IMPACT, a Protein Preferentially Expressed in the Mouse Brain, Binds GCN1 and Inhibits GCN2 Activation

Abstract

Translational control directed by the eukaryotic translation initiation factor 2 ␣-subunit (eIF2␣) kinase GCN2 is important for coordinating gene expression programs in response to nutritional deprivation. The GCN2 stress response, conserved from yeast to mammals, is critical for resistance to nutritional deficiencies and for the control of feeding behaviors in rodents. The mouse protein IMPACT has sequence similarities to the yeast YIH1 protein, an inhibitor of GCN2. YIH1 competes with GCN2 for binding to a positive regulator, GCN1. Here, we present evidence that IMPACT is the functional counterpart of YIH1. Overexpression of IM-PACT in yeast lowered both basal and amino acid starvation-induced levels of phosphorylated eIF2␣, as described for YIH1 (31). Overexpression of IMPACT in mouse embryonic fibroblasts inhibited phosphorylation of eIF2␣ by GCN2 under leucine starvation conditions, abolishing expression of its downstream target genes, ATF4 (CREB-2) and CHOP (GADD153). IMPACT bound to the minimal yeast GCN1 segment required for interaction with yeast GCN2 and YIH1 and to native mouse GCN1. At the protein level, IMPACT was detected mainly in the brain. IMPACT was found to be abundant in the majority of hypothalamic neurons. Scattered neurons expressing this protein at higher levels were detected in other regions such as the hippocampus and piriform cortex. The abundance of IMPACT correlated inversely with phosphorylated eIF2␣ levels in different brain areas. These results suggest that IMPACT ensures constant high levels of translation and low levels of ATF4 and CHOP in specific neuronal cells under amino acid starvation conditions.

Perturbations in actin dynamics reconfigure protein complexes that modulate GCN2 activity and promote an eIF2 response

“…Thus, actin and GCN1 might compete for the binding to Yih1. IMPACT shares a high degree of homology to Yih1, structurally and functionally (Cambiaghi et al, 2014;Pereira et al, 2005;Sattlegger et al, 2011), and the location of the binding sites determined for Yih1 might also apply to IMPACT. The data shown here support the notion that IMPACT might bind either GCN1 or G-actin.…”

“…+/+ and Gcn2 −/− MEFs were maintained in Dulbecco's Modified Eagle's medium (DMEM), high glucose, supplemented with 10% (v/v) fetal calf serum (FCS) (Gibco), as described (Pereira et al, 2005). Transfection of cells grown to ∼50% confluence with siRNAs was performed using Lipofectamine 2000 (Life Technologies), according to the manufacturer's instructions, for 5 h in Opti-MEM (Life Technologies).…”

“…Affinity-purified rabbit anti-GCN1 and anti-IMPACT, guinea pig anti-GCN2 and anti-IMPACT (Pereira et al, 2005), and rabbit serum against Saccharomyces cerevisiae eIF2α (Sui2) (Hashimoto et al, 2002) were previously described.…”

“…Like GCN2, Yih1/IMPACT also binds to GCN1, and this leads to reduced GCN1-GCN2 interaction in yeast and mammalian cells (Cambiaghi et al, 2014;Sattlegger et al, 2004). Overexpression of Yih1/IMPACT in yeast and mouse embryonic fibroblasts (MEFs) impairs GCN2 activation under different stress conditions (Cambiaghi et al, 2014;Pereira et al, 2005;Sattlegger et al, 2004). Depletion of IMPACT in neuron-like N2a cells, which express high levels of IMPACT in comparison with MEFs, leads to a higher basal level of GCN2 activity and to a stronger GCN2 activation under leucine deprivation (Roffe et al, 2013).…”

Mouse Pancreatic Islets Are Resistant to Indoleamine 2,3 Dioxygenase-Induced General Control Nonderepressible-2 Kinase Stress Pathway and Maintain Normal Viability and Function

“…This suggestion is based on the fact that Pereira et al recently showed that the mouse protein IMPACT, which binds GCN1 and inhibits GCN2 activation, abolishes the expression of its downstream target genes ATF4 and CHOP. 41 It should be emphasize that IMPACT is highly expressed in IDO-expressing tissues (eg, placenta and testis) and also in pancreas. 42 As such, the expression of IMPACT in pancreatic islet cells might function as a protective mechanism against IDOinduced GCN2 activation.…”