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Sumoylation of PES1 Upregulates Its Stability and Function Via Inhibiting Its Ubiquitination

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Sumoylation of PES1 Upregulates Its Stability and Function Via Inhibiting Its Ubiquitination www.impactjournals.com/oncotarget/ Oncotarget, Vol. 7, No. 31 Research Paper SUMOylation of PES1 upregulates its stability and function via inhibiting its ubiquitination Shujing Li1, Miao Wang1, Xinjian Qu2, Zhaowei Xu1, Yangyang Yang1, Qiming Su2, Huijian Wu1,2 1School of Life Science and Biotechnology, Dalian University of Technology, Dalian, China 2School of Life Science and Medicine, Dalian University of Technology, Panjin, China Correspondence to: Huijian Wu, email: [email protected] Keywords: PES1, SUMOylation, breast cancer, ubiquitination Received: July 21, 2015 Accepted: June 15, 2016 Published: July 08, 2016 ABSTRACT PES1 is a component of the PeBoW complex, which is required for the maturation of 28S and 5.8S ribosomal RNAs, as well as for the formation of the 60S ribosome. Deregulation of ribosomal biogenesis can contribute to carcinogenesis. In this study, we showed that PES1 could be modified by the small ubiquitin-like modifier (SUMO) SUMO-1, SUMO-2 and SUMO-3, and SUMOylation of PES1 was stimulated by estrogen (E2). One major SUMOylation site (K517) was identified in the C-terminal Glu-rich domain of PES1. Substitution of K517 with arginine abolished the SUMOylation of PES1. SUMOylation also stabilized PES1 through inhibiting its ubiquitination. In addition, PES1 SUMOylation positively regulated the estrogen signaling pathway. SUMOylation enhanced the ability of PES1 to promote estrogen receptor α (ERα)- mediated transcription by increasing the stability of ERα, both in the presence and absence of E2. Moreover, SUMOylation of PES1 also increased the proportion of S-phase cells in the cell cycle and promoted the proliferation of breast cancer cells both in vitro and in vivo. These findings showed that posttranslational modification of PES1 by SUMOylation may serve as a key factor that regulates the function of PES1 in vivo. INTRODUCTION The main function of PES1 is its involvement in the synthesis and maturation of ribosome and its effect PES1 is a nuclear protein discovered in 1996, on chromatin stretch. More and more research have and early studies have shown that several abnormal shown that PES1 is closely associated with tumorgenesis developments that occurred in the brain, eye, liver and via improving cell proliferation and participating in the other organs of zebrafish embryo as a result of PES1 progress of cell cycle in cancer cells. PES1 is mainly mutation can even cause the death of the embryo [1, 2]. expressed in developing tissues, but aberrant expression The human PES1 gene, which is located to 22q12.1 was of PES1 has been found in human brain tumors. first cloned in 2001 [3]. The PES1 protein contains 588 Neuroblastoma (NB) cases with MYCN amplification and amino acids and it has three nuclear localization signals international neuroblastoma staging system stage 4 (INSS (NLSs) that mediate the transport of the protein into stage 4) tend to show a higher expression level of PES1 the nucleus. PES1 is mainly localized in the nucleolus, [5]. In addition, high PES1 expression is associated with and rarely distributed in the cytoplasm. The C terminus the worst overall and relapse-free survival [5]. PES1 is (amino acids 315-412) of PES1 contains a typical BRCT also upregulated in several different carcinoma cell lines, domain that has been found in the carboxyl terminus of such as the colon cancer cell line SW480 and human one breast cancer suppressor protein, BRCA1. Many breast cancer cell line MCF-7 [6]. PES1 is upregulated proteins containing the BRCT domain play a role in DNA more in colon cancer tissues than in non-cancerous tissues, repair, remodeling and cell cycle. The BRCT domain of while down-regulation of PES1 in colon cancer cells can PES1 plays a crucial role in regulating the progress of cell lead to cell cycle arrest at the G1 phase, as well as reduced cycle [4]. In addition, PES1 also contains a glutamate rich proliferation and decreased growth of xenografts [7]. region, but its function is not clear. Clinical study has suggested that the expression of PES1 www.impactjournals.com/oncotarget 50522 Oncotarget in breast cancer tissues (stage I-IV is much higher than that of PES1 showed a band with a molecular mass much in normal breast tissues, while PES1 knockdown represses higher than that of PES1 when PES1 was coexpressed the growth and tumorgenesis of breast cancer cells [8]. with SUMO-1, -2 or -3 (Figure 1A), suggesting that PES1 One possible mechanism by which PES1 promotes the was modified by all three kinds of SUMO. Since PES1 development of breast cancer (ERα-positive) is through was mainly modified by SUMO-1, we therefore focused increasing the level of ERα while decreasing the level of mainly on the functions of PES1 modified by SUMO-1 ERβ [9]. ERα promotes the stimulating effect of estrogen in subsequent experiments. Moreover, analysis of PES1 on breast cancer cell