AP-SWATH Reveals Direct Involvement of VCP/p97 in Integrated Stress Response Signaling Through Facilitating CReP/PPP1R15B Degradation

Abstract

The ubiquitin-directed AAA-ATPase VCP/p97 facilitates degradation of damaged or misfolded proteins in diverse cellular stress response pathways. Resolving the complexity of its interactions with partner and substrate proteins and understanding its links to stress signaling is therefore a major challenge. Here, we used affinity-purification SWATH mass spectrometry (AP-SWATH) to identify proteins that specifically interact with the substrate-trapping mutant, p97-E578Q. AP-SWATH identified differential interactions over a large detection range from abundant p97 cofactors to pathway-specific partners and individual ligases such as RNF185 and MUL1 that were trapped in p97-E578Q complexes. In addition, we identified various substrate proteins and candidates including the PP1 regulator CReP/PPP1R15B that dephosphorylates eIF2α and thus counteracts attenuation of translation by stress-kinases. We provide evidence that p97 with its Ufd1-Npl4 adapter ensures rapid constitutive turnover and balanced levels of CReP in unperturbed cells. Moreover, we show that p97-mediated degradation, together with a reduction in CReP synthesis, is essential for timely stress-induced reduction of CReP levels and, consequently, for robust eIF2α phosphorylation to enforce the stress response. Thus, our results demonstrate that p97 not only facilitates bulk degradation of misfolded proteins upon stress, but also directly modulates the integrated stress response at the level of signaling.

Keywords: Chaperone*; Protein Degradation*; SWATH-MS; Stress response; Ubiquitin.

Fig. 1.

Comparative SWATH-MS of VCP/p97 wild-type and the substrate-trapping mutant, VCP/p97-E578Q, identifies differential binding of subsets of cofactors, ubiquitin ligases and potential substrates. A, Work flow. Isogenic stable HEK293 cells were induced to express strep-tagged VCP/p97 wild-type (p97-WT) or VCP/p97-E578Q (p97-EQ). p97 was affinity-purified from detergent extracts and associated proteins analyzed by SWATH-MS. Western blot analysis of overexpressed and endogenous p97 (indicated by arrows). B, Scatterplot of interactors as identified by SWATH analysis. The Y-axis represents the fold change ratio of VCP/p97-EQ versus VCP/p97-WT. The X-axis represents the fold change of VCP/p97-WT or VCP/p97-EQ versus GFP-pulldown controls. p values for contaminant filtering and EQ/WT comparison visualized as indicated. Individual candidates mentioned in the text are annotated. See supplemental Table S1 for full list. The proteins falling along the diagonal lines in either the upper right or lower right quadrant are those which have only been identified and quantified in either the VCP/p97-EQ, or VCP/p97-WT purifications, respectively. C, Plot representing the 108 high confidence hits arranged according to abundance in VCP/p97-WT and changes in VCP/p97-EQ are indicated by arrows. Note the high dynamic range over >4 log10. Individual candidates mentioned in the text are annotated. Observations of a given protein interaction in previous studies are annotated as indicated.

Fig. 2.

Validation of differential binding to cofactors and additional interactors. A, Gel filtration of lysate from cells expressing p97-WT or p97-EQ. Fraction numbers and antibodies used for Western blotting are indicated. Note the shift of p97, PLAA, UBXD1 and UBXD7 in p97-EQ lysate toward the high molecular weight fraction, whereas p47 remain unbound under the stringent conditions. B, Affinity purification of p97-WT or p97-EQ and detection of associated SEP-domain proteins p47, p37 and UBXD4. p97 variants were isolated using streptactin beads and associated proteins analyzed by Western blotting as indicated. C, MUL1-GFP, (D) RNF185-GFP, and (E) GFP-UBE4B were transiently expressed in p97-WT or p97-EQ cells, immunoprecipitated and associated p97 analyzed by Western blotting. Note that MUL1 and RNF185, but not UBE4B, showed increased binding of p97-EQ thus confirming SWATH data.

Fig. 3.

