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

2018; Elsevier BV; Volume: 17; Issue: 7 Linguagem: Inglês

10.1074/mcp.ra117.000471

ISSN

1535-9484

Autores

Julia Hülsmann, Bojana Kravić, Matthias Weith, Matthias Gstaiger, Ruedi Aebersold, Ben C. Collins, Hemmo Meyer,

Tópico(s)

Heat shock proteins research

Resumo

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. 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. Key elements of the response to cellular stresses that threaten protein homeostasis include the induction of protein folding factors such as chaperones, the efficient clearance of terminally misfolded proteins and the attenuation of protein synthesis (1.Harding H.P. Calfon M. Urano F. Novoa I. Ron D. Transcriptional and translational control in the Mammalian unfolded protein response.Annu. Rev. Cell Dev. Biol. 2002; 18: 575-599Crossref PubMed Scopus (808) Google Scholar). Inhibition of translation is achieved by various stress kinases including the endoplasmic reticulum (ER) 1The abbreviations used are: ER, endoplasmic reticulum; AAA, ATPases associated with diverse cellular activities; ALS, amyotrophic lateral sclerosis; AMFR, Autocrine motility factor receptor; AP, affinity purification; As, sodium arsenite; ASPSCR1, Alveolar soft part sarcoma chromosomal region candidate gene 1 protein; ATXN3, Ataxin-3; BCA, bicinchoninic acid; β-TrCP, beta-transducin repeat-containing protein; cdc, cell division cycle; Chk1, checkpoint kinase-1; CHX, cycloheximide; CReP, constitutive repressor of eIF2α phosphorylation; CUL, cullin; DDA, data dependent acquisition; DOX, doxycycline; E3, ubiquitin ligase; EGF, epidermal growth factor; eIF2α, eukaryotic translation initiation factor 2A; EPSTI1, Epithelial-stromal interaction protein 1; ERAD, ER-associated degradation; EQ, ATPase-deficient p97-E578Q mutant; exp, exposure; FAF, Fas-associated factor; FDR, False discovery rate; FEM1B, Fem-1 Homolog B; GADD34, Growth arrest and DNA damage-inducible protein; GFP, green fluorescent protein; GLUL, glutamine synthetase; HEK, human embryonic kidney; HRP, horseradish peroxidase; IBMPFD, inclusion body myopathy with early-onset Paget disease and frontotemporal dementia; IkBa, NF-kappa-B inhibitor alpha; IP, immunoprecipitation; iRD, indexed retention time; ISR, integrated stress response; log, logarithm; MUL1, Mitochondrial ubiquitin ligase activator of NFKB 1; MYO5C, Unconventional myosin-Vc; NFkB, nuclear factor NF-kappa-B; NGLY1, N-glycanase 1; Npl4, Nuclear protein localization protein 4 homolog; NSFL1C, N-ethylmaleimide-sensitive factor (NSF)L1 cofactor; OTU, ovarian tumor DUB domain; PD, pulldown; PERK, PKR-like ER kinase; PKR, protein kinase repressor; PLAA, phospholipase A-2-activating protein; PP1, Protein phosphatase-1; ppm, parts per million; PPP1R, Protein phosphatase 1 regulatory subunit; psm, peptide spectrum match; RNF31, RING finger protein 31; RNF185, RING finger protein 185; SCF, Skp1–Cullin–F-box; SEL1L, Protein sel-1 homolog 1; s.e.m., standard error of the mean; SEP, Shp1-eyc-p47 domain; SH3KBP1, SH3 Domain Containing Kinase Binding Protein 1; SHKBP1, SH3KBP1-binding protein 1; siRNA, small interfering RNA; SPRTN, SprT-like domain-containing protein Spartan; strep, streptavidin; SVIP, small VCP/p97-interacting protein; SWATH, Sequential