemistry, Vol. 277, No. 42, 18.10.2002, p. 40148-40155.

Research output: Contribution to journalArticle

@article{6fad9e5f4b05451fab3447ce6b9a74e3,
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.",
author = "Yellaturu, {Chandrahasa R.} and Manjula Bhanoori and Indira Neeli and Rao Gadiparthi",
year = "2002",
month = "10",
day = "18",
doi = "10.1074/jbc.M206376200",
language = "English (US)",
volume = "277",
pages = "40148--40155",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "42",

}

TY - JOUR

T1 - N-Ethylmaleimide inhibits platelet-derived growth factor BB-stimulated Akt phosphorylation via activation of protein phosphatase 2A

AU - Yellaturu, Chandrahasa R.

AU - Bhanoori, Manjula

AU - Neeli, Indira

AU - Gadiparthi, Rao

PY - 2002/10/18

Y1 - 2002/10/18

N2 - 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.

AB - 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.

UR - http://www.scopus.com/inward/record.url?scp=0037131377&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0037131377&partnerID=8YFLogxK

U2 - 10.1074/jbc.M206376200

DO - 10.1074/jbc.M206376200

M3 - Article

VL - 277

SP - 40148

EP - 40155

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 42

ER -