CReP/PPP1R15B is a direct target of the p97-Ufd1-Npl4 complex. A, Coimmunoprecipitation of endogenous CReP from lysate of p97-WT or p97-EQ cells. Cells were induced to express the p97 variants or left untreated (± DOX). Isolates were analyzed by Western blotting with indicated antibodies. Note the association of p97, Ufd1 and Npl4 in the presence of p97-EQ. B, CReP accumulates in ubiquitinated form upon inhibition of p97 or the proteasome. Flag-tagged CReP-mCherry or mCherry alone was transiently expressed, and cells were treated with the p97 inhibitor NMS-873 (5 μm), the proteasome inhibitor MG132 (20 μm), or vehicle alone (DMSO), for 1 h before lysis. CReP was immunoprecipitated and probed with ubiquitin antibodies by Western blotting.

Fig. 4.

p97 and Ufd1 are essential for constitutive degradation of CReP. A, Cycloheximide (CHX) chase experiments in the presence of the p97 inhibitor NMS-873 (5 μm) or DMSO alone. Cells were lysed at indicated time points after CHX addition and lysates subjected to Western blotting. Tubulin was probed as loading control. UT = untreated. B, Quantification of chemiluminescence in three independent experiments. CReP Western blotting signals were normalized to loading control and values displayed relative to those in untreated (UT) cells. Error bars, s.e.m., (*) p value < 0.05. C, As in (A), but with the unrelated competitive p97 inhibitor CB-5083 (5 μm). D, CHX chase experiments as in (A) were conducted in cells expressing p97-WT or the dominant-negative p97-EQ. Cells at time point 0 were mock (DMSO) treated. E, CHX chase experiments were perfomed in cells treated with control siRNA, or siRNA directed against Ufd1 or p47 for 48 h. Depletion efficiency of Ufd1 and p47 was monitored with specific antibodies as indicated. Cells at time point 0 were mock (DMSO) treated. F, Quantification of chemiluminescence in four independent experiments. CReP Western blotting signals were normalized to loading control and siRNA control at time point 0. Error bars, s.e.m., (*) p value < 0.05.

Fig. 5.

The p97-Ufd1-Npl4 complex is required for the stress-induced drop in CReP levels and the robust phosphorylation of eIF2α after UV irradiation. A, Quantitative removal of CReP after UV-induced stress. Cells were either mock-treated, or UV-irradiated in the presence of p97 inhibitor NMS-873 (5 μm) or vehicle alone (DMSO). Cells were lysed at indicated time points after irradiation, and lysates probed with indicated antibodies. Chk1 phosphorylation confirms UV-induced damage. Note the rapid decline of CReP after irradiation that was blocked by NMS-873, whereas DNA damage signaling was unaffected according to Chk1 phosphorylation. UT = untreated. B, Quantification of (A). CReP Western blotting signals were normalized to loading control and values were displayed relative to those in untreated cells. n = 3 independent experiments, error bars, s.e.m., (*) p value < 0.05. C, Ufd1 and β-TrCP depletion delays stress-induced degradation of CReP and prevents robust phosphorylation of eIF2α. Experiments as in (A) after treatment of cells with control, Ufd1 or β-TrCP siRNAs for 48 h. Note the reduced phosphorylation of eIF2α. UT = untreated (not irradiated). D, Quantification of stress-induced CReP degradation in experiments as in (C). CReP Western blotting signals were normalized to loading control and values were displayed relative to those in untreated cells. n = 3 independent experiments, error bars, s.e.m., (*) p value < 0.05, (**) p value < 0.01. E, Quantification of phospho-eIF2α signals in experiments as in (C). Values were normalized to loading control and displayed relative to the maximum stimulation in control cells. n = 4 independent experiments, error bars, s.e.m., (*) p value < 0.05, (**) p value < 0.01, (***) p value < 0.001. F, Model for how p97 affects eIF2α phosphorylation and protects cells from protein stress. p97 maintains protein homeostasis by bulk degradation of misfolded proteins in a variety of pathways and compartments including the ER. In addition, p97 facilitates CReP degradation. This is required to balance CReP levels in unperturbed cells and to ensure its quantitative removal after certain stresses in order to enforce stress-kinase mediated eIF2α phosphorylation.