Smurf1 polyubiquitinates on K285/K282 of the kinases Mst1/2 to attenuate their tumor-suppressor functions
2023; Elsevier BV; Volume: 299; Issue: 12 Linguagem: Inglês
10.1016/j.jbc.2023.105395
ISSN1083-351X
AutoresYana Xu, Meiyu Qu, Yangxun He, Qiangqiang He, Tingyu Shen, Jiahao Luo, Dan Tan, Hangyang Bao, Chengyun Xu, Xing Ji, Xinhua Hu, Muhammad Qasim Barkat, Linghui Zeng, Ximei Wu,
Tópico(s)Hippo pathway signaling and YAP/TAZ
ResumoSterile 20–like kinases Mst1 and Mst2 (Mst1/2) and large tumor suppressor 1/2 are core kinases to mediate Hippo signaling in maintaining tissue homeostasis. We have previously demonstrated that Smad ubiquitin (Ub) regulatory factor 1 (Smurf1), a HECT-type E3 ligase, ubiquitinates and in turn destabilizes large tumor suppressor 1/2 to induce the transcriptional output of Hippo signaling. Here, we unexpectedly find that Smurf1 interacts with and polyubiquitinates Mst1/2 by virtue of K27- and K29-linked Ub chains, resulting in the proteasomal degradation of Mst1/2 and attenuation of their tumor-suppressor functions. Among the potential Ub acceptor sites on Mst1/2, K285/K282 are conserved and essential for Smurf1-induced polyubiquitination and degradation of Mst1/2 as well as transcriptional output of Hippo signaling. As a result, K285R/K282R mutation of Mst1/2 not only negates the transcriptional output of Hippo signaling but enhances the tumor-suppressor functions of Mst1/2. Together, we demonstrate that Smurf1-mediated polyubiquitination on K285/K282 of Mst1/2 destabilizes Mst1/2 to attenuate their tumor-suppressor functions. Thus, the present study identifies Smurf1-mediated ubiquitination of Mst1/2 as a hitherto uncharacterized mechanism fine-tuning the Hippo signaling pathway and may provide additional targets for therapeutic intervention of diseases associated with this important pathway. Sterile 20–like kinases Mst1 and Mst2 (Mst1/2) and large tumor suppressor 1/2 are core kinases to mediate Hippo signaling in maintaining tissue homeostasis. We have previously demonstrated that Smad ubiquitin (Ub) regulatory factor 1 (Smurf1), a HECT-type E3 ligase, ubiquitinates and in turn destabilizes large tumor suppressor 1/2 to induce the transcriptional output of Hippo signaling. Here, we unexpectedly find that Smurf1 interacts with and polyubiquitinates Mst1/2 by virtue of K27- and K29-linked Ub chains, resulting in the proteasomal degradation of Mst1/2 and attenuation of their tumor-suppressor functions. Among the potential Ub acceptor sites on Mst1/2, K285/K282 are conserved and essential for Smurf1-induced polyubiquitination and degradation of Mst1/2 as well as transcriptional output of Hippo signaling. As a result, K285R/K282R mutation of Mst1/2 not only negates the transcriptional output of Hippo signaling but enhances the tumor-suppressor functions of Mst1/2. Together, we demonstrate that Smurf1-mediated polyubiquitination on K285/K282 of Mst1/2 destabilizes Mst1/2 to attenuate their tumor-suppressor functions. Thus, the present study identifies Smurf1-mediated ubiquitination of Mst1/2 as a hitherto uncharacterized mechanism fine-tuning the Hippo signaling pathway and may provide additional targets for therapeutic intervention of diseases associated with this important pathway. Hippo signaling is an evolutionarily conserved pathway that controls the organ size by regulating cell proliferation and apoptosis and regulates a variety of biological processes, such as organ development, tissue homeostasis, and tumorigenesis (1Zeybek N.D. Baysal E. Bozdemir O. Buber E. Hippo signaling: a stress response pathway in stem cells.Curr. Stem Cell Res. Ther. 2021; 16: 824-839Crossref PubMed Scopus (12) Google Scholar, 2Fallahi E. O'Driscoll N.A. Matallanas D. The MST/Hippo pathway and cell death: a non-canonical affair.Genes (Basel). 