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CRL4^(AMBRA1) targets Elongin C for ubiquitination and degradation to modulate CRL5 signaling

Chen, Si-Han and Jang, Gwendolyn M. and Hüttenhain, Ruth and Gordon, David E. and Du, Dan and Newton, Billy W. and Johnson, Jeffrey R. and Hiatt, Joseph and Hultquist, Judd F. and Johnson, Tasha L. and Liu, Yi-Liang and Burton, Lily A. and Ye, Jordan and Reichermeier, Kurt M. and Stroud, Robert M. and Marson, Alexander and Debnath, Jayanta and Gross, John D. and Krogan, Nevan J. (2018) CRL4^(AMBRA1) targets Elongin C for ubiquitination and degradation to modulate CRL5 signaling. EMBO Journal, 37 (18). Art. No. e97508. ISSN 0261-4189. doi:10.15252/embj.201797508.

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Multi‐subunit cullin‐RING ligases (CRLs) are the largest family of ubiquitin E3 ligases in humans. CRL activity is tightly regulated to prevent unintended substrate degradation or autocatalytic degradation of CRL subunits. Using a proteomics strategy, we discovered that CRL4^(AMBRA1) (CRL substrate receptor denoted in superscript) targets Elongin C (ELOC), the essential adapter protein of CRL5 complexes, for polyubiquitination and degradation. We showed that the ubiquitin ligase function of CRL4^(AMBRA1) is required to disrupt the assembly and attenuate the ligase activity of human CRL5^(SOCS3) and HIV‐1 CRL5^(VIF) complexes as AMBRA1 depletion leads to hyperactivation of both CRL5 complexes. Moreover, CRL4^(AMBRA1) modulates interleukin‐6/STAT3 signaling and HIV‐1 infectivity that are regulated by CRL5^(SOCS3) and CRL5^(VIF), respectively. Thus, by discovering a substrate of CRL4^(AMBRA1), ELOC, the shared adapter of CRL5 ubiquitin ligases, we uncovered a novel CRL cross‐regulation pathway.

Item Type:Article
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URLURL TypeDescription
Du, Dan0000-0002-3470-2890
Johnson, Jeffrey R.0000-0002-5586-4901
Gross, John D.0000-0002-1377-4705
Krogan, Nevan J.0000-0003-4902-337X
Alternate Title:CRL4AMBRA1 targets Elongin C for ubiquitination and degradation to modulate CRL5 signaling
Additional Information:© 2018 The Authors. Received 5 June 2017; Revised 26 July 2018; Accepted 1 August 2018. Published online 30.08.2018. We thank members of the Krogan laboratory for helpful discussion, especially Priya Shah, Manon Eckhardt, and Joshua Kane. Special thanks to Crystal Herron for manuscript editing and Mike Shales for help with figure layout and presentation. We also thank John Von Dollen and Erik Verschueren for technical assistance with implementing MS‐scoring algorithms in R environment; Sourav Bandyopadhyay (UCSF) for providing plasmids of SOCS2, SOCS3, VHL, and PPIL5; Rik Derynck (UCSF) for providing Hep3B cells; Reuben Harris (University of Minnesota) for providing SupT11‐APOBEC3G cell line; and Feng Zhang (MIT) for gifting pSpCas9(BB)‐2A‐GFP (PX458; Addgene plasmid # 48138). The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD008005. This research was supported by NIH funding for the HIV Accessory and Regulatory Complexes (HARC) Center and UCSF‐Gladstone Institute CFAR (P50GM082250, N.J.K., A.M., R.M.S. and J.D.G.; P30‐AI027763, R.H.). S.H.C. is recipient of a UCSF‐Zaffaroni fellowship and Ministry of Education fellowship (Taiwan). R.H. received postdoctoral fellowships from the Swiss National Science Foundation (P2EZP3_148742; P300P3_151154), the European Molecular Biology Organization (ALTF 1123‐2013), and the Human Frontiers in Science Program (LT000089/2014‐L). N.J.K. is supported by NIH funding (R01 AI120694). A.M. holds a Career Award for Medical Scientists from the Burroughs Wellcome Fund and is a Chan Zuckerberg Biohub Investigator. JH was supported by UCSF MSTP (T32GM007618). Author contributions: S‐HC and NJK conceived of the project. S‐HC designed and performed experiments, and NJK supervised the research. S‐HC and GMJ performed comparative proteomics on CRL4AMBRA1 with scoring assistance from RH. JRJ, BWN, and TLJ prepared MS samples, performed machine runs, and conducted database searching. JY generated and characterized ATG‐knockout lines. KMR generated and characterized CUL4A‐knockout lines. S‐HC generated stable cell lines, and performed tandem IPs, single IPs, cell‐based ubiquitination assays, immunoblotting, and co‐transfection experiments. S‐HC conducted IL‐6‐stimulation experiments, generated AMBRA‐knockout lines, and performed genotyping with assistance from DD. DEG, JH, and JFH isolated human primary T cells and performed HIV‐1 infection assays with support from AM. Y‐LL purified AMBRA1‐DDB1 recombinant proteins from insect cells under the supervision of RMS. LAB purified proteins and performed in vitro ubiquitination assays with the guidance of JDG. S‐HC wrote the manuscript with NJK with input from JD and JDG. S‐HC, GMJ, DD, and RH edited the manuscript for publication. Conflict of interest: A.M. serves as an advisor to Juno Therapeutics and PACT Therapeutics and is a founder of Spotlight Therapeutics, and the Marson laboratory has received sponsored research support from Juno Therapeutics and Epinomics. A patent has been filed on the use of Cas9 RNPs to edit the genome of human primary T cells (A.M.).
Funding AgencyGrant Number
University of California, San FranciscoUNSPECIFIED
Ministry of Education (Taipei)UNSPECIFIED
Swiss National Science Foundation (SNSF)P2EZP3_148742
Swiss National Science Foundation (SNSF)P300P3_151154
European Molecular Biology Organization (EMBO)ALTF 1123-2013
Human Frontier Science ProgramLT000089/2014-L
NIHR01 AI120694
Burroughs Wellcome FundUNSPECIFIED
Chan Zuckerberg BiohubUNSPECIFIED
NIH Predoctoral FellowshipT32GM007618
Subject Keywords:AMBRA1; cullin-RING ligase; HIV infection; interleukin-6; ubiquitin
Issue or Number:18
Record Number:CaltechAUTHORS:20180904-135202923
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Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:89361
Deposited By: Tony Diaz
Deposited On:04 Sep 2018 21:45
Last Modified:16 Nov 2021 00:34

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