IMPACT Is a Developmentally Regulated Protein in Neurons That Opposes the Eukaryotic Initiation Factor 2α Kinase GCN2 in the modulation of Neurite Outgrowth

Roffé, Martín; Hajj, Glaucia Noeli Maroso; Azevedo, Hátylas; Alves, Viviane S.; Castilho, Beatriz A.

doi:10.1074/jbc.m113.461970

Cited by 53 publications

(64 citation statements)

References 44 publications

(49 reference statements)

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“…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). Taken together, these data indicate that Yih1/IMPACT competitively inhibits the GCN1-mediated activation of GCN2.…”

Section: Introductionmentioning

confidence: 47%

“…Here, we made use of murineneuronal-like N2a cells, which express larger amounts of endogenous IMPACT compared with MEFs (Roffe et al, 2013). N2a cells were transfected with siRNA against IMPACT or with a control siRNA and then subjected to latrunculin-B treatment (Fig.…”

Section: Activation Of Gcn2 By Actin Depolymerization Requires Gcn1 Amentioning

confidence: 99%

“…Beads were resuspended in 20 µl of 1×Laemli's loading buffer (120 mM Tris pH 6.8, 4% SDS, 5% β-mercaptoethanol, 20% glycerol and 0.02% Bromophenol Blue), incubated at 90°C for 2 min, briefly centrifuged and supernatants resolved on SDS-PAGE. Immunoprecipitation of IMPACT-FLAG, expressed in N2a cells differentiated for 64 h (Roffe et al, 2013), was performed as described (Silva et al, 2015).…”

Section: Cell Extracts Immunoblots and Immunoprecipitationsmentioning

confidence: 99%

“…MEFs were subjected to leucine starvation by culturing in DMEM lacking L-leucine (Emcare, Brazil) supplemented with 10% dialyzed FCS (Gibco). N2a cells were grown and transfected with siIMPACT and siControl (scrambled for siIMPACT) as previously described (Roffe et al, 2013).…”

Section: Gcn2mentioning

confidence: 99%

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

Silva

Sattlegger

Castilho

2016

Journal of Cell Science

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Genetic and pharmacological interventions in yeast and mammalian cells have suggested a cross-talk between the actin cytoskeleton and protein synthesis. Regulation of the activity of the translation initiation factor 2 (eIF2) is a paramount mechanism for cells to rapidly adjust the rate of protein synthesis and to trigger reprogramming of gene expression in response to internal and external cues. Here, we show that disruption of F-actin in mammalian cells inhibits translation in a GCN2-dependent manner, correlating with increased levels of uncharged tRNA. GCN2 activation increased phosphorylation of its substrate eIF2α and the induction of the integrated stress response master regulator, ATF4. GCN2 activation by latrunculin-B is dependent on GCN1 and inhibited by IMPACT. Our data suggest that GCN2 occurs in two different complexes, GCN2-eEF1A and GCN2-GCN1. Depolymerization of F-actin shifts GCN2 to favor the complex with GCN1, concomitant with GCN1 being released from its binding to IMPACT, which is sequestered by G-actin. These events might further contribute to GCN2 activation. Our findings indicate that GCN2 is an important sensor of the state of the actin cytoskeleton.

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Section: Introductionmentioning

confidence: 47%

Section: Activation Of Gcn2 By Actin Depolymerization Requires Gcn1 Amentioning

confidence: 99%

Section: Cell Extracts Immunoblots and Immunoprecipitationsmentioning

confidence: 99%

Section: Gcn2mentioning

confidence: 99%

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

Silva

Sattlegger

Castilho

2016

Journal of Cell Science

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“…Other studies suggest that through GCN2, IDO suppresses T-cell differentiation by blocking key enzymes for fatty acid synthesis (Eleftheriadis et al , 2015). Leucine deprivation has also been suggested to inhibit differentiation of myoblasts through GCN2 (Averous et al , 2012) and GCN2 is specifically inhibited by the protein IMPACT in differentiated neural cells to allow for high levels of translation (Pereira et al , 2005; Roffe et al , 2013). Based on these earlier observations and our findings, GCN2 is suggested to play critical roles in cellular differentiation among diverse tissues.…”

Section: Discussionmentioning

confidence: 99%

Human Keratinocyte Differentiation Requires Translational Control by the eIF2α Kinase GCN2

Collier

Wek

Spandau

2017

Journal of Investigative Dermatology

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Appropriate and sequential differentiation of keratinocytes is essential for all functions of the human epidermis. While transcriptional regulation has proven to be important for keratinocyte differentiation, little is known about the role of translational control. A key mechanism for modulating translation is through phosphorylation of the α subunit of eIF2. A family of different eIF2α kinases function in the integrative stress response to inhibit general protein synthesis coincident with preferential translation of select mRNAs that participate in stress alleviation. Here we demonstrate that translational control through eIF2α phosphorylation is required for normal keratinocyte differentiation. Analyses of polysome profiles revealed that key differentiation genes, including involucrin, are bound to heavy polysomes during differentiation, despite decreased general protein synthesis. Induced eIF2α phosphorylation by the GCN2 protein kinase facilitated translational control and differentiation-specific protein expression during keratinocyte differentiation. Furthermore, loss of GCN2 thwarted translational control, normal epidermal differentiation, and differentiation gene expression in organotypic skin culture. These findings underscore a previously unknown function for GCN2 phosphorylation of eIF2α and translational control in the formation of an intact human epidermis.

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Experimentally based structural model of Yih1 provides insight into its function in controlling the key translational regulator Gcn2

Harjes

Jameson

et al. 2020

FEBS Letters

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Yeast impact homolog 1 (Yih1), or IMPACT in mammals, is part of a conserved regulatory module controlling the activity of General Control Nonderepressible 2 (Gcn2), a protein kinase that regulates protein synthesis. Yih1/IMPACT is implicated not only in many essential cellular processes, such as neuronal development, immune system regulation and the cell cycle, but also in cancer. Gcn2 must bind to Gcn1 in order to impair the initiation of protein translation. Yih1 hinders this key Gcn1‐Gcn2 interaction by binding to Gcn1, thus preventing Gcn2‐mediated inhibition of protein synthesis. Here, we solved the structures of the two domains of Saccharomyces cerevisiae Yih1 separately using Nuclear Magnetic Resonance and determined the relative positions of the two domains using a range of biophysical methods. Our findings support a compact structural model of Yih1 in which the residues required for Gcn1 binding are buried in the interface. This model strongly implies that Yih1 undergoes a large conformational rearrangement from a latent closed state to a primed open state to bind Gcn1. Our study provides structural insight into the interactions of Yih1 with partner molecules.

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IMPACT Is a Developmentally Regulated Protein in Neurons That Opposes the Eukaryotic Initiation Factor 2α Kinase GCN2 in the modulation of Neurite Outgrowth

Cited by 53 publications

References 44 publications

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

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

Human Keratinocyte Differentiation Requires Translational Control by the eIF2α Kinase GCN2

Experimentally based structural model of Yih1 provides insight into its function in controlling the key translational regulator Gcn2

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