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Ribosomal protein S2 interplays with MDM2 to induce p53
Biochemical and Biophysical Research Communications xxx (xxxx) xxx
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Ribosomal protein S2 interplays with MDM2 to induce p53 Jinhong Cho a, b, Jinyoung Park c, Sang Chul Shin a, Jae-Hong Kim b, Eunice EunKyeong Kim a, Eun Joo Song d, * a
Biomedical Research Institute, Korea Institute of Science and Technology, Hwarangno 14-gil 5, Seongbuk-gu, Seoul, 02792, Republic of Korea Department of Biotechnology, College of Life Sciences and Biotechnology, Korea University, 5-1 Anam-dong, Sungbuk-gu, Seoul, 02841, Republic of Korea c Molecular Recognition Research Center, Korea Institute of Science and Technology, Hwarangno 14-gil 5, Seongbuk-gu, Seoul, 02792, Republic of Korea d Graduate School of Pharmaceutical Sciences, College of Pharmacy, Ewha Womans University, Seoul, 03760, Republic of Korea b
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Article history: Received 25 December 2019 Accepted 5 January 2020 Available online xxx
The MDM2-p53 pathway is crucial for maintenance of p53 homeostasis. Some ribosomal proteins (RPs) play critical roles in regulating p53 by interacting with MDM2. However, the role and functional mechanism of each RP in MDM2-p53 pathway still remain unknown. In this study, we found that Ribosomal Protein S2 (RPS2) is a new regulator of MDM2-P53 signaling pathway to regulate p53 protein level. Here, we characterized that RPS2 interacts with MDM2 through the RING finger domain of MDM2. RPS2 is ubiquitinated by MDM2 and the ubiquitinated status of RPS2 regulates the stability of p53, which is activated in response to cellular stresses such as DNA damage, oxidative stress, and especially ribosomal stress. In addition, p53 is not induced in RPS2 knockdown even in the ribosomal stressed condition, indicating that RPS2 is essential for the stabilization of p53. Collectively, our data suggest that RPS2 plays a critical role in the regulation of p53 signaling including the ribosomal stress response. © 2020 Elsevier Inc. All rights reserved.
Keywords: Ribosomal protein S2 MDM2 p53 Ubiquitination
1. Introduction The tumor suppressor p53 is a key protein for cell cycle control and apoptosis [1,2]. Numerous cellular stresses such as DNA damage, oxidative stress, and ribosome biogenesis stress trigger the induction of p53, which in turn induces its target genes related to cell cycle arrest, apoptosis, and senescence [3e5]. The expression of p53 is regulated by a ubiquitin-proteasome system, and MDM2, an E3 ligase, is known as the primary regulator of p53 [6]. Under normal conditions, p53 is ubiquitinated by MDM2 and degraded by the proteasome to maintain low levels. However, p53 is activated via the inhibition of MDM2 under stress conditions. Ribosomal stress is also a well-known condition in which p53 is activated to mediate cellular responses [5,7,8]. Ribosome biogenesis is a highly delicate cellular process that produces mature ribosomes [7,8]. Transcription and processing of rRNA, proper synthesis of rRNA and ribosomal proteins (RPs), and coordinated assembly of pre-ribosome subunits occur in the
* Corresponding author. Graduate School of Pharmaceutical Sciences and College of Pharmacy, Ewha Womans University 52, Ewhayeodae-gil, Seodaemun-gu, Seoul 03760 Republic of Korea. E-mail address: [email protected] (E.J. Song).
nucleolus. Afterward, pre-ribosome subunits are exported to the nucleoplasm and cytoplasm, where mature ribosomes are generated. The import of nuclear RPs from the cytoplasm to the nucleus is also involved in ribosome biogenesis. Ribosomal stress is induced by the impairment of ribosome biogenesis. For example, the inhibition of rRNA transcription, rRNA processing, and pre-ribosome subunit assembly induce ribosomal stress. The inhibition of preribosome subunit export and the inhibition of RP nuclear import also induce ribosomal stress [8]. Ribosomal stress is also induced by the exposure of genotoxic reagents like doxorubicin or actinomycin D (ActD). Interestingly, ribosomal stress induces the accumulation of ribosome-free RPs and these RPs are involved in various cellular processes such as the MDM2-p53 signaling pathway [9]. However, the function and its mechanism of each RP in MDM2-p53 pathway are still unknown. Recently, with increased understanding of the RP-MDM2-p53 pathway, it has been conducted to determine the correlations between MDM2 and RPs and an underlying mechanism for the regulation of p53 and MDM2 by RPs has been described [9]. RPL11 and RPL5 bind to MDM2 and block MDM2-mediated p53 ubiquitination and degradation, thereby resulting in p53-dependent cell cycle arrest during ribosomal stress [10]. In addition, RPS7 and RPS27a are ubiquitinated by MDM2 and the ubiquitinated RPs regulate MDM2 activity in response to ribosomal stress as a mutual
https://doi.org/10.1016/j.bbrc.2020.01.038 0006-291X/© 2020 Elsevier Inc. All rights reserved.
Please cite this article as: J. Cho et al., Ribosomal protein S2 interplays with MDM2 to induce p53, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.01.038
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Fig. 1. RPS2 interacts with MDM2 via the RING finger domain of MDM2. (A) HEK293T cells were overexpressed with HA-RPS2 and Myc-MDM2. Cell lysates were harvested after 24 h and then were immunoprecipitated with HA agarose. Immunoprecipitated proteins were detected with the indicated antibodies. (B) HEK293T cells were overexpressed with the pCS2 vector or HA-RPS2, and the cell lysates were immunoprecipitated with anti-HA agarose and detected with the indicated antibodies. (C) Wild-type (WT) Myc-MDM2 and its truncations including Myc-MDM21222, Myc-MDM2223339, and Myc-MDM2340441 were co-overexpressed in HEK293T cells with HA-RPS2 and immunoprecipitation was performed with anti-HA agarose. Bound proteins were immunoblotted with the indicated antibodies. The non-specific band is denoted by an asterisk.
regulatory loop [11,12]. Therefore, the RP-MDM2-p53 signaling pathway is known to serve as a molecular switch for monitoring the integrity of ribosomal biogenesis. In this study, we thought that other RPs beside RPL11 and RPL5 might be involved in the MDM2-p53 pathway, because the ribosome consists of more than 70 proteins and needs tight regulation for its function. Here, we found that RPS2 interacts with MDM2 through the RING finger domain of MDM2. Moreover, we report that RPS2 is ubiquitinated by MDM2 and increases MDM2 ubiquitination. Especially, we characterized that the ubiquitinated status of RPS2 by MDM2 functions as an intracellular signal rather than proteasomal degradation. By this interplay between RPS2 and MDM2, RPS2 induces and stabilizes p53. In this study, we show that RPS2 plays a critical role in regulating p53 signaling including the ribosomal stress response. These findings suggest that RPS2 could be a delicate regulator to maintain the p53 protein level.