Window Acquisition of all Theoretical Mass Spectra; Tg, thapsigargin; Tm, tunicamycin; TRAFD1, TRAF-type zinc finger domain containing protein 1; TRIC, TRansfer of Identification Confidence; Ub, ubiquitin; UBAC2, Ubiquitin-associated domain-containing protein 2; UBX, ubiquitin domain-X; UBE, ubiquitin-conjugating enzyme; UBXD, UBX domain-containing protein; Ufd, Ubiquitin fusion degradation protein; UPS, ubiquitin-proteasome system; UT, untreated; VCP, Valosin-containing protein; VCPIP1, Valosin-containing protein p97/p47 complex-interacting protein 1; WLS, wntless Wnt ligand secretion mediator; WT, wild-type; YOD1, yeast OTU1 deubiquitinating enzyme 1 homolog; ZFAND2B, AN1-type zinc finger protein 2B. 1The abbreviations used are: ER, endoplasmic reticulum; AAA, ATPases associated with diverse cellular activities; ALS, amyotrophic lateral sclerosis; AMFR, Autocrine motility factor receptor; AP, affinity purification; As, sodium arsenite; ASPSCR1, Alveolar soft part sarcoma chromosomal region candidate gene 1 protein; ATXN3, Ataxin-3; BCA, bicinchoninic acid; β-TrCP, beta-transducin repeat-containing protein; cdc, cell division cycle; Chk1, checkpoint kinase-1; CHX, cycloheximide; CReP, constitutive repressor of eIF2α phosphorylation; CUL, cullin; DDA, data dependent acquisition; DOX, doxycycline; E3, ubiquitin ligase; EGF, epidermal growth factor; eIF2α, eukaryotic translation initiation factor 2A; EPSTI1, Epithelial-stromal interaction protein 1; ERAD, ER-associated degradation; EQ, ATPase-deficient p97-E578Q mutant; exp, exposure; FAF, Fas-associated factor; FDR, False discovery rate; FEM1B, Fem-1 Homolog B; GADD34, Growth arrest and DNA damage-inducible protein; GFP, green fluorescent protein; GLUL, glutamine synthetase; HEK, human embryonic kidney; HRP, horseradish peroxidase; IBMPFD, inclusion body myopathy with early-onset Paget disease and frontotemporal dementia; IkBa, NF-kappa-B inhibitor alpha; IP, immunoprecipitation; iRD, indexed retention time; ISR, integrated stress response; log, logarithm; MUL1, Mitochondrial ubiquitin ligase activator of NFKB 1; MYO5C, Unconventional myosin-Vc; NFkB, nuclear factor NF-kappa-B; NGLY1, N-glycanase 1; Npl4, Nuclear protein localization protein 4 homolog; NSFL1C, N-ethylmaleimide-sensitive factor (NSF)L1 cofactor; OTU, ovarian tumor DUB domain; PD, pulldown; PERK, PKR-like ER kinase; PKR, protein kinase repressor; PLAA, phospholipase A-2-activating protein; PP1, Protein phosphatase-1; ppm, parts per million; PPP1R, Protein phosphatase 1 regulatory subunit; psm, peptide spectrum match; RNF31, RING finger protein 31; RNF185, RING finger protein 185; SCF, Skp1–Cullin–F-box; SEL1L, Protein sel-1 homolog 1; s.e.m., standard error of the mean; SEP, Shp1-eyc-p47 domain; SH3KBP1, SH3 Domain Containing Kinase Binding Protein 1; SHKBP1, SH3KBP1-binding protein 1; siRNA, small interfering RNA; SPRTN, SprT-like domain-containing protein Spartan; strep, streptavidin; SVIP, small VCP/p97-interacting protein; SWATH, Sequential Window Acquisition of all Theoretical Mass Spectra; Tg, thapsigargin; Tm, tunicamycin; TRAFD1, TRAF-type zinc finger domain containing protein 1; TRIC, TRansfer of Identification Confidence; Ub, ubiquitin; UBAC2, Ubiquitin-associated domain-containing protein 2; UBX, ubiquitin