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. 2015 Jul 2;59(1):35-49.
doi: 10.1016/j.molcel.2015.04.026. Epub 2015 Jun 4.

The Unfolded Protein Response Triggers Site-Specific Regulatory Ubiquitylation of 40S Ribosomal Proteins

Reneé Higgins #  1 , Joshua M Gendron #  1 , Lisa Rising  1 , Raymond Mak  1 , Kristofor Webb  1 , Stephen E Kaiser  2 , Nathan Zuzow  1 , Paul Riviere  1 , Bing Yang  1 , Emma Fenech  3 , Xin Tang  1 , Scott A Lindsay  1 , John C Christianson  3 , Randolph Y Hampton  1 , Steven A Wasserman  1 , Eric J Bennett  1
Affiliations

The Unfolded Protein Response Triggers Site-Specific Regulatory Ubiquitylation of 40S Ribosomal Proteins

Reneé Higgins et al. Mol Cell. .

Abstract

Insults to ER homeostasis activate the unfolded protein response (UPR), which elevates protein folding and degradation capacity and attenuates protein synthesis. While a role for ubiquitin in regulating the degradation of misfolded ER-resident proteins is well described, ubiquitin-dependent regulation of translational reprogramming during the UPR remains uncharacterized. Using global quantitative ubiquitin proteomics, we identify evolutionarily conserved, site-specific regulatory ubiquitylation of 40S ribosomal proteins. We demonstrate that these events occur on assembled cytoplasmic ribosomes and are stimulated by both UPR activation and translation inhibition. We further show that ER stress-stimulated regulatory 40S ribosomal ubiquitylation occurs on a timescale similar to eIF2α phosphorylation, is dependent upon PERK signaling, and is required for optimal cell survival during chronic UPR activation. In total, these results reveal regulatory 40S ribosomal ubiquitylation as an important facet of eukaryotic translational control.