according to the manufacturer’s instructions. The control siRNA sequence was 50 -CCUACGCCACCAAUUUCGU-3’. The RPS2 siRNA sequences were #1 50 -TAAAGTGAATTAAGCGTGA-30 and #2 50 GAGGCAAGGCCGAGGATAA-3’ (Genolution). 2.2. Plasmids The coding sequence for human RPS2, were amplified by PCR and cloned into the pCS2 expression plasmid, incorporating HA or Myc tag at the N terminus. Deletion mutants of MDM2 including MDM21222, MDM2223339, and MDM2340491, were amplified by PCR and cloned into the same vectors as previously described. pCS2-Myc-MDM2, pCMV-MDM2, and pCMV-MDM2C464A were a kind gift from Dr. SH Baek [14]. Ubiquitin and various mutants were prepared as previously described [15]. 2.3. Antibodies and reagents
2. Materials and methods 2.1. Cell lines and transfection HEK293T cells, U2OS cells, A549 cells, H1299 cells, and HeLa cells were purchased from Korea Cell Line Bank (KCLB). HEK293T cells and HeLa cells were grown in Dulbecco’s Modified Eagle’s Medium (DMEM). U2OS cells, A549 cells, and H1299 cells were grown in Roswell Park Memorial Institute 1640 (RPMI 1640). Both media contained 10% fetal bovine serum, 100 units/ml penicillin and 100 mg/ml streptomycin. All of the cell lines were grown at 37 C in 5% CO2 and 95% air. Transfection in cells was performed with either Mirus LT-1 (Mirus) or Effectene (Qiagen) following the manufacturer’s instructions. Plasmids were transfected into HEK 293T cells, using the calcium phosphate/DNA co-precipitation method [13]. siRNA was transfected into cells using Lipofectamine2000 (Invitrogen)
The following antibodies were purchased and used for immunostaining and western blotting. Antibodies for RPS2 were purchased from Bethyl Laboratories. Antibodies for p53, p21, HA, and Myc were provided from Santa Cruz Biotechnology. Flag antibody was purchased from Sigma Aldrich. MDM2 antibody was purchased from Calbiochem and b-actin antibody was provided from Ab Frontier. Cells were treated with MG132 (A G scientific) at a 10 mM concentration to inhibit proteasomal degradation. Cells were treated with Actinomycin D (Merck) at a 5 nM concentration to induce ribosomal stress. Cells were treated with cycloheximide at a 100 mg/ml concentration to inhibit protein synthesis. 2.4. Immunoprecipitation Cells were harvested and lysed with cell lysis buffer (50 mM Tris-Cl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.5% NP-40, protease
Please cite this article as: J. Cho et al., Ribosomal protein S2 interplays with MDM2 to induce p53, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.01.038
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Fig. 2. RPS2 is ubiquitinated by MDM2. (A) HA-RPS2 was co-overexpressed with His-ubiquitin, MDM2, or MDM2C464A in HEK293T cells. After treatment with MG132 (10 mM) for 4 h, His-ubiquitin and covalently modified proteins were purified from the cells under denaturing conditions on Ni-NTA agarose. Ubiquitinated RPS2 was immunoblotted with antiHA antibody. (B) HA-RPS2 and Myc-MDM2 were co-overexpressed in HEK293T cells in the presence of His-ubiquitin WT, His-ubiquitinK11R, His-ubiquitinK48R, or His-ubiquitinK63R. After treatment with MG132 (10 mM) for 4 h, ubiquitin conjugates were purified on Ni-NTA agarose under denaturing conditions, and ubiquitinated RPS2 was detected with the anti-HA antibody. (C) HEK293T cells were overexpressed with the HA-RPS2, MDM2, ubiquitin, ubiquitinK11only, ubiquitinK48only, ubiquitinK63only. After treatment with MG132 (10 mM) for 4 h, the cell lysates were immunoprecipitated with anti-HA agarose and detected with the indicated antibodies. (D) HA-RPS2 was co-expressed with Myc-MDM2 in HeLa cells. After 24 h, the cells were treated with either DMSO (control) or MG132 (10 mM) for 4 h. Cell lysates were immunoblotted with the indicated antibodies.
inhibitor cocktail (Roche) and 1 mM sodium orthovanadate), incubated for 10 min and centrifuged at 12,000 RPM for 15 min at 4 C. Protein concentrations in the supernatants of the cell lysates were measured using a BCA kit (Thermo Scientific). 2 mg of cell lysates were incubated with agarose for 4 h at 4 C, and the agarose was washed three times with lysis buffer. Monoclonal anti-HA agarose conjugate (Sigma Aldrich) was used for HA-IP. After the last wash step, the supernatant was removed carefully with a syringe. For HA-IP, the agarose was eluted with 2X SDS sample buffer by boiling at 95 C and then separated by SDS-PAGE and subjected to western blotting.
2.5. Ni-NTA pulldown assay Transfected cells were harvested with urea lysis buffer (8 M urea, 300 mM NaCl, 50 mM Na2HPO4, 50 mM Tris, 1 mM phenylmethylsulfonyl fluoride (PMSF) and 10 mM imidazole, pH 8.0) by scraping. Harvested cells were carefully lysed by sonication. Each sample was incubated with Ni-NTA agarose (Qiagen) for 4 h at 4 C in a rotator. The agarose was washed five times with urea lysis buffer (8 M urea, 300 mM NaCl, 50 mM Na2HPO4, 50 mM Tris, 1 mM PMSF and 20 mM imidazole, pH 8.0). After the last wash step, the supernatants were carefully removed with a syringe. The agarose was eluted with 2X SDS sample buffer by boiling at 95 C. The samples were separated by SDS-PAGE for detection and subjected
Please cite this article as: J. Cho et al., Ribosomal protein S2 interplays with MDM2 to induce p53, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.01.038
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Fig. 3. RPS2 induces and stabilizes p53 protein level. (A) HeLa cells transfected with siRNA against control or RPS2 were treated with either DMSO (control) or MG132 (10 mM) for 4 h. Cell lysates were immunoblotted with the indicated antibodies. (B) U2OS cells overexpressed with pCS2 vector or HA-RPS2 were treated with either DMSO (control) or MG132 (10 mM) for 4 h. Cell lysates were immunoblotted with the indicated antibodies. (C) U2OS cells were overexpressed with increasing amounts of HA-RPS2 (0, 1, and 2 mg). After 24 h, cell lysates were immunoblotted with the indicated antibodies. (D) U2OS cells overexpressed with the pCS2 vector or HA-RPS2 were treated with 100 mg/ml cycloheximide and harvested at the indicated times. Cell lysates were immunoblotted with the indicated antibodies.
