The Scfβ-Trcp E3 Ubiquitin Ligase Regulates Immune Receptor

Home , FBXO5, Ubiquitin ligase

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). Germline deletion of TIPE2 results in fatal systemic in- Dr. P. Wang (Shandong University, Jinan, China). Expression plasmids for Myc-tagged TAK1 and TAB1 expression plasmids and NF-kB re- flammation and hypersensitivity to TLR and TCR stimulation porter plasmid were provided by Dr. W. Zhao (Shandong University, Jinan, (14). Mechanistically, TIPE2 controls innate immunity to bacteria China). Expression plasmids for HA-tagged Ub-related plasmids were and dsRNA virus through targeting Rac GTPases, and TIPE2 provided by B. Yang (Xinxiang Medical University, Xinxiang, China). Downloaded from links inflammation to cancer by targeting RalGDS, a guanine AP-1 reporter plasmid was purchased from Beyotime Biotechnology. nucleotide exchange factor for the small GTPases RalA and Cell culture and transfection RalB (17–20). Recently, we reported that TIPE2 functions as phosphatidylinositol4,5-bisphosphate [PtdIns(4,5)P2]–transfer Bone marrow–derived macrophages (BMDMs) were generated as de- protein, which controls leukocytes polarization and migration scribed (17, 29). The purity of BMDMs was more than 95% as determined by CD11b+ and F4/80+ flow cytometry staining. Thioglycollate-elicited (21). In addition, many inflammatory factors, including bacteria, peritoneal macrophages were prepared as described (22). THP-1 cells http://www.jimmunol.org/ TLR ligands and oxidized low-density lipoproteins, could signif- were obtained from American Type Culture Collection and were main- icantly downregulate TIPE2 expression (17, 19, 22). Furthermore, tained in RMPI 1640 supplemented with 10% (v/v) FBS (HyClone), downregulation of TIPE2 has also been observed in patients with HEPES (10 mM), penicillin (100 U/ml), and streptomycin (100 mg/ml) (Life Technologies). THP-1 cells were differentiated into macrophage-like systemic lupus erythematosus, hepatitis B, hepatitis C, diabetic cells by incubation in the presence of PMA (100 ng/ml) for 48 h, as de- nephropathy, childhood asthma, and human hepatocellular scribed previously (30). Mouse macrophage cell line Raw264.7 and human carcinoma (18, 23–27). However, how TIPE2 expression is embryonic kidney (HEK293T) cells were obtained from American Type regulated by inflammatory signals has not been investigated. Culture Collection. Primary macrophages, Raw264.7, and HEK293T cells In this study, we identify TIPE2 as a Ub substrate of b-TrCP. were maintained in complete DMEM supplement with 10% (v/v) FBS (Life Technologies), penicillin (100 U/ml), and streptomycin (100 mg/ml) by guest on September 27, 2021 TIPE2 ubiquitination and subsequent degradation are temporally (Life Technologies) at 37˚C under 5% CO . For transient transfection of b 2 regulated by the E3 Ub ligase SCF -TrCP in a TGF-b–activated plasmids into HEK293T cells, polyethyleneimine reagent (Polysciences) kinase 1 (TAK1)–dependent manner. It in turn promotes immune was used according to the manufacturer’s instructions. Plasmids or du- receptor responses to inflammatory factors. plexes of small interfering RNA (siRNA) were transfected into BMDMs or Raw264.7 cells with jetPRIME reagent (Polyplus Transfection) according to the manufacturer’s protocol, as described previously (31). Target se- Materials and Methods quences for transient silencing were 59-GCUUUACCGCGUGUCCAAA- Mice 39 (siRNA 1), 59-CCAUGACCGCACUUAGCUU-39 (siRNA 2), and 59-CCAAGUCACAUGACCGCAU-39 (siRNA 3) for TIPE2, and the The C57BL/6J background wild-type (WT) mice were obtained from Vital “scrambled” control sequence was 59-UUCUCCGAACGUGUCACGU-39, River Laboratory Animal Technology (Beijing, China). All mice used were which were obtained from GenePharma (Shanghai, China). Cul1 siRNA 8- to 12-wk-old and were maintained under pathogen-free conditions in the and b-TrCP1 siRNA, a pool of three target-specific siRNAs with respective Xinxiang Medical University Animal Care Facilities. All animal procedures control siRNA, were obtained from Santa Cruz Biotechnology. were undertaken in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals, with the approval by the Institutional Real-time RT-PCR Animal Care and Use Committee of the Xinxiang Medical University. Total RNA was extracted from cells with TRIzol reagent (Invitrogen). Five Reagents and Abs hundred nanograms of total RNA were reversely transcribed using RT LPS (Escherichia coli O111:B4), poly(I:C), and (5Z)-7-oxozeaenol were Master Mix (TakaraBio). Quantitative real-time PCR (qPCR) was carried from Sigma-Aldrich. Pam3CSK4 was purchased from InvivoGen. MG-132 out in an Applied Biosystems 7500 System with TB Green Premix Ex Taq II was from Merck-Calbiochem. Staurosporine was obtained from Abcam. (TakaraBio), as previously described (32). Specific primers used for PCR L Phosphatase (l–phosphatase) was obtained from Santa Cruz Biotech- assays were 59-GCCTTCTTGGGACTGATGCT-39 and 59-CTGCAAG- nology. PMA was obtained from Beyotime Biotechnology. Anti–p-IkBa TGCATCATCGTTGT-39 for mIL-6, 59-TCAGAAACATCCAAGGCCA- (9246), anti–p-p38 (9215), anti–p-JNK (9251), anti–p-ERK (4377), anti–p- GAC-39 and 59-CGGACCGACCAGCCATTTTAC-39 for mTIPE2, and IKKa/b (Ser176/180, 2697), IkBa (4814), and IKKa (11930) were from 59-CCACACCCGCCACCAGTTCG-39 and 59-TACAGCCCGGGGAGC- ATCGT-39 for mb-actin. The relative changes in gene expression were Cell Signaling Technology. Anti-p38 (A11340), anti-JNK1 (A0288), anti- 2DDct ERK (A10613) and anti-Myc (AE010) were from Abclonal Technology. analyzed by the 2 method, and a melting-curve analysis was per- Anti-Ub (sc-8017) and protein A/G agarose (sc-2003) that were used formed to ensure the specificity of the products. Each sample was run in for immunoprecipitation were from Santa Cruz Biotechnology. Anti-Flag triplicate. The relative changes in gene expression were calculated using (F3165) was from Sigma-Aldrich. Anti-HA (901513, previously Covance b-actin as the loading control. The specific primers used were synthesized catalog no. MMS-101R) was from BioLegend. Anti-TIPE2 (15940-1-AP), by GENEWIZ. anti–b-actin (66009-1-Ig) and the respective HRP-conjugated secondary Abs were from Proteintech. Immunoprecipitation and Western blot analysis Sequences and plasmid constructs For immunoprecipitation, whole-cell extracts were collected and lysed with cell lysis buffer containing 1% (v/v) Nonidet P-40, 50 mM Tris-HCL Human b-TrCP2 ORF (NM_033644) was obtained from Vigene Biosci- (pH 7.6), 50 mM EDTA, 150 mM NaCl, and protease inhibitor mixture ences and subcloned into the pcDNA3.1 eukaryotic expression vector tablets (Roche). The lysates were cleared by centrifugation for 15 min at The Journal of Immunology 3

