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Deubiquitinase USP33 is negatively regulated by β-TrCP through ubiquitin-dependent proteolysis
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Deubiquitinase USP33 is negatively regulated by β-TrCP through ubiquitindependent proteolysis Qiao Chenga,b,1, Yukang Yuana,b,1, Lemin Lia,b,1, Tingting Guoa,b, Ying Miaoa,b, Ying Rena,b, Jin Liua,b, Qian Fenga,b, Xiaofang Wanga,b, Peng Zhaoa,b, Yibo Zuoa,b, Liping Qiana,b, ⁎ Liting Zhanga,b, Hui Zhenga,b, a b
Institutes of Biology and Medical Sciences, Soochow University, Suzhou, Jiangsu Province 215123, China Jiangsu Key Laboratory of Infection and Immunity, Soochow University, Suzhou, Jiangsu Province 215123, China
A R T I C L E I N F O
A BS T RAC T
Keywords: Protein ubiquitination E3 ligase β-TrCP USP33
Ubiquitin-mediated proteolysis regulates cellular levels of various proteins, and therefore plays important roles in controlling cell signaling and disease progression. The Skp1-Cul1-F-box ubiquitin ligase β-TrCP is recognized as an important negative regulator for numerous key signaling proteins. Recently, the deubiquitinases (DUBs) have turned out to be essential to regulate signaling pathways related to human diseases. However, whether βTrCP is able to regulate the deubiquitinase family members remains largely unexplored. Here, we found that βTrCP downregulated cellular levels of endogenous USP33. We also revealed that β-TrCP interacted with USP33 independently of the classic binding motif for β-TrCP, and mediated USP33 degradation via the ubiquitin proteasome pathway. Furthermore, we found that the WD40 motif of β-TrCP and 201–400 amino acid motif of USP33 are required for the interaction between β-TrCP and USP33. Consequently, β-TrCP attenuated USP33mediated inhibition of cell proliferation and cell invasion. Taken together, our study clarified that the E3 ligase β-TrCP regulates cellular USP33 levels by the ubiquitin-proteasomal proteolysis.
1. Introduction
tinases (DUBs). So far, approximately 100 human DUBs have been identified, and are subdivided into five families: ubiquitin-specific protease (USPs), ubiquitin C-terminal hydrolases (UCHs), ovarian tumor proteases (OTUs), Josephin domain family (MJD) and JAB1/ MPN/Mov34 metalloenzymes (JAMMs) [12,13]. Although some members of these DUBs have been studied, the functions and detailed regulatory mechanisms of many DUBs members are still unclear. Among these DUBs families, the USPs family is the biggest one and comprises of over fifty members. So far, the regulation of the USPs family members mediated by β-TrCP remains largely unexplored. By analysis of amino acid sequences of 56 USPs family members, we found that USP33 contains a typical binding motif for β-TrCP. Thus, we sought to determine the role of β-TrCP in USP33 regulation. USP33 is associated with several important biological functions. It has been reported that USP33 is a previously unknown tumorsuppressing gene for colorectal cancers by deubiquitinating and stabilizing Robo1 [14]. Similarly, USP33 is necessary for Slit signaling in breast cancer cells [15]. In addition, USP33 plays an essential role in centrosome amplification by deubiquitinating the centriolar protein CP110 [16,17]. It has also been demonstrated that USP33 deubiqui-
Ubiquitin-mediated proteolysis plays crucial roles in regulating many biological processes including cell proliferation, apoptosis, and immune responses [1]. The conjugation of ubiquitin is catalyzed by three types of enzymes: ubiquitin-activating enzyme (E1), ubiquitinconjugating enzyme (E2) and ubiquitin ligases (E3). The ubiquitin E3 ligases are determiners for the substrate specificity, and have been extensively studied during the past decades. β-TrCP is one of these E3 ubiquitin ligases, which has been demonstrated to be an important regulator for many key signaling proteins, including IκB [2,3], βCatenin [3,4], IFNAR1 [5], ATF4 [6], and Emil [7]. All of these protein substrates contain the conserved DSG(XX)2+nS motif, which is a “typical” binding motif for β-TrCP. However, recent studies reported several “atypical” substrates of β-TrCP including STAT1 [8], Wee1 [9], GHR [10], ELAVL1/huR [11], CDC25B [11], and TP53 [11], which do not contain the classic DSG(XX)2+nS motif. Deubiquitination, as a reverse process of ubiquitination, has recently been shown to be essential for controlling disease-related signaling pathways. Protein deubiquitination is mediated by deubiqui-
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Corresponding author at: Institutes of Biology and Medical Sciences, Soochow University, Suzhou, Jiangsu Province 215123, China. E-mail address: [email protected] (H. Zheng). These authors contributed equally to this study.
http://dx.doi.org/10.1016/j.yexcr.2017.05.011 Received 10 January 2017; Received in revised form 3 May 2017; Accepted 12 May 2017 0014-4827/ © 2017 Elsevier Inc. All rights reserved.
Please cite this article as: Cheng, Q., Experimental Cell Research (2017), http://dx.doi.org/10.1016/j.yexcr.2017.05.011
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Fig. 1. β-TrCP downregulates USP33 protein levels. (A) DSG(X)2+nS motif is evolutionarily conserved from Zebrafish to Homo sapiens. (B) Immunoblots were used to detect the level of HA-β-TrCP and endogenous USP33 in HEK293T cells that were transfected with either an empty vector or increasing doses of β-TrCP. Probing with β-actin served as loading control. (C) HEK293T cells were transfected with or without shβ-TrCP, the levels of β-actin, endogenous USP33 and β-TrCP were measured by immunoblotting with indicated antibodies. (D) Bar diagram showed the mRNA level of endogenous USP33 as measured by qRT-PCR in HEK293T cells transfected with empty vector or increasing doses of β-TrCP.