to western blotting and chemiluminescence was measured using the Ez-Capture MG imaging system (ATTO Corporation). 2.6. Quantitative reverse transcription-PCR analysis Total RNA was extracted using RNeasy Mini Kit (Qiagen) from cells. cDNAs were synthesized from total RNA using ReverTra Ace qPCR RT Master Mix (Toyobo) and analyzed using SYBR Green Realtime PCR Master Mix (Toyobo). All results were normalized to bactin expression. Used primer sequences are given as follows. The RPS2 primers were (F) 50 -GGAACAAGATCGGCAAGC-30 and (R) 50 GGAGACGATGCCAGTGCCC-3’. The p21 primers were (F) 50 -CACCACTGGAGGGTGACTTC-30 and (R) 50 -ATCTGTCATGCTGGTCTGCC-3’. The E2F7 primers were (F) 50 -GGAAAGGCAACAGCAAACTCT-30 and (R) 50 -GAGAGCACCAAGAGTAGAAGA-3’. The GADD45a primers were (F) 50 -TGCGAGAACGACATCAACAT-30 and (R) 50 -TCCC GGCAAAAACAAATAAG-3’. The b-actin primers were (F) 50 CTCTTCCAGCCTTCCTTCCT-30 and (R) 50 -AGCACTGTGTTGGCGTACAG-3’. 3. Results and discussion 3.1. RPS2 interacts with MDM2 via RING finger domain of MDM2 Based on the fact that MDM2 is reported to function as an E3 ligase of other RPs [10,11], we speculated that MDM2 might interact with RPS2. To examine this possibility, we performed immunoprecipitation experiments as shown in Fig. 1. When the possible interactions between the two were examined by transfecting MDM2 and RPS2, we were able to observe the binding between MDM2 and RPS2 (Fig. 1A). Overexpressed HA-RPS2 interacted with myc-MDM2. Moreover, the interaction between HA-RPS2 and endogenous MDM2 in cells was observed (Fig. 1B). Next, to determine the region required for the interaction between MDM2 and RPS2, truncated MDM2 was overexpressed. We made three kinds of truncated MDM2: MDM21222, MDM2223339, and MDM2340491. MDM21222 includes the p53 binding domain, MDM2223339
includes an acidic and zinc finger domain, and MDM2340491 includes the RING finger domain. Further interaction analysis revealed that amino acids 340e491, which include the RING finger domain of MDM2, were required for the interaction with RPS2 (Fig. 1C), suggesting that MDM2 might function as an E3 ligase of RPS2. Therefore, we next examined whether RPS2 is a substrate of MDM2. 3.2. RPS2 is ubiquitinated by MDM2 Since several RPs such as RPS7 [11], RPS27a [12], and RPL6 [16] are known to be ubiquitinated by MDM2, we next assessed the RPS2 ubiquitination with the Ni-NTA assay. When RPS2 was coexpressed with MDM2 and ubiquitin, high-molecular-weight forms of RPS2 were detected as shown in Fig. 2A. These variants represented a covalent modification of RPS2 with ubiquitin, as confirmed through denaturing Ni-NTA pull-down assays. RPS2 was ubiquitinated by MDM2 but not by the E3 ligase-inactive mutant, MDM2C464A, indicating that MDM2 is an E3 ligase of RPS2 (Fig. 2A). Next, we investigated the linkage specificity of the RPS2-bound ubiquitin chain by co-overexpressing RPS2 with Lys-to-Arg mutants of ubiquitin. The result showed that RPS2 ubiquitination was only slightly affected by the overexpression of a K48R mutant of ubiquitin and was especially decreased by the overexpression of a K63R mutant (Fig. 2B). To make sure this result, we used another type of ubiquitin mutants; of which all Lys are replaced by Arg except K11 or K48 or K63. For example, Ubiquitin K63-only allows the formation of only K63-linked ubiquitin chains, precluding another chain-linked ubiquitination. Consistent with Fig. 2B, RPS2 ubiquitination was not occurred by the ubiquitin K11-only and slightly decreased by the ubiquitin K48-only. However, RPS2 ubiquitination was especially increased by the ubiquitin K63-only (Fig. 2C). These results indicate that MDM2-mediated RPS2 ubiquitination is mainly related to the K63-linked ubiquitin chain. Substrates have different fates depending on the ubiquitin-linked chain specificity. K48-linked ubiquitination of the substrate leads to proteasomal degradation. However, K63-linked ubiquitination
Please cite this article as: J. Cho et al., Ribosomal protein S2 interplays with MDM2 to induce p53, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.01.038
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Fig. 4. RPS2 inhibits MDM2, thereby inducing both p53 and its downstream pathway. (A) HEK293T cells were co-overexpressed with His-ubiquitin, p53, MDM2, and HA-RPS2 for 24 h, then treated with MG132 (10 mM) for 4 h. Ubiquitin conjugates were purified on Ni-NTA-agarose under denaturing conditions, and ubiquitinated p53 was detected with anti-p53 antibody. (B) H1299 cells were co-overexpressed with MDM2, His-ubiquitin, and HA-RPS2, then treated with 10 mM MG132 for 4 h. Ubiquitin conjugates were purified on Ni-NTA-agarose under denaturing conditions, and ubiquitinated MDM2 was detected by anti-MDM2 antibodies. (C) Real-time PCR analysis of p53 downstream genes in A549 cells transfected with siRNA against control or RPS2 for 72 h. Relative expression values are normalized against b-actin RNA levels and graphs are shown as fold induction over control siRNA-transfected cells. (D) A549 cells transfected with siRNA against control or RPS2 were treated with either DMSO or 5 nM actinomycin D for 4 h. Cell lysates were immunoblotted with the indicated antibodies.
has essential roles in cellular signaling [17]. Accordingly, this result indicates that a major portion of ubiquitinated RPS2 by MDM2 has essential roles in cellular signaling rather than proteasomal degradation. To confirm this result, we compared the RPS2 protein level. The expression of RPS2 was not affected by co-overexpression with MDM2 with or without MG132 treatment (Fig. 2D). Taken together, these results indicate that RPS2 is a substrate of MDM2, and its ubiquitination, which is mainly associated with the K63linked ubiquitin chain, may act as a signal in a cellular pathway, rather than inducing degradation by the proteasome. 3.3. RPS2 induces and stabilizes p53 protein level It has demonstrated that several types of RPs are involved in the regulation of p53 activation and we newly confirmed the interaction between MDM2 and RPS2 as described above. Therefore, we first examined whether RPS2 is involved in the p53 signaling pathway. As shown in Fig. 3A, the protein level of p53 was downregulated in RPS2-knockdown cells and this was rescued by treatment with the proteasome inhibitor MG132. Conversely, p53 was slightly increased in the overexpression of HA-RPS2 but remained unchanged even in the presence of RPS2 following treatment with
MG132 (Fig. 3B). These results indicate that RPS2 upregulates the p53 protein level in a proteasome-dependent manner. To clarify the effect of RPS2 on the p53 level, we transiently overexpressed increasing amounts of HA-RPS2. The p53 level increased in a dosedependent manner, and p21, a downstream gene of p53, showed the same increasing pattern as p53 (Fig. 3C). To further investigate the changes in the p53 levels induced by RPS2, we treated cells with cycloheximide and harvested the cells for a time course analysis. The degradation assay showed that p53 degradation was delayed when HA-RPS2 was overexpressed (Fig. 3D). These results suggest that RPS2 regulates the stability of p53 in a proteasome-dependent manner. 3.4. RPS2 inhibits MDM2, thereby inducing both p53 and its downstream pathway Because the stability of p53 is mainly regulated by MDM2dependent ubiquitination, we examined whether RPS2 has an effect on the MDM2-mediated ubiquitination of p53 using a Ni-NTA pull-down assay. As shown in Fig. 4A, MDM2 increased the ubiquitination of p53 but overexpression of RPS2 attenuated the ubiquitination of p53 by MDM2. It has been reported that RPL11 inhibits
Please cite this article as: J. Cho et al., Ribosomal protein S2 interplays with MDM2 to induce p53, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.01.038
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MDM2 function by increasing the ubiquitination of MDM2 [18]. Therefore, we examined whether RPS2 has an effect on the ubiquitination of MDM2. MDM2 is a crucial down-regulator of p53 but p53 upregulates MDM2 in a positive feedback loop. To exclude the positive feedback effect on MDM2 ubiquitination from the increased p53, we performed the Ni-NTA pull-down assays with a p53-deficient H1299 cell line (Fig. 4B). Consistent with previous reports about other RPs, RPS2 also increased the ubiquitination of MDM2. These results indicate that RPS2 inhibits MDM2 by inducing MDM2 ubiquitination. Since RPS2 regulates p53 stability with this mechanism, it can also regulate the transcriptional activity of p53. To elucidate the effect of RPS2 on p53 activity, we performed realtime PCR to compare the expression level of p53 target genes under RPS2 knockdown. As expected, RPS2 knockdown downregulated the transcriptional level of p53 downstream genes, such as p21, E2F7, and GADD45a (Fig. 4C). These data indicate that RPS2 regulates not only the protein levels of p53 but also p53 activity for the downstream pathway. Based on these results, we concluded that RPS2 stabilizes p53 by inhibiting MDM2-dependent ubiquitination of p53. RPs are essential factors in ribosome biogenesis, and perturbations in ribosome biogenesis induce ribosomal stress, which can trigger activation of the p53 signaling pathway [5]. Since the upregulation of p53 by RPs mainly occurred under ribosomal stress, we checked the protein level of p53 in the depletion of RPS2 to investigate the effect of RPS2 under ribosomal stress. As shown in Fig. 4D, ribosomal stress increased p53 but did not increase p53 under RPS2 depletion. These results suggest that RPS2 is required for p53 stabilization under ribosomal stress. Thus far, there have only been a few reports that show the posttranslational modifications (PTMs) of RPS2. RPS2 interacts with protein arginine methyltransferase 3 (PRMT3). PRMT3 reduces the ubiquitination of RPS2 and stabilizes RPS2 protein [19] and RPS2 is methylated by PRMT3 [20]. Furthermore, ZNF598 and RACK1 mediate the ubiquitination of ribosomal small subunit proteins including RPS2 in mature ribosomes, thereby participating in mammalian ribosome-associated quality control through regulatory ribosomal ubiquitination (RRub) [21]. These studies suggest that RPS2 can have different roles in the cell depending on the PTM. Here, we found the novel PTM of RPS2 involved in the p53 signaling pathway. We showed that the RPS2 is ubiquitinated by MDM2 and MDM2-mediated RPS2 ubiquitination is mainly associated with the K63-linked ubiquitin chain, which means it can be used as a regulatory signal for cellular mechanisms, not for proteasomal degradation [17]. In addition, this modification of RPS2 is required for the regulation of the MDM2-p53 signaling pathway by inhibiting MDM2 through an increase in ubiquitinated MDM2, leading to p53 accumulation. In conclusion, we provide evidence that RPS2 is both a regulator and a substrate of MDM2. Therefore, RPS2 interplays with MDM2 to regulate p53.