14000 3 g and incubated with 0.5 mg of monoclonal anti-Flag or 0.5 mg ionomycin plus PMA (Supplemental Fig. 1). To identify which of monoclonal anti-HA together with protein A/G plus-agarose immuno- pathway was involved in this process, BMDMs were treated with precipitation reagent (Santa Cruz Biotechnology) at 4˚C overnight. After proteasomal inhibitor MG132 prior to LPS stimulation. We found incubation, the beads were washed three times with lysis buffer and boiled for 10 min in 30 mlof23 SDS sample buffer. For endogenous immuno- that pretreatment of BMDMs with vehicle control and subsequent precipitation experiments, the whole-cell lysates were incubated with in- stimulation with LPS for 4 h resulted in a slight reduction of dicated Abs, as described above. Protein concentrations in the extracts TIPE2 and a marked decrease at 6 h after stimulation (Fig. 1D). were determined with a bicinchoninic acid assay (Beyotime Biotechnol- MG132 had reversed the ability of LPS to suppress TIPE2 ex- ogy). For Western blot analysis, immunoprecipitates or whole-cell lysates were loaded and subjected to SDS-PAGE, transferred onto polyvinylidene pression at 4 and 6 h after stimulation (Fig. 1D, 1E). However, difluoride membrane (MilliporeSigma), and then blotted by the Amersham because prolonged MG132 treatment is toxic to primary cells, Imager 600RGB detection system (GE Healthcare), as described previ- the effect of MG132 on the decrease at 24 h noted before could ously (22). Some blots were chosen to be stripped of bound Abs and then not be examined. Nevertheless, it was obvious that the proteasome reincubated with other Abs to analyze their protein contents. did affect the amount of TIPE2 protein after LPS stimulation. Ubiquitination assays Proteasome is one of the two major routes for elimination of target proteins. We next assessed whether TIPE2 was modified by For analysis of the ubiquitination of TIPE2 in macrophages, cells were stimulated with LPS, and then whole-cell extracts were immunoprecipitated ubiquitination as a sorting signal for the target of proteasome. with anti-TIPE2 and analyzed by Western blot with anti-Ub. For analysis of Indeed, stimulation of MG132-pretreated BMDMs with LPS in- the ubiquitination of TIPE2 in 293T cells, 293T cells were transfected creased the polyubiquitinated forms of TIPE2 (Fig. 1F). Among with Flag-TIPE2, HA-Ub (WT), or HA-Ub mutants, and then whole-cell the Ub linkages, K48- and K63-linked polyubiquitin chains are the extracts were immunoprecipitated with the Flag-specific Ab and analyzed by Western blot with anti-HA. For analysis of b-TrCP1–mediated two most abundant polyubiquitin chains. In general, K48-linked Downloaded from ubiquitination and degradation of TIPE2 in 293T cells, 293T cells Ub chains are the most prevalent proteasome-targeting signal, were transfected with Myc-TAB1, HA-Ub (WT), Flag-TIPE2 or its mu- whereas K63-linked chains regulate “proteasome-independent” tant Myc-TAK1, and Myc–b-TrCP1, and then whole-cell extracts were processes such as protein kinases activation and endocytosis immunoprecipitated with the Flag-specific Ab and analyzed by Western (34). Ub mutants K48R and K63R, which contain a single lysine- blot with anti-HA. to-arginine mutation at positions 48 and 63, respectively, and

Luciferase assay knockout (KO) mutant, which contains arginine substitutions of http://www.jimmunol.org/ Raw264.7 cells were cotransfected with the mixture of NF-kB or AP-1 all of its lysine residues, were used to study the form of TIPE2 reporter plasmid, pRL-TK-Renilla-luciferase internal control plasmid, polyubiquitination. As shown in Fig. 1G, overexpressed TIPE2 and different amounts of TIPE2 expression plasmid with jetPRIME was not conjugated to the Ub mutants K48R and KO, but was transfection reagent (Polyplus Transfection). Total amounts of DNA were robustly conjugated to the Ub mutants WT, K63R, and K48 in maintained constantly by supplementing with pcDNA3.1 empty vector. Twenty-four hours after transfection, cells were stimulated with LPS which contains arginine substitutions of all of its lysine residues for 6 h and luciferase activities were measured with a Dual-Luciferase except the one at position 48. These results indicate that TIPE2 is Reporter Assay System (Promega), according to the manufacturer’s in- degraded in a Ub K48-linked, proteasome-dependent pathway. structions. Data were acquired by GloMax EXPLORER (Promega) and

were normalized for transfection efficiency by dividing firefly luciferase TIPE2 interacts with b-TrCP through the WD-40 domain by guest on September 27, 2021 activity with that of Renilla luciferase. To investigate how TIPE2 protein is regulated in activation-induced Statistical analysis signaling events, we attempted to identify TIPE2-interacting All quantitative data are presented as mean 6 SEM of two or three ex- proteins by performing large-scale coimmunoprecipitation and periments. A two-tailed Student t test was used to determine the signi- mass spectrometry screenings using Raw264.7 cells (17, 18). ficance of the data between experimental groups, with a p value ,0.05 Among the peptides identified, b-TrCP1 attracts our attention. considered statistically significant. All statistical analyses were performed b-TrCP1, one of the important F-box proteins, serves as the with the Prism 5.0 for Windows (GraphPad Software). subunit recruiting substrates of SCF protein type of E3 Ub ligase complex. The interaction between TIPE2 and b-TrCP1 observed Results by mass spectrometry suggests that SCFb-TrCP may be the Ub li- TIPE2 protein level is regulated in a gase targeting TIPE2 for degradation. We thus validated whether proteasome-dependent manner TIPE2 bound to the TrCP complex, and found that TIPE2 inter- We previously established that TIPE2 is an essential negative acted with both b-TrCP1 and b-TrCP2 when these proteins were regulator of inflammation and immunity (14). We and others overexpressed in transfected 293T cells (Fig. 2A, 2B). b-TrCP1 previously reported that many inflammatory factors could down- and b-TrCP2 share identical biochemical properties and sub- regulate TIPE2 expression at the levels of both transcription and strates, so b-TrCP1 was used for the following studies unless translation (33). In addition, upon treatment with protein synthesis specified otherwise. b-TrCP1, a member of the FBXW subfamily inhibitor cycloheximide (CHX), TIPE2 protein half-life is de- (contains WD-40 substrate binding domains), contains a Tr do- creased in LPS-stimulated macrophages (18), indicating that the main (D domain of b-TrCP1), an F-box domain, and a C-terminal posttranslational modification may exist on the TIPE2 expression WD-40 domain (Fig. 2C). To search for the domain of b-TrCP1 regulation. To further investigate the mechanisms, we first ex- that was responsible for the interaction with TIPE2, we con- amined its expression in murine BMDMs, peritoneal macrophages structed deletion mutants and found that the WD-40 repeat do- from WT mice, and PMA-differentiated human THP-1 cells be- main of b-TrCP1 was required for its interaction with TIPE2 fore and after stimulation with different TLR agonists. Upon (Fig. 2D). Endogenous interaction was also confirmed in macro- stimulation with Pam3CSK4 (TLR2 ligand), poly(I:C) (TLR3 li- phages stimulated with LPS for various time points (Fig. 2E), gand), and LPS (TLR4 ligand), the expression of TIPE2 protein suggesting that the interaction between b-TrCP1 and TIPE2 is the was significantly decreased by all of these ligands, although it signal dependent. To gain more insights into the nature of the appeared that PMA-differentiated THP-1 cell line was not more b-TrCP1–TIPE2 interaction, we used a b-TrCP1 mutant (R474A) sensitive than primary cells (Fig. 1A–C). The effect of TIPE2 harboring a point mutation in the WD-40 b–propeller structure of decrease was not limited to macrophages because its expression b-TrCP1 that interacts with the substrate destruction motif (35). was also significantly decreased in Jurkat T cell responses to Whereas WT b-TrCP1 immunoprecipitated TIPE2, the interaction 4 TIPE2 IS A NOVEL SUBSTRATE OF THE SCFb-TrCP COMPLEX Downloaded from http://www.jimmunol.org/

