Phosphorylation of eIF2 Directs ATF5 Translational Control in Response to Diverse Stress Conditions*

Phosphorylation of eukaryotic initiation factor 2 (eIF2) is an important mechanism regulating global and gene-specific translation in response to different environmental stresses. Central to the eIF2 kinase response is the preferential translation of ATF4 mRNA, encoding a transcriptional activator of genes involved in stress remediation. In this report, we addressed whether there are additional transcription factors whose translational expression is regulated by eIF2 kinases. We show that the expression of the basic zipper transcriptional regulator ATF5 is induced in response to many different stresses, including endoplasmic reticulum stress, arsenite exposure, and proteasome inhibition, by a mechanism requiring eIF2 phosphorylation. ATF5 is subject to translational control as illustrated by the preferential association of ATF5 mRNA with large polyribosomes in response to stress. ATF5 translational control involves two upstream open reading frames (uORFs) located in the 5′-leader of the ATF5 mRNA, a feature shared with ATF4. Mutational analyses of the 5′-leader of ATF5 mRNA fused to a luciferase reporter suggest that the 5′-proximal uORF1 is positive-acting, allowing scanning ribosomes to reinitiate translation of a downstream ORF. During non-stressed conditions, when eIF2 phosphorylation is low, ribosomes reinitiate translation at the next ORF, the inhibitory uORF2. Phosphorylation of eIF2 during stress delays translation reinitiation, allowing scanning ribosomes to bypass uORF2, and instead translate the ATF5 coding region. In addition to translational control, ATF5 mRNA levels are significantly reduced in ATF4-/- mouse embryo fibroblasts, suggesting that ATF4 contributes to basal ATF5 transcription. These results demonstrate that eIF2 kinases direct the translational expression of multiple transcription regulators by a mechanism involving delayed translation reinitiation.

[1]  L. Hendershot,et al.  Two distinct stress signaling pathways converge upon the CHOP promoter during the mammalian unfolded protein response. , 2002, Journal of molecular biology.

[2]  T. E. Dever,et al.  Gene-Specific Regulation by General Translation Factors , 2002, Cell.

[3]  J. Alexander,et al.  An improved, rapid Northern protocol. , 1997, Biochemical and biophysical research communications.

[4]  C. Vinson,et al.  Classification of Human B-ZIP Proteins Based on Dimerization Properties , 2002, Molecular and Cellular Biology.

[5]  Shigeru Takahashi,et al.  Stress-induced Translation of ATF5 mRNA Is Regulated by the 5′-Untranslated Region* , 2008, Journal of Biological Chemistry.

[6]  D. Ron,et al.  CHOP induces death by promoting protein synthesis and oxidation in the stressed endoplasmic reticulum. , 2004, Genes & development.

[7]  M. Schapira,et al.  Regulated translation initiation controls stress-induced gene expression in mammalian cells. , 2000, Molecular cell.

[8]  C. Yanofsky,et al.  The cross-pathway control gene of Neurospora crassa, cpc-1, encodes a protein similar to GCN4 of yeast and the DNA-binding domain of the oncogene v-jun-encoded protein. , 1988, Proceedings of the National Academy of Sciences of the United States of America.

[9]  Yuan-Xiang Pan,et al.  Amino Acid Deprivation Induces the Transcription Rate of the Human Asparagine Synthetase Gene through a Timed Program of Expression and Promoter Binding of Nutrient-responsive Basic Region/Leucine Zipper Transcription Factors as Well as Localized Histone Acetylation* , 2004, Journal of Biological Chemistry.

[10]  S. Macaskill,et al.  Gcn4 co‐ordinates morphogenetic and metabolic responses to amino acid starvation in Candida albicans , 2002, The EMBO journal.

[11]  Tsonwin Hai,et al.  Activating Transcription Factor 3 Is Integral to the Eukaryotic Initiation Factor 2 Kinase Stress Response , 2004, Molecular and Cellular Biology.

[12]  Hong Chen,et al.  Nutritional control of gene expression: how mammalian cells respond to amino acid limitation. , 2005, Annual review of nutrition.