Author contributions J.C., J.H.K., E.E.K., and E.J.S. conceived and designed the experiments. J.C. and J.P. performed the majority of experiments and S.C.S and E.E.K carried out biochemical assay and analyzed the data. J.C., E.E.K, and E.J.S. wrote the manuscript.
Declaration of competing interest The authors declare no potential conflicts of interests. Acknowledgements This work was supported by a National Research Foundation of Korea (NRF) grant funded by the Korean government (MSIP) (2019R1A2C2004052 and 2017R1A2B3007224) and the R&D Convergence Program of NST (National Research Council of Science & Technology) of Republic of Korea (CAP-16-03-KRIBB). References [1] F. Bunz, A. Dutriaux, C. Lengauer, T. Waldman, S. Zhou, J.P. Brown, J.M. Sedivy, K.W. Kinzler, B. Vogelstein, Requirement for p53 and p21 to sustain G2 arrest after DNA damage, Science (N.Y.) 282 (1998) 1497e1501. [2] C.E. Canman, C.Y. Chen, M.H. Lee, M.B. Kastan, DNA damage responses: p53 induction, cell cycle perturbations, and apoptosis, Cold Spring Harbor Symp. Quant. Biol. 59 (1994) 277e286. [3] D. Liu, Y. Xu, p53, oxidative stress, and aging, Antioxidants Redox Signal. 15 (2011) 1669e1678. [4] K.H. Vousden, C. Prives, Blinded by the light: the growing complexity of p53, Cell 137 (2009) 413e431. [5] S. Bursac, M.C. Brdovcak, G. Donati, S. Volarevic, Activation of the tumor suppressor p53 upon impairment of ribosome biogenesis, Biochim. Biophys. Acta 1842 (2014) 817e830. [6] J.P. Kruse, W. Gu, Modes of p53 regulation, Cell 137 (2009) 609e622. [7] S. Boulon, B.J. Westman, S. Hutten, F.M. Boisvert, A.I. Lamond, The nucleolus under stress, Mol. Cell 40 (2010) 216e227. [8] L. Golomb, S. Volarevic, M. Oren, p53 and ribosome biogenesis stress: the essentials, FEBS Lett. 588 (2014) 2571e2579. [9] X. Zhou, W.J. Liao, J.M. Liao, P. Liao, H. Lu, Ribosomal proteins: functions beyond the ribosome, J. Mol. Cell Biol. 7 (2015) 92e104. [10] S. Bursac, M.C. Brdovcak, M. Pfannkuchen, I. Orsolic, L. Golomb, Y. Zhu, C. Katz, L. Daftuar, K. Grabusic, I. Vukelic, V. Filic, M. Oren, C. Prives, S. Volarevic, Mutual protection of ribosomal proteins L5 and L11 from degradation is essential for p53 activation upon ribosomal biogenesis stress, Proc. Natl. Acad. Sci. U.S.A. 109 (2012) 20467e20472. [11] Y. Zhu, M.V. Poyurovsky, Y. Li, L. Biderman, J. Stahl, X. Jacq, C. Prives, Ribosomal protein S7 is both a regulator and a substrate of MDM2, Mol. Cell 35 (2009) 316e326. [12] X.X. Sun, T. DeVine, K.B. Challagundla, M.S. Dai, Interplay between ribosomal protein S27a and MDM2 protein in p53 activation in response to ribosomal stress, J. Biol. Chem. 286 (2011) 22730e22741. [13] R.E. Kingston, C.A. Chen, H. Okayama, Calcium Phosphate Transfection, Current Protocols in Cell Biology, 2003 (Chapter 20), Unit 20.23. [14] H. Kim, J.M. Lee, G. Lee, J. Bhin, S.K. Oh, K. Kim, K.E. Pyo, J.S. Lee, H.Y. Yim, K.I. Kim, D. Hwang, J. Chung, S.H. Baek, DNA damage-induced RORalpha is crucial for p53 stabilization and increased apoptosis, Mol. Cell 44 (2011) 797e810. [15] E.J. Song, S.L. Werner, J. Neubauer, F. Stegmeier, J. Aspden, D. Rio, J.W. Harper, S.J. Elledge, M.W. Kirschner, M. Rape, The Prp19 complex and the Usp4Sart3 deubiquitinating enzyme control reversible ubiquitination at the spliceosome, Genes Dev. 24 (2010) 1434e1447. [16] D. Bai, J. Zhang, W. Xiao, X. Zheng, Regulation of the HDM2-p53 pathway by ribosomal protein L6 in response to ribosomal stress, Nucleic Acids Res. 42 (2014) 1799e1811. [17] Y. Kulathu, D. Komander, Atypical ubiquitylation - the unexplored world of polyubiquitin beyond Lys48 and Lys63 linkages, Nature reviews, Mol. Cell. Biol. 13 (2012) 508e523. [18] M.S. Dai, D. Shi, Y. Jin, X.X. Sun, Y. Zhang, S.R. Grossman, H. Lu, Regulation of the MDM2-p53 pathway by ribosomal protein L11 involves a postubiquitination mechanism, J. Biol. Chem. 281 (2006) 24304e24313. [19] S. Choi, C.R. Jung, J.Y. Kim, D.S. Im, PRMT3 inhibits ubiquitination of ribosomal protein S2 and together forms an active enzyme complex, Biochim. Biophys. Acta 1780 (2008) 1062e1069. [20] R. Swiercz, D. Cheng, D. Kim, M.T. Bedford, Ribosomal protein rpS2 is hypomethylated in PRMT3-deficient mice, J. Biol. Chem. 282 (2007) 16917e16923. [21] E. Sundaramoorthy, M. Leonard, R. Mak, J. Liao, A. Fulzele, E.J. Bennett, ZNF598 and RACK1 regulate mammalian ribosome-associated quality control function by mediating regulatory 40S ribosomal ubiquitylation, Mol. Cell 65 (2017) 751e760, e754.