FIGURE 1. TIPE2 degradation is mediated through the Ub-proteasome pathway. (A–C) BMDMs (A), peritoneal macrophages (B) from WT mice, and PMA-differentiated THP-1 cells (C) were treated with or without Pam3CSK4 (100 ng/ml), poly(I:C) (10 mg/ml), or LPS (500 ng/ml) for the indicated times. Western blot analysis of TIPE2 protein expression levels. (D and E) Western blot analysis of TIPE2 in BMDMs pretreated with MG132 (10 mM) or DMSO control and then treated for the indicated times with LPS (500 ng/ml) (D). Graphs below the Western blot show the relative abundances of TIPE2 over time by guest on September 27, 2021 (E). (F) BMDMs were treated as in (D) and then stimulated with LPS (500 ng/ml) for the indicated times. Cell lysates were subjected to immunoprecipitation (IP) with Ab against TIPE2 and analyzed by Western blot for the indicated proteins. (G) HEK293T cells were transfected with TIPE2-Flag and HA-tagged Ub expression vectors encoding either WT, Lys48 → Arg48 (K48R) mutant, Lys63 → Arg63 (K63R) mutant, lysine-less (KO) mutant, or K48-only Ub mutant (K48). Cell lysates were subjected to immunoprecipitation with Flag Ab and analyzed by Western blot with HA Ab to detect Ub. b-actin was used as a loading control. Data are representative of three experiments [mean 6 SEM in (E)]. Except for (A)–(C), which are representative of two experiments, similar results were obtained from three independent experiments (D, F, and G). of b-TrCP1 (R474A) mutant with TIPE2 was significantly reduced polyubiquitination. We pretreated BMDMs with staurosporine or (Fig. 2F). It is well characterized that proper phosphorylation of left them untreated and stimulated the cells with LPS, and then substrates by certain kinases is required for SCFb-TrCP-mediated examined LPS-induced polyubiquitination of TIPE2. Indeed, the ubiquitination and degradation (36). In keeping with this notion, protein kinase inhibitor staurosporine substantially suppressed we found that the interaction between b-TrCP1 and TIPE2 was the LPS-induced polyubiquitination of TIPE2 (Fig. 3C). Taken markedly abolished by the phosphatase treatment (Fig. 2G). All together, these data demonstrated that LPS stimulation resulted together, these data indicate that b-TrCP1 interacts with TIPE2 in phosphorylation-dependent polyubiquitination and degradation through the WD-40 domain and TIPE2 is likely to be the target of of TIPE2 in macrophages. the SCFb-TrCP complex. b-TrCP is responsible for TIPE2 degradation LPS-induced TIPE2 degradation is phosphorylation dependent The SCF E3 ligase complex recognizes and binds specific sub- We observed that phosphatase treatment significantly reduced the strates. Among them, Cul1 is an essential adaptor for the assembly interaction between b-TrCP1 and TIPE2 (Fig. 2G), supporting a of the SCFb-TrCP complex, and F-box protein recognizes substrates phosphorylation-dependent degradation of TIPE2 protein. To de- within the SCFb-TrCP complex (36). Having shown that TIPE2 was termine whether phosphorylation was required for TIPE2 degra- recruited to the b-TrCP complex upon LPS stimulation (Fig. 2E), dation after activation, we pretreated BMDMs with staurosporine we next determined whether TIPE2 was a target of this complex. or left them untreated and then stimulated the cells with LPS to We first tested the function of Cul1 in LPS-induced degradation induce TIPE2 degradation. As shown in Fig. 3A, 3B, the protein of TIPE2. siRNAs, which were a pool of three target-specific kinase inhibitor staurosporine significantly blocked LPS-induced siRNAs, were used to suppress endogenous Cul1 expression. degradation of TIPE2, indicating that phosphorylation of TIPE2 The Cul1-specific siRNA efficiently knocked down the protein was likely crucial for its degradation. Previously we found in BMDMs (Fig. 4A). These Cul1-knockdown cells were then that TIPE2 underwent K48-linked polyubiquitination (Fig. 1G). stimulated with LPS for different times, and cell lysates were We next asked if TIPE2 phosphorylation was also critical for its examined by Western blot. Notably, we found that silence of The Journal of Immunology 5 Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021 FIGURE 2. TIPE2 interacts with b-TrCP. (A and B) Lysates from 293T cells transiently cotransfected with HA-TIPE2 and Flag–b-TrCP1 (A) or Flag-b- TrCP2 (B) expression plasmids were immunoprecipitated (IP) with anti-Flag Ab or control IgG and subjected to SDS-PAGE and immunoblotting (IB). (C) Schematic diagram of b-TrCP1 WT and mutant constructs. b-TrCP1 WT contains a Tr domain (D domain of b-TrCP1), an F-box domain, and a C-terminal WD-40 domain. In mutant R474A, the arginine residue at position 474 within the WD-40 domain was substituted with alanine. (D) Lysates of 293T cells transiently cotransfected with HA-b-TrCP1 constructs and a Flag-TIPE2 construct were immunoprecipitated with anti-Flag and subjected to SDS-PAGE and IB. (E) BMDMs were left untreated or were treated with LPS (500 ng/ml) for the indicated times. Cell lysates were immunoprecipitated with anti- TIPE2 Ab or control IgG and subjected to SDS-PAGE and IB. (F) Lysates of 293T cells transiently cotransfected with HA-TIPE2 and Flag-tagged WT or R474 mutant b-TrCP1 constructs were immunoprecipitated with anti-Flag and subjected to SDS-PAGE and IB. Empty vector (EV) was used as a negative control. (G) Lysates of 293T cells transiently transfected with HA-TIPE2 and Flag-tagged b-TrCP1 constructs or Flag-tagged b-TrCP1 alone were immunoprecipitated with anti-Flag and subjected to SDS-PAGE and IB as indicated. Where indicated, cell lysates were pretreated with l-phosphatase before the immunoprecipitation procedure. Similar results were obtained from three independent experiments. endogenous Cul1 led to an elevation of TIPE2 protein levels IkBa was also significantly impaired in b-TrCP1–knockdown (Fig. 4B, 4C). One of the most important targets degraded by the cells (Supplemental Fig. 2B). All together, these results demon- SCFb-TrCP complex is the inhibitor of NF-kB(IkBa) protein (9). strate that TIPE2 protein stability is negatively controlled by the Many stimuli, including LPS, lead to the degradation of IkBa SCFb-TrCP E3 ligase complex. via the SCFb-TrCP complex to transduce downstream signals. Consistent with this result, upon stimulation of Cul1 knockdown TAK1 induces phosphorylation-dependent TIPE2 degradation macrophages with LPS, the degradation of IkBa was markedly Emerging evidences have demonstrated that proper phosphoryla- impaired compared with cell transfection with control siRNA tion of substrates by specific kinase(s) is critical for SCFb-TrCP to (Supplemental Fig. 2A). recognize its substrates for subsequent ubiquitination and degra- To further determine whether TIPE2 was a bona fide substrate dation (36). Consistent with a crucial role of substrate phosphor- of the SCFb-TrCP complex, we next assessed TIPE2 protein ylation for recognition by SCFb-TrCP, phosphatase treatment abundance in BMDMs after silencing endogenous b-TrCP1 with resulted in a significant reduction in interaction between b-TrCP1 siRNAs, which were also a pool of three target-specific siRNAs. and TIPE2 (Fig. 2G). To identify the upstream kinase(s) that could The abundance of b-TrCP1 was efficiently knocked down by be responsible for regulating TIPE2 phosphorylation, we then siRNAs (Fig. 4D). These knockdown cells were treated with LPS, screened a panel of kinases for their effects on the degradation of and then the degradation of TIPE2 was examined. After knock- TIPE2. We found that the IKK complex, which was essential for down of b-TrCP1, TIPE2 protein abundance was increased in phosphorylation and degradation of IkBa (including IKKa, response to LPS (Fig. 4E, 4F), suggesting that b-TrCP1 pri- IKKb, and Nemo [IKKg]), was not required for TIPE2 degrada- marily governs TIPE2 abundance. Similarly, the degradation of tion (Supplemental Fig. 3A). In addition, the IKK-related kinases 6 TIPE2 IS A NOVEL SUBSTRATE OF THE SCFb-TrCP COMPLEX

