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Mol Cell Proteomics. 2018 Jul;17(7):1295-1307. doi: 10.1074/mcp.RA117.000471. Epub 2018 Mar 29.

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

Author information

1
From the ‡Molecular Biology I, Centre for Medical Biotechnology, Faculty of Biology, University of Duisburg-Essen, 45141 Essen, Germany.
2
§Department of Biology, Institute of Molecular Systems Biology, ETH Zurich, 8093 Zurich, Switzerland.
3
¶Faculty of Science, University of Zurich, Zurich, Switzerland.
4
§Department of Biology, Institute of Molecular Systems Biology, ETH Zurich, 8093 Zurich, Switzerland; hemmo.meyer@uni-due.de collins@imsb.biol.ethz.ch.
5
From the ‡Molecular Biology I, Centre for Medical Biotechnology, Faculty of Biology, University of Duisburg-Essen, 45141 Essen, Germany; hemmo.meyer@uni-due.de collins@imsb.biol.ethz.ch.

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

PMID:
29599191
PMCID:
PMC6030730
DOI:
10.1074/mcp.RA117.000471
[Indexed for MEDLINE]
Free PMC Article

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