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Figures

Figure 1
Figure 1. Characterization of alterations in protein ubiquitylation upon UPR activation
A) Whole cell lysates of HCT116 cells treated with dithiothreitol (DTT), tunicamycin (Tm), or epoxomicin (Epox) were immunoblotted with the indicated antibodies. XBP1 splicing was detected by PCR from cDNA produced from total mRNA. B) SILAC-based quantitative proteomic analysis of total proteins (bottom) or diGly-enriched fractions (top) from metabolically heavy labeled cells, measured as the Log2 heavy to light (H:L) peptide or protein intensity ratio. C) The fraction of quantified diGly-modified peptides (top) or total proteins (bottom) whose abundance increased (white bars), decreased (grey bars), or was unchanged (black bars) by the indicated treatment is depicted. The standard deviation (SD) derived from untreated controls provided the basis for determining the fraction of diGly-modified peptides or total proteins that were altered in response to the indicated treatment. Log2 ratios greater than one SD from the untreated mean are plotted separately as increased and decreased. D) Gene ontology (GO) enrichment analysis of proteins containing diGly-modified peptides which increased (up, open bars) or decreased (down, filled bars) in abundance upon ER stress. The fold enrichment of selected biological processes (BP, top) and cellular compartments (CC, bottom) is depicted. See also Figure S1 and Tables S1-S4
Figure 2
Figure 2. UPR stimulation induces site-specific ubiquitylation of 40S ribosomal proteins
A) Heat map of the Log2 H:L ratios from all SILAC experiments for all quantified diGly-modified peptides arising from 40S or 60S ribosomal proteins. Unt=untreated; 1D and 4D =1 and 4hr DTT; 2T and 4T=2 and 4 hr Tm; 8E=8 hr Epox. B) The Log2 ratio of individual diGly-modified lysine residues from representative 60S proteins (top), or 40S proteins (bottom) in each experimental condition. The positions of the diGly-modified lysines in each protein are indicated. Error bars = SEM. C) Left: 80S ribosome structure with the positions of RPS2 (orange), RPS3 (yellow), RPS20 (pale green) and RACK1 (green) indicated. Ribosomal RNAs are shown in ribbon representation in light orange (60S) or gray (40S) (PDB: 4V6X with mRNA superimposed from PDB: 4KZZ). Middle: view of the isolated 40S from the perspective of the 60S interface (left), or the solvent exposed surface distal to the 60S interface (right) is shown. Right: magnified view of the solvent exposed surface of the 40S subunit. Side chain coloring indicates lysine residues within 40S ribosomal proteins for which a 1.5 fold increase (red) or decrease (blue) in diGly modification was detected upon proteasome inhibition. D) Immunoblot of HCT116 whole cell lysates from cells immunoblotted with antibodies against RPS2 or RPS3 with short (s) or long (l) exposures. E) Quantification of the fraction of RPS2 (blue bars) or RPS3 (red bars) that is ub-modified in untreated cells or cells treated as indicated. Error bars = SEM from triplicate experiments. * indicates a p-value of < 0.05 using Student's t-test. See also Figure S2 and Table S5
Figure 3
Figure 3. Regulatory RPS2 and RPS3 ubiquitylation is induced by diverse but specific cell stressors
A) HCT116 cells were treated with a panel of cellular stressors. Whole cell lysates were analyzed by SDS-PAGE and immunoblotted with the indicated antibodies. (s) and (l) denote short and long exposures, respectively. B) Quantification of the percent RPS3 (left) or RPS2 (right) that is ub-modified from untreated cells or cells treated with the indicated cellular stressor. Error bars represent SEM from combined time course treatments for each cellular stressor. * indicates a p-value of < 0.05 comparing untreated cells to the combined time points for each cell stressor using Student's t-test. ER stress – DTT (D), Tunicamycin (Tm, T); Mitochondrial Stress – Antimycin A (AMA, A), Rotenone (Rot, R); Translation inhibitors – Cycloheximide (CHX, C), Anisomycin (ANS, A), Harringtonine (HTN, H); Translation initiation inhibitors – 4E1RCat (4E1R, 4), Torin-1 (TRN, T), Sodium Arsenite (NaAsO2, A); DNA damage inducers – Etoposide (Etop, E), Mitomycin-C (MMC, M), Cisplatin (Cptn, C), UV (U); Protein folding stressors – Azetidine-2-carboxylic acid (AZC, A), Canavanine (CAN, C), Pifithrin-μ (HSP70i, 7), Exposure to 42°C (Heat shock, H), Serum starvation overnight followed by re-addition of full media (SS Recovery). See also Figure S3
Figure 4
Figure 4. Cytosolic elongating ribosomes and free 40S subunits contain ubiquitylated RPS2 and RPS3