FIGURE 3. Requirement for phosphorylation of TIPE2 for activation-induced degradation. (A and B) BMDMs were left untreated or were pretreated with staurosporine and then stimulated with LPS (500 ng/ml) for the indicated times. Cell lysates were analyzed by Western blot for the indicated proteins (A). The quantiﬁcation of the amounts of TIPE2 detected by Western blot (B). (C) BMDMs were pretreated with MG132 (10 mM) for 2 h and then were left untreated or were pretreated with staurosporine and stimulated with LPS (500 ng/ml) for the indicated times. Lysates were immunoprecipitated with anti- TIPE2 and subjected to SDS-PAGE and IB. Data are representative of three experiments [mean 6 SEM in (B)]. Similar results were obtained from three A C independent experiments in ( ) and ( ). Downloaded from

TBK1 and IKKi were also not involved in degrading TIPE2 cotransfected TAB1 (Fig. 5A). In further support of a physiolog- (Supplemental Fig. 3B). Surprisingly, TAK1 (a crucial regulatory ical role of TAK1 in governing TIPE2 stability, inactivating TAK1 kinase of inﬂammatory and immune signals), but not any other with a selective TAK1 inhibitor, (5Z)-7-oxozeaenol, led to an el- kinases examined, could decrease TIPE2 protein levels in the evation of TIPE2 abundance after LPS stimulation (Fig. 5B, 5C).

presence of endogenous b-TrCP (Supplemental Fig. 3B). Indeed, The inhibitory effect of (5Z)-7-oxozeaenol was validated by http://www.jimmunol.org/ a previous study showed that TIPE2 interacted with TAK1 but not detecting the activation of downstream signaling kinases, because IRAK4, IRAK-M, and IKKb through binding with its internal the phosphorylation of IKKa/b was markedly inhibited by adding kinase domain (37), raising a possibility that TIPE2 may be a (5Z)-7-oxozeaenol (Supplemental Fig. 4A). These findings co- novel substrate of phosphorylation. herently support the notion that TAK1 is crucially involved in the We next sought to explore whether TAK1 was the upstream destruction of TIPE2 protein by SCFb-TrCP. kinase that phosphorylated TIPE2 and subsequently triggered its It is well established that the WD40 b–propeller structure of degradation by SCFb-TrCP. We first explored a transient transfec- b-TrCP binds its substrates through a diphosphorylated degradation model to see whether TAK1 overexpression could be able to tion motif (phosphodegron) within the consensus DpSGXX(X)pS, induce TIPE2 degradation. Indeed, the expression of TAK1, but EpSGXX(X)pS, or pSpSGXX(X)pS, all of which are canonical by guest on September 27, 2021 not its kinase-deficient mutant (K63A) (38), caused drastic loss of degron motifs recognized by b-TrCP (39, 40). Upon scanning the exogenously expressed TIPE2, which was blocked by the pro- protein sequences, we found putative evolutionarily conserved teasome inhibitor MG132 (Fig. 5A). However, it did not affect the noncanonical b-TrCP degron motif in the N-terminal region of

FIGURE 4. TIPE2 protein stability is controlled by the SCFb-TrCP1 E3 ligase complex. (A) Western blot analysis of Cul1 expression in BMDMs transfected with control siRNA or Cul1 siRNA for 36 h. (B and C) BMDMs were treated as in (A) and then stimulated with LPS (500 ng/ml) for the indicated times. Western blot analysis of TIPE2 protein expression levels (B). The quantiﬁcation of the amounts of TIPE2 detected by Western blot (C), as presented in (B). (D) Western blot analysis of b-TrCP1 expression in BMDMs transfected with control siRNA or b-TrCP1 siRNA for 36 h. (E and F) BMDMs were treated as in (D) and then stimulated with LPS (500 ng/ml) for the indicated times. Western blot analysis of TIPE2 protein expression levels (E). The quantiﬁcation of the amounts of TIPE2 detected by Western blot (F), as presented in (E). Data are representative of three experiments [mean 6 SEM in (C) and (F)]. Similar results were obtained from three independent experiments in (A), (B), (D), and (E). Ctrl, control. The Journal of Immunology 7 Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021

FIGURE 5. TAK1-mediated phosphorylation of TIPE2 at Ser3 triggers its association with b-TrCP1 for subsequent ubiquitination and degradation. (A) Lysates of 293T cells transiently cotransfected with HA-TIPE2, Flag-b-TrCP1, Myc-TAB1, and indicated kinases were subjected to SDS-PAGE and IB. Where indicated, cells were pretreated with MG132 (10 mM). (B and C) BMDMs were left untreated or were pretreated with (5Z)-7-oxozeaenol (oxozeaenol) (100 nM) for 30 min and then stimulated with LPS (500 ng/ml) for the indicated times. Cell lysates were analyzed by Western blot for the indicated proteins (B). The quantiﬁcation of the amounts of TIPE2 detected by Western blot (C), as presented in (B). (D) Alignment of the candidate phosphodegron sequence in TIPE2 from different species (top). Amino acid sequence of the TIPE2 mutant is shown (bottom). (E) Lysates of 293T cells transiently cotransfected with Flag–b-TrCP1 and HA-tagged WT or S3A mutant TIPE2 constructs were immunoprecipitated with anti-Flag and subjected to SDS-PAGE and IB. Empty vector (EV) was used as a negative control. (F) IB analysis of 293T cells transfected with Flag–b-TrCP1, Myc-TAB1 and HA-tagged WT or S3A mutant TIPE2 constructs, as indicated. Where indicated, cells were cotransfected with Myc-TAK1, or treated with the proteasome inhibitor MG132 (10 mM). (G) IB analysis of cell lysates and immunoprecipitation (IP) derived from 293T cells cotransfected with HA-Ub, Myc-b–TrCP1, Myc-TAB1, Myc-TAK1, and Flag-tagged WT or S3A mutant TIPE2 constructs as indicated. Data are representative of three experiments [mean 6 SEM in (C)]. Similar results were obtained from three independent experiments in (A), (B), and (D)–(G).