HA-β-TrCP. HA-Ub was a gift from Dr. Lingqiang Zhang (State Key Laboratory of Proteomics, China). FH-USP33 was from Dr. J. Wade Harper (Harvard Medical School, Addgene plasmids). Flag-USP33△201–836 and Flag-USP33-△401–836 plasmids were generated using PCR amplified from FH-USP33. ShUSP33 and shβ-TrCP were purchased from GENECHEM (Shanghai, China). The following antibodies were used: anti-Flag (Sigma, F7425), anti-HA (Abcam, ab9110), antiUbiquitin (Santa Cruz, sc-8017), anti-β-TrCP (Cell Signaling, #11984), anti-USP33 (Proteintech, 20445-1-Ap), anti-β-actin (Proteintech, 66009-1-Ig). Secondary antibodies for Western blot were goat antirabbit and anti-mouse conjugates (Bioworld).
tinates RAS-like GTPase RALB and therefore promoting autophagosome formation [18]. However, how cellular USP33 levels are regulated remains largely unexplored. Here we discovered that the E3 ligase β-TrCP did not reduce USP33 mRNA levels, but significantly downregulated USP33 protein levels. βTrCP is able to interact with USP33, and promotes the ubiquitination and proteasomal degradation of USP33. Interestingly, the effect of βTrCP on USP33 is independent of the typical DSG(XX)2+nS motif in USP33. The 201–400 amino acid motif of USP33 is important for the interaction with β-TrCP. And the WD40 repeat motif of β-TrCP is required for the interaction with USP33. Furthermore, we found that βTrCP attenuated USP33-mediated inhibition of cell proliferation and cell invasion. Thus, our data revealed a new regulatory mechanism of the deubiquitinase USP33, and uncovered an additional signaling pathway, that is, β-TrCP downregulates USP33 to promote cell proliferation.
2.3. Western blot All cells were lysed on ice in lysis buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% NP-40, 0.5 mM EDTA and 50 μg/ml PMSF supplemented with protease inhibitor mixture cocktail (Sigma). N-ethylmaleimide (10 mM) was added to the above lysis buffer when protein ubiquitination was detected. Equivalent quantity of proteins was subjected to SDS-PAGE followed by transferring to PVDF membranes (Millipore). Membranes were blocked with 5% non-fat milk or 5% BSA, and then incubated with the corresponding primary antibodies, followed by either HRP-conjugated Goat anti-mouse or Goat anti-rabbit secondary antibodies. Immunoreactive bands were detected with ECL-Prime (Thermo Scientific).
2. Materials and methods 2.1. Cell culture and reagents HeLa cells, human embryonic kidney cells (HEK293T) and human colorectal cancer cells (HCT116) were maintained in Dulbecco's modified Eagle's medium (DMEM, Hyclone) supplemented with 10% FBS (GIBCO, Life Technologies), 100 U/ml penicillin and 100 μg/ml streptomycin. Plasmids were transfected into cells by using Longtrans (Ucallm) according to the manufacturer's recommendations. Protein synthesis was blocked by treating cells for the indicated time with 50 μg/ml of cycloheximide (Solarbio). Proteasomal degradation of proteins was blocked by treating cells with 10 μM of the proteasome inhibitor MG132 (Sigma).
2.4. Immunoprecipitation assay Cells were lysed on ice. After 12,000g centrifugation for 15 min, the supernatant was collected and subjected to immunoprecipitation with specific antibodies overnight on a rotor at 4 ℃. Protein A/G agarose beads (Millipore, #16-266) were washed twice and then added into cell lysates. The mixture was incubated for an additional 3 h. The immunoprecipitation of FLAG-tagged proteins was performed by using FLAG (M2) affinity gel (Sigma). The unbound proteins were washed away and specific interaction or ubiquitination was analyzed by western blot.
2.2. Plasmids and antibodies Plasmids expressing β-TrCP and HA-β-TrCP were gifts from Dr. Serge Y. Fuchs (University of Pennsylvania). Flag-β-TrCP and Flag-βTrCP-△WD40 plasmids were generated using PCR amplified from 2
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Fig. 2. β-TrCP promotes USP33 ubiquitin-dependent degradation which is independent of DSG motif. HEK293T cells transfected with β-TrCP were treated with or without MG132 (10 μM) for 12 h before harvest. Endogenous USP33 levels were analyzed by immunoblotting as indicated. (B) HEK293T cells were transfected with HA-Ub together with or without β-TrCP. Cell lysates were collected and subjected to IP analysis using an anti-USP33 antibody, followed by immunoblotting as indicated. (C) HEK293T cells were transfected with HA-Ub alone, or HA-Ub plus shβ-TrCP. Cell lysates were collected for IP analysis using an anti-USP33 antibody, followed by immunoblotting as indicated. (D) HEK293T cells were transfected with FH-USP33(△DSG), together with or without β-TrCP. FH-USP33(△DSG) was pull downed by an anti-Flag antibody and analyzed by immunoblotting as indicated. (E) HEK293T cells were transfected with FH-USP33(△DSG), together with shβ-TrCP. Cell lysates were subjected to immunoprecipitation with an antiFlag antibody. The ubiquitination levels were measured by an anti-Ub antibody. (F) HEK293T cells were transfected with FH-USP33(△DSG), as well as increasing doses of β-TrCP. Cells were collected and cell lysates were subjected to immunoblotting with indicated antibodies.
analyzed from representatives of three independent experiments and shown as the average mean ± standard deviation (SD).
2.5. RNA isolation and real-time PCR Total RNAs were isolated from cells using Trizol reagent (Invitrogen). cDNA was synthesized by reverse transcription using oligo (dT) and subjected to quantitative real-time PCR with USP33 and β-actin primers in the presence of SYBR Green Supermix (BIO-RAD). The primer sequences were listed as following: USP33 (Forward primer: 5′-CAGTCTGCATCTCCAAAGAGAA-3′; Reverse primer: 5′ACTACTGGACCCCAAAACCA-3′); β-actin (Forward primer: 5′ACCAACTGGGACGACATGGAGAAA-3′ and Reverse primer: 5′ATAGCACAGCCTGGATAGCAACG-3′). The relative expression levels of the target genes were normalized to β-actin mRNA. The results were
2.6. Cycloheximide chase assay Stability of USP33 proteins was determined by cycloheximide (CHX) chase assay. Briefly, HEK293T cells were seeded in cell culture plates, and then transfected with or without shβ-TrCP for 48 h. Cells were treated with DMSO or CHX (50 μg/ml) for the indicated times and were subsequently harvested and subjected to analysis by western blots. 3
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Fig. 3. The WD40 motif of β-TrCP and 201–400 amino acid motif of USP33 are necessary for their interaction. HEK293T cells were transfected with HA-β-TrCP, and cell lysates were collected and subjected to IP analysis using an anti-HA antibody. Immunoblotting was performed using indicated antibodies. (B) HEK293T cells were transfected with FH-USP33(△DSG), together with or without β-TrCP. Cell lysates were harvested for IP analysis using an anti-Flag antibody. (C) HEK293T cells were transfected with β-TrCP, together with either Flag-USP33-△201–836 or Flag-USP33-△401–836. Cell lysates were collected for IP analysis using an anti-Flag antibody, followed by immunoblotting as indicated. (D) HEK293T cells transfected with either Flag-β-TrCP-WT or Flag-β-TrCP-△WD40 were lysed for IP analysis using an anti-USP33 antibody, followed by immunoblotting as indicated.
