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Roles of mitogen-activated protein kinase signal-integrating kinases 1 and 2 in oxidant-mediated eIF4E phosphorylation.

ABSTRACT: Oxidative stress alters cellular metabolic processes including protein synthesis. The eukaryotic initiation factor, eIF4E, acts in the rate-limiting steps of initiation and promotes nuclear export. Phosphorylation of eIF4E by mitogen activated protein kinase signal-integrating kinases 1 and 2 (Mnk) influences the affinity of eIF4E for the 5'-mRNA cap and fosters nuclear export activity. Although phosphorylation of eIF4E on Ser209 is observed following oxidant exposure, the contribution of Mnk isoforms and the significance of phosphorylation remain elusive. Using a Mnk inhibitor and fibroblasts derived from Mnk knockout mice, we demonstrate that that H2O2 enhances eIF4E phosphorylation in cells containing Mnk1. In contrast, cells containing only Mnk2 show little change or a decrease in eIF4E phosphorylation in response to H2O2. H2O2 also shifted eIF4GI protein from the nucleus to the cytoplasm suggesting that the increases in eIF4E phosphorylation may reflect enhanced substrate availability to cytoplasmic Mnk1. In Mnk1(+/+) cells, H2O2 also enhanced eIF4E phosphorylation in the nucleus to a greater degree than in the cytoplasm, an effect not observed in cells containing Mnk2. In response to H2O2, all MEFs showed increased eIF4E:4E-BP1 and 4E-BP2:eIF4E binding and reduced eIF4E:eIF4GI binding. We also observed a dramatic increase in the amount of Mnk1 associated with eIF4E following affinity chromatography. These changes coincided with a smaller reduction in global protein synthesis in response to H2O2 in the DKO cells. These findings suggest that changes in eIF4GI distribution may enhance eIF4E phosphorylation and that the presence of either Mnk1 or 2 or any degree of eIF4E phosphorylation negatively regulates global protein synthesis in response to oxidant stress.

SUBMITTER: Shenberger JS

PROVIDER: S-EPMC2001257 | biostudies-literature | 2007

REPOSITORIES: biostudies-literature

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Roles of mitogen-activated protein kinase signal-integrating kinases 1 and 2 in oxidant-mediated eIF4E phosphorylation.

Shenberger Jeffrey S JS Zhang Lianqin L Hughlock Mariah K MK Ueda Takeshi T Watanabe-Fukunaga Rie R Fukunaga Rikiro R

The international journal of biochemistry & cell biology 20070510 10

Oxidative stress alters cellular metabolic processes including protein synthesis. The eukaryotic initiation factor, eIF4E, acts in the rate-limiting steps of initiation and promotes nuclear export. Phosphorylation of eIF4E by mitogen activated protein kinase signal-integrating kinases 1 and 2 (Mnk) influences the affinity of eIF4E for the 5'-mRNA cap and fosters nuclear export activity. Although phosphorylation of eIF4E on Ser209 is observed following oxidant exposure, the contribution of Mnk is ...[more]

PMID: 17689282

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Project description:INTRODUCTION:We have previously demonstrated that transforming growth factor-? (TGF-?) in the presence of elevated levels of Smad3, its primary signaling protein, stimulates rat vascular smooth muscle cell (VSMC) proliferation and intimal hyperplasia. The mechanism is partly through the nuclear exportation of phosphorylated cyclin-dependent kinase inhibitor p27. The objective of this study is to clarify the downstream pathways through which Smad3 produces its proliferative effect. Specifically, we evaluated the role of extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) in TGF-?-induced VSMC proliferation. METHODS:Cultured rat aortic VSMCs were incubated with TGF-? at varying concentrations and times, and phosphorylated ERK was measured by Western blotting. Smad3 was enhanced in VSMCs using an adenovirus expressing Smad3 or inhibited with small interfering RNA (siRNA). For in vivo experiments, male Sprague-Dawley rats underwent carotid balloon injury, followed by intraluminal infection with an adenovirus expressing Smad3. Arteries were harvested at 3 days and subjected to immunohistochemistry for Smad3, phospho-ERK MAPK, and proliferating cell nuclear antigen. RESULTS:In cultured VSMCs, TGF-? induced activation and phosphorylation of ERK MAPK in a time-dependent and concentration-dependent manner. Overexpression of the signaling protein Smad3 enhanced TGF-?-induced activation of ERK MAPK, whereas inhibition of Smad3 with a siRNA blocked ERK MAPK phosphorylation in response to TGF-?. These data suggest that Smad3 acts as a signaling intermediate between TGF-? and ERK MAPK. Inhibition of ERK MAPK activation with PD98059 completely blocked the ability of TGF-?/Smad3 to stimulate VSMC proliferation, demonstrating the importance of ERK MAPK in this pathway. Immunoprecipitation of phospho-ERK MAPK and blotting with Smad3 revealed a physical association, suggesting that activation of ERK MAPK by Smad3 requires a direct interaction. In an in vivo rat carotid injury model, overexpression of Smad3 resulted in an increase in phosphorylated ERK MAPK as well as increased VSMC proliferation as measured by proliferating cell nuclear antigen. CONCLUSIONS:Our findings demonstrate a mechanism through which TGF-? stimulates VSMC proliferation. Although TGF-? has been traditionally identified as an inhibitor of proliferation, our data suggest that TGF-? enhances VSMC proliferation through a Smad3/ERK MAPK signaling pathway. These findings at least partly explain the mechanism by which TGF-? enhances intimal hyperplasia. Knowledge of this pathway provides potential novel targets that may be used to prevent restenosis.

Dataset Information

Roles of mitogen-activated protein kinase signal-integrating kinases 1 and 2 in oxidant-mediated eIF4E phosphorylation.

Publications

Roles of mitogen-activated protein kinase signal-integrating kinases 1 and 2 in oxidant-mediated eIF4E phosphorylation.

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