domain-X; UBE, ubiquitin-conjugating enzyme; UBXD, UBX domain-containing protein; Ufd, Ubiquitin fusion degradation protein; UPS, ubiquitin-proteasome system; UT, untreated; VCP, Valosin-containing protein; VCPIP1, Valosin-containing protein p97/p47 complex-interacting protein 1; WLS, wntless Wnt ligand secretion mediator; WT, wild-type; YOD1, yeast OTU1 deubiquitinating enzyme 1 homolog; ZFAND2B, AN1-type zinc finger protein 2B. stress sensor PERK (2.Harding H.P. Zhang Y. Bertolotti A. Zeng H. Ron D. Perk is essential for translational regulation and cell survival during the unfolded protein response.Mol. Cell. 2000; 5: 897-904Abstract Full Text Full Text PDF PubMed Scopus (1550) Google Scholar, 3.Scheuner D. Song B. McEwen E. Liu C. Laybutt R. Gillespie P. Saunders T. Bonner-Weir S. Kaufman R.J. Translational control is required for the unfolded protein response and in vivo glucose homeostasis.Mol. Cell. 2001; 7: 1165-1176Abstract Full Text Full Text PDF PubMed Scopus (1086) Google Scholar, 4.Harding H.P. Zhang Y. Zeng H. Novoa I. Lu P.D. Calfon M. Sadri N. Yun C. Popko B. Paules R. Stojdl D.F. Bell J.C. Hettmann T. Leiden J.M. Ron D. An integrated stress response regulates amino acid metabolism and resistance to oxidative stress.Mol. Cell. 2003; 11: 619-633Abstract Full Text Full Text PDF PubMed Scopus (2364) Google Scholar). As elements of the integrated stress response (ISR) these kinases converge on phosphorylating the eukaryotic initiation factor 2 alpha (eIF2α) at serine-51 to block global translation at the ribosome (5.Pavitt G.D. Ron D. New insights into translational regulation in the endoplasmic reticulum unfolded protein response.Cold Spring Harb. Perspect. Biol. 2012; 4: 1-12Crossref Scopus (109) Google Scholar, 6.Holcik M. Sonenberg N. Translational control in stress and apoptosis.Nat. Rev. Mol. Cell Biol. 2005; 6: 318-327Crossref PubMed Scopus (1029) Google Scholar). The phosphorylation of eIF2α is antagonized by protein phosphatase-1 (PP1) complexes with one of two alternative regulatory subunits. Gadd34/PPP1R15A is induced after stress, and shuts down the stress response to resume protein synthesis (7.Connor J.H. Weiser D.C. Li S. Hallenbeck J.M. Shenolikar S. Growth arrest and DNA damage-inducible protein GADD34 assembles a novel signaling complex containing protein phosphatase 1 and inhibitor 1.Mol. Cell. Biol. 2001; 21: 6841-6850Crossref PubMed Scopus (223) Google Scholar, 8.Novoa I. Zhang Y. Zeng H. Jungreis R. Harding H.P. Ron D. Stress-induced gene expression requires programmed recovery from translational repression.EMBO J. 2003; 22: 1180-1187Crossref PubMed Scopus (349) Google Scholar). In contrast, CReP/PPP1R15B is constitutively expressed and balances eIF2α phosphorylation in unperturbed cells (9.Jousse C. Oyadomari S. Novoa I. Lu P. Zhang Y. Harding H.P. Ron D. Inhibition of a constitutive translation initiation factor 2alpha phosphatase, CReP, promotes survival of stressed cells.J. Cell Biol. 2003; 163: 767-775Crossref PubMed Scopus (237) Google Scholar). The clearance of misfolded proteins, on the other hand, is mediated by the ubiquitin-proteasome system (UPS) and autophagy. The AAA+-type ATPase Valosin-containing protein (VCP)/p97 (also called Cdc48 or Ter94) is a critical bottleneck in these processes (10.Stolz A. Hilt W. Buchberger A. Wolf