2.7. Cell proliferation assay
2.10. Transwell assay
HeLa cells were transfected with shUSP33. Different hours (48 h, 72 h, 96 h and 120 h) after transfection, cells were harvested and counted under an upright microscope, respectively. The numbers of cells were analyzed by GraphPad Prism 5 (San Diego, CA, USA).
HCT116 cells (2×105 cells/200 μl) transfected with plasmids expressing FH-USP33 and/or β-TrCP were seeded in the upper chamber of 24-well. Medium containing 20% FBS was added to the lower wells. 24 h after incubation in 37 ℃, 5% CO2, membranes were fixed with Methanol for 30 min following by staining with crystal violet. The number of invading cells was quantified from five randomly selected visual fields.
2.8. CCK8 (cell counting kit) assay HCT116 cells transfected with plasmids expressing shUSP33 or FH-USP33 and/or β-TrCP were seeded in 96-well plate at a density of 3×103 cells/well. To measure cell viability, CCK8 was added to each well and the plate was further incubated for 4 h at 37 ℃. The number of living cells was evaluated at absorbance of 450 nm. The data were analyzed by using GraphPad Prism 5 (San Diego, CA, USA).
2.11. Statistical analysis All data were shown as means ± standard deviations (SD). Statistical significance was tested by the two-tailed Student t-test. P < 0.05 was regarded as statistically significant. 3. Results
2.9. Wound-healing assay 3.1. β-TrCP downregulates USP33 protein levels HCT116 cells transfected with plasmids expressing FH-USP33 and/ or β-TrCP were cultured to full confluence in 6-well plate and scratched by using sterile pipette tips. After scratching, the cells were gently washed twice with PBS, then medium containing 2% FBS was added. Scratched cells were photographed under an inverted microscope after 0 h, 12 h and 24 h. Migration of cells was assessed by the width of the scratched area.
Through analyzing the amino acid sequence of USP33, we noted that there is a putative DSG(X)2+nS motif in USP33 conserved from Zebrafish to Homo sapiens (Fig. 1A). The DSG(X)2+nS motif has been demonstrated to be an important characteristic of substrates for E3 ubiquitin ligase β-TrCP. Accordingly, we speculated that USP33 is a potential substrate of β-TrCP. To address this hypothesis, we used an 4
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Fig. 4. USP33 protein stability is regulated by β-TrCP. (A) HEK293T cells transfected with either shβ-TrCP or an empty vector (shCON) were treated with cycloheximide (50 µg/ ml) for 0, 6 and 12 h. The level of endogenous USP33 was evaluated by immunoblotting. (B) HEK293T cells were transfected with FH-USP33(△DSG), together with or without shβTrCP. Then cells were treated with CHX as in (A). Immunoblotting was performed to detect the level of FH-USP33 (△DSG) and endogenous β-TrCP with indicated antibodies. Data were analyzed by GraphPad Prism 5. *p < 0.05 by Student's t-test.
anti-USP33 antibody to evaluate endogenous USP33 protein levels in HEK293T cells transfected with HA-β-TrCP. We noticed that endogenous USP33 protein levels were gradually downregulated by HA-βTrCP in a dose-dependent manner (Fig. 1B). In contrast, knockdown of β-TrCP in HEK293T cells upregulated endogenous USP33 protein levels (Fig. 1C). These data suggest that β-TrCP might mediate USP33 downregulation at the protein level. To further determine whether βTrCP affects the transcription of USP33, we transfected HEK293T cells with increasing amount of β-TrCP and examined the mRNA level of USP33. We found that overexpression of β-TrCP did not decrease, but slightly increased USP33 mRNA levels (Fig. 1D). Taken together, these results suggest that β-TrCP downregulates USP33 expression at the protein level but not at the mRNA level.
noticed that Flag-HA-USP33 expressing plasmid in Addgene is actually the isoform 3 of USP33. Thus, we obtained this Flag-HA-USP33, termed FH-USP33(△DSG), for further studies. Unexpectedly, we found that overexpression of β-TrCP can also increase the ubiquitination of FH-USP33(△DSG) (Fig. 2D), and knockdown of β-TrCP also decreased the ubiquitination of FH-USP33(△DSG) (Fig. 2E). Furthermore, overexpression of β-TrCP can also promote FHUSP33(△DSG) degradation in a dose-dependent manner (Fig. 2F). These results suggest that the effect of β-TrCP on USP33 is independent of the classic DSG motif.