TIPE2, in which glycine residues (G) were replaced by phenyl- that ectopic expression of b-TrCP1 and TAK1 led to a rapid alanine (F). Importantly, this degron was highly conserved in disappearance of the WT, but not the S3A mutant, form of TIPE2 vertebrate orthologs of TIPE2 (Fig. 5D). To further determine that was deficient in associating with b-TrCP1 in vivo degradation whether this putative phosphodegron was important for TIPE2 assays (Fig. 5E, 5F). Moreover, the proteasome inhibitor MG132 degradation, we mutated the Ser3 residue to alanine (thereafter completely prevented the degradation of TIPE2, indicating the called the S3A mutant) (Fig. 5D). Notably, unlike the WT form of involvement of the 26S proteasome pathway in this process TIPE2, the S3A mutant could largely abolish the interaction with (Fig. 5F). In further keeping with this finding, mutating Ser3 to b-TrCP1 (Fig. 5E), indicating that b-TrCP1 interacted with TIPE2 alanine markedly abolished TAK1-dependent, b-TrCP1–mediated in a phosphodegron-dependent manner. In support of the critical polyubiquitination of TIPE2 in 293T cells (Fig. 5G), suggesting role of Ser3 in b-TrCP1–mediated destruction of TIPE2, we found that the inefficient destruction of S3A by SCFb-TrCP could be 8 TIPE2 IS A NOVEL SUBSTRATE OF THE SCFb-TrCP COMPLEX likely due to deficient ubiquitination of TIPE2. Taken together, abundance of TIPE2 correlated with the strength of LPS signaling, these results demonstrate that TAK1-induced TIPE2 phosphory- as was demonstrated by the activation kinetics of the MAPKs and lation at serine-3 plays an important role in regulating the fate the NF-kB pathways (Fig. 6A, 6B), and by the expression of of TIPE2. the gene encoding the proinflammatory cytokine Il6 (Fig. 6C). Furthermore, compared with cells expressing empty vector, cells TIPE2 downregulation sensitizes macrophages to expressing TIPE2 inhibited the LPS-induced responses in a dose- LPS-induced activation dependent manner, as shown by AP-1 and NF-kB luciferase as- To investigate the functional significance of TIPE2 degradation in says (Fig. 6D, 6E). These results suggest that the amount of TIPE2 the signaling pathway, synthesized interfering RNAs targeting in macrophages determines the strength of LPS-induced signaling mouse TIPE2 were used to inhibit endogenous TIPE2 expression. and gene expression. Taken together, we propose a model for We found that TIPE2 siRNA 1 and siRNA 2 had a higher efficiency the intrinsic mechanisms that control the TLR signaling pathway than siRNA 3 to suppress the expression of TIPE2, which mimics by TIPE2, as shown in Fig. 7. the selective degradation of TIPE2. TIPE2 was decreased to different amounts in BMDMs that were expressing the different Discussion siRNAs, as determined by qPCR (Supplemental Fig. 4B) and We previously observed that TIPE2 downregulation in the tumor Western blot (Supplemental Fig. 4C) analysis. TIPE2 knockdown occurred at the protein level but not in the Tipe2 gene transcript, macrophages were then stimulated with LPS. Significant increases and TIPE2 protein was heavily ubiquitinated in cells (18), indi- in the phosphorylation of JNK, p38, and IkBa were observed cating that TIPE2 is required to be maintained at precise level.

30–60 min after LPS stimulation in TIPE2-efficient knockdown However, the biological significance of ubiquitination and degra- Downloaded from cells when compared with those in cells transfected with siRNA 3 dation of TIPE2 and the identity of E3 Ub ligase involved are un- and control siRNA (Fig. 6A). Similar results were observed in the known. In this study, we report SCFb-TrCP serves as an E3 ligase phosphorylation of IKKa/b, the key upstream kinases of IkBa that controls TIPE2 destruction to govern macrophage activation. In (Fig. 6B). However, no significant differences in ERK phos- addition, we discovered TAK1 as the upstream kinase that phosphorylation between these groups were noted at any time points phorylates TIPE2 at Ser3 to trigger its ubiquitination and subsequent b-TrCP examined (Fig. 6A), consistent with a previous report in which degradation by SCF . In line with these findings, TIPE2 WT, http://www.jimmunol.org/ TIPE2 knockdown or KO did not affect ERK activation in but not TIPE2 S3A mutant, interacted with b-TrCP1. Furthermore, macrophages (14). With this siRNA approach, we found that the TIPE2 S3A mutant displayed resistant to TAK1/b-TrCP1–mediated by guest on September 27, 2021

FIGURE 6. The abundance of TIPE2 protein correlates with the strength of LPS signaling. (A) Western blot analysis of p-IkBa, p-JNK, p-p38, p-ERK, and total JNK1, p38, and ERK in BMDMs transfected with control siRNA or TIPE2 siRNA for 36 h and then stimulated with LPS (100 ng/ml) for the indicated times. (B) Western blot analysis of p-IKKa/b and total IKKa in BMDMs transfected with control siRNA or TIPE2 siRNA for 36 h and then stimulated with LPS (100 ng/ml) for the indicated times. (C) BMDMs were treated as in (A) and then stimulated with LPS for 4 h. qPCR analysis of Il6 mRNA in macrophages. (D and E) Raw264.7 cells were transfected with NF-kB or AP-1 reporter plasmid together with increasing amount of TIPE2 expression plasmids (TIPE2 no.1: 200 ng; TIPE2 no. 2: 400 ng) or a control plasmid, and luciferase activities were analyzed after treatment with LPS for 6 h. Data are representative of three experiments [mean 6 SEM of three samples in (C), *p , 0.05, **p , 0.01; mean 6 SEM of four samples in (D) and (E), *p , 0.05]. Similar results were obtained from three independent experiments in (A) and (B). The Journal of Immunology 9 Downloaded from

