MEK Signaling Is Required for Phosphorylation of eIF2α following Amino Acid Limitation of HepG2 Human Hepatoma Cells

Michelle M Thiaville,Yuan-Xiang Pan,Altin Gjymishka,Can Zhong,Randal J Kaufman,Michael S Kilberg

doi:10.1074/jbc.m708320200

Abstract

The mammalian amino acid response (AAR) pathway is up-regulated by protein or amino acid depletion. This pathway involves detection of uncharged tRNA by the GCN2 kinase, phosphorylation of the translation initiation factor eIF2alpha (eukaryotic initiation factor 2alpha), and, through subsequent translational control, enhanced de novo synthesis of the transcription factor ATF4. The present studies demonstrate that inhibition of MEK activation in HepG2 human hepatoma cells by PD98059 or U0126 blocked the increased phosphorylation of eIF2alpha and ATF4 synthesis triggered by amino acid limitation, showing that the AAR requires activation of the MEK-ERK pathway. Inhibitors of the JNK or p38 MAPK pathways were ineffective. Consequently, inhibition of MEK activation blocked transcriptional induction of ATF4 target genes, but the induction was rescued by overexpression of ATF4 protein. Furthermore, the enhanced ERK phosphorylation following amino acid deprivation required GCN2 kinase activity and eIF2alpha phosphorylation. Inhibition of protein phosphatase 1 action on phospho-eIF2alpha by knockdown of GADD34 did not block the sensitivity to PD98059, suggesting that MEK functions to enhance GCN2-dependent eIF2alpha phosphorylation rather than suppressing dephosphorylation. Collectively, these results document a critical interdependence between the MEK-ERK MAPK signaling pathway and the amino acid stress-activated pathway.

Highlights

Rivation in vivo [1,2,3,4] as well as to amino acid limitation in vitro
The de novo synthesis of ATF4 and its subsequent transcription program is dependent on activation of the MAPK/ERK kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling mitogen-activated protein kinase (MAPK) pathway
The results suggest that the site of MEK action is to permit and/or enhance GCN2-mediated eIF2␣ phosphorylation, not PP1GADD34-mediated dephosphorylation

Summary

Introduction

Rivation in vivo [1,2,3,4] as well as to amino acid limitation in vitro (reviewed in Ref. 5). Elevated ATF4 protein results in the activation of a wide array of stress-induced genes [12], including those that contain an amino acid response element (AARE) [5]. The enhanced Raf-1 kinase activity triggers the MEK/ERK signaling cascade, which in turn stimulates autophagy in an amino acid-dependent manner via a G␣-interacting protein that regulates the rate of GTP hydrolysis by G␣i3 protein [17, 19]. Inhibition of eIF2␣ phosphatase activity by siRNA against GADD34 (growth arrest and DNA damage-inducible gene 34) did not alter the sensitivity to MEK inhibition, indicating that MEK functions to enhance GCN2-dependent eIF2␣ phosphorylation rather than to suppress the dephosphorylation step. The data show that MEK is activated by a GCN2-dependent process and that there is an absolute requirement for MEK/ERK signaling upstream of eIF2␣-mediated translational control in the mammalian AAR pathway

Methods

Results

Conclusion