Control of cellular GADD34 levels by the 26S proteasome

Abstract

GADD34, the product of a growth arrest and DNA damage-inducible gene, is expressed at low levels in unstressed cells. In response to stress, the cellular content of GADD34 protein increases and, on termination of stress, rapidly declines. We investigated the mechanisms that control GADD34 levels in human cells. GADD34 proteins containing either an internal FLAG or a C-terminal green fluorescent protein epitope were degraded at rates similar to endogenous GADD34. However, the addition of epitopes at the N terminus or deletion of N-terminal sequences stabilized GADD34. N-terminal peptides of GADD34, either alone or fused to heterologous proteins, exhibited rapid degradation similar to wild-type GADD34, thereby identifying an N-terminal degron. Deletion of internal PEST repeats had no impact on GADD34 stability but modulated the binding and activity of protein phosphatase 1. Proteasomal but not lysosomal inhibitors enhanced GADD34 stability and eukaryotic initiation factor 2alpha (eIF-2alpha) dephosphorylation, a finding consistent with GADD34's role in assembling an eIF-2alpha phosphatase. GADD34 was polyubiquitinated, and this modification enhanced its turnover in cells. A stabilized form of GADD34 promoted the accumulation and aggregation of the mutant cystic fibrosis transmembrane conductance regulator (CFTRDeltaF508), highlighting the physiological importance of GADD34 turnover in protein processing in the endoplasmic reticulum and the potential impact of prolonged GADD34 expression in human disease.

FIG. 1.

GADD34 expression in human cell lines subjected to various forms of stress. A549, HeLa, and HEK293T cells were exposed to various stress, including ER stress, nutrient deprivation, DNA damage, apoptosis, oxidative stress, and inhibition of protein phosphatase or the proteasome. Specifically, cells were treated with either the vehicle (0.1% [vol/vol] dimethyl sulfoxide [DMSO]), 1 μM thapsigargin (TG), 10 μg/ml (wt/vol) tunicamycin (TN), leucine-free DMEM (-Leu); 125 μg/ml (wt/vol) methyl methanesulfonate (MMS), 6 μM etoposide (Etop), 0.5 μg/ml (wt/vol) staurosporine (Staur), 50 μM C2-ceramide (C2), 1 mM hydrogen peroxide (H₂O₂), 75 μM sodium arsenite (Ars), 100 nM OA, or 1 μM Z-Leu-Leu-Leu-B(OH)₂ (MG262) for 5 h. Cells were lysed, and the lysates were analyzed by immunoblotting with an anti-GADD34 antibody. Actin levels were used to equalize for protein loading.

FIG. 2.

Cellular GADD34 levels following various stress. (A) Time-dependent accumulation of endogenous GADD34 was analyzed in SW480 cells during continued exposure to 1 μM thapsigargin (Tg), 50 μM arsenite (Ars), leucine-deficient DMEM (-Leu), or 5 μM MG132 by immunoblotting with anti-GADD34 antibody as described in Materials and Methods. (B) The schematic shows the experimental design in which cells were subjected to various stress stimuli for 8 h to induce GADD34 (stage I). Then, replacement with complete medium was used to promote the recovery of cells from the stress stimuli (stage II). (C) A549 cells were incubated in media lacking either leucine (Leu) or glucose (Glc) or containing 5 μM MG132 to elicit GADD34 induction. At the times indicated (0, 2, 4, and 8 h) following their transfer into complete media, cells were lysed and GADD34 levels analyzed by immunoblotting as described in Materials and Methods. Immunoblotting with antitubulin antibody was used to assess protein loading.

FIG. 3.

GADD34 modulates eIF-2α phosphorylation. (A) HeLa cells either uninduced (lanes 1 to 3) or exposed to 5 μM MG132 for 8 h to induce GADD34 expression (lanes 4 to 6) were subjected to brief (0.5 h) treatment with either OA or CA at the times indicated. Lysates were subjected to immunoblotting with anti-GADD34 and anti-phospho-eIF-2α antibodies. The schematic (inset) shows the kinase and phosphatase that control eIF-2α phosphorylation and the proposed mode of action of OA and CA, which target PP2A and/or PP1 to modulate eIF-2α phosphorylation. (B) The schematic shows the experimental design by which MG132 treatment for 8 h was used to induce GADD34 (stage I), followed by the washout of drug with fresh media lacking MG132 and cell recovery (stage II). At the times indicated, cells were treated with OA or CA for 30 min (stage III) to elicit eIF-2α phosphorylation and monitor the activity of the residual GADD34. (C) Cells at stages I, II, and III were subjected to immunoblotting to monitor GADD34 expression and eIF-2α phosphorylation. Parallel immunoblots for tubulin established equivalent protein loading.

FIG. 4.