FIGURE 7. A proposed model for the intrinsic mechanisms that control the TLR signaling pathway. At steady state, endogenous TIPE2 interacts with TAK1 through binding to the region of the internal kinase domain of TAK1, resulting in interfering with the formation of the TAK1-TAB1-TAB2 complex and subsequently inhibiting activation of TAK1 and its downstream molecules. Upon TLR activation, TIPE2 is subjected to phosphorylation by TAK1, which is activated by K63 polyubiquitin chains through upstream regulator TRAF6, which leads to SCFb-TrCP-mediated ubiquitination and degradation. TIPE2 degradation may remove sequestration of TAK1-TAB1-TAB2 complex and trigger conformational changes in TAK1 that leads to autophosphorylation- induced activation of TAK1. Then TAK1 phosphorylates the IKK complex, which consists of IKKa, IKKb, and IKKg, ultimately resulting in activation of http://www.jimmunol.org/ the NF-kB and the MAPK signaling pathways. The coordination of the degradation of IkBa and TIPE2 by the same SCFb-TrCP complex provides an intrinsic regulatory mechanism to prevent persistent inflammatory responses. The graphs are adapted from QIAGEN with some modifications. degradation. Therefore, our results reveal a crucial role of Ser3 of NF-kB activity (45). However, in contrast to IkBa, we found phosphorylation in controlling TIPE2 stability. Because TIPE2 is that TIPE2 was continuously decreased when TLR ligands were an important negative regulator in immunity, inflammation, and present. The differential regulation of the inhibitory protein IkBa cancer, regulation of TAK1 and b-TrCP1 could govern these and the negative regulator TIPE2 may protect the host to efficiently processes via control of TIPE2 stability. More importantly, as control unnecessary inflammatory responses via both feedback by guest on September 27, 2021 TAK1 has been reported to be activated by various inflammatory regulation of IkBa and continuous degradation of TIPE2. There- signals (41, 42), our data provide an important molecular link fore, the molecular mechanism by which TIPE2 is degraded appears between inflammatory factor–induced kinase cascades and the to be similar to the one which affects the members of the IkB temporal destruction of TIPE2 (Fig. 7). family, with regard to the phosphorylation, ubiquitination, and It has been shown that miR-21 could regulate Tipe2 expression proteolysis, although the efficiency of phosphorylation, as well as in a Tipe2-coding region-dependent manner. NF-kB activation the kinetics of degradation, appears to be different. induces miR-21, and then miR-21 targets Tipe2 to mediate its We further demonstrated that LPS-induced degradation of TIPE2 mRNA degradation (43), suggesting that miR-21 governs Tipe2 was phosphorylation dependent, similar to the polyubiquitination abundance through a posttranscriptional mechanism. However, and degradation of other target proteins, such as b-catenin, upon treatment with protein synthesis inhibitor CHX, TIPE2 FBXO5, IkB, and DEP domain–containing mTOR-interacting protein half-life is decreased in LPS-stimulated macrophages (18), protein (DEPTOR) (7, 46, 47). The kinase inhibitor staur- indicating that the posttranslational modification could also exist osporine impaired LPS-induced polyubiquitination and degrada- on TIPE2 expression regulation. In line with this result, in the tion of TIPE2 (Fig. 3). However, the kinases that are involved in current study, we demonstrated that activation-induced downreg- TLR signaling, such as IKKa, IKKb, Nemo (IKKg), TBK1, and ulation of TIPE2 occurred through the proteasome-mediated IKKi, are not responsible for LPS-induced degradation of TIPE2. degradation pathway. The proteasomal inhibitor MG132 largely To our surprise, TAK1, an essential component in inflammatory blocked TIPE2 degradation. Although we cannot completely ex- signaling, is responsible for the phosphorylation and degradation clude the contribution of such a pathway under certain conditions of TIPE2. TAK1 is a multifunctional kinase that can be activated (i.e., NF-kB–miR-21 axis in the regulation of TIPE2), our data by a wide variety of proinflammatory mediators, such as TNF-a, clearly demonstrate that TIPE2 degradation is almost completely IL-1b, TGF-b, TLR ligands, TCR, and B cell receptor Ags (48). prevented by proteasome inhibitor in LPS-activated macrophages In line with TAK1 as the upstream kinase that phosphorylates (Fig. 1). We further found that stimulation of cells with LPS TIPE2, many inflammatory factors that can activate TAK1, in- resulted in Lys48-linked polyubiquitination of TIPE2, which cluding bacteria, TLR ligands, oxidized low-density lipoproteins, marked it for degradation. It is well known that the degradation of and anti-CD3 plus anti-CD28, could significantly downregulate IkBa is required for activation of the NF-kB signaling cascades, TIPE2 expression (17–19, 22, 43). Indeed, a previous study reported which enable the translocation of NF-kB to the nucleus where it that TIPE2 interacted with TAK1 through binding with its internal induces genes transcription (44). Interestingly, NF-kB activation kinase domain (37). Therefore our results support that TIPE2 is simultaneously induces expression of its own repressor, IkBa. The a novel substrate of TAK1 and TAK1 is a general regulator for newly synthesized IkBa then suppresses NF-kB and thus forms an TIPE2 degradation downstream of various inflammatory factor– auto-feedback regulatory loop, which results in oscillating levels induced signaling. 10 TIPE2 IS A NOVEL SUBSTRATE OF THE SCFb-TrCP COMPLEX