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Ribosomal protein S2 interplays with MDM2 to induce p53 Jinhong Cho a, b, Jinyoung Park c, Sang Chul Shin a, Jae-Hong Kim b, Eunice EunKyeong Kim a, Eun Joo Song d, * a
Biomedical Research Institute, Korea Institute of Science and Technology, Hwarangno 14-gil 5, Seongbuk-gu, Seoul, 02792, Republic of Korea Department of Biotechnology, College of Life Sciences and Biotechnology, Korea University, 5-1 Anam-dong, Sungbuk-gu, Seoul, 02841, Republic of Korea c Molecular Recognition Research Center, Korea Institute of Science and Technology, Hwarangno 14-gil 5, Seongbuk-gu, Seoul, 02792, Republic of Korea d Graduate School of Pharmaceutical Sciences, College of Pharmacy, Ewha Womans University, Seoul, 03760, Republic of Korea b
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Article history: Received 25 December 2019 Accepted 5 January 2020 Available online xxx
The MDM2-p53 pathway is crucial for maintenance of p53 homeostasis. Some ribosomal proteins (RPs) play critical roles in regulating p53 by interacting with MDM2. However, the role and functional mechanism of each RP in MDM2-p53 pathway still remain unknown. In this study, we found that Ribosomal Protein S2 (RPS2) is a new regulator of MDM2-P53 signaling pathway to regulate p53 protein level. Here, we characterized that RPS2 interacts with MDM2 through the RING finger domain of MDM2. RPS2 is ubiquitinated by MDM2 and the ubiquitinated status of RPS2 regulates the stability of p53, which is activated in response to cellular stresses such as DNA damage, oxidative stress, and especially ribosomal stress. In addition, p53 is not induced in RPS2 knockdown even in the ribosomal stressed condition, indicating that RPS2 is essential for the stabilization of p53. Collectively, our data suggest that RPS2 plays a critical role in the regulation of p53 signaling including the ribosomal stress response. © 2020 Elsevier Inc. All rights reserved.
Keywords: Ribosomal protein S2 MDM2 p53 Ubiquitination
1. Introduction The tumor suppressor p53 is a key protein for cell cycle control and apoptosis [1,2]. Numerous cellular stresses such as DNA damage, oxidative stress, and ribosome biogenesis stress trigger the induction of p53, which in turn induces its target genes related to cell cycle arrest, apoptosis, and senescence [3e5]. The expression of p53 is regulated by a ubiquitin-proteasome system, and MDM2, an E3 ligase, is known as the primary regulator of p53 [6]. Under normal conditions, p53 is ubiquitinated by MDM2 and degraded by the proteasome to maintain low levels. However, p53 is activated via the inhibition of MDM2 under stress conditions. Ribosomal stress is also a well-known condition in which p53 is activated to mediate cellular responses [5,7,8]. Ribosome biogenesis is a highly delicate cellular process that produces mature ribosomes [7,8]. Transcription and processing of rRNA, proper synthesis of rRNA and ribosomal proteins (RPs), and coordinated assembly of pre-ribosome subunits occur in the
* Corresponding author. Graduate School of Pharmaceutical Sciences and College of Pharmacy, Ewha Womans University 52, Ewhayeodae-gil, Seodaemun-gu, Seoul 03760 Republic of Korea. E-mail address: [email protected] (E.J. Song).
nucleolus. Afterward, pre-ribosome subunits are exported to the nucleoplasm and cytoplasm, where mature ribosomes are generated. The import of nuclear RPs from the cytoplasm to the nucleus is also involved in ribosome biogenesis. Ribosomal stress is induced by the impairment of ribosome biogenesis. For example, the inhibition of rRNA transcription, rRNA processing, and pre-ribosome subunit assembly induce ribosomal stress. The inhibition of preribosome subunit export and the inhibition of RP nuclear import also induce ribosomal stress [8]. Ribosomal stress is also induced by the exposure of genotoxic reagents like doxorubicin or actinomycin D (ActD). Interestingly, ribosomal stress induces the accumulation of ribosome-free RPs and these RPs are involved in various cellular processes such as the MDM2-p53 signaling pathway [9]. However, the function and its mechanism of each RP in MDM2-p53 pathway are still unknown. Recently, with increased understanding of the RP-MDM2-p53 pathway, it has been conducted to determine the correlations between MDM2 and RPs and an underlying mechanism for the regulation of p53 and MDM2 by RPs has been described [9]. RPL11 and RPL5 bind to MDM2 and block MDM2-mediated p53 ubiquitination and degradation, thereby resulting in p53-dependent cell cycle arrest during ribosomal stress [10]. In addition, RPS7 and RPS27a are ubiquitinated by MDM2 and the ubiquitinated RPs regulate MDM2 activity in response to ribosomal stress as a mutual
https://doi.org/10.1016/j.bbrc.2020.01.038 0006-291X/© 2020 Elsevier Inc. All rights reserved.
Please cite this article as: J. Cho et al., Ribosomal protein S2 interplays with MDM2 to induce p53, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.01.038
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Fig. 1. RPS2 interacts with MDM2 via the RING finger domain of MDM2. (A) HEK293T cells were overexpressed with HA-RPS2 and Myc-MDM2. Cell lysates were harvested after 24 h and then were immunoprecipitated with HA agarose. Immunoprecipitated proteins were detected with the indicated antibodies. (B) HEK293T cells were overexpressed with the pCS2 vector or HA-RPS2, and the cell lysates were immunoprecipitated with anti-HA agarose and detected with the indicated antibodies. (C) Wild-type (WT) Myc-MDM2 and its truncations including Myc-MDM21222, Myc-MDM2223339, and Myc-MDM2340441 were co-overexpressed in HEK293T cells with HA-RPS2 and immunoprecipitation was performed with anti-HA agarose. Bound proteins were immunoblotted with the indicated antibodies. The non-specific band is denoted by an asterisk.
regulatory loop [11,12]. Therefore, the RP-MDM2-p53 signaling pathway is known to serve as a molecular switch for monitoring the integrity of ribosomal biogenesis. In this study, we thought that other RPs beside RPL11 and RPL5 might be involved in the MDM2-p53 pathway, because the ribosome consists of more than 70 proteins and needs tight regulation for its function. Here, we found that RPS2 interacts with MDM2 through the RING finger domain of MDM2. Moreover, we report that RPS2 is ubiquitinated by MDM2 and increases MDM2 ubiquitination. Especially, we characterized that the ubiquitinated status of RPS2 by MDM2 functions as an intracellular signal rather than proteasomal degradation. By this interplay between RPS2 and MDM2, RPS2 induces and stabilizes p53. In this study, we show that RPS2 plays a critical role in regulating p53 signaling including the ribosomal stress response. These findings suggest that RPS2 could be a delicate regulator to maintain the p53 protein level.
