scholarly journals Growth Arrest and DNA Damage-Inducible Protein GADD34 Targets Protein Phosphatase 1α to the Endoplasmic Reticulum and Promotes Dephosphorylation of the α Subunit of Eukaryotic Translation Initiation Factor 2

2003 ◽  
Vol 23 (4) ◽  
pp. 1292-1303 ◽  
Author(s):  
Matthew H. Brush ◽  
Douglas C. Weiser ◽  
Shirish Shenolikar
Keyword(s):  
Protein Phosphatase ◽  
Growth Arrest ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Α Subunit ◽  
Eukaryotic Translation ◽  
Initiation Factor 2 ◽  
Eukaryotic Translation Initiation ◽  
Inducible Protein ◽  
Translation Initiation Factor 2

ABSTRACT The growth arrest and DNA damage-inducible protein, GADD34, associates with protein phosphatase 1 (PP1) and promotes in vitro dephosphorylation of the α subunit of eukaryotic translation initiation factor 2, (eIF-2α). In this report, we show that the expression of human GADD34 in cultured cells reversed eIF-2α phosphorylation induced by thapsigargin and tunicamycin, agents that promote protein unfolding in the endoplasmic reticulum (ER). GADD34 expression also reversed eIF-2α phosphorylation induced by okadaic acid but not that induced by another phosphatase inhibitor, calyculin A (CA), which is a result consistent with PP1 being a component of the GADD34-assembled eIF-2α phosphatase. Structure-function studies identified a bipartite C-terminal domain in GADD34 that encompassed a canonical PP1-binding motif, KVRF, and a novel RARA sequence, both of which were required for PP1 binding. N-terminal deletions of GADD34 established that while PP1 binding was necessary, it was not sufficient to promote eIF-2α dephosphorylation in cells. Imaging of green fluorescent protein (GFP)-GADD34 proteins showed that the N-terminal 180 residues directed the localization of GADD34 at the ER and that GADD34 targeted the α isoform of PP1 to the ER. These data provide new insights into the mode of action of GADD34 in assembling an ER-associated eIF-2α phosphatase that regulates protein translation in mammalian cells.

scholarly journals Snf1 Promotes Phosphorylation of the α Subunit of Eukaryotic Translation Initiation Factor 2 by Activating Gcn2 and Inhibiting Phosphatases Glc7 and Sit4

2010 ◽  
Vol 30 (12) ◽  
pp. 2862-2873 ◽  
Author(s):  
Vera Cherkasova ◽  
Hongfang Qiu ◽  
Alan G. Hinnebusch
Keyword(s):  
Translation Initiation ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Activation Loop ◽  
Α Subunit ◽  
Eukaryotic Translation ◽  
Eif2α Phosphorylation ◽  
Initiation Factor 2 ◽  
Eukaryotic Translation Initiation ◽  
Translation Initiation Factor 2

ABSTRACT Snf1 is the ortholog of mammalian AMP-activated kinase and is responsible for activation of glucose-repressed genes at low glucose levels in budding yeast. We show that Snf1 promotes the formation of phosphorylated α subunit of eukaryotic translation initiation factor 2 (eIF2α-P), a regulator of general and gene-specific translation, by stimulating the function of eIF2α kinase Gcn2 during histidine starvation of glucose-grown cells. Thus, eliminating Snf1 or mutating its activation loop lowers Gcn2 kinase activity, reducing the autophosphorylation of Thr-882 in the Gcn2 activation loop, and decreases eIF2α-P levels in starved cells. Consistently, eliminating Reg1, a negative regulator of Snf1, provokes Snf1-dependent hyperphosphorylation of both Thr-882 and eIF2α. Interestingly, Snf1 also promotes eIF2α phosphorylation in the nonpreferred carbon source galactose, but this occurs by inhibition of protein phosphatase 1α (PP1α; Glc7) and the PP2A-like enzyme Sit4, rather than activation of Gcn2. Both Glc7 and Sit4 physically interact with eIF2α in cell extracts, supporting their direct roles as eIF2α phosphatases. Our results show that Snf1 modulates the level of eIF2α phosphorylation by different mechanisms, depending on the kind of nutrient deprivation existing in cells.