b-TrCP, a well-studied SCF E3 ligase subunit, regulates cell 5. Jin, W., M. Chang, and S. C. Sun. 2012. Peli: a family of signal-responsive E3 ubiquitin ligases mediating TLR signaling and T-cell tolerance. Cell. Mol. functions by mediating intracellular protein degradation. It has Immunol. 9: 113–122. been characterized that substrates of b-TrCP contain the consen- 6. Jiang, X., and Z. J. Chen. 2011. The role of ubiquitylation in immune defence sus DpSGXX(X)pS, EpSGXX(X)pS or pSpSGXX(X)pS, all of and pathogen evasion. Nat. Rev. Immunol. 12: 35–48. 7. Zheng, N., Z. Wang, and W. Wei. 2016. Ubiquitination-mediated degradation which are canonical degron motifs recognized by b-TrCP (39, 40). of cell cycle-related proteins by F-box proteins. Int. J. Biochem. Cell Biol. 73: The serine residues in this degron could be phosphorylated by 99–110. 8. Wang, D., L. Ma, B. Wang, J. Liu, and W. Wei. 2017. E3 ubiquitin ligases in certain protein kinases, which results in b-TrCP recognition and cancer and implications for therapies. Cancer Metastasis Rev. 36: 683–702. subsequent ubiquitination of the substrates. In this study, we 9. Spencer, E., J. Jiang, and Z. J. Chen. 1999. Signal-induced ubiquitination of found that TIPE2 does possess a potential EpSF(X)2+nS motif. IkappaBalpha by the F-box protein Slimb/beta-TrCP. Genes Dev. 13: 284–294. 10. Fong, A., and S. C. Sun. 2002. Genetic evidence for the essential role of beta- However, this evolutionarily conserved EpSF(X)2+nS motif in the transducin repeat-containing protein in the inducible processing of NF-kappa N-terminal region of TIPE2 is not a canonical motif for b-TrCP B2/p100. J. Biol. Chem. 277: 22111–22114. binding. As a matter of fact, some reports have demonstrated 11. Lang, V., J. Janzen, G. Z. Fischer, Y. Soneji, S. Beinke, A. Salmeron, H. Allen, R. T. Hay, Y. Ben-Neriah, and S. C. Ley. 2003. betaTrCP-mediated proteolysis several “atypical” substrates of b-TrCP, including CYLD, GHR, of NF-kappaB1 p105 requires phosphorylation of p105 serines 927 and 932. p53, and Mcl-1 (49–52), which do not harbor the classic motif. In Mol. Cell. Biol. 23: 402–413. 12. Li, J., Q. Y. Chai, and C. H. Liu. 2016. The ubiquitin system: a critical regulator this study, we uncover a novel “atypical” degron motif in TIPE2 of innate immunity and pathogen-host interactions. Cell. Mol. Immunol. 13: for b-TrCP recognition. Indeed, our data showed that TIPE2 560–576. interacted with b-TrCP1 in an LPS-dependent manner, whereas 13. Sun, S. C. 2017. The non-canonical NF-kB pathway in immunity and inflammation. Nat. Rev. Immunol. 17: 545–558. this interaction was impaired by pretreatment with l-phosphatase, 14. Sun, H., S. Gong, R. J. Carmody, A. Hilliard, L. Li, J. Sun, L. Kong, L. Xu, suggesting that the SCFb-TrCP complex was likely responsible for B. Hilliard, S. Hu, et al. 2008. TIPE2, a negative regulator of innate and adaptive Downloaded from the ubiquitination of TIPE2. We further confirmed this with immunity that maintains immune homeostasis. Cell 133: 415–426. 15. Zhang, G., C. Hao, Y. Lou, W. Xi, X. Wang, Y. Wang, Z. Qu, C. Guo, Y. Chen, siRNA-mediated knockdown of Cul1 or b-TrCP1. Although we Y. Zhang, and S. Liu. 2010. Tissue-specific expression of TIPE2 provides in- cannot exclude the possible involvement of other E3 Ub ligases, sights into its function. Mol. Immunol. 47: 2435–2442. b-TrCP 16. Zhang, L., Y. Shi, Y. Wang, F. Zhu, Q. Wang, C. Ma, Y. H. Chen, and L. Zhang. our data show that the SCF complex is responsible for the 2011. The unique expression profile of human TIPE2 suggests new functions ubiquitination of TIPE2. Importantly, we found that the amount of beyond its role in immune regulation. Mol. Immunol. 48: 1209–1215. 17. Wang, Z., S. Fayngerts, P. Wang, H. Sun, D. S. Johnson, Q. Ruan, W. Guo, TIPE2 in macrophages determined the strength of LPS-induced http://www.jimmunol.org/ and Y. H. Chen. 2012. TIPE2 protein serves as a negative regulator of signaling and gene expression. Downregulation of TIPE2 has been phagocytosis and oxidative burst during infection. Proc. Natl. Acad. Sci. observed in patients with systemic lupus erythematosus, hepatitis USA 109: 15413–15418. B, hepatitis C, diabetic nephropathy, childhood asthma, and hu- 18. Gus-Brautbar, Y., D. Johnson, L. Zhang, H. Sun, P. Wang, S. Zhang, L. Zhang, and Y. H. Chen. 2012. The anti-inflammatory TIPE2 is an inhibitor of the on- man hepatocellular carcinoma (18, 23–27), raising the possibility cogenic Ras. Mol. Cell 45: 610–618. that the expression level of TIPE2 may represent a threshold factor 19. Sun, H., G. Zhuang, L. Chai, Z. Wang, D. Johnson, Y. Ma, and Y. H. Chen. 2012. TIPE2 controls innate immunity to RNA by targeting the phosphatidylinositol for inflammatory diseases and cancer. Given all the evidences 3-kinase-Rac pathway. J. Immunol. 189: 2768–2773. obtained so far, our results suggest that TIPE2 more likely oper- 20. Lou, Y., H. Sun, S. Morrissey, T. Porturas, S. Liu, X. Hua, and Y. H. Chen. 2014. ates at an early “time window” of immune responses and the Critical roles of TIPE2 protein in murine experimental colitis. J. Immunol. 193: 1064–1070. by guest on September 27, 2021 decrease of TIPE2 protein may play an important role in the 21. Fayngerts, S. A., Z. Wang, A. Zamani, H. Sun, A. E. Boggs, T. P. Porturas, pathogenesis of these diseases. W. Xie, M. Lin, T. Cathopoulis, J. R. Goldsmith, et al. 2017. Direction of leu- In summary, our results provide mechanistic insights into a kocyte polarization and migration by the phosphoinositide-transfer protein TIPE2. Nat. Immunol. 18: 1353–1360. degradation pathway for the frequent reduction in the expression 22. Lou, Y., S. Liu, C. Zhang, G. Zhang, J. Li, M. Ni, G. An, M. Dong, X. Liu, of TIPE2 protein in various types of human immunological F. Zhu, et al. 2013. Enhanced atherosclerosis in TIPE2-deficient mice is associated with increased macrophage responses to oxidized low-density lipoprotein. disorders and cancer. The basal level of TIPE2 may represent a J. Immunol. 191: 4849–4857. threshold for maintaining a non-/anti-inflammatory environment. 23. Ma, Y., X. Liu, Z. Wei, X. Wang, Z. Wang, W. Zhong, Y. Li, F. Zhu, C. Guo, Our work further suggests that restoring TIPE2 expression may L. Zhang, and X. Wang. 2013. The expression and significance of TIPE2 in peripheral blood mononuclear cells from asthmatic children. Scand. J. Immunol. inhibit inflammation. This finding advances our understanding of 78: 523–528. the regulatory mechanisms of inflammation-associated diseases, 24. Kong, L., K. Liu, Y. Z. Zhang, M. Jin, B. R. Wu, W. Z. Wang, W. Li, Y. M. Nan, and modulation of TIPE2 expression may be beneficial in treating and Y. H. Chen. 2013. Downregulation of TIPE2 mRNA expression in peripheral blood mononuclear cells from patients with chronic hepatitis C. Hepatol. Int. 7: patients with autoimmunity, chronic inflammation, or infectious 844–849. diseases. 25. Xi, W., Y. Hu, Y. Liu, J. Zhang, L. Wang, Y. Lou, Z. Qu, J. Cui, G. Zhang, X. Liang, et al. 2011. Roles of TIPE2 in hepatitis B virus-induced hepatic inflammation in humans and mice. Mol. Immunol. 48: 1203–1208. Acknowledgments 26. Li, D., L. Song, Y. Fan, X. Li, Y. Li, J. Chen, F. Zhu, C. Guo, Y. Shi, and We thank Wang Laboratory members for reagents and/or valuable advice. L. Zhang. 2009. Down-regulation of TIPE2 mRNA expression in peripheral blood mononuclear cells from patients with systemic lupus erythematosus. Clin. We also thank Drs. Suxia Liu, Wei Zhao, Peihui Wang, and Bo Yang for Immunol. 133: 422–427. providing plasmids. 27. Zhang, S., Y. Zhang, X. Wei, J. Zhen, Z. Wang, M. Li, W. Miao, H. Ding, P. Du, W. Zhang, et al. 2010. Expression and regulation of a novel identified TNFAIP8 family is associated with diabetic nephropathy. Biochim. Biophys. Acta 1802: Disclosures 1078–1086. The authors have no financial conflicts of interest. 28. Zhong, J., S. Shaik, L. Wan, A. E. Tron, Z. Wang, L. Sun, H. Inuzuka, and W. Wei. 2013. SCF b-TRCP targets MTSS1 for ubiquitination-mediated destruction to regulate cancer cell proliferation and migration. Oncotarget 4: 2339–2353. References 29. Lou, Y., M. Han, H. Liu, Y. Niu, Y. Liang, J. Guo, W. Zhang, and H. Wang. 2019. 1. Hu, H., and S. C. Sun. 2016. Ubiquitin signaling in immune responses. Cell Res. Essential roles of S100A10 in Toll-like receptor signaling and immunity to in- 26: 457–483. fection. Cell. Mol. Immunol. DOI: 10.1038/s41423-019-0278-1. 2. Zheng, N., Q. Zhou, Z. Wang, and W. Wei. 2016. Recent advances in SCF 30. Starr, T., T. J. Bauler, P. Malik-Kale, and O. Steele-Mortimer. 2018. The ubiquitin ligase complex: clinical implications. Biochim. Biophys. Acta 1866: phorbol 12-myristate-13-acetate differentiation protocol is critical to the in- 12–22. teraction of THP-1 macrophages with Salmonella Typhimurium. PLoS One 3. Bhattacharyya, S., H. Yu, C. Mim, and A. Matouschek. 2014. Regulated protein 13: e0193601. turnover: snapshots of the proteasome in action. Nat. Rev. Mol. Cell Biol. 15: 31. Song, H., B. Liu, W. Huai, Z. Yu, W. Wang, J. Zhao, L. Han, G. Jiang, L. Zhang, 122–133. C. Gao, and W. Zhao. 2016. The E3 ubiquitin ligase TRIM31 attenuates NLRP3 4. Skaar, J. R., J. K. Pagan, and M. Pagano. 2014. SCF ubiquitin ligase-targeted inflammasome activation by promoting proteasomal degradation of NLRP3. Nat. therapies. Nat. Rev. Drug Discov. 13: 889–903. Commun. 7: 13727. The Journal of Immunology 11

