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Activation of GCN2 by the ribosomal P-stalk.

ABSTRACT: Cells dynamically adjust their protein translation profile to maintain homeostasis in changing environments. During nutrient stress, the kinase general control nonderepressible 2 (GCN2) phosphorylates translation initiation factor eIF2?, initiating the integrated stress response (ISR). To examine the mechanism of GCN2 activation, we have reconstituted this process in vitro, using purified components. We find that recombinant human GCN2 is potently stimulated by ribosomes and, to a lesser extent, by tRNA. Hydrogen/deuterium exchange-mass spectrometry (HDX-MS) mapped GCN2-ribosome interactions to domain II of the uL10 subunit of the ribosomal P-stalk. Using recombinant, purified P-stalk, we showed that this domain of uL10 is the principal component of binding to GCN2; however, the conserved 14-residue C-terminal tails (CTTs) in the P1 and P2 P-stalk proteins are also essential for GCN2 activation. The HisRS-like and kinase domains of GCN2 show conformational changes upon binding recombinant P-stalk complex. Given that the ribosomal P-stalk stimulates the GTPase activity of elongation factors during translation, we propose that the P-stalk could link GCN2 activation to translational stress, leading to initiation of ISR.

SUBMITTER: Inglis AJ

PROVIDER: S-EPMC6421407 | biostudies-literature | 2019 Mar

REPOSITORIES: biostudies-literature

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Activation of GCN2 by the ribosomal P-stalk.

Inglis Alison J AJ Masson Glenn R GR Shao Sichen S Perisic Olga O McLaughlin Stephen H SH Hegde Ramanujan S RS Williams Roger L RL

Proceedings of the National Academy of Sciences of the United States of America 20190225 11

Cells dynamically adjust their protein translation profile to maintain homeostasis in changing environments. During nutrient stress, the kinase general control nonderepressible 2 (GCN2) phosphorylates translation initiation factor eIF2α, initiating the integrated stress response (ISR). To examine the mechanism of GCN2 activation, we have reconstituted this process in vitro, using purified components. We find that recombinant human GCN2 is potently stimulated by ribosomes and, to a lesser extent, ...[more]

PMID: 30804176

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Project description:The effect of targeted therapeutics on anticancer immune responses is poorly understood. The BRAF inhibitor dabrafenib has been reported to activate the integrated stress response (ISR) kinase GCN2, and the therapeutic effect has been partially attributed to GCN2 activation. Because ISR signaling is a key component of myeloid-derived suppressor cell (MDSC) development and function, we measured the effect of dabrafenib on MDSC differentiation and suppressive activity. Our data showed that dabrafenib attenuated MDSC ability to suppress T-cell activity, which was associated with a GCN2-dependent block of the transition from monocytic progenitor to polymorphonuclear (PMN)-MDSCs and proliferative arrest resulting in PMN-MDSC loss. Transcriptional profiling revealed that dabrafenib-driven GCN2 activation altered metabolic features in MDSCs enhancing oxidative respiration, and attenuated transcriptional programs required for PMN development. Moreover, we observed a broad downregulation of transcriptional networks associated with PMN developmental pathways, and increased activity of transcriptional regulons driven by Atf5, Mafg, and Zbtb7a. This transcriptional program alteration underlies the basis for PMN-MDSC developmental arrest, skewing immature MDSC development toward monocytic lineage cells. In vivo, we observed a pronounced reduction in PMN-MDSCs in dabrafenib-treated tumor-bearing mice suggesting that dabrafenib impacts MDSC populations systemically and locally, in the tumor immune infiltrate. Thus, our data reveal transcriptional networks that govern MDSC developmental programs, and the impact of GCN2 stress signaling on the innate immune landscape in tumors, providing novel insight into potentially beneficial off-target effects of dabrafenib.SignificanceAn important, but poorly understood, aspect of targeted therapeutics for cancer is the effect on antitumor immune responses. This article shows that off-target effects of dabrafenib activating the kinase GCN2 impact MDSC development and function reducing PMN-MDSCs in vitro and in vivo. This has important implications for our understanding of how this BRAF inhibitor impacts tumor growth and provides novel therapeutic target and combination possibilities.

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Activation of GCN2 by the ribosomal P-stalk.

Publications

Activation of GCN2 by the ribosomal P-stalk.

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