3.2. β-TrCP promotes USP33 ubiquitin-dependent degradation which is independent of the recognition of DSG motif
Since β-TrCP promotes USP33 ubiquitin-dependent degradation, then we assessed the interaction between β-TrCP and USP33. Coimmunoprecipitation assays showed that β-TrCP was able to interact with endogenous USP33 (Fig. 3A). β-TrCP can also interact with FHUSP33(△DSG) (Fig. 3B), indicating that the interaction between βTrCP and USP33 is independent of DSG motif of USP33. In order to determine what regions of USP33 are necessary for interaction with βTrCP. We constructed different mutants of USP33 including FlagUSP33-△201–836 and Flag-USP33-△401–836. The interaction of β-TrCP with these USP33 mutants was evaluated. Our data showed that βTrCP interacted with Flag-USP33-△401–836. When the 201–400 amino acid motif of USP33 was further deleted (Flag-USP33-△201–836), the interaction with β-TrCP was abolished (Fig. 3C), suggesting that 201– 400 amino acid motif of USP33 is required for the interaction with βTrCP. Given that the WD40 β-propeller domain of β-TrCP is important for the interaction between β-TrCP and its substrates, we further constructed Flag-β-TrCP-WT and Flag-β-TrCP-△WD40 plasmids. Our data showed that USP33 was able to interact with β-TrCP-WT, but unable to interact with β-TrCP-△WD40 (Fig. 3D). Collectively, we
3.3. WD40 motif of β-TrCP and 201–400 amino acid motif of USP33 are necessary for the interaction between β-TrCP and USP33
To uncover the mechanism of USP33 downregulation mediated by β-TrCP, we took advantage of proteasomal degradation inhibitor MG132. Our results showed that MG132 blocked β-TrCP-mediated USP33 downregulation (Fig. 2A), suggesting that β-TrCP promotes USP33 downregulation via the proteasome pathway. To validate that βTrCP is indeed involved in the ubiquitin-proteasome degradation pathway of USP33, we performed ubiquitination analysis by transfecting cells with HA-Ub, together with β-TrCP or shβ-TrCP. The results showed that overexpression of β-TrCP substantially increased the polyubiquitination of endogenous USP33 (Fig. 2B). Conversely, knockdown of β-TrCP significantly decreased the ubiquitination levels of endogenous USP33 (Fig. 2C). USP33 has three different isoforms identified so far. Among them, the isoform 3 (transcript variant 3) lacks the last 109 amino acids (△834–942) of USP33, and therefore lacks the DSG(X)2+nS motif. Therefore, USP33 isoform 3 is suitable for the analysis of interaction between β-TrCP and USP33-DSG motif. We 5
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Fig. 5. β-TrCP attenuates USP33-mediated suppression of cell proliferation and invasion. HeLa cells transfected with shUSP33 were collected at 48 h, 72 h, 96 h, 120 h post-transfection respectively, and counted under an upright microscope; numbers of cells were added up and analyzed by GraphPad Prism 5. (B, C) HCT116 cells transfected with shUSP33 (B) or FH-USP33(△DSG) together with or without β-TrCP (C) were seeded into 96-well plate. CCK8 was added to each well and the plate was further incubated for 4 h at 37 ℃. The absorbance at 450 nm was measured and analyzed by GraphPad Prism 5. (D) HCT116 cells transfected with FH-USP33(△DSG) together with or without β-TrCP were scratched. Scratched cells were photographed under an inverted microscope. Migration of cells was assessed by the width of the scratched area. (E) HCT116 cells transfected with FHUSP33(△DSG) together with or without β-TrCP were seeded in the upper chamber of 24-well. Medium containing 20% FBS was added to the lower wells. The number of invading cells was quantified from five randomly selected visual fields after 24 h incubation. *p < 0.05, **p < 0.01 and ***p < 0.001 by Student's t-test.
found that the WD40 motif of β-TrCP and 201–400 amino acid motif of USP33 are necessary for the interaction between β-TrCP and USP33.
(shCON) were treated with protein synthesis inhibitor CHX (50 µg/ ml). The results showed that knockdown of β-TrCP in cells slowed down the degradation rate of endogenous USP33 and therefore enhanced USP33 protein stability (Fig. 4A). Moreover, knockdown of β-TrCP can also slow down the degradation of exogenous FH-USP33 (△DSG) (Fig. 4B). Collectively, these data suggest that β-TrCP can regulate USP33 protein stability.
3.4. β-TrCP regulates USP33 protein stability Given that β-TrCP is able to promote the ubiquitination and proteasomal degradation of USP33, we speculated that the protein stability of USP33 could be regulated by β-TrCP. To address this hypothesis, we conducted cycloheximide (CHX) pulse chase analysis of USP33 protein. Cells transfected with shβ-TrCP or control vector 6
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3.5. β-TrCP attenuates USP33-mediated inhibition of cell proliferation and invasion
Foundation of China (31370873 and 31570865), the Program of 1000 Young Talents, and Jiangsu Provincial Distinguished Young Scholars (BK20130004). We also thank the program for Changjiang Scholars and Innovative Research Team in University of Ministry of Education of China (PCSIRT-IRT1075), a project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD)，and the Jiangsu Provincial Innovative Research Team.
USP33 has turned out to be a previously unknown tumor-suppressing gene. Thus, we firstly analyzed the effect of USP33 on cell proliferation. The results showed that the proliferation of HeLa cells was considerably accelerated when USP33 was knocked down (Fig. 5A). Furthermore, we evaluated the effect of USP33 on the proliferation of human colorectal cancer cells HCT116 by CCK8 assay. Data showed that the proliferation of HCT116 cells was promoted when USP33 was knocked down (Fig. 5B). In contrast, when USP33 was over expressed, the proliferation of HCT116 cells was inhibited. However, the inhibitory effect of USP33 on cell proliferation was attenuated by β-TrCP overexpression (Fig. 5C). These results indicate that β-TrCP attenuates USP33-mediated inhibition of cell proliferation. Furthermore, both wound-healing assay and Transwell assay were used to assess the cell invasion. Our data showed that the inhibitory effect of USP33 on cell invasion was attenuated by β-TrCP overexpression (Fig. 5D, E). Taken together, we found that β-TrCP attenuates USP33-mediated inhibition of cell proliferation and invasion.