32. Wang, J., L. Kang, D. Song, L. Liu, S. Yang, L. Ma, Z. Guo, H. Ding, H. Wang, 43. Ruan, Q., P. Wang, T. Wang, J. Qi, M. Wei, S. Wang, T. Fan, D. Johnson, and B. Yang. 2017. Ku70 senses HTLV-1 DNA and modulates HTLV-1 repli- X. Wan, W. Shi, et al. 2014. MicroRNA-21 regulates T-cell apoptosis by directly cation. J. Immunol. 199: 2475–2482. targeting the tumor suppressor gene Tipe2. Cell Death Dis. 5: e1095. 33. Goldsmith, J. R., and Y. H. Chen. 2017. Regulation of inflammation and tu- 44. Hayden, M. S., and S. Ghosh. 2011. NF-kB in immunobiology. Cell Res. 21: morigenesis by the TIPE family of phospholipid transfer proteins. Cell. Mol. 223–244. Immunol. 14: 1026. 45. Nelson, D. E., A. E. Ihekwaba, M. Elliott, J. R. Johnson, C. A. Gibney, 34. Ohtake, F., H. Tsuchiya, Y. Saeki, and K. Tanaka. 2018. K63 ubiquitylation B. E. Foreman, G. Nelson, V. See, C. A. Horton, D. G. Spiller, et al. 2004. triggers proteasomal degradation by seeding branched ubiquitin chains. Proc. Oscillations in NF-kappaB signaling control the dynamics of gene expression. Natl. Acad. Sci. USA 115: E1401–E1408. Science 306: 704–708. 35. Wu, G., G. Xu, B. A. Schulman, P. D. Jeffrey, J. W. Harper, and N. P. Pavletich. 46. Guardavaccaro, D., Y. Kudo, J. Boulaire, M. Barchi, L. Busino, M. Donzelli, 2003. Structure of a beta-TrCP1-Skp1-beta-catenin complex: destruction motif F. Margottin-Goguet, P. K. Jackson, L. Yamasaki, and M. Pagano. 2003. Control binding and lysine specificity of the SCF(beta-TrCP1) ubiquitin ligase. Mol. Cell of meiotic and mitotic progression by the F box protein beta-Trcp1 in vivo. Dev. 11: 1445–1456. Cell 4: 799–812. 36. Wang, Z., P. Liu, H. Inuzuka, and W. Wei. 2014. Roles of F-box proteins in 47. Gao, D., H. Inuzuka, M. K. Tan, H. Fukushima, J. W. Locasale, P. Liu, L. Wan, cancer. Nat. Rev. Cancer 14: 233–247. B. Zhai, Y. R. Chin, S. Shaik, et al. 2011. mTOR drives its own activation via 37. Oho, M., R. Nakano, R. Nakayama, W. Sakurai, A. Miyamoto, Y. Masuhiro, and SCF(bTrCP)-dependent degradation of the mTOR inhibitor DEPTOR. Mol. Cell S. Hanazawa. 2016. TIPE2 (tumor necrosis factor a-induced protein 8-like 2) is 44: 290–303. a novel negative regulator of TAK1 signal. J. Biol. Chem. 291: 22650–22660. 48. Hirata, Y., M. Takahashi, T. Morishita, T. Noguchi, and A. Matsuzawa. 2017. 38. Blonska, M., P. B. Shambharkar, M. Kobayashi, D. Zhang, H. Sakurai, B. Su, Post-translational modifications of the TAK1-TAB complex. Int. J. Mol. Sci. and X. Lin. 2005. TAK1 is recruited to the tumor necrosis factor-alpha (TNF- 18: E205. alpha) receptor 1 complex in a receptor-interacting protein (RIP)-dependent 49.Xia,Y.,R.C.Padre,T.H.DeMendoza, V. Bottero, V. B. Tergaonkar, and manner and cooperates with MEKK3 leading to NF-kappaB activation. J. Biol. I. M. Verma. 2009. Phosphorylation of p53 by IkappaB kinase 2 pro- Chem. 280: 43056–43063. motes its degradation by beta-TrCP. Proc. Natl. Acad. Sci. USA 106: 39. Shimizu, K., H. Fukushima, K. Ogura, E. C. Lien, N. T. Nihira, J. Zhang, 2629–2634. B. J. North, A. Guo, K. Nagashima, T. Nakagawa, et al. 2017. The SCFb-TRCP 50. Ding, Q., X. He, J. M. Hsu, W. Xia, C. T. Chen, L. Y. Li, D. F. Lee, J. C. Liu, E3 ubiquitin ligase complex targets Lipin1 for ubiquitination and degradation to Q. Zhong, X. Wang, and M. C. Hung. 2007. Degradation of Mcl-1 by beta-TrCP Downloaded from promote hepatic lipogenesis. Sci. Signal. 10: eaah4117. mediates glycogen synthase kinase 3-induced tumor suppression and chemo- 40. Ci, Y., X. Li, M. Chen, J. Zhong, B. J. North, H. Inuzuka, X. He, Y. Li, J. Guo, sensitization. Mol. Cell. Biol. 27: 4006–4017. and X. Dai. 2018. SCFb-TRCP E3 ubiquitin ligase targets the tumor suppressor 51. Wu, X., H. Fukushima, B. J. North, Y. Nagaoka, K. Nagashima, F. Deng, ZNRF3 for ubiquitination and degradation. Protein Cell 9: 879–889. K. Okabe, H. Inuzuka, and W. Wei. 2014. SCFb-TRCP regulates osteoclasto- 41. Ajibade, A. A., H. Y. Wang, and R. F. Wang. 2013. Cell type-specific function of genesis via promoting CYLD ubiquitination. Oncotarget 5: 4211–4221. TAK1 in innate immune signaling. Trends Immunol. 34: 307–316. 52. van Kerkhof, P., J. Putters, and G. J. Strous. 2007. The ubiquitin ligase 42. Dai, L., C. Aye Thu, X. Y. Liu, J. Xi, and P. C. Cheung. 2012. TAK1, more than SCF(betaTrCP) regulates the degradation of the growth hormone receptor.

just innate immunity. IUBMB Life 64: 825–834. J. Biol. Chem. 282: 20475–20483. http://www.jimmunol.org/ by guest on September 27, 2021

Supplementary Materials

Fig. S1 TIPE2 expression is decreased upon activation in Jurkat T cells. Jurkat cells were treated with or without ionomycin (1 μM) plus PMA (50 ng/ml) for the indicated times. Western blot analysis of TIPE2 protein expression levels. Similar results were obtained from two independent experiments.

Fig. S2 Cul1 or β-TrCP1 Knockdown significantly inhibits degradation of IκBα. (A) Bone marrow-derived macrophages were transfected with control siRNA or Cul1 siRNA for 36 h and then stimulated with LPS (500 ng/ml) for the indicated times. Western blot analysis of IκBα degradation. (B) Bone marrow-derived macrophages were transfected with control siRNA or β-TrCP1 siRNA for 36 h and then stimulated with LPS (500 ng/ml) for the indicated times. Western blot analysis of IκBα degradation. Similar results were obtained from three independent experiments in panels A and B.

Fig. S3 The specific kinase TAK1 is involved in the degradation of TIPE2. (A-B) HEK 293T cells were transiently transfected with Flag-TIPE2 expression plasmid and increasing amount of HA-tagged kinases expression plasmids as indicated, TIPE2 expression changes were analyzed by Western blot. Similar results were obtained from three independent experiments in panels A and B.

Fig. S4 The effect of TAK1 inhibitor (5Z)-7-oxozeaenol (oxozeaenol) on the LPS-induced phosphorylation of IKKα/β and TIPE2 knockdown efficiency confirmation. (A) Bone marrow-derived macrophages were left untreated or were pre-treated with (5Z)-7-oxozeaenol (oxozeaenol) (100 nM) for 30 min and then stimulated with LPS (100 ng/ml) for the indicated times. Cell lysates were analyzed by Western blot for the indicated proteins. Similar results were obtained from two independent experiments. (B-C) Bone marrow-derived macrophages were transfected with control siRNA or TIPE2 siRNA for 36 h. Quantitative real-time PCR analysis of Tipe2 mRNA in macrophages (B). Western blot analysis of TIPE2 protein expression levels in macrophages (C). Similar results were obtained from three independent experiments.