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The SCFβ-TrCP E3 Ubiquitin Ligase Regulates Immune Receptor Signaling by Targeting the Negative Regulatory Protein TIPE2 This information is current as of September 27, 2021. Yunwei Lou, Meijuan Han, Yaru Song, Jiateng Zhong, Wen Zhang, Youhai H. Chen and Hui Wang J Immunol published online 18 March 2020 http://www.jimmunol.org/content/early/2020/03/17/jimmun ol.1901142 Downloaded from Supplementary http://www.jimmunol.org/content/suppl/2020/03/17/jimmunol.190114 Material 2.DCSupplemental http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication by guest on September 27, 2021 *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2020 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published March 18, 2020, doi:10.4049/jimmunol.1901142 The Journal of Immunology The SCFb-TrCP E3 Ubiquitin Ligase Regulates Immune Receptor Signaling by Targeting the Negative Regulatory Protein TIPE2 Yunwei Lou,*,† Meijuan Han,*,† Yaru Song,‡ Jiateng Zhong,x Wen Zhang,† Youhai H. Chen,{ and Hui Wang*,† TNFAIP8-like 2 (TIPE2) is a negative regulator of immune receptor signaling that maintains immune homeostasis. Dysregulated TIPE2 expression has been observed in several types of human immunological disorders. However, how TIPE2 expression is regulated remains to be determined. We report in this study that the SCFb-TrCP E3 ubiquitin ligase regulates TIPE2 protein abundance by targeting it for ubiquitination and subsequent degradation via the 26S proteasome. Silencing of either cullin-1 or 3 b-TrCP1 resulted in increased levels of TIPE2 in immune cells. TAK1 phosphorylated the Ser in the noncanonical degron Downloaded from motif of TIPE2 to trigger its interaction with b-TrCP for subsequent ubiquitination and degradation. Importantly, the amount of TIPE2 protein in immune cells determined the strength of TLR 4–induced signaling and downstream gene expression. Thus, our study has uncovered a mechanism by which SCFb-TrCP E3 ubiquitin ligase regulates TLR responses. The Journal of Immunology, 2020, 204: 000–000. he immune system is a complex network of cells, tissues, and an Ub ligase (E3) (1). Among them, E3 ligases specifically http://www.jimmunol.org/ and organs, which must be capable of eradicating a vast bind to target proteins and confer specificity of the reaction. To T variety of invading microorganisms but must avoid date, more than 600 E3 ligases have been discovered in the human attacking the self. To inhibit harmful pathology caused by un- genome (2). According to their protein sequence homology, the E3 controlled inflammatory responses, the immune system has evolved Ub ligases are generally classified into three main families: the comprehensive and multiple-level mechanisms to stringently homology to the E6-AP C terminus (HECT), the really interesting regulate immune receptor signaling. As one of the most important new gene (RING), and the ring-between-ring (RBR) family (3, 4). regulatory mechanisms, protein ubiquitination, a form of conven- Ub attached to substrates can form polymeric chains and affect the tional posttranslational modification, is extensively used to orches- expression or function of substrates through different mechanisms. trate appropriate immune responses. The protein ubiquitination In particular, lysine 48 (K48)–linked polyubiquitination primarily by guest on September 27, 2021 requires the sequential action of three enzymes: a ubiquitin targets proteins for proteasomal degradation, whereas K63-linked (Ub)-activating enzyme (E1), an Ub-conjugating enzyme (E2), polyubiquitination mediates nonproteolytic functions, including assembly of signaling complexes and activation of protein kinases *Henan Collaborative Innovation Center of Molecular Diagnosis and Laboratory (5). Accumulating evidences show that many E3 ligases have key Medicine, School of Laboratory Medicine, Xinxiang Medical University, Xinxiang, roles in regulating immune responses (6). † Henan 453003, People’s Republic of China; Henan Key Laboratory of Immunology As compared with the typical single-subunit E3 ligases (such as and Targeted Drugs, Xinxiang Medical University, Xinxiang, Henan 453003, People’s Republic of China; ‡Department of Pulmonary Medicine, The Affiliated RING-finger family and HECT family E3 ligases), the Cullin-RING Renmin Hospital of Xinxiang Medical University, Xinxiang, Henan 453100, People’s multisubunit ligase complexes are assembled on the cullin backbones Republic of China; xDepartment of Pathology, Xinxiang Medical University, { (4). Among these family members, the Skp1–cullin-1–F-box (SCF) Xinxiang, Henan 453003, People’s Republic of China; and Department of Pathology and Laboratory Medicine, University of Pennsylvania Perelman School of Medicine, protein is the