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4. Discussion So far, the study of USP33 is still very limited. USP33 has been reported to be a previously unknown tumor-suppressing gene which inhibits cell migration of colorectal cancers and breast cancers. Furthermore, USP33 is involved in the prognosis of lung cancers [19]. The in vivo substrates of USP33 identified so far include Robo1, CP110 and RALB. Nevertheless, regulation of USP33 protein itself remains largely unexplored. A recent study showed that USP33 can be regulated by P97 and HERC2 [20], which are the only report on the regulation of USP33 protein so far. Here, our study identified a new regulator, the E3 ligase β-TrCP, which could regulate the protein stability and function of USP33 (Figs. 4 and 5). Interestingly, our data demonstrated that the effect of β-TrCP on USP33 is independent of the DSG motif in USP33, which is supposed to be a classic binding motif for β-TrCP. We found that USP33(△DSG) can also interact with β-TrCP (Fig. 3B). Furthermore, β-TrCP also increased the ubiquitination of USP33(△DSG) (Fig. 2D) and decreased the protein stability of USP33(△DSG) (Fig. 4B). These results suggest that β-TrCP affects USP33 levels by an atypical manner. As a matter of fact, there have been many atypical substrates of β-TrCP identified so far. Furthermore, we identified that the 201–400 amino acid region of USP33 is important for interaction with β-TrCP. And the WD40 repeat motif of β-TrCP is required for the interaction with USP33. The E3 ligase β-TrCP regulated not only the protein stability but also the function of USP33. USP33 has been recognized as a novel tumor-suppressing gene. Thus, we are quite interested in the effect of USP33 on tumor cell proliferation. Using HeLa or HCT116 cells as a model, we analyzed the cell proliferation mediated by USP33. Our data showed that knockdown of USP33 promoted the proliferation of both HeLa and HCT116 cells. Furthermore, we demonstrated that overexpression of β-TrCP attenuated USP33-mediated inhibition of cell proliferation and invasion (Fig. 5). Here, our study for the first time uncovered that β-TrCP plays the roles in tumor cell proliferation and invasion by promoting ubiquitination and degradation of USP33. Acknowledgements This work was supported by grants from National Natural Science
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Experimental Cell Research journal homepage: www.elsevier.com/locate/yexcr
Deubiquitinase USP33 is negatively regulated by β-TrCP through ubiquitindependent proteolysis Qiao Chenga,b,1, Yukang Yuana,b,1, Lemin Lia,b,1, Tingting Guoa,b, Ying Miaoa,b, Ying Rena,b, Jin Liua,b, Qian Fenga,b, Xiaofang Wanga,b, Peng Zhaoa,b, Yibo Zuoa,b, Liping Qiana,b, ⁎ Liting Zhanga,b, Hui Zhenga,b, a b
Institutes of Biology and Medical Sciences, Soochow University, Suzhou, Jiangsu Province 215123, China Jiangsu Key Laboratory of Infection and Immunity, Soochow University, Suzhou, Jiangsu Province 215123, China
A R T I C L E I N F O
A BS T RAC T
Keywords: Protein ubiquitination E3 ligase β-TrCP USP33
Ubiquitin-mediated proteolysis regulates cellular levels of various proteins, and therefore plays important roles in controlling cell signaling and disease progression. The Skp1-Cul1-F-box ubiquitin ligase β-TrCP is recognized as an important negative regulator for numerous key signaling proteins. Recently, the deubiquitinases (DUBs) have turned out to be essential to regulate signaling pathways related to human diseases. However, whether βTrCP is able to regulate the deubiquitinase family members remains largely unexplored. Here, we found that βTrCP downregulated cellular levels of endogenous USP33. We also revealed that β-TrCP interacted with USP33 independently of the classic binding motif for β-TrCP, and mediated USP33 degradation via the ubiquitin proteasome pathway. Furthermore, we found that the WD40 motif of β-TrCP and 201–400 amino acid motif of USP33 are required for the interaction between β-TrCP and USP33. Consequently, β-TrCP attenuated USP33mediated inhibition of cell proliferation and cell invasion. Taken together, our study clarified that the E3 ligase β-TrCP regulates cellular USP33 levels by the ubiquitin-proteasomal proteolysis.
1. Introduction
tinases (DUBs). So far, approximately 100 human DUBs have been identified, and are subdivided into five families: ubiquitin-specific protease (USPs), ubiquitin C-terminal hydrolases (UCHs), ovarian tumor proteases (OTUs), Josephin domain family (MJD) and JAB1/ MPN/Mov34 metalloenzymes (JAMMs) [12,13]. Although some members of these DUBs have been studied, the functions and detailed regulatory mechanisms of many DUBs members are still unclear. Among these DUBs families, the USPs family is the biggest one and comprises of over fifty members. So far, the regulation of the USPs family members mediated by β-TrCP remains largely unexplored. By analysis of amino acid sequences of 56 USPs family members, we found that USP33 contains a typical binding motif for β-TrCP. Thus, we sought to determine the role of β-TrCP in USP33 regulation. USP33 is associated with several important biological functions. It has been reported that USP33 is a previously unknown tumorsuppressing gene for colorectal cancers by deubiquitinating and stabilizing Robo1 [14]. Similarly, USP33 is necessary for Slit signaling in breast cancer cells [15]. In addition, USP33 plays an essential role in centrosome amplification by deubiquitinating the centriolar protein CP110 [16,17]. It has also been demonstrated that USP33 deubiqui-
Ubiquitin-mediated proteolysis plays crucial roles in regulating many biological processes including cell proliferation, apoptosis, and immune responses [1]. The conjugation of ubiquitin is catalyzed by three types of enzymes: ubiquitin-activating enzyme (E1), ubiquitinconjugating enzyme (E2) and ubiquitin ligases (E3). The ubiquitin E3 ligases are determiners for the substrate specificity, and have been extensively studied during the past decades. β-TrCP is one of these E3 ubiquitin ligases, which has been demonstrated to be an important regulator for many key signaling proteins, including IκB [2,3], βCatenin [3,4], IFNAR1 [5], ATF4 [6], and Emil [7]. All of these protein substrates contain the conserved DSG(XX)2+nS motif, which is a “typical” binding motif for β-TrCP. However, recent studies reported several “atypical” substrates of β-TrCP including STAT1 [8], Wee1 [9], GHR [10], ELAVL1/huR [11], CDC25B [11], and TP53 [11], which do not contain the classic DSG(XX)2+nS motif. Deubiquitination, as a reverse process of ubiquitination, has recently been shown to be essential for controlling disease-related signaling pathways. Protein deubiquitination is mediated by deubiqui-
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1
Corresponding author at: Institutes of Biology and Medical Sciences, Soochow University, Suzhou, Jiangsu Province 215123, China. E-mail address: [email protected] (H. Zheng). These authors contributed equally to this study.
http://dx.doi.org/10.1016/j.yexcr.2017.05.011 Received 10 January 2017; Received in revised form 3 May 2017; Accepted 12 May 2017 0014-4827/ © 2017 Elsevier Inc. All rights reserved.
Please cite this article as: Cheng, Q., Experimental Cell Research (2017), http://dx.doi.org/10.1016/j.yexcr.2017.05.011
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Fig. 1. β-TrCP downregulates USP33 protein levels. (A) DSG(X)2+nS motif is evolutionarily conserved from Zebrafish to Homo sapiens. (B) Immunoblots were used to detect the level of HA-β-TrCP and endogenous USP33 in HEK293T cells that were transfected with either an empty vector or increasing doses of β-TrCP. Probing with β-actin served as loading control. (C) HEK293T cells were transfected with or without shβ-TrCP, the levels of β-actin, endogenous USP33 and β-TrCP were measured by immunoblotting with indicated antibodies. (D) Bar diagram showed the mRNA level of endogenous USP33 as measured by qRT-PCR in HEK293T cells transfected with empty vector or increasing doses of β-TrCP.