D.H. Cdc48: a power machine in protein degradation.Trends Biochem. Sci. 2011; 36: 515-523Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar, 11.Meyer H. Bug M. Bremer S. Emerging functions of the VCP/p97 AAA-ATPase in the ubiquitin system.Nat. Cell Biol. 2012; 14: 117-123Crossref PubMed Scopus (581) Google Scholar). It helps mobilize and unfold large cohorts of ubiquitin-modified substrates to facilitate their degradation in the proteasome in diverse pathways and compartments ranging from ER-associated degradation (ERAD), ribosomal quality control, mitochondrial stress response to macroautophagy and degradation of chromatin-associated proteins (11.Meyer H. Bug M. Bremer S. Emerging functions of the VCP/p97 AAA-ATPase in the ubiquitin system.Nat. 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Biophys. Acta. 2008; 1782: 744-748Crossref PubMed Scopus (171) Google Scholar, 16.Meyer H. Weihl C.C. The VCP/p97 system at a glance: connecting cellular function to disease pathogenesis.J. Cell Sci. 2014; 127: 3877-3883Crossref PubMed Scopus (276) Google Scholar). Moreover, p97 inhibitors are being explored as cancer drugs based on the rationale that they induce a proteostasis crisis and cell death in cancer cells with extra burden of misfolded proteins, and first inhibitors are moving into clinical trials (17.Anderson D.J. Le Moigne R. Djakovic S. Kumar B. Rice J. Wong S. Wang J. Yao B. Valle E. Kiss von Soly S. Madriaga A. Soriano F. Menon M.K. Wu Z.Y. Kampmann M. Chen Y. Weissman J.S. Aftab B.T. Yakes F.M. Shawver L. Zhou H.J. Wustrow D. Rolfe M. Targeting the AAA ATPase p97 as an Approach to Treat Cancer through Disruption of Protein Homeostasis.Cancer Cell. 2015; 28: 653-665Abstract Full Text Full Text PDF PubMed Scopus (244) Google Scholar, 18.Magnaghi P. D'Alessio R. Valsasina B. Avanzi N. Rizzi S. Asa D. Gasparri F. Cozzi L. Cucchi U. Orrenius C. Polucci P. Ballinari D. Perrera C. Leone A. Cervi G. Casale E. Xiao Y. Wong C. Anderson D.J. Galvani A. Donati D. O'Brien T. Jackson P.K. Isacchi A. Covalent and allosteric inhibitors of the ATPase VCP/p97 induce cancer cell death.Nat. Chem. Biol. 2013; 9: 548-556Crossref PubMed Scopus (256) Google Scholar). p97 has two AAA+ domains, D1 and D2, that form two stacked hexameric rings around a central channel (13.Xia D. Tang W.K. Ye Y. Structure and function of the AAA+ ATPase p97/Cdc48p.Gene. 2016; 583: 64-77Crossref PubMed Scopus (110) Google Scholar). ATP hydrolysis in D2 is most critical for unfolding (19.Bodnar N.O. Rapoport T.A. Molecular mechanism of substrate processing by the Cdc48 ATPase complex.Cell. 2017; 169 (e729): 722-735Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar, 20.Ye Y. Meyer H.H. Rapoport T.A. Function of the p97-Ufd1-Npl4 complex in retrotranslocation from the ER to the cytosol: dual recognition of nonubiquitinated polypeptide segments and polyubiquitin chains.J. Cell Biol. 2003; 162: 71-84Crossref PubMed Scopus (501) Google Scholar, 21.Blythe E.E. Olson K.C. Chau V. Deshaies R.J. Ubiquitin- and ATP-dependent unfoldase activity of P97/VCP*NPLOC4*UFD1L is enhanced by a mutation that causes multisystem proteinopathy.Proc. Natl. Acad. Sci. U.S.A. 2017; 114: E4380-E4388Crossref PubMed Scopus (94) Google Scholar). Consistent with this, a variant harboring the E578Q mutation in the