according to the manufacturer’s instructions. The control siRNA sequence was 50 -CCUACGCCACCAAUUUCGU-3’. The RPS2 siRNA sequences were #1 50 -TAAAGTGAATTAAGCGTGA-30 and #2 50 GAGGCAAGGCCGAGGATAA-3’ (Genolution). 2.2. Plasmids The coding sequence for human RPS2, were amplified by PCR and cloned into the pCS2 expression plasmid, incorporating HA or Myc tag at the N terminus. Deletion mutants of MDM2 including MDM21222, MDM2223339, and MDM2340491, were amplified by PCR and cloned into the same vectors as previously described. pCS2-Myc-MDM2, pCMV-MDM2, and pCMV-MDM2C464A were a kind gift from Dr. SH Baek [14]. Ubiquitin and various mutants were prepared as previously described [15]. 2.3. Antibodies and reagents
2. Materials and methods 2.1. Cell lines and transfection HEK293T cells, U2OS cells, A549 cells, H1299 cells, and HeLa cells were purchased from Korea Cell Line Bank (KCLB). HEK293T cells and HeLa cells were grown in Dulbecco’s Modified Eagle’s Medium (DMEM). U2OS cells, A549 cells, and H1299 cells were grown in Roswell Park Memorial Institute 1640 (RPMI 1640). Both media contained 10% fetal bovine serum, 100 units/ml penicillin and 100 mg/ml streptomycin. All of the cell lines were grown at 37 C in 5% CO2 and 95% air. Transfection in cells was performed with either Mirus LT-1 (Mirus) or Effectene (Qiagen) following the manufacturer’s instructions. Plasmids were transfected into HEK 293T cells, using the calcium phosphate/DNA co-precipitation method [13]. siRNA was transfected into cells using Lipofectamine2000 (Invitrogen)
The following antibodies were purchased and used for immunostaining and western blotting. Antibodies for RPS2 were purchased from Bethyl Laboratories. Antibodies for p53, p21, HA, and Myc were provided from Santa Cruz Biotechnology. Flag antibody was purchased from Sigma Aldrich. MDM2 antibody was purchased from Calbiochem and b-actin antibody was provided from Ab Frontier. Cells were treated with MG132 (A G scientific) at a 10 mM concentration to inhibit proteasomal degradation. Cells were treated with Actinomycin D (Merck) at a 5 nM concentration to induce ribosomal stress. Cells were treated with cycloheximide at a 100 mg/ml concentration to inhibit protein synthesis. 2.4. Immunoprecipitation Cells were harvested and lysed with cell lysis buffer (50 mM Tris-Cl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.5% NP-40, protease
Please cite this article as: J. Cho et al., Ribosomal protein S2 interplays with MDM2 to induce p53, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.01.038
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Fig. 2. RPS2 is ubiquitinated by MDM2. (A) HA-RPS2 was co-overexpressed with His-ubiquitin, MDM2, or MDM2C464A in HEK293T cells. After treatment with MG132 (10 mM) for 4 h, His-ubiquitin and covalently modified proteins were purified from the cells under denaturing conditions on Ni-NTA agarose. Ubiquitinated RPS2 was immunoblotted with antiHA antibody. (B) HA-RPS2 and Myc-MDM2 were co-overexpressed in HEK293T cells in the presence of His-ubiquitin WT, His-ubiquitinK11R, His-ubiquitinK48R, or His-ubiquitinK63R. After treatment with MG132 (10 mM) for 4 h, ubiquitin conjugates were purified on Ni-NTA agarose under denaturing conditions, and ubiquitinated RPS2 was detected with the anti-HA antibody. (C) HEK293T cells were overexpressed with the HA-RPS2, MDM2, ubiquitin, ubiquitinK11only, ubiquitinK48only, ubiquitinK63only. After treatment with MG132 (10 mM) for 4 h, the cell lysates were immunoprecipitated with anti-HA agarose and detected with the indicated antibodies. (D) HA-RPS2 was co-expressed with Myc-MDM2 in HeLa cells. After 24 h, the cells were treated with either DMSO (control) or MG132 (10 mM) for 4 h. Cell lysates were immunoblotted with the indicated antibodies.
inhibitor cocktail (Roche) and 1 mM sodium orthovanadate), incubated for 10 min and centrifuged at 12,000 RPM for 15 min at 4 C. Protein concentrations in the supernatants of the cell lysates were measured using a BCA kit (Thermo Scientific). 2 mg of cell lysates were incubated with agarose for 4 h at 4 C, and the agarose was washed three times with lysis buffer. Monoclonal anti-HA agarose conjugate (Sigma Aldrich) was used for HA-IP. After the last wash step, the supernatant was removed carefully with a syringe. For HA-IP, the agarose was eluted with 2X SDS sample buffer by boiling at 95 C and then separated by SDS-PAGE and subjected to western blotting.
2.5. Ni-NTA pulldown assay Transfected cells were harvested with urea lysis buffer (8 M urea, 300 mM NaCl, 50 mM Na2HPO4, 50 mM Tris, 1 mM phenylmethylsulfonyl fluoride (PMSF) and 10 mM imidazole, pH 8.0) by scraping. Harvested cells were carefully lysed by sonication. Each sample was incubated with Ni-NTA agarose (Qiagen) for 4 h at 4 C in a rotator. The agarose was washed five times with urea lysis buffer (8 M urea, 300 mM NaCl, 50 mM Na2HPO4, 50 mM Tris, 1 mM PMSF and 20 mM imidazole, pH 8.0). After the last wash step, the supernatants were carefully removed with a syringe. The agarose was eluted with 2X SDS sample buffer by boiling at 95 C. The samples were separated by SDS-PAGE for detection and subjected
Please cite this article as: J. Cho et al., Ribosomal protein S2 interplays with MDM2 to induce p53, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.01.038
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Fig. 3. RPS2 induces and stabilizes p53 protein level. (A) HeLa cells transfected with siRNA against control or RPS2 were treated with either DMSO (control) or MG132 (10 mM) for 4 h. Cell lysates were immunoblotted with the indicated antibodies. (B) U2OS cells overexpressed with pCS2 vector or HA-RPS2 were treated with either DMSO (control) or MG132 (10 mM) for 4 h. Cell lysates were immunoblotted with the indicated antibodies. (C) U2OS cells were overexpressed with increasing amounts of HA-RPS2 (0, 1, and 2 mg). After 24 h, cell lysates were immunoblotted with the indicated antibodies. (D) U2OS cells overexpressed with the pCS2 vector or HA-RPS2 were treated with 100 mg/ml cycloheximide and harvested at the indicated times. Cell lysates were immunoblotted with the indicated antibodies.