2016; 7: 28Crossref PubMed Scopus (64) Google Scholar, 3Meng Z. Moroishi T. Guan K.L. Mechanisms of Hippo pathway regulation.Genes Dev. 2016; 30: 1-17Crossref PubMed Scopus (1048) Google Scholar). Sterile 20–like kinases Mst1 and Mst2 (Mst1/2), mammalian homologs of Drosophila Hippo, are core kinases of Hippo signaling pathway and share ∼75% identical amino acid sequence (4Galan J.A. Avruch J. MST1/MST2 protein kinases: regulation and physiologic roles.Biochemistry. 2016; 55: 5507-5519Crossref PubMed Scopus (56) Google Scholar, 5Du X. Yu A. Tao W. The non-canonical Hippo/Mst pathway in lymphocyte development and functions.Acta Biochim. Biophys. Sin. 2015; 47: 60-64Crossref Scopus (24) Google Scholar). Activation of Mst1/2 phosphorylates the large tumor suppressor 1 and 2 (Lats1/2)–Mps 1 binder (Mob1), Lats1/2–Mob1 in turn phosphorylates Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ), resulting in their ubiquitination and proteasomal degradation. In contrast, inactivation of either Mst1/2 or Lats1/2–Mob1 stabilizes YAP/TAZ and induces YAP/TAZ nuclear translocation to bind to the TEA domain transcription factor (TEAD), resulting in the transcriptional output of proproliferative and prosurvival genes, such as the connective tissue growth factor (Ctgf) and cysteine-rich angiogenic inducer 61 (Cyr61) (3Meng Z. Moroishi T. Guan K.L. Mechanisms of Hippo pathway regulation.Genes Dev. 2016; 30: 1-17Crossref PubMed Scopus (1048) Google Scholar, 6Ma S. Meng Z. Chen R. Guan K.L. The hippo pathway: biology and pathophysiology.Annu. Rev. Biochem. 2019; 88: 577-604Crossref PubMed Scopus (581) Google Scholar). As a result, genetic ablation of both Mst1 and Mst2 causes the enlarged livers and spontaneous hepatocellular carcinoma (HCC) in mice (7Lu L. Li Y. Kim S.M. Bossuyt W. Liu P. Qiu Q. et al.Hippo signaling is a potent in vivo growth and tumor suppressor pathway in the mammalian liver.Proc. Natl. Acad. Sci. U. S. A. 2010; 107: 1437-1442Crossref PubMed Scopus (589) Google Scholar, 8Zhou D. Conrad C. Xia F. Park J.S. Payer B. Yin Y. et al.Mst1 and Mst2 maintain hepatocyte quiescence and suppress hepatocellular carcinoma development through inactivation of the Yap1 oncogene.Cancer Cell. 2009; 16: 425-438Abstract Full Text Full Text PDF PubMed Scopus (730) Google Scholar, 9Zinatizadeh M.R. Miri S.R. Zarandi P.K. Chalbatani G.M. Rapôso C. Mirzaei H.R. et al.The Hippo tumor suppressor pathway (YAP/TAZ/TEAD/MST/LATS) and EGFR-RAS-RAF-MEK in cancer metastasis.Genes Dis. 2021; 8: 48-60Crossref PubMed Scopus (31) Google Scholar). Ubiquitination is a universal protein post-translational modification and involved in a variety of biological processes, such as inflammation, metabolism, DNA damage and repair, autophagy, and tumorigenesis (10Li S. Zhao J. Shang D. Kass D.J. Zhao Y. Ubiquitination and deubiquitination emerge as players in idiopathic pulmonary fibrosis pathogenesis and treatment.JCI Insight. 2018; 3e120362Crossref Scopus (28) Google Scholar, 11Popovic D. Vucic D. Dikic I. Ubiquitination in disease pathogenesis and treatment.Nat. Med. 2014; 20: 1242-1253Crossref PubMed Scopus (734) Google Scholar, 12Shaid S. Brandts C.H. Serve H. Dikic I. Ubiquitination and selective autophagy.Cell Death Differ. 