Walker B motif of D2 traps substrate proteins (13.Xia D. Tang W.K. Ye Y. Structure and function of the AAA+ ATPase p97/Cdc48p.Gene. 2016; 583: 64-77Crossref PubMed Scopus (110) Google Scholar, 20.Ye Y. Meyer H.H. Rapoport T.A. Function of the p97-Ufd1-Npl4 complex in retrotranslocation from the ER to the cytosol: dual recognition of nonubiquitinated polypeptide segments and polyubiquitin chains.J. Cell Biol. 2003; 162: 71-84Crossref PubMed Scopus (501) Google Scholar). A host of accessory proteins cooperate with p97. They include more than thirty cofactor proteins that directly interact with the regulatory N-domain or the C-terminal tail of p97 through dedicated interaction domains, and assist p97 as ubiquitin adapters, targeting factors or regulators (16.Meyer H. Weihl C.C. The VCP/p97 system at a glance: connecting cellular function to disease pathogenesis.J. Cell Sci. 2014; 127: 3877-3883Crossref PubMed Scopus (276) Google Scholar, 22.Buchberger A. Schindelin H. Hanzelmann P. Control of p97 function by cofactor binding.FEBS Lett. 2015; 589: 2578-2589Crossref PubMed Scopus (136) Google Scholar). However, it is often unclear if and how binding of accessory factors in diverse pathways is linked to substrate unfolding. A second challenge is the identification of critical substrate proteins. Although p97 targets large cohorts of misfolded proteins in established stress pathways, only few regulatory degradation substrates are known so far, and it is unclear if and how p97 governs stress signaling by directly targeting stress regulators. To tackle the complexity of p97 interactions, we compared p97 wild-type (wt) complexes with complexes containing the p97 substrate-trapping mutant p97-E578Q using affinity purification followed by SWATH mass spectrometry (AP-SWATH). AP-SWATH identified and quantified differential interactions over a large range of interactor abundance. This revealed subsets of cofactors, accessory factors and ubiquitin ligases that were specifically enriched with p97-E578Q indicating that their binding is linked to substrate unfolding. In addition, we isolated a set of p97 substrate candidates. Among them, we identified the PP1 regulator CReP/PPP1R15B as a direct target of the p97-Ufd1-Npl4 cofactor complex. We provide evidence that p97 balances CReP levels in unperturbed cells. Moreover, we demonstrate that p97-mediated degradation of CReP is an important element for the timely reduction of CReP levels after severe stresses and thus for ensuring robust eIF2α phosphorylation to enforce the ISR. pcDNA5-p97-WT/E578Q-myc-strep/FRT/TO were described previously (23.Ritz D. Vuk M. Kirchner P. Bug M. Schütz S. Hayer A. Bremer S. Lusk C. Baloh R.H. Lee H. Glatter T. Gstaiger M. Aebersold R. Weihl C.C. Meyer H. Endolysosomal sorting of ubiquitinated caveolin-1 is regulated by VCP/p97 and UBXD1 and impaired by VCP disease mutations.Nat. Cell Biol. 2011; 13: 1116-1123Crossref PubMed Scopus (158) Google Scholar). Expression constructs coding for RNF185 and MUL1 with C-terminal GFP tag were generated by gateway cloning (Invitrogen, Carlsbad, CA) using human ORFeome library vectors (hORFeome V5.1, Open Biosystems; internal IDs: 10236 (RNF185), 6868 (MUL1)). LR recombination was performed with the pcDNA5/FRT/TO/GFP/GW destination vector obtained from pcDNA5/FRT/TO/cSH/GW (24.Varjosalo M. Sacco R. Stukalov A. van Drogen A. Planyavsky M. Hauri S. Aebersold R. Bennett K.L. Colinge J. Gstaiger M. Superti-Furga G. Interlaboratory reproducibility of large-scale human protein-complex analysis by standardized AP-MS.Nat. Methods. 