to western blotting and chemiluminescence was measured using the Ez-Capture MG imaging system (ATTO Corporation). 2.6. Quantitative reverse transcription-PCR analysis Total RNA was extracted using RNeasy Mini Kit (Qiagen) from cells. cDNAs were synthesized from total RNA using ReverTra Ace qPCR RT Master Mix (Toyobo) and analyzed using SYBR Green Realtime PCR Master Mix (Toyobo). All results were normalized to bactin expression. Used primer sequences are given as follows. The RPS2 primers were (F) 50 -GGAACAAGATCGGCAAGC-30 and (R) 50 GGAGACGATGCCAGTGCCC-3’. The p21 primers were (F) 50 -CACCACTGGAGGGTGACTTC-30 and (R) 50 -ATCTGTCATGCTGGTCTGCC-3’. The E2F7 primers were (F) 50 -GGAAAGGCAACAGCAAACTCT-30 and (R) 50 -GAGAGCACCAAGAGTAGAAGA-3’. The GADD45a primers were (F) 50 -TGCGAGAACGACATCAACAT-30 and (R) 50 -TCCC GGCAAAAACAAATAAG-3’. The b-actin primers were (F) 50 CTCTTCCAGCCTTCCTTCCT-30 and (R) 50 -AGCACTGTGTTGGCGTACAG-3’. 3. Results and discussion 3.1. RPS2 interacts with MDM2 via RING finger domain of MDM2 Based on the fact that MDM2 is reported to function as an E3 ligase of other RPs [10,11], we speculated that MDM2 might interact with RPS2. To examine this possibility, we performed immunoprecipitation experiments as shown in Fig. 1. When the possible interactions between the two were examined by transfecting MDM2 and RPS2, we were able to observe the binding between MDM2 and RPS2 (Fig. 1A). Overexpressed HA-RPS2 interacted with myc-MDM2. Moreover, the interaction between HA-RPS2 and endogenous MDM2 in cells was observed (Fig. 1B). Next, to determine the region required for the interaction between MDM2 and RPS2, truncated MDM2 was overexpressed. We made three kinds of truncated MDM2: MDM21222, MDM2223339, and MDM2340491. MDM21222 includes the p53 binding domain, MDM2223339
includes an acidic and zinc finger domain, and MDM2340491 includes the RING finger domain. Further interaction analysis revealed that amino acids 340e491, which include the RING finger domain of MDM2, were required for the interaction with RPS2 (Fig. 1C), suggesting that MDM2 might function as an E3 ligase of RPS2. Therefore, we next examined whether RPS2 is a substrate of MDM2. 3.2. RPS2 is ubiquitinated by MDM2 Since several RPs such as RPS7 [11], RPS27a [12], and RPL6 [16] are known to be ubiquitinated by MDM2, we next assessed the RPS2 ubiquitination with the Ni-NTA assay. When RPS2 was coexpressed with MDM2 and ubiquitin, high-molecular-weight forms of RPS2 were detected as shown in Fig. 2A. These variants represented a covalent modification of RPS2 with ubiquitin, as confirmed through denaturing Ni-NTA pull-down assays. RPS2 was ubiquitinated by MDM2 but not by the E3 ligase-inactive mutant, MDM2C464A, indicating that MDM2 is an E3 ligase of RPS2 (Fig. 2A). Next, we investigated the linkage specificity of the RPS2-bound ubiquitin chain by co-overexpressing RPS2 with Lys-to-Arg mutants of ubiquitin. The result showed that RPS2 ubiquitination was only slightly affected by the overexpression of a K48R mutant of ubiquitin and was especially decreased by the overexpression of a K63R mutant (Fig. 2B). To make sure this result, we used another type of ubiquitin mutants; of which all Lys are replaced by Arg except K11 or K48 or K63. For example, Ubiquitin K63-only allows the formation of only K63-linked ubiquitin chains, precluding another chain-linked ubiquitination. Consistent with Fig. 2B, RPS2 ubiquitination was not occurred by the ubiquitin K11-only and slightly decreased by the ubiquitin K48-only. However, RPS2 ubiquitination was especially increased by the ubiquitin K63-only (Fig. 2C). These results indicate that MDM2-mediated RPS2 ubiquitination is mainly related to the K63-linked ubiquitin chain. Substrates have different fates depending on the ubiquitin-linked chain specificity. K48-linked ubiquitination of the substrate leads to proteasomal degradation. However, K63-linked ubiquitination
Please cite this article as: J. Cho et al., Ribosomal protein S2 interplays with MDM2 to induce p53, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.01.038
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Fig. 4. RPS2 inhibits MDM2, thereby inducing both p53 and its downstream pathway. (A) HEK293T cells were co-overexpressed with His-ubiquitin, p53, MDM2, and HA-RPS2 for 24 h, then treated with MG132 (10 mM) for 4 h. Ubiquitin conjugates were purified on Ni-NTA-agarose under denaturing conditions, and ubiquitinated p53 was detected with anti-p53 antibody. (B) H1299 cells were co-overexpressed with MDM2, His-ubiquitin, and HA-RPS2, then treated with 10 mM MG132 for 4 h. Ubiquitin conjugates were purified on Ni-NTA-agarose under denaturing conditions, and ubiquitinated MDM2 was detected by anti-MDM2 antibodies. (C) Real-time PCR analysis of p53 downstream genes in A549 cells transfected with siRNA against control or RPS2 for 72 h. Relative expression values are normalized against b-actin RNA levels and graphs are shown as fold induction over control siRNA-transfected cells. (D) A549 cells transfected with siRNA against control or RPS2 were treated with either DMSO or 5 nM actinomycin D for 4 h. Cell lysates were immunoblotted with the indicated antibodies.
has essential roles in cellular signaling [17]. Accordingly, this result indicates that a major portion of ubiquitinated RPS2 by MDM2 has essential roles in cellular signaling rather than proteasomal degradation. To confirm this result, we compared the RPS2 protein level. The expression of RPS2 was not affected by co-overexpression with MDM2 with or without MG132 treatment (Fig. 2D). Taken together, these results indicate that RPS2 is a substrate of MDM2, and its ubiquitination, which is mainly associated with the K63linked ubiquitin chain, may act as a signal in a cellular pathway, rather than inducing degradation by the proteasome. 3.3. RPS2 induces and stabilizes p53 protein level It has demonstrated that several types of RPs are involved in the regulation of p53 activation and we newly confirmed the interaction between MDM2 and RPS2 as described above. Therefore, we first examined whether RPS2 is involved in the p53 signaling pathway. As shown in Fig. 3A, the protein level of p53 was downregulated in RPS2-knockdown cells and this was rescued by treatment with the proteasome inhibitor MG132. Conversely, p53 was slightly increased in the overexpression of HA-RPS2 but remained unchanged even in the presence of RPS2 following treatment with
MG132 (Fig. 3B). These results indicate that RPS2 upregulates the p53 protein level in a proteasome-dependent manner. To clarify the effect of RPS2 on the p53 level, we transiently overexpressed increasing amounts of HA-RPS2. The p53 level increased in a dosedependent manner, and p21, a downstream gene of p53, showed the same increasing pattern as p53 (Fig. 3C). To further investigate the changes in the p53 levels induced by RPS2, we treated cells with cycloheximide and harvested the cells for a time course analysis. The degradation assay showed that p53 degradation was delayed when HA-RPS2 was overexpressed (Fig. 3D). These results suggest that RPS2 regulates the stability of p53 in a proteasome-dependent manner. 3.4. RPS2 inhibits MDM2, thereby inducing both p53 and its downstream pathway Because the stability of p53 is mainly regulated by MDM2dependent ubiquitination, we examined whether RPS2 has an effect on the MDM2-mediated ubiquitination of p53 using a Ni-NTA pull-down assay. As shown in Fig. 4A, MDM2 increased the ubiquitination of p53 but overexpression of RPS2 attenuated the ubiquitination of p53 by MDM2. It has been reported that RPL11 inhibits
Please cite this article as: J. Cho et al., Ribosomal protein S2 interplays with MDM2 to induce p53, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.01.038
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MDM2 function by increasing the ubiquitination of MDM2 [18]. Therefore, we examined whether RPS2 has an effect on the ubiquitination of MDM2. MDM2 is a crucial down-regulator of p53 but p53 upregulates MDM2 in a positive feedback loop. To exclude the positive feedback effect on MDM2 ubiquitination from the increased p53, we performed the Ni-NTA pull-down assays with a p53-deficient H1299 cell line (Fig. 4B). Consistent with previous reports about other RPs, RPS2 also increased the ubiquitination of MDM2. These results indicate that RPS2 inhibits MDM2 by inducing MDM2 ubiquitination. Since RPS2 regulates p53 stability with this mechanism, it can also regulate the transcriptional activity of p53. To elucidate the effect of RPS2 on p53 activity, we performed realtime PCR to compare the expression level of p53 target genes under RPS2 knockdown. As expected, RPS2 knockdown downregulated the transcriptional level of p53 downstream genes, such as p21, E2F7, and GADD45a (Fig. 4C). These data indicate that RPS2 regulates not only the protein levels of p53 but also p53 activity for the downstream pathway. Based on these results, we concluded that RPS2 stabilizes p53 by inhibiting MDM2-dependent ubiquitination of p53. RPs are essential factors in ribosome biogenesis, and perturbations in ribosome biogenesis induce ribosomal stress, which can trigger activation of the p53 signaling pathway [5]. Since the upregulation of p53 by RPs mainly occurred under ribosomal stress, we checked the protein level of p53 in the depletion of RPS2 to investigate the effect of RPS2 under ribosomal stress. As shown in Fig. 4D, ribosomal stress increased p53 but did not increase p53 under RPS2 depletion. These results suggest that RPS2 is required for p53 stabilization under ribosomal stress. Thus far, there have only been a few reports that show the posttranslational modifications (PTMs) of RPS2. RPS2 interacts with protein arginine methyltransferase 3 (PRMT3). PRMT3 reduces the ubiquitination of RPS2 and stabilizes RPS2 protein [19] and RPS2 is methylated by PRMT3 [20]. Furthermore, ZNF598 and RACK1 mediate the ubiquitination of ribosomal small subunit proteins including RPS2 in mature ribosomes, thereby participating in mammalian ribosome-associated quality control through regulatory ribosomal ubiquitination (RRub) [21]. These studies suggest that RPS2 can have different roles in the cell depending on the PTM. Here, we found the novel PTM of RPS2 involved in the p53 signaling pathway. We showed that the RPS2 is ubiquitinated by MDM2 and MDM2-mediated RPS2 ubiquitination is mainly associated with the K63-linked ubiquitin chain, which means it can be used as a regulatory signal for cellular mechanisms, not for proteasomal degradation [17]. In addition, this modification of RPS2 is required for the regulation of the MDM2-p53 signaling pathway by inhibiting MDM2 through an increase in ubiquitinated MDM2, leading to p53 accumulation. In conclusion, we provide evidence that RPS2 is both a regulator and a substrate of MDM2. Therefore, RPS2 interplays with MDM2 to regulate p53.