2013; 20: 21-30Crossref PubMed Scopus (512) Google Scholar). Ubiquitination is initiated by transferring ubiquitin (Ub) from an Ub-activating enzyme (E1) to an Ub-conjugating enzyme (E2) and produces a covalently linked intermediate (E2–Ub). Ub protein ligases (E3 ligases) determine the substrate specificity of ubiquitination by the covalent attachment of Ub to substrate proteins (13Akimov V. Barrio-Hernandez I. Hansen S.V.F. Hallenborg P. Pedersen A.K. Bekker-Jensen D.B. et al.UbiSite approach for comprehensive mapping of lysine and N-terminal ubiquitination sites.Nat. Struct. Mol. Biol. 2018; 25: 631-640Crossref PubMed Scopus (276) Google Scholar). Currently, there are over 600 putative E3 ligases classified into three families, homologous to E6-AP carboxy terminus (HECT), RING, and ring-between ring–ring families (14Buetow L. Huang D.T. Structural insights into the catalysis and regulation of E3 ubiquitin ligases.Nat. Rev. Mol. Cell Biol. 2016; 17: 626-642Crossref PubMed Google Scholar, 15Morreale F.E. Walden H. Types of ubiquitin ligases.Cell. 2016; 165: 248Abstract Full Text PDF PubMed Scopus (258) Google Scholar). Smad Ub regulatory factor 1 (Smurf1), a HECT-type E3 ligase, is initially believed to regulate Smad1/5 protein stability in the transforming growth factor-β and bone morphogenic protein signaling pathways (16Sapkota G. Alarcon C. Spagnoli F.M. Brivanlou A.H. Massague J. Balancing BMP signaling through integrated inputs into the Smad1 linker.Mol. Cell. 2007; 25: 441-454Abstract Full Text Full Text PDF PubMed Scopus (327) Google Scholar). Smurf1 ubiquitinates a variety of substrates that are involved in the cell proliferation and differentiation, cell stemness, chromatin organization and dynamics, DNA damage and repair, genomic integrity, gene expression, and cell migration and invasion (17Koganti P. Levy-Cohen G. Blank M. Smurfs in protein homeostasis, signaling, and cancer.Front. Oncol. 2018; 8: 295Crossref PubMed Scopus (68) Google Scholar, 18Jiang M. Shi L. Yang C. Ge Y. Lin L. Fan H. et al.miR-1254 inhibits cell proliferation, migration, and invasion by down-regulating Smurf1 in gastric cancer.Cell Death Dis. 2019; 10: 32Crossref PubMed Scopus (70) Google Scholar). All these events are inextricably linked to the tumorigenesis. Smurf1 is also highly expressed in a variety of tumors and functions as a tumor promoter by ubiquitinating tumor suppressors (19Fu L. Cui C.P. Zhang X. Zhang L. The functions and regulation of Smurfs in cancers.Semin. Cancer Biol. 2020; 67: 102-116Crossref PubMed Scopus (52) Google Scholar, 20Xia Q. Li Y. Han D. Dong L. SMURF1, a promoter of tumor cell progression?.Cancer Gene Ther. 2021; 28: 551-565Crossref PubMed Scopus (15) Google Scholar). We have previously demonstrated that Smurf1 ubiquitinates Lats1/2 to induce the transcriptional output of Hippo (21Qu M. Gong Y. Jin Y. Gao R. He Q. Xu Y. et al.HSP90beta chaperoning SMURF1-mediated LATS proteasomal degradation in the regulation of bone formation.Cell. Signal. 