[13]  D. Steindler,et al.  Regulated Expression of ATF5 Is Required for the Progression of Neural Progenitor Cells to Neurons , 2003, The Journal of Neuroscience.

[14]  Krishnamurthy Natarajan,et al.  Gcn4p, a Master Regulator of Gene Expression, Is Controlled at Multiple Levels by Diverse Signals of Starvation and Stress , 2002, Eukaryotic Cell.

[15]  D. Ron,et al.  Translation reinitiation at alternative open reading frames regulates gene expression in an integrated stress response , 2004, The Journal of cell biology.

[16]  P. Walter,et al.  Signal integration in the endoplasmic reticulum unfolded protein response , 2007, Nature Reviews Molecular Cell Biology.

[17]  R. Wek,et al.  Translational control and the unfolded protein response. , 2007, Antioxidants & redox signaling.

[18]  N. Sonenberg,et al.  Translational control in stress and apoptosis , 2005, Nature Reviews Molecular Cell Biology.

[19]  R. Wek,et al.  Reinitiation involving upstream ORFs regulates ATF4 mRNA translation in mammalian cells. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[20]  A. Hinnebusch.  Translational regulation of GCN4 and the general amino acid control of yeast. , 2005, Annual review of microbiology.

[21]  K. Ligon,et al.  Histology-Based Expression Profiling Yields Novel Prognostic Markers in Human Glioblastoma , 2005, Journal of neuropathology and experimental neurology.

[22]  L. Greene,et al.  Selective destruction of glioblastoma cells by interference with the activity or expression of ATF5 , 2006, Oncogene.

[23]  Xiaozhong Wang,et al.  CHOP is implicated in programmed cell death in response to impaired function of the endoplasmic reticulum. , 1998, Genes & development.

[24]  E McEwen,et al.  Translational control is required for the unfolded protein response and in vivo glucose homeostasis. , 2001, Molecular cell.

[25]  L. Greene,et al.  ATF5 regulates the proliferation and differentiation of oligodendrocytes , 2005, Molecular and Cellular Neuroscience.

[26]  R. Wek,et al.  Dimerization Is Required for Activation of eIF2 Kinase Gcn2 in Response to Diverse Environmental Stress Conditions* , 2004, Journal of Biological Chemistry.

[27]  R. Paules,et al.  An integrated stress response regulates amino acid metabolism and resistance to oxidative stress. , 2003, Molecular cell.

[28]  R. Wek,et al.  7 Regulation of the yeast general amino acid control pathway in response to nutrient stress , 2004 .

[29]  T. Anthony,et al.  Coping with stress: eIF2 kinases and translational control. , 2006, Biochemical Society transactions.

[30]  L. Greene,et al.  Downregulation of Activating Transcription Factor 5 Is Required for Differentiation of Neural Progenitor Cells into Astrocytes , 2005, The Journal of Neuroscience.

[31]  R. Kaufman,et al.  The mammalian unfolded protein response. , 2003, Annual review of biochemistry.

[32]  D. Ron,et al.  Endoplasmic reticulum stress signaling in disease. , 2006, Physiological reviews.

[33]  D. Scheuner,et al.  Phosphorylation of the α Subunit of Eukaryotic Initiation Factor 2 Is Required for Activation of NF-κB in Response to Diverse Cellular Stresses , 2003, Molecular and Cellular Biology.

[34]  Yuan-Xiang Pan,et al.  Human CCAAT/Enhancer-binding Protein β Gene Expression Is Activated by Endoplasmic Reticulum Stress through an Unfolded Protein Response Element Downstream of the Protein Coding Sequence* , 2004, Journal of Biological Chemistry.

[35]  V. Carraro,et al.  Induction of CHOP Expression by Amino Acid Limitation Requires Both ATF4 Expression and ATF2 Phosphorylation* , 2004, Journal of Biological Chemistry.

[36]  G. Braus,et al.  Transcriptional autoregulation and inhibition of mRNA translation of amino acid regulator gene cpcA of filamentous fungus Aspergillus nidulans. , 2001, Molecular biology of the cell.

[37]  C. Mitchelmore,et al.  Mouse Atf5: molecular cloning of two novel mRNAs, genomic organization, and odorant sensory neuron localization. , 2002, Genomics.