Mol Cell Biol. 2004 Feb;24(3):1365-77.

Activating transcription factor 3 is integral to the eukaryotic initiation factor 2 kinase stress response.

Jiang HY, Wek SA, McGrath BC, Lu D, Hai T, Harding HP, Wang X, Ron D, Cavener DR, Wek RC.

Source

Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, Indiana 46202, USA.

Abstract

In response to environmental stress, cells induce a program of gene expression designed to remedy cellular damage or, alternatively, induce apoptosis. In this report, we explore the role of a family of protein kinases that phosphorylate eukaryotic initiation factor 2 (eIF2) in coordinating stress gene responses. We find that expression of activating transcription factor 3 (ATF3), a member of the ATF/CREB subfamily of basic-region leucine zipper (bZIP) proteins, is induced in response to endoplasmic reticulum (ER) stress or amino acid starvation by a mechanism requiring eIF2 kinases PEK (Perk or EIF2AK3) and GCN2 (EIF2AK4), respectively. Increased expression of ATF3 protein occurs early in response to stress by a mechanism requiring the related bZIP transcriptional regulator ATF4. ATF3 contributes to induction of the CHOP transcriptional factor in response to amino acid starvation, and loss of ATF3 function significantly lowers stress-induced expression of GADD34, an eIF2 protein phosphatase regulatory subunit implicated in feedback control of the eIF2 kinase stress response. Overexpression of ATF3 in mouse embryo fibroblasts partially bypasses the requirement for PEK for induction of GADD34 in response to ER stress, further supporting the idea that ATF3 functions directly or indirectly as a transcriptional activator of genes targeted by the eIF2 kinase stress pathway. These results indicate that ATF3 has an integral role in the coordinate gene expression induced by eIF2 kinases. Given that ATF3 is induced by a very large number of environmental insults, this study supports involvement of eIF2 kinases in the coordination of gene expression in response to a more diverse set of stress conditions than previously proposed.

PMID:: 14729979; [PubMed - indexed for MEDLINE]
PMCID:: PMC321431

Free PMC Article

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FIG. 1.

Enhanced expression of ATF3, ATF4, and CHOP in response to ER stress requires PEK activity. PEK^+/+ and PEK^−/− MEF cells were exposed to the presence of thapsigargin (Tg) for the indicated number of hours or to the absence of this ER stress agent (0 h). (A) Whole-cell lysates were prepared from the cultured cells, and the levels of eIF2α specifically phosphorylated at Ser-51 (eIF2α-P) or total eIF2α were measured by immunoblot analysis. The same amount of protein lysate was analyzed in each lane. Relative levels of phosphorylated eIF2α were normalized to levels of total eIF2α in each lysate preparation. (B) MEF cells containing functional eIF2 kinases (wildtype, lanes 1 to 4 and 13; PEK^−/− GCN2^−/−, lanes 5 to 8; PEK^−/− GCN2^−/− PKR^−/−, lanes 9 to 12; PEK^−/−, lane 14) were treated with thapsigargin for the indicated number of hours. Levels of phosphorylated eIF2α were measured by immunoblot assay. (C) Levels of ATF3, ATF4, CHOP, GRP78, and actin were measured by immunoblot analysis with an antibody specific to each protein. To facilitate normalization between PEK^+/+ and PEK^−/− panels, the lysate from PEK^−/− cells that were stressed for 6 h was included in lane C of the PEK^+/+ panel. Similarly, lane C in the PEK^−/− immunoblot panel was an analysis of lysate prepared from PEK^+/+ MEF cells treated with thapsigargin for 6 h. (D) A plasmid expressing PEK was transiently transfected into PEK^−/− MEF cells (lanes 5 and 6), and PEK^+/+ and transfected cells were subjected to the presence (+) or absence (−) of ER stress for 6 h. Vec indicates that the parent expression plasmid alone was introduced into the PEK^−/− MEF cells (lanes 3 and 4). Whole-cell lysates were prepared from these transfected cells, and ATF3, ATF4, CHOP, PEK, and actin were measured by immunoblot analysis.

Activating Transcription Factor 3 Is Integral to the Eukaryotic Initiation Factor 2 Kinase Stress Response

Mol Cell Biol. Mol Cell Biol;24(3):1365-1377.