2023; 102110523Crossref PubMed Scopus (1) Google Scholar). Here, we have further identified that Smurf1 ubiquitinates Mst1/2 predominantly on K285/K282 to promote their proteasomal degradation and thereby attenuates their tumor-suppressor functions. Thus, the present study has uncovered Smurf1 in conjunction with Mst1/2 ubiquitination as a hitherto uncharacterized mechanism controlling Hippo signaling. Knockdown of dSmurf1 (Drosophila homolog of Smurf1/2) stabilizes Wts (Drosophila homolog of Lats1/2) and increases the phosphorylation levels of Yki (Drosophila homolog of YAP and TAZ) and ultimately regulates Hippo signaling transduction. In contrast, overexpression of dSmurf1 has no effect on modulating Wts stability (22Cao L. Wang P. Gao Y. Lin X. Wang F. Wu S. Ubiquitin E3 ligase dSmurf is essential for Wts protein turnover and Hippo signaling.Biochem. Biophys. Res. Commun. 2014; 454: 167-171Crossref PubMed Scopus (13) Google Scholar). In order to determine whether Smurf1 regulates Hippo signaling in mammalian cells, we performed Tead4-luciferase reporter and quantitative PCR (qPCR) analyses in 293T cells. Knockdown of Smurf1 significantly decreased the Tead4-luciferase reporter activities as well as the mRNA levels of Ctgf and Cyr61, target genes of Hippo signaling pathway (Fig. 1, A and B). In contrast, overexpression of Smurf1 in 293T cells unexpectedly and robustly increased the Tead4-luciferase activities and the mRNA levels of Ctgf and Cyr61 (Fig. 1, C and D), which appears to be at odds with the result in Drosophila (22Cao L. Wang P. Gao Y. Lin X. Wang F. Wu S. Ubiquitin E3 ligase dSmurf is essential for Wts protein turnover and Hippo signaling.Biochem. Biophys. Res. Commun. 2014; 454: 167-171Crossref PubMed Scopus (13) Google Scholar). To determine the exact role of Smurf1 in the regulation of Hippo signaling, we next performed Western blotting analyses in 293T cells. Overexpression of Smurf1 robustly and moderately negated the protein levels of p-Lats1/2, p-YAP, p-TAZ, and of Mst1/2, p-Mst1/2, Lats1/2, respectively, whereas it noticeably enhanced the protein levels of YAP and TAZ (Fig. 1E). In contrast, Smurf1 siRNA robustly and moderately induced the protein levels of p-Lats1/2, p-YAP, p-TAZ, and of Mst1/2, p-Mst1/2, Lats1/2, respectively, whereas it considerably reduced the protein levels of YAP and TAZ (Fig. 1F). To confirm the universality, we cultured primary mouse embryonic fibroblasts (MEFs) from WT and Smurf1−/− (KO) embryos and infected them with or without Smurf1-expressing lentiviruses. Western blotting analyses indicated that ablation of Smurf1 exhibited more robustly than Smurf1 siRNA in regulating the expression of these key components of Hippo signaling pathway, whereas lentiviral overexpression of Smurf1 completely restored the effects of Smurf1 ablation in MEFs (Fig. 1G). In line with these observations, qPCR analyses revealed that Smurf1 ablation in MEFs significantly decreased the mRNA levels of Ctgf and Cyr61, whereas lentiviral overexpression of Smurf1 not only restored the Smurf1 ablation–negated mRNA levels but also increased the basal mRNA levels (Fig. 1H). Either upregulation (overexpression) or downregulation (knockdown/KO) of Smurf1 expression affects the Mst1/2 and p-Mst1/2 protein levels to the same extent (Fig. 1, E–G); this finding suggests that Smurf1-mediating Mst1/2 protein level changes cause the corresponding changes in p-Mst1/2 protein levels. Notably, either upregulation or downregulation of Smurf1 expression affects p-Lats1/2 levels more profoundly than Lats1/2 levels (Fig. 1, E–G). As Mst1/2 lie on the upstream of Lats1/2 (3Meng Z. Moroishi T. Guan K.L. Mechanisms of Hippo pathway regulation.Genes Dev. 2016; 30: 1-17Crossref PubMed Scopus (1048) Google Scholar, 6Ma S. Meng Z. Chen R. Guan K.L. The hippo pathway: biology and pathophysiology.Annu. Rev. Biochem. 