proliferation, whereas ERβ inhibits SUMOylation in COS-7 cells cotransfected with Flag- the proliferation of breast cancer cells. All these data show PES1 and Myc-SUMO-1 showed that the overexpressed that abnormality associated with PES1 may be a common PES1 was modified by SUMO-1 (Figure 1B). To further feature of malignancy. confirm the SUMOylation of PES1, we cotransfected Small ubiquitin like-modifier (SUMO) modification COS-7 cells with Flag-PES1 and GFP-SUMO-1 or GFP- is a crucial post-translational modification for regulating SUMO-1 GA and analyzed the SUMOylated products. protein activity, stability, alteration of protein-protein SUMO-1 GA is a SUMO mutant that lacks the ability interaction and transcriptional activity as well as protein to join with the substrate due to a C-terminal diglycine sublocalization in the cells. A consensus sequence ΨKXE substitution (GG to GA). A band of higher molecular mass exists in most proteins that are modified by SUMOylation. indicative of PES1-SUMO1 was detected only when PES1 In this sequence, Ψ stands for a hydrophobic amino acid was coexpressed with wild-type SUMO-1 in the cells and K is the conjugation site for SUMO. SUMOylation (Figure 1C). The effect of UBC9, a SUMO E2 conjugate modification is a dynamic process, in which the modified enzyme, on the SUMOylation of PES1 was also examined. proteins can be deSUMOylated by SUMO-specific Cells transfected with UBC9 and SUMO-1 showed the proteases (SENPs) [10]. Many proteins with important highest intensity for the PES1-SUMO-1 band (Figure roles in cellular processes have been identified as targets 1D). In addition, a SUMOylation assay conducted with that are modified by SUMO. For examples, SUMOylation purified recombinant SUMO-conjugating enzymes and of DEC1 increases its ability to repress the transcriptional GST-PES1 clearly showed that PES1 could be modified by activity of CLOCK/BMAL1 [11], and SUMOylation SUMO-1 in vitro (Figure 1E). The effect of estrogen (E2) of GPS2 promotes its ability to inhibit ERα-mediated on endogenous SUMOylated PES1 was also investigated. transcription [12]. Sequence analysis of PES1 revealed PES1 immunoprecipitated from MCF-7 cells was detected two potential SUMO conjugation sites, K249 and K517, by anti-SUMO1 antibody, and its level increased when both of which were contained within the consensus the cells were treated with E2 (Figure 1F). To further sequence ΨKXE, so we wanted to examine whether PES1 confirm this result, MCF-7 cells were transfected with is a target protein of SUMO. Myc-SUMO-1, and PES1 was then immunoprecipitated In this study, we demonstrated that PES1 from the cells and probed with anti-Myc antibody. The could be SUMOylated by SUMO-1, -2 and -3, and result showed that the intensity of the band corresponding showed that K517 was the main SUMOylation site of to SUMOylated PES1 increased with E2 treatment (Figure PES1. SUMOylation stabilized PES1 byinhibiting its 1G). The protein level of PES1 was upregulated when the ubiquitination. PES1 SUMOylation also promoted the E2 treatment lasted for 8 h, but remained unchanged when transcriptional activity of ERα by enhancing its stability. the E2 treatment lasted for 4 h (Figure 1H). These data SUMOylation of PES1 inhibited the association of suggested that PES1 could be modified by SUMO, and E2 ERα with its ubiquitination E3 ligase ChIP and as well may upregulate the SUMOylation of PES1. as inhibiting the ubiquitination of ERα. Moreover, SUMOylation of PES1 increased the percentage of MCF- K517 is the primary site for PES1 SUMOylation 7 cells in the S phase and promoted the proliferation of breast cancer cells as well as the formation of xenograft Analysis of the sequence of PES1 revealed two tumor. Taken together, our data suggested that post- conserved lysine residues in the Glu-rich domain: one translation modification of PES1 via SUMOylation might corresponding to K249 and the other to K517 (Figure play important roles in ERα-related estrogen signaling 2A). Furthermore, these two sites are conserved in pathway and in the growth of breast cancer cells. different species (homo, mouse, marmoset and xenopus) (Figure 2A). Changing K249 to arginine (K249R) RESULTS resulted in nearly no change in the SUMOylation status of PES1, whereas changing K517 to arginine (K517R) PES1 is modified by SUMO or both K249 and K517 to arginine (2KR) appeared to abolish the SUMOylation of PES1 (Figure 2B). To further To identify whether PES1 is a SUMOylated define the SUMOylation sites in PES1, COS-7 cells substrate, COS-7 cells were cotransfected with GFP- were transfected with Myc-SUMO1 together with GFP- PES1 and Myc-SUMO-1, -2 or -3. Western blot analysis PES1 or GFP-K517R. GFP-PES1 immunoprecipitated www.impactjournals.com/oncotarget 50523 Oncotarget from the cell extract was detected by anti-Myc antibody, Effect of SUMOylation on the localization and while the immunoprecipitated GFP-K517R did not show stability of PES1 any reaction with the anti-Myc antibody, indicating that the SUMO-1 did not conjugate with the mutant PES1 As reported in previous study [13], PES1 is (Figure 2C).
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