Author contributions J.C., J.H.K., E.E.K., and E.J.S. conceived and designed the experiments. J.C. and J.P. performed the majority of experiments and S.C.S and E.E.K carried out biochemical assay and analyzed the data. J.C., E.E.K, and E.J.S. wrote the manuscript.
Declaration of competing interest The authors declare no potential conflicts of interests. Acknowledgements This work was supported by a National Research Foundation of Korea (NRF) grant funded by the Korean government (MSIP) (2019R1A2C2004052 and 2017R1A2B3007224) and the R&D Convergence Program of NST (National Research Council of Science & Technology) of Republic of Korea (CAP-16-03-KRIBB). References [1] F. Bunz, A. Dutriaux, C. Lengauer, T. Waldman, S. Zhou, J.P. Brown, J.M. Sedivy, K.W. Kinzler, B. Vogelstein, Requirement for p53 and p21 to sustain G2 arrest after DNA damage, Science (N.Y.) 282 (1998) 1497e1501. [2] C.E. Canman, C.Y. Chen, M.H. Lee, M.B. Kastan, DNA damage responses: p53 induction, cell cycle perturbations, and apoptosis, Cold Spring Harbor Symp. Quant. Biol. 59 (1994) 277e286. [3] D. Liu, Y. Xu, p53, oxidative stress, and aging, Antioxidants Redox Signal. 15 (2011) 1669e1678. [4] K.H. Vousden, C. Prives, Blinded by the light: the growing complexity of p53, Cell 137 (2009) 413e431. [5] S. Bursac, M.C. Brdovcak, G. Donati, S. Volarevic, Activation of the tumor suppressor p53 upon impairment of ribosome biogenesis, Biochim. Biophys. Acta 1842 (2014) 817e830. [6] J.P. Kruse, W. Gu, Modes of p53 regulation, Cell 137 (2009) 609e622. [7] S. Boulon, B.J. Westman, S. Hutten, F.M. Boisvert, A.I. Lamond, The nucleolus under stress, Mol. Cell 40 (2010) 216e227. [8] L. Golomb, S. Volarevic, M. Oren, p53 and ribosome biogenesis stress: the essentials, FEBS Lett. 588 (2014) 2571e2579. [9] X. Zhou, W.J. Liao, J.M. Liao, P. Liao, H. Lu, Ribosomal proteins: functions beyond the ribosome, J. Mol. Cell Biol. 7 (2015) 92e104. [10] S. Bursac, M.C. Brdovcak, M. Pfannkuchen, I. Orsolic, L. Golomb, Y. Zhu, C. Katz, L. Daftuar, K. Grabusic, I. Vukelic, V. Filic, M. Oren, C. Prives, S. Volarevic, Mutual protection of ribosomal proteins L5 and L11 from degradation is essential for p53 activation upon ribosomal biogenesis stress, Proc. Natl. Acad. Sci. U.S.A. 109 (2012) 20467e20472. [11] Y. Zhu, M.V. Poyurovsky, Y. Li, L. Biderman, J. Stahl, X. Jacq, C. Prives, Ribosomal protein S7 is both a regulator and a substrate of MDM2, Mol. Cell 35 (2009) 316e326. [12] X.X. Sun, T. DeVine, K.B. Challagundla, M.S. Dai, Interplay between ribosomal protein S27a and MDM2 protein in p53 activation in response to ribosomal stress, J. Biol. Chem. 286 (2011) 22730e22741. [13] R.E. Kingston, C.A. Chen, H. Okayama, Calcium Phosphate Transfection, Current Protocols in Cell Biology, 2003 (Chapter 20), Unit 20.23. [14] H. Kim, J.M. Lee, G. Lee, J. Bhin, S.K. Oh, K. Kim, K.E. Pyo, J.S. Lee, H.Y. Yim, K.I. Kim, D. Hwang, J. Chung, S.H. Baek, DNA damage-induced RORalpha is crucial for p53 stabilization and increased apoptosis, Mol. Cell 44 (2011) 797e810. [15] E.J. Song, S.L. Werner, J. Neubauer, F. Stegmeier, J. Aspden, D. Rio, J.W. Harper, S.J. Elledge, M.W. Kirschner, M. Rape, The Prp19 complex and the Usp4Sart3 deubiquitinating enzyme control reversible ubiquitination at the spliceosome, Genes Dev. 24 (2010) 1434e1447. [16] D. Bai, J. Zhang, W. Xiao, X. Zheng, Regulation of the HDM2-p53 pathway by ribosomal protein L6 in response to ribosomal stress, Nucleic Acids Res. 42 (2014) 1799e1811. [17] Y. Kulathu, D. Komander, Atypical ubiquitylation - the unexplored world of polyubiquitin beyond Lys48 and Lys63 linkages, Nature reviews, Mol. Cell. Biol. 13 (2012) 508e523. [18] M.S. Dai, D. Shi, Y. Jin, X.X. Sun, Y. Zhang, S.R. Grossman, H. Lu, Regulation of the MDM2-p53 pathway by ribosomal protein L11 involves a postubiquitination mechanism, J. Biol. Chem. 281 (2006) 24304e24313. [19] S. Choi, C.R. Jung, J.Y. Kim, D.S. Im, PRMT3 inhibits ubiquitination of ribosomal protein S2 and together forms an active enzyme complex, Biochim. Biophys. Acta 1780 (2008) 1062e1069. [20] R. Swiercz, D. Cheng, D. Kim, M.T. Bedford, Ribosomal protein rpS2 is hypomethylated in PRMT3-deficient mice, J. Biol. Chem. 282 (2007) 16917e16923. [21] E. Sundaramoorthy, M. Leonard, R. Mak, J. Liao, A. Fulzele, E.J. Bennett, ZNF598 and RACK1 regulate mammalian ribosome-associated quality control function by mediating regulatory 40S ribosomal ubiquitylation, Mol. Cell 65 (2017) 751e760, e754.
Please cite this article as: J. Cho et al., Ribosomal protein S2 interplays with MDM2 to induce p53, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.01.038