2013; 10: 307-314Crossref PubMed Scopus (147) Google Scholar) by substitution of the strep-HA with GFP coding sequence via Gibson Assembly cloning. Full length UBE4B cDNA was amplified from an IMAGE clone (ID 7939541) and cloned into pEGFP-C1 using XhoI and BamHI. Flag-tagged PPP1R15B-mCherry (FLAG_hPPP1R15B_4–713_mCherry_UK1298) was a gift from David Ron (Addgene plasmid # 80707) (25.Chen R. Rato C. Yan Y. Crespillo-Casado A. Clarke H.J. Harding H.P. Marciniak S.J. Read R.J. Ron D. G-actin provides substrate-specificity to eukaryotic initiation factor 2alpha holophosphatases.eLife. 2015; 4: 1-28Crossref Scopus (21) Google Scholar). HEK293 and HeLa cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum (FCS) in the presence of penicillin/streptomycin. Stable inducible HEK293 cell lines expressing p97-WT-myc-strep or p97-EQ-myc-strep were generated with the Flp-In T-Rex system (Invitrogen) according to the manufacturer's protocol. Stable cell lines were maintained in culture medium as described above, supplemented with 15 μg/ml blasticidin S and 100 μg/ml hygromycin B. Expression was induced with 1 μg/ml doxycycline. HEK293 and HeLa cells were transiently transfected with plasmids using JetPRIME (Polyplus, Illkirch-Graffenstaden, France) or Lipofectamine2000 reagents (Invitrogen), respectively, 1 day after seeding. Medium was changed 4 h after transfection. Cells were analyzed after 24 h. The siRNA oligomers targeting Ufd1 (#1: GUGGCCACCUACUCCAAAUTT; #2: CUACAAAGAACCCGAAAGATT), p47 (AGCCAGCUCUUCCAUCUUATT), β-TrCP 1/2 (GUGGAAUUUGUGGAACAUCTT), CReP (AAGGGAUGGAUGCAGGUUCCATT) and a nontargeting control oligomer (UUCUCCGAACGUGUCACGUTT) were purchased from Microsynth (Balgach, Switzerland) and were characterized previously (9.Jousse C. Oyadomari S. Novoa I. Lu P. Zhang Y. Harding H.P. Ron D. Inhibition of a constitutive translation initiation factor 2alpha phosphatase, CReP, promotes survival of stressed cells.J. Cell Biol. 2003; 163: 767-775Crossref PubMed Scopus (237) Google Scholar, 23.Ritz D. Vuk M. Kirchner P. Bug M. Schütz S. Hayer A. Bremer S. Lusk C. Baloh R.H. Lee H. Glatter T. Gstaiger M. Aebersold R. Weihl C.C. Meyer H. Endolysosomal sorting of ubiquitinated caveolin-1 is regulated by VCP/p97 and UBXD1 and impaired by VCP disease mutations.Nat. Cell Biol. 2011; 13: 1116-1123Crossref PubMed Scopus (158) Google Scholar, 26.Dobrynin G. Popp O. Romer T. Bremer S. Schmitz M.H. Gerlich D.W. Meyer H. Cdc48/p97-Ufd1-Npl4 antagonizes Aurora B during chromosome segregation in HeLa cells.J. Cell Sci. 2011; 124: 1571-1580Crossref PubMed Scopus (43) Google Scholar, 27.Busino L. Donzelli M. Chiesa M. Guardavaccaro D. Ganoth D. Dorrello N.V. Hershko A. Pagano M. Draetta G.F. Degradation of Cdc25A by beta-TrCP during S phase and in response to DNA damage.Nature. 