2019; 88: 577-604Crossref PubMed Scopus (581) Google Scholar) and we have previously demonstrated that Smurf1 ubiquitinates and destabilizes Lats1/2 to decrease the p-Lats1/2 levels (21Qu M. Gong Y. Jin Y. Gao R. He Q. Xu Y. et al.HSP90beta chaperoning SMURF1-mediated LATS proteasomal degradation in the regulation of bone formation.Cell. Signal. 2023; 102110523Crossref PubMed Scopus (1) Google Scholar), the corresponding p-Lats1/2 level changes in response to Smurf1 could be resulted from Smurf1-mediating regulation of both Mst1/2 and Lats1/2. This notion was supported further by the Tead4-luciferase reporter assays, which showed overexpression of Smurf1 but not its inactive mutant (C699A [CA]) (23Wang H.R. Ogunjimi A.A. Zhang Y. Ozdamar B. Bose R. Wrana J.L. Degradation of RhoA by Smurf1 ubiquitin ligase.Methods Enzymol. 2006; 406: 437-447Crossref PubMed Scopus (58) Google Scholar) not only restored the Mst1/2 negated Tead4-luciferase activities but also increased the basal levels of them (Fig. 1I). Thus, Smurf1 induces the transcriptional output of Hippo signaling by targeting not only Lats1/2 but also Mst1/2. To determine the potential role of Smurf1 in destabilizing Mst1/2, we overexpressed or knocked down Smurf1 in 293T cells. Overexpression of Smurf1 did not affect the mRNA levels of Mst1/2 but decreased the exogenous and endogenous protein levels of Mst1/2 in a dose-dependent manner (Figs. 2, A–C and S1A). Whereas the E3 ligase inactive mutant (CA) of Smurf1 had no effect on Mst1/2 protein levels (Fig. 2, D and E), suggesting that E3 ligase activity of Smurf1 is required for negating Mst1/2 expression. To investigate whether endogenous Smurf1 is sufficient to regulate the abundance of Mst1/2, we knocked down Smurf1 in 293T cells and performed Western blotting analyses. As expected, Smurf1 siRNA or shRNA had no effect on mRNA levels of Mst1/2 but significantly upregulated the protein levels of Mst1/2 (Figs. 2F and S1B). However, treatment of cells with MG132, a proteosome inhibitor, but not with chloroquine, a lysosomal inhibitor, completely reversed the Smurf1's effects on Mst1/2 protein levels (Figs. 2, G and H and S1C). To investigate whether Smurf1 downregulates Mst1/2 by reducing their stability, we performed cycloheximide (CHX) chase assays (24Lignitto L. Arcella A. Sepe M. Rinaldi L. Delle Donne R. Gallo A. et al.Proteolysis of MOB1 by the ubiquitin ligase praja2 attenuates Hippo signalling and supports glioblastoma growth.Nat. Commun. 2013; 4: 1822Crossref PubMed Scopus (90) Google Scholar) and measured the degradation rate of Mst1/2. In the absence of Smurf1, the protein half-life (t1/2) values of Mst1/2 were approximately 7.5 and 7.0 h, respectively, whereas in the presence of Smurf1, the t1/2 values of Mst1/2 were approximately 3.8 and 4.0 h, respectively (Fig. 2I). In contrast, Smurf1 siRNA increased the t1/2 values of Mst1/2 by approximately 90% and 58% (3.3 and 4.7 h to 7.3 and 7.4 h), as compared with scramble siRNA, respectively (Fig. 2J). Likewise, the t1/2 values of Mst1/2 in WT MEFs were approximately 2.9 and 1.8 h, respectively, whereas in Smurf1-ablated MEFs, the t1/2 values of Mst1/2 were increased to approximately 13.8 and 6.5 h, respectively (Fig. 2K). Thus, Smurf1 destabilizes Mst1/2 by promoting their proteasomal degradation. To investigate the potential interactions between Mst1/2 and Smurf1, we performed