HA-β-TrCP. HA-Ub was a gift from Dr. Lingqiang Zhang (State Key Laboratory of Proteomics, China). FH-USP33 was from Dr. J. Wade Harper (Harvard Medical School, Addgene plasmids). Flag-USP33△201–836 and Flag-USP33-△401–836 plasmids were generated using PCR amplified from FH-USP33. ShUSP33 and shβ-TrCP were purchased from GENECHEM (Shanghai, China). The following antibodies were used: anti-Flag (Sigma, F7425), anti-HA (Abcam, ab9110), antiUbiquitin (Santa Cruz, sc-8017), anti-β-TrCP (Cell Signaling, #11984), anti-USP33 (Proteintech, 20445-1-Ap), anti-β-actin (Proteintech, 66009-1-Ig). Secondary antibodies for Western blot were goat antirabbit and anti-mouse conjugates (Bioworld).
tinates RAS-like GTPase RALB and therefore promoting autophagosome formation [18]. However, how cellular USP33 levels are regulated remains largely unexplored. Here we discovered that the E3 ligase β-TrCP did not reduce USP33 mRNA levels, but significantly downregulated USP33 protein levels. βTrCP is able to interact with USP33, and promotes the ubiquitination and proteasomal degradation of USP33. Interestingly, the effect of βTrCP on USP33 is independent of the typical DSG(XX)2+nS motif in USP33. The 201–400 amino acid motif of USP33 is important for the interaction with β-TrCP. And the WD40 repeat motif of β-TrCP is required for the interaction with USP33. Furthermore, we found that βTrCP attenuated USP33-mediated inhibition of cell proliferation and cell invasion. Thus, our data revealed a new regulatory mechanism of the deubiquitinase USP33, and uncovered an additional signaling pathway, that is, β-TrCP downregulates USP33 to promote cell proliferation.
2.3. Western blot All cells were lysed on ice in lysis buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% NP-40, 0.5 mM EDTA and 50 μg/ml PMSF supplemented with protease inhibitor mixture cocktail (Sigma). N-ethylmaleimide (10 mM) was added to the above lysis buffer when protein ubiquitination was detected. Equivalent quantity of proteins was subjected to SDS-PAGE followed by transferring to PVDF membranes (Millipore). Membranes were blocked with 5% non-fat milk or 5% BSA, and then incubated with the corresponding primary antibodies, followed by either HRP-conjugated Goat anti-mouse or Goat anti-rabbit secondary antibodies. Immunoreactive bands were detected with ECL-Prime (Thermo Scientific).
2. Materials and methods 2.1. Cell culture and reagents HeLa cells, human embryonic kidney cells (HEK293T) and human colorectal cancer cells (HCT116) were maintained in Dulbecco's modified Eagle's medium (DMEM, Hyclone) supplemented with 10% FBS (GIBCO, Life Technologies), 100 U/ml penicillin and 100 μg/ml streptomycin. Plasmids were transfected into cells by using Longtrans (Ucallm) according to the manufacturer's recommendations. Protein synthesis was blocked by treating cells for the indicated time with 50 μg/ml of cycloheximide (Solarbio). Proteasomal degradation of proteins was blocked by treating cells with 10 μM of the proteasome inhibitor MG132 (Sigma).
2.4. Immunoprecipitation assay Cells were lysed on ice. After 12,000g centrifugation for 15 min, the supernatant was collected and subjected to immunoprecipitation with specific antibodies overnight on a rotor at 4 ℃. Protein A/G agarose beads (Millipore, #16-266) were washed twice and then added into cell lysates. The mixture was incubated for an additional 3 h. The immunoprecipitation of FLAG-tagged proteins was performed by using FLAG (M2) affinity gel (Sigma). The unbound proteins were washed away and specific interaction or ubiquitination was analyzed by western blot.
2.2. Plasmids and antibodies Plasmids expressing β-TrCP and HA-β-TrCP were gifts from Dr. Serge Y. Fuchs (University of Pennsylvania). Flag-β-TrCP and Flag-βTrCP-△WD40 plasmids were generated using PCR amplified from 2
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Fig. 2. β-TrCP promotes USP33 ubiquitin-dependent degradation which is independent of DSG motif. HEK293T cells transfected with β-TrCP were treated with or without MG132 (10 μM) for 12 h before harvest. Endogenous USP33 levels were analyzed by immunoblotting as indicated. (B) HEK293T cells were transfected with HA-Ub together with or without β-TrCP. Cell lysates were collected and subjected to IP analysis using an anti-USP33 antibody, followed by immunoblotting as indicated. (C) HEK293T cells were transfected with HA-Ub alone, or HA-Ub plus shβ-TrCP. Cell lysates were collected for IP analysis using an anti-USP33 antibody, followed by immunoblotting as indicated. (D) HEK293T cells were transfected with FH-USP33(△DSG), together with or without β-TrCP. FH-USP33(△DSG) was pull downed by an anti-Flag antibody and analyzed by immunoblotting as indicated. (E) HEK293T cells were transfected with FH-USP33(△DSG), together with shβ-TrCP. Cell lysates were subjected to immunoprecipitation with an antiFlag antibody. The ubiquitination levels were measured by an anti-Ub antibody. (F) HEK293T cells were transfected with FH-USP33(△DSG), as well as increasing doses of β-TrCP. Cells were collected and cell lysates were subjected to immunoblotting with indicated antibodies.
analyzed from representatives of three independent experiments and shown as the average mean ± standard deviation (SD).
2.5. RNA isolation and real-time PCR Total RNAs were isolated from cells using Trizol reagent (Invitrogen). cDNA was synthesized by reverse transcription using oligo (dT) and subjected to quantitative real-time PCR with USP33 and β-actin primers in the presence of SYBR Green Supermix (BIO-RAD). The primer sequences were listed as following: USP33 (Forward primer: 5′-CAGTCTGCATCTCCAAAGAGAA-3′; Reverse primer: 5′ACTACTGGACCCCAAAACCA-3′); β-actin (Forward primer: 5′ACCAACTGGGACGACATGGAGAAA-3′ and Reverse primer: 5′ATAGCACAGCCTGGATAGCAACG-3′). The relative expression levels of the target genes were normalized to β-actin mRNA. The results were
2.6. Cycloheximide chase assay Stability of USP33 proteins was determined by cycloheximide (CHX) chase assay. Briefly, HEK293T cells were seeded in cell culture plates, and then transfected with or without shβ-TrCP for 48 h. Cells were treated with DMSO or CHX (50 μg/ml) for the indicated times and were subsequently harvested and subjected to analysis by western blots. 3
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Fig. 3. The WD40 motif of β-TrCP and 201–400 amino acid motif of USP33 are necessary for their interaction. HEK293T cells were transfected with HA-β-TrCP, and cell lysates were collected and subjected to IP analysis using an anti-HA antibody. Immunoblotting was performed using indicated antibodies. (B) HEK293T cells were transfected with FH-USP33(△DSG), together with or without β-TrCP. Cell lysates were harvested for IP analysis using an anti-Flag antibody. (C) HEK293T cells were transfected with β-TrCP, together with either Flag-USP33-△201–836 or Flag-USP33-△401–836. Cell lysates were collected for IP analysis using an anti-Flag antibody, followed by immunoblotting as indicated. (D) HEK293T cells transfected with either Flag-β-TrCP-WT or Flag-β-TrCP-△WD40 were lysed for IP analysis using an anti-USP33 antibody, followed by immunoblotting as indicated.