2003; 426: 87-91Crossref PubMed Scopus (358) Google Scholar). Reverse transfection into HEK293 cells was performed with 10 nm final concentration using Lipofectamine RNAiMax (Invitrogen). Cells were analyzed after 48 h or as indicated. For cycloheximide chase experiments, HEK293 cells were treated with 50 μg/ml CHX (Sigma, St. Louis, MO) for indicated times. p97 and proteasome inhibitors were added for 1 h (or as indicated) using NMS-873 (5 μm; Sigma) and CB-5083 (5 μm; Selleckchem, Houston, TX) or MG132 (20 μm; Merck Millipore, Darmstadt, Germany). For pharmacological stress induction, cells were exposed to 0.5 mm sodium arsenite, 50 μg/ml tunicamycin (both Sigma) or 10 μm thapsigargin (Diagonal, Münster, Germanyl) for the indicated period. UV irradiation was performed by removing medium from adherent cells and covering them in 37 °C PBS. Cells were exposed to 300 J/m2 UV-C (254 nm) and PBS was replaced by the previously removed media (for simultaneous inhibitor treatment drugs were added directly after UV exposure). Cells were analyzed at indicated time points after irradiation. Cells were harvested 24 h after transient transfection and/or 20 h after induction with doxycycline in IP buffer (150 mm KCl, 5 mm MgCl2, 50 mm Tris-HCl pH 7.4, 1% Triton X-100, 5% glycerol, 2 mm β-mercaptoethanol supplemented with cOmplete EDTA-free protease inhibitors and PhosSTOP (Roche, Basel, Switzerland)) and incubated on ice for 20 min. To stabilize ubiquitination of CReP, the IP buffer was additionally supplemented with 10 mm N-ethylmaleimide (Sigma). Lysates were cleared by centrifugation (15 min, 17,000 × g, 4 °C) and protein concentration was determined in a BCA assay (Interchim, Montlucon, France). For degradation assays, 25 μg protein/sample were directly resolved by SDS-PAGE and analyzed by Western blotting. Immunoprecipitation and pulldown experiments were performed using Strep-Tactin Sepharose (IBA BioTAGnology, Göttingen, Germany), anti-GFP nanobodies or specific antibodies and Protein G Sepharose (GE Healthcare, Chicago, IL). Affinity purifications were performed for 2 h at 4 °C rotating (strep-/GFP pulldown assays) or 2 h incubation with the indicated primary antibodies on ice and additionally with Protein G Sepharose for 1 h rotating at 4 °C to capture antigen-antibody complexes. Beads were washed five times in IP buffer and eluted by boiling in 2 × SDS sample buffer. Samples were resolved by SDS-PAGE and transferred to nitrocellulose membranes (Amersham Biosciences, GE Healthcare). Immunoblot analysis was performed with the indicated antibodies and visualized by chemiluminescence. Quantification of Western Blots was performed by capturing antibody signal either with a CCD camera (Amersham Biosciences Imager 600 (GE Healthcare)) and/or with an Odyssey CLx fluorescence reader (LI-COR, Lincoln, NE) whereas image analysis was performed with the TotalLab Quant software. HeLa or HEK293 cells were fixed in 4% paraformaldehyde, permeabilized with 0.1% Triton X-100 in PBS, blocked by 3% bovine serum albumin in PBS containing 0.1% Triton X-100 and 0.1% saponin. Confocal laser scanning microscopy was performed on a TCS SP5 AOBS system equipped with standard PMT detectors as well as sensitive HyD detectors (Leica Microsystems, Wetzlar, Germany). Images were acquired using an HCX PL APO 63×/1.4NA oil-immersion objective. Lasers used for excitation were DPSS 561 nm (Alexa Fluor® 568, mCherry), Ar 488 nm (GFP) and Diode 405 nm (DAPI). Acquisition and hardware was controlled by LAS AF software (Leica