coimmunoprecipitation in 293T cells transiently expressing FLAG-Mst1/2. The immunocomplexes precipitated by an anti-FLAG antibody but not its immunoglobulin G control contained a large amount of Smurf1 in addition to FLAG-Mst1/2 as expected (Fig. 3A). Likewise, immunofluorescence staining in 293T cells transfected with FLAG-Mst1/2 and Myc-Smurf1 consistently indicated that FLAG- and Myc-derived immunosignals were detected predominantly in the cytosol, and the apparently overlapping signals were readily observed in cytosol either (Fig. 3B). To further determine the interaction between Smurf1 and Mst1/2, we performed bioinformatics analysis by using UbiBrowser database (http://ubibrowser.ncpsb.org.cn). Smurf1 as well as Smurf2, a closely related homolog of Smurf1 (19Fu L. Cui C.P. Zhang X. Zhang L. The functions and regulation of Smurfs in cancers.Semin. Cancer Biol. 2020; 67: 102-116Crossref PubMed Scopus (52) Google Scholar), was most likely to interact with and ubiquitinated Mst1/2 (Fig. 3C). To explore whether Smurf2 interacts with Mst1/2 either, we performed coimmunoprecipitation in 293T cells transfected with Myc-Smurf2 and FLAG-Mst1/2. Immunocomplexes precipitated by an anti-FLAG antibody contained no Myc-Smurf2 in addition to FLAG-Mst1/2 as expected (Fig. S2, A and B). Moreover, Western blotting analyses in 293T cells transfected with FLAG-Mst1/2, and the increasing doses of Myc-Smurf2 consistently revealed that Smurf2 had no effect on Mst1/2 protein levels (Fig. S2, C and D). Thus, Smurf1 instead of Smurf2 interacts with and destabilizes Mst1/2. We next examined whether Smurf1 directly ubiquitinated Mst1/2. Coimmunoprecipitation analyses indicated that Myc-Smurf1 but not its inactive mutant (CA) significantly promoted Mst1/2 polyubiquitination in 293T cells transiently expressing Myc-Smurf1, FLAG-Mst1/2, and hemagglutinin (HA)-Ub (Fig. 3D). However, knockdown of Smurf1 or ablation of Smurf1 significantly reduced the ubiquitination levels of Mst1/2 in 293T cells or MEFs, respectively (Fig. 3, E and F). To validate that Smurf1 directly ubiquitinates Mst1/2, we performed in vitro ubiquitination reaction followed by Western blotting analyses. The apparent polyubiquitination of Mst1/2 was observed in the reactive mixture with FLAG-Smurf1 but not without FLAG-Smurf1 (Fig. 3G). To further determine the type of the polyubiquitination chain linked to Mst1/2, we generated a series of Ub mutants including K0, K6, K11, K27, K29, K33, K48, and K63. Each of them could only express a single linkage type of Ub and where K0 means that all lysine residues have been changed to arginine (R) and K6 means that all lysine residues except K6 have been changed to R (25Feng X. Jia Y. Zhang Y. Ma F. Zhu Y. Hong X. et al.Ubiquitination of UVRAG by SMURF1 promotes autophagosome maturation and inhibits hepatocellular carcinoma growth.Autophagy. 2019; 15: 1130-1149Crossref PubMed Scopus (64) Google Scholar, 26van Wijk S.J. Fulda S. Dikic I. Heilemann M. Visualizing ubiquitination in mammalian cells.EMBO Rep. 2019; 20e46520Crossref PubMed Scopus (60) Google Scholar). We then performed coimmunoprecipitation assays in 293T cells transfected with FLAG-Mst1/2, Myc-Smurf1, and HA-Ub variants. Smurf1 robustly increased K27- and K29 chain–linked polyubiquitination of Mst1/2 (Fig. 3H), whereas K27R and K29R mutants largely diminished the Smurf1-induced polyubiquitination of Mst1/2 (Fig. 3I). Thus, Smurf1 induces the polyubiquitination of