Figure 6.

Ubiquitination and stability of HRI are governed by the BIRC6 complex. A, Proteomic changes following BIRC6 depletion in the presence and absence of ISRIB. HCC202-Cas9 cells were transduced with either a control sgRNA (sgCh2-2) or an sgRNA targeting BIRC6 (sgBIRC6-4). Four days later, cells were harvested and subjected to LC/MS-MS. The magnitude [LogFC (sgBIRC6/sgCh2-2)] and significance [−log₁₀ (P)] of the difference in protein expression between the control and BIRC6 knockout samples were plotted. Here and in B, the products of the genes that are transcriptionally regulated by ISR are indicated by the orange dots, while HRI is indicated by the green dot. B, Comparison of the BIRC6 depletion–induced proteomic changes in the presence and absence of ISRIB treatment. C, Elevated expression of HRI protein after depleting individual components of the BIRC6 complex. HCC202-Cas9 and SNU503-Cas9 cells were transduced with the indicated sgRNA, and their lysates were harvested 4 days later. Lysates of the cells treated with MG132 (10 μmol/L) or a vehicle control for 6 hours were also analyzed by immunoblotting. D, Stabilization of HRI following BIRC6 depletion. HCC202-Cas9 cells, transduced with either sgCh2-2 or sgBIRC6-4, were transiently transfected with a plasmid expressing HRI-V5. These cells were subsequently treated with cycloheximide (CHX; 50 μg/mL) and harvested at the indicated time points. Changes in the relative intensity between V5 and β-actin signals were plotted (right). Values = means ± SEM (n = 4). ****, P < 0.0001. E, Reduced HRI ubiquitination following BIRC6 depletion. HCC202-Cas9 cells that constitutively express HA-tagged Ubiquitin (HA-Ubiquitin) were further engineered to express HRI-V5 in a doxycycline (DOX)-inducible manner and then transduced with sgCh2-2 or sgBIRC6-4. These cells were subsequently treated with DOX (1 μg/mL, 48 hours), ISRIB (1 μmol/L, 48 hours), and/or MG132 (10 μmol/L, 6 hours), and their lysates were immunoprecipitated (IP) with anti-V5 followed by immunoblotting. The ubiquitin chains attached to HRI-V5 were clearly detected in the control (sgCh2-2) sample treated with all the three reagents (DOX, ISRIB, MG132) but was less clear in the BIRC6 knockout (sgBIRC6-4) sample. The relative intensity between HA(-ubiquitin) and (HRI-)V5 signals for the samples cotreated with DOX, ISRIB, and MG132 was plotted (right). Values = means ± SD (n = 5). F, A physical interaction between UBR4 and HRI. HCC202-Cas9 cells were engineered to express HRI-V5 in a DOX-inducible manner. Following treatment with DOX (1 μg/mL, 48 hours), ISRIB (1 μmol/L, 48 hours), and/or MG132 (10 μmol/L, 6 hours), cells were harvested, and the lysates were subjected to anti-V5 IP and analysis by immunoblotting. G, Analysis of HRI phosphorylation status using a Phos-tag gel. HCC202-Cas9 cells, transduced with either sgCh2-2 or sgBIRC6-4, were transiently transfected with a plasmid expressing HRI-V5. HCC202-Cas9 cells without sgRNA transduction were also transfected with an HRI-V5–expressing plasmid and subsequently treated with either arsenite (300 μmol/L, 3 hours) or vehicle control (mock). Lysates of these cells were either treated with lambda phosphatase (+λPP) or left untreated (+λPP) and analyzed by immunoblotting using a Phos-tag gel and a standard protein (regular) gel. The knockout of BIRC6 resulted in the upregulation of phosphorylated and nonphosphorylated forms of HRI. H, Changes in expression of ISR markers upon HRI depletion. The Cas9-expressing derivatives of the indicated cells were transduced with either an sgRNA against HRI or a control sgRNA (sgCh2-2). Four days later, their lysates were harvested and analyzed for the expression levels of various ISR marker proteins. Relative intensity of the ATF3 and SESN2 bands, both of which were normalized to the intensity of the corresponding β-actin band, between sgCh2-2 and sgHRI samples were plotted. Values = means ± SD (n = 3). ****,P < 0.0001 (dependent vs. nondependent). The experiment shown in A and B was conducted once, the experiments shown in C and F were conducted twice, the experiments shown in G and H were conducted three times, the experiment shown in D was conducted four times, and the experiment shown in E was conducted five times.