scholarly journals Activation-dependent substrate recruitment by the eukaryotic translation initiation factor 2 kinase PERK

2006 ◽  
Vol 172 (2) ◽  
pp. 201-209 ◽  
Author(s):  
Stefan J. Marciniak ◽  
Lidia Garcia-Bonilla ◽  
Junjie Hu ◽  
Heather P. Harding ◽  
David Ron
Keyword(s):  
Er Stress ◽  
Translation Initiation ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Eukaryotic Translation Initiation Factor ◽  
Α Subunit ◽  
Eukaryotic Translation ◽  
Initiation Factor 2 ◽  
Eukaryotic Translation Initiation ◽  
Translation Initiation Factor 2

Regulated phosphorylation of the α subunit of eukaryotic translation initiation factor 2 (eIF2α) by the endoplasmic reticulum (ER) stress-activated protein kinase PERK modulates protein synthesis and couples the production of ER client proteins with the organelle's capacity to fold and process them. PERK activation by ER stress is known to involve transautophosphorylation, which decorates its unusually long kinase insert loop with multiple phosphoserine and phosphothreonine residues. We report that PERK activation and phosphorylation selectively enhance its affinity for the nonphosphorylated eIF2 complex. This switch correlates with a marked change to the protease sensitivity pattern, which is indicative of a major conformational change in the PERK kinase domain upon activation. Although it is dispensable for catalytic activity, PERK's kinase insert loop is required for substrate binding and for eIF2α phosphorylation in vivo. Our findings suggest a novel mechanism for eIF2 recruitment by activated PERK and for unidirectional substrate flow in the phosphorylation reaction.

scholarly journals Control of α Subunit of Eukaryotic Translation Initiation Factor 2 (eIF2α) Phosphorylation by the Human Papillomavirus Type 18 E6 Oncoprotein: Implications for eIF2α-Dependent Gene Expression and Cell Death

2004 ◽  
Vol 24 (8) ◽  
pp. 3415-3429 ◽  
Author(s):  
Shirin Kazemi ◽  
Stavroula Papadopoulou ◽  
Suiyang Li ◽  
Qiaozhu Su ◽  
Shuo Wang ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Α Subunit ◽  
Eukaryotic Translation ◽  
Eif2α Phosphorylation ◽  
E6 Oncoprotein ◽  
Initiation Factor 2 ◽  
Eukaryotic Translation Initiation ◽  
Translation Initiation Factor 2

ABSTRACT Phosphorylation of the α subunit of eukaryotic translation initiation factor 2 (eIF2α) at serine 51 inhibits protein synthesis in cells subjected to various forms of stress including virus infection. The human papillomavirus (HPV) E6 oncoprotein contributes to virus-induced pathogenicity through multiple mechanisms including the inhibition of apoptosis and the blockade of interferon (IFN) action. We have investigated a possible functional relationship between the E6 oncoprotein and eIF2α phosphorylation by an inducible-dimerization form of the IFN-inducible protein kinase PKR. Herein, we demonstrate that HPV type 18 E6 protein synthesis is rapidly repressed upon eIF2α phosphorylation caused by the conditional activation of the kinase. The remainder of E6, however, can rescue cells from PKR-mediated inhibition of protein synthesis and induction of apoptosis. E6 physically associates with GADD34/PP1 holophosphatase complex, which mediates translational recovery, and facilitates eIF2α dephosphorylation. Inhibition of eIF2α phosphorylation by E6 mitigates eIF2α-dependent responses to transcription and translation of proapoptotic genes. These findings demonstrate, for the first time, a role of the oncogenic E6 in apoptotic signaling induced by PKR and eIF2α phosphorylation. The functional interaction between E6 and the eIF2α phosphorylation pathway may have important implications for HPV infection and associated pathogenesis.

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