2.7. Cell proliferation assay
2.10. Transwell assay
HeLa cells were transfected with shUSP33. Different hours (48 h, 72 h, 96 h and 120 h) after transfection, cells were harvested and counted under an upright microscope, respectively. The numbers of cells were analyzed by GraphPad Prism 5 (San Diego, CA, USA).
HCT116 cells (2×105 cells/200 μl) transfected with plasmids expressing FH-USP33 and/or β-TrCP were seeded in the upper chamber of 24-well. Medium containing 20% FBS was added to the lower wells. 24 h after incubation in 37 ℃, 5% CO2, membranes were fixed with Methanol for 30 min following by staining with crystal violet. The number of invading cells was quantified from five randomly selected visual fields.
2.8. CCK8 (cell counting kit) assay HCT116 cells transfected with plasmids expressing shUSP33 or FH-USP33 and/or β-TrCP were seeded in 96-well plate at a density of 3×103 cells/well. To measure cell viability, CCK8 was added to each well and the plate was further incubated for 4 h at 37 ℃. The number of living cells was evaluated at absorbance of 450 nm. The data were analyzed by using GraphPad Prism 5 (San Diego, CA, USA).
2.11. Statistical analysis All data were shown as means ± standard deviations (SD). Statistical significance was tested by the two-tailed Student t-test. P < 0.05 was regarded as statistically significant. 3. Results
2.9. Wound-healing assay 3.1. β-TrCP downregulates USP33 protein levels HCT116 cells transfected with plasmids expressing FH-USP33 and/ or β-TrCP were cultured to full confluence in 6-well plate and scratched by using sterile pipette tips. After scratching, the cells were gently washed twice with PBS, then medium containing 2% FBS was added. Scratched cells were photographed under an inverted microscope after 0 h, 12 h and 24 h. Migration of cells was assessed by the width of the scratched area.
Through analyzing the amino acid sequence of USP33, we noted that there is a putative DSG(X)2+nS motif in USP33 conserved from Zebrafish to Homo sapiens (Fig. 1A). The DSG(X)2+nS motif has been demonstrated to be an important characteristic of substrates for E3 ubiquitin ligase β-TrCP. Accordingly, we speculated that USP33 is a potential substrate of β-TrCP. To address this hypothesis, we used an 4
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Fig. 4. USP33 protein stability is regulated by β-TrCP. (A) HEK293T cells transfected with either shβ-TrCP or an empty vector (shCON) were treated with cycloheximide (50 µg/ ml) for 0, 6 and 12 h. The level of endogenous USP33 was evaluated by immunoblotting. (B) HEK293T cells were transfected with FH-USP33(△DSG), together with or without shβTrCP. Then cells were treated with CHX as in (A). Immunoblotting was performed to detect the level of FH-USP33 (△DSG) and endogenous β-TrCP with indicated antibodies. Data were analyzed by GraphPad Prism 5. *p < 0.05 by Student's t-test.
anti-USP33 antibody to evaluate endogenous USP33 protein levels in HEK293T cells transfected with HA-β-TrCP. We noticed that endogenous USP33 protein levels were gradually downregulated by HA-βTrCP in a dose-dependent manner (Fig. 1B). In contrast, knockdown of β-TrCP in HEK293T cells upregulated endogenous USP33 protein levels (Fig. 1C). These data suggest that β-TrCP might mediate USP33 downregulation at the protein level. To further determine whether βTrCP affects the transcription of USP33, we transfected HEK293T cells with increasing amount of β-TrCP and examined the mRNA level of USP33. We found that overexpression of β-TrCP did not decrease, but slightly increased USP33 mRNA levels (Fig. 1D). Taken together, these results suggest that β-TrCP downregulates USP33 expression at the protein level but not at the mRNA level.
noticed that Flag-HA-USP33 expressing plasmid in Addgene is actually the isoform 3 of USP33. Thus, we obtained this Flag-HA-USP33, termed FH-USP33(△DSG), for further studies. Unexpectedly, we found that overexpression of β-TrCP can also increase the ubiquitination of FH-USP33(△DSG) (Fig. 2D), and knockdown of β-TrCP also decreased the ubiquitination of FH-USP33(△DSG) (Fig. 2E). Furthermore, overexpression of β-TrCP can also promote FHUSP33(△DSG) degradation in a dose-dependent manner (Fig. 2F). These results suggest that the effect of β-TrCP on USP33 is independent of the classic DSG motif.
3.2. β-TrCP promotes USP33 ubiquitin-dependent degradation which is independent of the recognition of DSG motif
Since β-TrCP promotes USP33 ubiquitin-dependent degradation, then we assessed the interaction between β-TrCP and USP33. Coimmunoprecipitation assays showed that β-TrCP was able to interact with endogenous USP33 (Fig. 3A). β-TrCP can also interact with FHUSP33(△DSG) (Fig. 3B), indicating that the interaction between βTrCP and USP33 is independent of DSG motif of USP33. In order to determine what regions of USP33 are necessary for interaction with βTrCP. We constructed different mutants of USP33 including FlagUSP33-△201–836 and Flag-USP33-△401–836. The interaction of β-TrCP with these USP33 mutants was evaluated. Our data showed that βTrCP interacted with Flag-USP33-△401–836. When the 201–400 amino acid motif of USP33 was further deleted (Flag-USP33-△201–836), the interaction with β-TrCP was abolished (Fig. 3C), suggesting that 201– 400 amino acid motif of USP33 is required for the interaction with βTrCP. Given that the WD40 β-propeller domain of β-TrCP is important for the interaction between β-TrCP and its substrates, we further constructed Flag-β-TrCP-WT and Flag-β-TrCP-△WD40 plasmids. Our data showed that USP33 was able to interact with β-TrCP-WT, but unable to interact with β-TrCP-△WD40 (Fig. 3D). Collectively, we
3.3. WD40 motif of β-TrCP and 201–400 amino acid motif of USP33 are necessary for the interaction between β-TrCP and USP33
To uncover the mechanism of USP33 downregulation mediated by β-TrCP, we took advantage of proteasomal degradation inhibitor MG132. Our results showed that MG132 blocked β-TrCP-mediated USP33 downregulation (Fig. 2A), suggesting that β-TrCP promotes USP33 downregulation via the proteasome pathway. To validate that βTrCP is indeed involved in the ubiquitin-proteasome degradation pathway of USP33, we performed ubiquitination analysis by transfecting cells with HA-Ub, together with β-TrCP or shβ-TrCP. The results showed that overexpression of β-TrCP substantially increased the polyubiquitination of endogenous USP33 (Fig. 2B). Conversely, knockdown of β-TrCP significantly decreased the ubiquitination levels of endogenous USP33 (Fig. 2C). USP33 has three different isoforms identified so far. Among them, the isoform 3 (transcript variant 3) lacks the last 109 amino acids (△834–942) of USP33, and therefore lacks the DSG(X)2+nS motif. Therefore, USP33 isoform 3 is suitable for the analysis of interaction between β-TrCP and USP33-DSG motif. We 5
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Fig. 5. β-TrCP attenuates USP33-mediated suppression of cell proliferation and invasion. HeLa cells transfected with shUSP33 were collected at 48 h, 72 h, 96 h, 120 h post-transfection respectively, and counted under an upright microscope; numbers of cells were added up and analyzed by GraphPad Prism 5. (B, C) HCT116 cells transfected with shUSP33 (B) or FH-USP33(△DSG) together with or without β-TrCP (C) were seeded into 96-well plate. CCK8 was added to each well and the plate was further incubated for 4 h at 37 ℃. The absorbance at 450 nm was measured and analyzed by GraphPad Prism 5. (D) HCT116 cells transfected with FH-USP33(△DSG) together with or without β-TrCP were scratched. Scratched cells were photographed under an inverted microscope. Migration of cells was assessed by the width of the scratched area. (E) HCT116 cells transfected with FHUSP33(△DSG) together with or without β-TrCP were seeded in the upper chamber of 24-well. Medium containing 20% FBS was added to the lower wells. The number of invading cells was quantified from five randomly selected visual fields after 24 h incubation. *p < 0.05, **p < 0.01 and ***p < 0.001 by Student's t-test.