best-characterized mammalian RING-finger Ub ligase Philadelphia, PA 19104 composed of the cullin scaffold cullin-1 (Cul1), which simulta- ORCIDs: 0000-0002-9695-6757 (Y.H.C.); 0000-0002-2454-3814 (H.W.). neously interacts with the RING subunit Rbx1, and the adaptor Received for publication September 19, 2019. Accepted for publication February 7, protein Skp1. Skp1, in turn, binds to the F-box domain of many 2020. F-box proteins, serving as the receptor subunit that recruits This work was supported by National Natural Science Foundation of China Grants specific substrate proteins. It has been validated that the human 81871309, 31500707, and U1804190. Y.L. was also supported by start-up funds from Xinxiang Medical University (Grant 505248). genome encodes 69 F-box proteins. Those proteins have been classified into three categories: those with WD40 domains (Fbxw), Y.L., M.H., Y.S., and J.Z. performed experiments; H.W. and Y.L. conceived the study and wrote the manuscript; W.Z. provided expertise and advice; Y.H.C. contributed to those with leucine-rich repeats (Fbxl), and those with other diverse the discussion of the results; and H.W. supervised the project. domains (Fbxo) (7). Although great achievements have been Address correspondence and reprint requests to Prof. Hui Wang, School of Laboratory made for elucidating the roles of SCF complexes in cancer (8), Medicine, Xinxiang Medical University, 601 Jinsui Road, Xinxiang, Henan Province the functions of SCF in immunity remain poorly understood. 453003, People’s Republic of China. E-mail address: [email protected] b-Transducin repeat–containing protein (b-TrCP), the best- The online version of this article contains supplemental material. characterized F-box protein associated with immunity, exists as Abbreviations used in this article: BMDM, bone marrow–derived macrophage; Cul1, cullin-1; IKK, IkB kinase; KO, knockout; qPCR, quantitative real-time PCR; RING, two distinct paralogs: b-TrCP1 and b-TrCP2. The SCF com- really interesting new gene; SCF, Skp1–cullin-1–F-box; siRNA, small interfering plex, which contains the F-box protein b-TrCP, is referred to as RNA; TAK1, TGF-b–activated kinase 1; TIPE2, TNFAIP8-like 2; b-TrCP, b-transducin SCFb-TrCP. SCFb-TrCP has been implicated in regulating NF-kB repeat–containing protein; Ub, ubiquitin; WT, wild-type. signaling by degrading IkBa or processing of p100 (also known as Copyright Ó 2020 by The American Association of Immunologists, Inc. 0022-1767/20/$37.50 NF-kB2) and p105 (also known as NF-kB1) (9–11). For example, www.jimmunol.org/cgi/doi/10.4049/jimmunol.1901142 2 TIPE2 IS A NOVEL SUBSTRATE OF THE SCFb-TrCP COMPLEX various inflammatory stimuli, such as LPS, TNF-a, or IL-17A, (Invitrogen) with Flag tag at the C terminus. Recombinant vectors result in the degradation of IkBa via the SCFb-TrCP complex, encoding Nemo and IKKi were constructed by PCR-based amplification of which enables the translocation of the NF-kB p65:p50 hetero- cDNA from THP-1 cells or PMA-differentiated THP-1 cells and then cloned into pcDNA3.0 eukaryotic expression vector (Invitrogen) with HA dimer into the nucleus, where it controls inflammatory genes tag at the C terminus. The Flag-tagged b-TrCP1 and b-TrCP1 R474A transcription. It is noted that specific phosphorylation of the mutant expression plasmids were described previously (28). HA-tagged substrate is required for recognition by b-TrCP through certain b-TrCP1 WT and domain deletion plasmids b-TrCP1 D F-box, b-TrCP1 protein kinases, such as IkB kinases (IKKs), to mediate the ubiq- D WD-40, and Myc-tagged b-TrCP1 were constructed using PCR- generated fragments, which were cloned into pcDNA3.0 or pcDNA3.1 uitination and sequential degradation (12, 13). Although several eukaryotic expression vector (Invitrogen) with HA tag or Myc tag at the substrates of b-TrCP have been identified to be involved in regu- C terminus. Flag-tagged TIPE2 expression plasmid was provided by lation of immunity, it still remains unclear whether any of the other Dr. S. Liu (Shandong University, Jinan, China). HA-tagged TIPE2 plasmid candidates exist in the immune system. was constructed using a PCR-generated fragment, which was cloned into TNFAIP8-like 2 (TIPE2), is a member of the TNFAIP8 family. pcDNA3.0 eukaryotic expression vector (Invitrogen) with HA tag at the C terminus. The TIPE2 S3A mutation was generated using the Mut Ex- TIPE2 is preferentially expressed by myeloid and lymphoid cells press II Fast Mutagenesis Kit V2 (Vazyme, Nanjing, China). All constructs (14–16). We previously identified TIPE2 as a negative regulator of were confirmed by DNA sequencing. Expression plasmids for HA-tagged immunity and inflammation that maintains immune homoeostasis TBK1, TAK1, IKKa, and IKKb expression plasmids were gifts from (14).