Proteasomal degradation of GADD34. (A) SW480 cells exposed to 1 μM thapsigargin or 25 μM arsenite for 5 h to induce GADD34 were treated with CHX (30 μg/ml) either alone or in combination with MG262 (5 μM) or CQ (200 μM), and GADD34 levels were analyzed at selected times by immunoblotting. (B) GADD34 fused at its C terminus to GFP (GADD34-GFP) or containing an internal FLAG epitope, FLAG(109)GADD34, were expressed in HEK293T cells. After CHX treatment to inhibit protein synthesis, degradation of the expressed GADD34 proteins was analyzed by immunoblotting with anti-GFP or anti-FLAG antibodies. Cells were treated with either MG132 or CQ to investigate the role of proteasome or lysosome in GADD34 degradation. Tubulin levels monitored in all immunoblots ensured equal protein loading.

FIG. 5.

GADD34 is polyubiquitinated. (A) HeLa cells expressing either FLAG(109)GADD34 (lanes 1 and 2), FLAG-RFP (lane 3), GADD34-GFP (lane 4), or both FLAG-RFP and GFP-GADD34 (lane 5) were treated with or without MG262 (5 μM, 8 h). Cell lysates were subjected to immunoprecipitation using agarose-conjugated anti-FLAG antibody. Lysates and immunoprecipitates were subjected to SDS-PAGE and immunoblotted with antiubiquitin, anti-FLAG, and anti-GFP antibodies. Asterisks indicate the location of heavy and light chains of anti-FLAG IgG in all immunoprecipitates. Molecular mass markers (in kilodaltons) migrated as indicated on each blot. (B) GADD34-GFP was expressed in HEK293T cells in combination with empty vector, with vector encoding wild-type myc-tagged ubiquitin (myc-Ub), or with a mutant myc-ubiquitin (myc-Ub K48R). After 24 h, protein synthesis was inhibited with CHX, and GADD34 levels analyzed at the indicated times by immunoblotting. Tubulin levels established equivalent protein loading. (C) The results from three independent experiments (shown in panel B) were quantified by laser scanning and are shown with standard errors.

FIG. 6.

N-terminal degron in GADD34. (A) Schematic represents all GADD34 proteins analyzed in turnover studies. The positions of a putative ER membrane-association domain (MEMB), PEST, and PP1-binding domains are shown in boxes. (B) C-terminally GFP-tagged GADD34 proteins were expressed in HEK293T cells and subjected to turnover studies. (C) GADD34 proteins fused at the N terminus (GFP-GADD34) or the C terminus (GADD34-GFP) with GFP were expressed in HEK293T cells, and their rate of degradation analyzed as described in Materials and Methods. Similarly, turnover of an N-terminally tagged FLAG-GADD34 was compared to that of the internally tagged FLAG(109)GADD34. (D) Quantification of degradation of all GFP-fusion proteins analyzed in the present study is shown and represents the summary of three independent experiments. All data are shown with standard errors. In panels B and C, tubulin immunoblots served as loading controls.

FIG. 7.

Internal lysines and GADD34 degradation. (A) C-terminal HA-tagged wild-type (WT) and lysineless (0K) GADD34 polypeptides, residues 1 to 155, were expressed in HEK293T cells and subjected to turnover analyses in the absence or presence of 5 μM MG262. Lysates were generated at 0, 1.5, and 3 h following the addition of CHX; subjected to SDS-PAGE; and immunoblotted for HA and tubulin.

FIG. 8.

Stabilized GADD34 facilitates protein aggregation. (A) GFP-CFTRΔF508 was coexpressed in HEK293T cells with either cytosolic (RFP) or ER (DsRed2-RFP) proteins. Cells were untreated (control), exposed to 5 μM MG262 for 8 h, or cotransfected with FLAG-GADD34. Cells were analyzed by fluorescence microscopy. (B) GFP-CFTRΔF508 or GFP-GRP94 were expressed in HEK293T cells that were either untreated (control), exposed to 5 μM MG262 for 8 h, or cotransfected with FLAG-GADD34. At 24 h posttransfection, cells were lysed in RIPA buffer. After removing an aliquot (T), the lysates were subjected to centrifugation to separate soluble (S) and insoluble (I) fractions. Proteins were solubilized by heating and sonication in RIPA containing 1% SDS. T, S, and I fractions were subjected to SDS-PAGE and analyzed by immunoblotting. Double asterisks (**) indicate faster-migrating band glycosylation intermediates of CFTR, and a single asterisk (*) indicates HMW aggregated forms of CFTR. (C) Distribution of FLAG-GADD34 in HEK293T cells cotransfected with GFP-GRP94, CFTRΔF508, or GFP-250 was analyzed in the T, S, and I fractions by immunoblotting. Tubulin and GM130 immunoblots served as controls.