found that the WD40 motif of β-TrCP and 201–400 amino acid motif of USP33 are necessary for the interaction between β-TrCP and USP33.
(shCON) were treated with protein synthesis inhibitor CHX (50 µg/ ml). The results showed that knockdown of β-TrCP in cells slowed down the degradation rate of endogenous USP33 and therefore enhanced USP33 protein stability (Fig. 4A). Moreover, knockdown of β-TrCP can also slow down the degradation of exogenous FH-USP33 (△DSG) (Fig. 4B). Collectively, these data suggest that β-TrCP can regulate USP33 protein stability.
3.4. β-TrCP regulates USP33 protein stability Given that β-TrCP is able to promote the ubiquitination and proteasomal degradation of USP33, we speculated that the protein stability of USP33 could be regulated by β-TrCP. To address this hypothesis, we conducted cycloheximide (CHX) pulse chase analysis of USP33 protein. Cells transfected with shβ-TrCP or control vector 6
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3.5. β-TrCP attenuates USP33-mediated inhibition of cell proliferation and invasion
Foundation of China (31370873 and 31570865), the Program of 1000 Young Talents, and Jiangsu Provincial Distinguished Young Scholars (BK20130004). We also thank the program for Changjiang Scholars and Innovative Research Team in University of Ministry of Education of China (PCSIRT-IRT1075), a project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD)，and the Jiangsu Provincial Innovative Research Team.
USP33 has turned out to be a previously unknown tumor-suppressing gene. Thus, we firstly analyzed the effect of USP33 on cell proliferation. The results showed that the proliferation of HeLa cells was considerably accelerated when USP33 was knocked down (Fig. 5A). Furthermore, we evaluated the effect of USP33 on the proliferation of human colorectal cancer cells HCT116 by CCK8 assay. Data showed that the proliferation of HCT116 cells was promoted when USP33 was knocked down (Fig. 5B). In contrast, when USP33 was over expressed, the proliferation of HCT116 cells was inhibited. However, the inhibitory effect of USP33 on cell proliferation was attenuated by β-TrCP overexpression (Fig. 5C). These results indicate that β-TrCP attenuates USP33-mediated inhibition of cell proliferation. Furthermore, both wound-healing assay and Transwell assay were used to assess the cell invasion. Our data showed that the inhibitory effect of USP33 on cell invasion was attenuated by β-TrCP overexpression (Fig. 5D, E). Taken together, we found that β-TrCP attenuates USP33-mediated inhibition of cell proliferation and invasion.
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4. Discussion So far, the study of USP33 is still very limited. USP33 has been reported to be a previously unknown tumor-suppressing gene which inhibits cell migration of colorectal cancers and breast cancers. Furthermore, USP33 is involved in the prognosis of lung cancers [19]. The in vivo substrates of USP33 identified so far include Robo1, CP110 and RALB. Nevertheless, regulation of USP33 protein itself remains largely unexplored. A recent study showed that USP33 can be regulated by P97 and HERC2 [20], which are the only report on the regulation of USP33 protein so far. Here, our study identified a new regulator, the E3 ligase β-TrCP, which could regulate the protein stability and function of USP33 (Figs. 4 and 5). Interestingly, our data demonstrated that the effect of β-TrCP on USP33 is independent of the DSG motif in USP33, which is supposed to be a classic binding motif for β-TrCP. We found that USP33(△DSG) can also interact with β-TrCP (Fig. 3B). Furthermore, β-TrCP also increased the ubiquitination of USP33(△DSG) (Fig. 2D) and decreased the protein stability of USP33(△DSG) (Fig. 4B). These results suggest that β-TrCP affects USP33 levels by an atypical manner. As a matter of fact, there have been many atypical substrates of β-TrCP identified so far. Furthermore, we identified that the 201–400 amino acid region of USP33 is important for interaction with β-TrCP. And the WD40 repeat motif of β-TrCP is required for the interaction with USP33. The E3 ligase β-TrCP regulated not only the protein stability but also the function of USP33. USP33 has been recognized as a novel tumor-suppressing gene. Thus, we are quite interested in the effect of USP33 on tumor cell proliferation. Using HeLa or HCT116 cells as a model, we analyzed the cell proliferation mediated by USP33. Our data showed that knockdown of USP33 promoted the proliferation of both HeLa and HCT116 cells. Furthermore, we demonstrated that overexpression of β-TrCP attenuated USP33-mediated inhibition of cell proliferation and invasion (Fig. 5). Here, our study for the first time uncovered that β-TrCP plays the roles in tumor cell proliferation and invasion by promoting ubiquitination and degradation of USP33. Acknowledgements This work was supported by grants from National Natural Science
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