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  • Mdm2-Mediated Ubiquitylation: P53 and Beyond

    Mdm2-Mediated Ubiquitylation: P53 and Beyond

    Cell Death and Differentiation (2010) 17, 93–102 & 2010 Macmillan Publishers Limited All rights reserved 1350-9047/10 $32.00 www.nature.com/cdd Review Mdm2-mediated ubiquitylation: p53 and beyond J-C Marine*,1 and G Lozano2 The really interesting genes (RING)-finger-containing oncoprotein, Mdm2, is a promising drug target for cancer therapy. A key Mdm2 function is to promote ubiquitylation and proteasomal-dependent degradation of the tumor suppressor protein p53. Recent reports provide novel important insights into Mdm2-mediated regulation of p53 and how the physical and functional interactions between these two proteins are regulated. Moreover, a p53-independent role of Mdm2 has recently been confirmed by genetic data. These advances and their potential implications for the development of new cancer therapeutic strategies form the focus of this review. Cell Death and Differentiation (2010) 17, 93–102; doi:10.1038/cdd.2009.68; published online 5 June 2009 Mdm2 is a key regulator of a variety of fundamental cellular has also emerged from recent genetic studies. These processes and a very promising drug target for cancer advances and their potential implications for the development therapy. It belongs to a large family of (really interesting of new cancer therapeutic strategies form the focus of this gene) RING-finger-containing proteins and, as most of its review. For a more detailed discussion of Mdm2 and its other members, Mdm2 functions mainly, if not exclusively, as various functions an interested reader should also consult an E3 ligase.1 It targets various substrates for mono- and/or references9–12. poly-ubiquitylation thereby regulating their activities; for instance by controlling their localization, and/or levels by The p53–Mdm2 Regulatory Feedback Loop proteasome-dependent degradation.
  • A Systems-Wide Screen Identifies Substrates of the SCF Ubiquitin Ligase

    A Systems-Wide Screen Identifies Substrates of the SCF Ubiquitin Ligase

    RESEARCH RESOURCE PROTEOMICS A systems-wide screen identifies substrates of the SCFbTrCP ubiquitin ligase Teck Yew Low,1,2 Mao Peng,1,2* Roberto Magliozzi,3* Shabaz Mohammed,1,2† Daniele Guardavaccaro,3 Albert J. R. Heck1,2‡ Cellular proteins are degraded by the ubiquitin-proteasome system (UPS) in a precise and timely fashion. Such precision is conferred by the high substrate specificity of ubiquitin ligases. Identification of substrates of ubiquitin ligases is crucial not only to unravel the molecular mechanisms by which the UPS controls protein degradation but also for drug discovery purposes because many established UPS substrates are implicated in disease. We developed a combined bioinformatics and affinity purification– mass spectrometry (AP-MS) workflow for the system-wide identification of substrates of SCFbTrCP,a member of the SCF family of ubiquitin ligases. These ubiquitin ligases are characterized by a multi- subunit architecture typically consisting of the invariable subunits Rbx1, Cul1, and Skp1, and one of 69 F-box proteins. The F-box protein of this member of the family is bTrCP. SCFbTrCP binds, through the WD40 b repeats of TrCP, to the DpSGXX(X)pS diphosphorylated motif in its substrates. We recovered 27 pre- Downloaded from viously reported SCFbTrCP substrates, of which 22 were verified by two independent statistical proto- cols, thereby confirming the reliability of this approach. In addition to known substrates, we identified 221 proteins that contained the DpSGXX(X)pS motif and also interacted specifically with the WD40 repeats of bTrCP. Thus, with SCFbTrCP, as the example, we showed that integration of structural infor- mation, AP-MS, and degron motif mining constitutes an effective method to screen for substrates of ubiquitin ligases.