A) 293T cells treated as indicated were separated into cytoplasmic (cyto), endoplasmic reticulum (ER), and nuclear (nuc) enriched fractions using differential detergent-based fractionation. Whole cell lysates and separated fractions were blotted with the indicated antibodies. B) The percent RPS3 (left) or RPS2 (right) that is ubiquitin-modified in each fraction was quantified at the 120 minute time point from the immunoblots depicted in panel A. C) HCT116 cells were untreated or treated with DTT, Tm, or MG132. Whole cell lysates were separated on a 10-50% linear sucrose gradient. The rRNA absorbance (A254) reading during subsequent fraction collection is depicted. The blue diamonds indicate the position of each fraction. D) Sucrose gradient fractions from cells treated as indicated were analyzed by SDS-PAGE and immunoblotted with the indicated antibodies. IN represents the whole cell lysate input. Arrows depict the position of the 40S and 80S particles. See also Figure S4
Figure 5
Figure 5. PERK mediated eIF2α phosphorylation is necessary but not sufficient for UPR-induced regulatory 40S ribosomal ubiquitylation
A) Whole cell lysates from immortalized mouse embryonic fibroblasts derived from wildtype or PERK knockout mice treated with DTT, harringtonine (HTN), or anisomycin (ANS) were analyzed by SDS-PAGE and immunoblotted with the indicated antibodies. B) Whole cell lysates from HCT116 cells treated with DTT or HTN alone or in combination with a PERK inhibitor were analyzed by SDS-PAGE and immunoblotted with the indicated antibodies. C) HEK293 cells expressing Fv2E-PERK were treated with DTT or HTN for 4 hrs, or the dimerizing ligand AP20187 (AP) for 4 or 24 hrs. Whole cell lysates were analyzed by SDS-PAGE and immunoblotted with the indicated antibodies. D) Whole cell lysates from immortalized mouse embryonic fibroblasts derived from mice expressing wildtype eIF2α (S/S) or S51A mutant eIF2α (A/A) were treated as indicated and analyzed by SDS-PAGE and immunoblotted with the indicated antibodies. E) HCT116 cells were treated with a panel of individual eIF2α kinase inducers. Whole cell lysates were analyzed by SDS-PAGE and immunoblotted with the indicated antibodies. See also Figure S5
Figure 6
Figure 6. Loss of site-specific regulatory ubiquitylation of RPS2 and RPS20 enhances ER-stress induced cell death
A) 293T cells stably expressing Flag-HA (FH) tagged wild type or mutant RPS2 were untreated or treated with DTT. Whole cell lysates (inputs) or TUBE-enriched fractions were analyzed by SDS-PAGE and immunoblotted with the indicated antibodies. B) Whole cell lysates from 293T cells stably expressing Flag-HA (FH) tagged wild type RPS2 (WT), RPS20 (WT), RPS2K58R, RPS2K275R, RPS2K58RK275R (2KR), or RPS20K8R were analyzed by SDS-PAGE and immunoblotted with the indicated antibodies. P=parental 293T cell line. C) 293T cells with stable expression of the indicated wild type or mutant ribosomal proteins were either untreated (Unt) or treated with thapsigargin (Tg, left) or tunicamycin (Tm,right) at the indicated concentrations for 48hrs. Cell viability was measured using Celltiter-Glo. Error bars represent SEM from triplicate measurements for each condition. *, and ** indicates a p-value of < 0.05 and 0.01, respectively, compared to the respective wild type control cell line using Student's t-test. See also Figure S6
Figure 7
Figure 7. 40S regulatory ubiquitylation is conserved across eukaryotes
A) Multiple sequence alignments of segments of RPS2, RPS3, and RPS20 from H. sapiens, D. melanogaster, and S. cerevisiae that contain ub-modified lysine residues. The highlighted lysine positions indicate lysine residues observed to be ub-modified in human cells (position is human numbering). B) Structural representation of the solvent exposed surface of the 40S subunit from H. sapiens (PDB:4V6X), D. melanogaster (PDB:1V6W), and S. cerevisiae (PDB:4V7R). Representations and coloring are as in Fig 2C. C) Top: SILAC (H:L) Log2 ratios for selected RPS2, RPS3, and RPS20 ub-modified peptides from HCT116 cells treated with bortezomib for 8 hours. Primary data is extracted from published datasets (Kim et al., 2011). Error bars represent SEM from multiple peptide MS quantifications. Middle: Spectral counts (SCs) for ub-modified peptides from untreated (blue bars) and epoxomicin treated (red bars) Drosophila S2 cells. Error bars represent SEM from triplicate measurements. * indicates a p-value of < 0.05 using Student's t-test. Bottom: SILAC (H:L) Log2 ratios for selected diGly-modified peptides from heavy labeled yeast cultures treated with MG132 prior to mixing with untreated, unlabeled control cells. Error bars represent SEM from multiple peptide MS quantifications. The position of the ub-modified lysine is aligned to the analogous human position. The lysine position within the D. melanogaster or S. cerevisiae 40S protein sequences is shown below. Lysine residues not found to be ub-modified or conserved are indicated. See also Figure S7 and Table S6
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