Microsystems). Antibodies used for Western blot analysis against p97 (HME8), UBXD1 (E43), Ufd1 (HME14), Npl4 (HME18), p47 (HME22) and p37 (20880) were described previously (23.Ritz D. Vuk M. Kirchner P. Bug M. Schütz S. Hayer A. Bremer S. Lusk C. Baloh R.H. Lee H. Glatter T. Gstaiger M. Aebersold R. Weihl C.C. Meyer H. Endolysosomal sorting of ubiquitinated caveolin-1 is regulated by VCP/p97 and UBXD1 and impaired by VCP disease mutations.Nat. Cell Biol. 2011; 13: 1116-1123Crossref PubMed Scopus (158) Google Scholar, 28.Meyer H.H. Shorter J.G. Seemann J. Pappin D. Warren G. A complex of mammalian Ufd1 and Npl4 links the AAA-ATPase, p97, to ubiquitin and nuclear transport pathways.EMBO J. 2000; 19: 2181-2192Crossref PubMed Scopus (382) Google Scholar, 29.Kress E. Schwager F. Holtackers R. Seiler J. Prodon F. Zanin E. Eiteneuer A. Toya M. Sugimoto A. Meyer H. Meraldi P. Gotta M. The UBXN-2/p37/p47 adaptors of CDC-48/p97 regulate mitosis by limiting the centrosomal recruitment of Aurora A.J. Cell Biol. 2013; 201: 559-575Crossref PubMed Scopus (21) Google Scholar). Anti-UBXD4 (25272) antibody was raised in rabbits against purified recombinant GST-tagged proteins (BioGenes, Berlin, Germany). Anti-UBXD7 (S409D) antibody was generated and obtained from the MRCprotein phosphorylation unit, Dundee (G. Alexandru). Anti-PLAA (Y102) was purchased from Abcam (Cambridge, UK). Mouse anti-ubiquitin (clone FK2, 04–263) and rabbit anti-Histone H3 (06–755) were purchased from Millipore. Anti-PPP1R15B (CReP) was from Proteintech (Rosemont, IL) (14634–1-AP). Rabbit monoclonal anti-phosphoS51-eIF2α (#3398) and anti-phosphoS317Chk1 (#2344) antibodies were purchased from Cell Signaling (Danvers, MA). Mouse monoclonal anti-eIF2α (d-3, sc-133132) and anti-HSC 70 (B-6, sc-7298) antibodies were from Santa Cruz Biotechnology (Dallas, TX). Monoclonal anti-Flag M2 (F3165), anti-myc (M4439), anti-α-tubulin (T5168) and anti-GAPDH (G8795) were from Sigma-Aldrich. Anti-GFP was from Roche. HRP-coupled secondary antibodies were purchased from Bio-Rad (Hercules, CA) and IRDye®-conjugated secondary antibodies from LI-COR. Antibodies used for immunofluorescence stainings were anti-Tom20 (Santa Cruz, sc-11415) and anti-Calnexin (Enzo Life Sciences, ADI-SPA-860-F), and secondary antibodies conjugated to Alexa Fluor® 568 fluorophore (Invitrogen). All experiments were performed in triplicate or as indicated, with error bars denoting s.e.m. Statistical analyses were carried out with the unpaired two-sided t test. p < 0.05 (*) was considered statistically significant. Size exclusion chromatography was performed on a Superose 6 10/300 GL column at 20 °C in 50 mm HEPES pH 7.5, 150 mm NaCl, 5 mm MgCl2, 0.5% NP40, 1 mm DTT, 25 mm β-Glycerophosphate, 10 μm leupeptin/pepstatin. 3.5 mg cleared cell lysates from stable HEK293 p97-WT/EQ cell lines (24 h after induction with doxycycline) were separated into 13 fractions and analyzed by Western blotting. Cells were lysed on ice in HNN lysis buffer (0.5% IGEPAL ca-630, 50 mm HEPES, pH 7.5, 150 mm NaCl, 50 mm NaF, 200 μm NaVO3, 0.5 mm PMSF, 1.2 μm Avidin, and protease inhibitor mixture (Sigma)) and centrifuged at 16,100 × g for 15 min at 4 °C. Single step affinity purification from the supernatants via the streptavidi

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