Mst1/2 via the atypical K27- and K29-linked Ub chains. To determine the potential lysine residues that are essential for Smurf1-induced ubiquitination and degradation of Mst1/2, mass spectrometry analysis (PhosphoSitePlus, https://www.phosphosite.org) was performed and showed that there were 18 and 15 potential Ub acceptor sites on Mst1 and Mst2, respectively (Fig. S3, A and B). We then constructed Mst1/2 deubiquitinated variants harboring mutations at the consensus lysine residues (Lys to Arg) individually and evaluated their capacity to mediate Smurf1-induced ubiquitination in 293T cells. Both K285R of Mst1 and K282R of Mst2 were apparently resistant to Smurf1-mediated ubiquitination (Fig. 4, A and B), whereas overexpression of Smurf1 but not its inactive mutant (CA) significantly negated the protein levels of WT Mst1/2 but not their K285R/K282R mutants (Fig. 4, C and D). Moreover, CHX chase assays demonstrated that K285R/K282R mutants of Mst1/2 had a significantly longer half-life than WT Mst1/2: the mutants increased the t1/2 values of Mst1/2 by approximately 100% and 170% (2 and 3.6 h to 4 and 9.7 h), respectively (Fig. 4, E and F). Consistently, qPCR analyses indicated that overexpression of K285R/K282R mutants of Mst1/2 more robustly negated the mRNA levels of Ctgf and Cyr61 than WT Mst1/2, respectively (Fig. 4, G and H). Whereas analyses of subcellular distribution revealed that K285R/K282R mutants more significantly increased and decreased the cytosolic and nuclear YAP/TAZ protein levels than WT Mst1/2, respectively (Fig. 4, I and J). Thus, K285/K282 residues of Mst1/2 are critical Ub acceptor sites for Smurf1-mediating ubiquitination and degradation of Mst1/2 in the regulation of Hippo signaling. Mst1/2 loss of functions are closely related to the tumorigenesis of HCC (7Lu L. Li Y. Kim S.M. Bossuyt W. Liu P. Qiu Q. et al.Hippo signaling is a potent in vivo growth and tumor suppressor pathway in the mammalian liver.Proc. Natl. Acad. Sci. U. S. A. 2010; 107: 1437-1442Crossref PubMed Scopus (589) Google Scholar, 8Zhou D. Conrad C. Xia F. Park J.S. Payer B. Yin Y. et al.Mst1 and Mst2 maintain hepatocyte quiescence and suppress hepatocellular carcinoma development through inactivation of the Yap1 oncogene.Cancer Cell. 2009; 16: 425-438Abstract Full Text Full Text PDF PubMed Scopus (730) Google Scholar, 27Song H. Mak K.K. Topol L. Yun K. Hu J. Garrett L. et al.Mammalian Mst1 and Mst2 kinases play essential roles in organ size control and tumor suppression.Proc. Natl. Acad. Sci. U. S. A. 2010; 107: 1431-1436Crossref PubMed Scopus (448) Google Scholar), and Smurf1 has been reported as a tumor promoter in diverse cancers, such as pancreatic cancer, gastric cancer, and lung cancer (20Xia Q. Li Y. Han D. Dong L. SMURF1, a promoter of tumor cell progression?.Cancer Gene Ther. 2021; 28: 551-565Crossref PubMed Scopus (15) Google Scholar). To determine the potential relationship between Smurf1 and tumorigenesis of HCC, we performed the bioinformatics and statistical analyses of cancer-related gene expression from The Human Protein Atlas database and found that Smurf1 was highly expressed in the HCC (Fig. 5A). We compared the protein levels of Smurf1 and Mst1/2 in human normal hepatocyte L02 cells and human HCC HepG2 cells and found that Smurf1 and Mst1/2 protein levels were significantly higher and lower in HepG2 cells than in L02 cells, respectively (Fig. 5B). We next examine the ubiquitination levels of Mst1/2 in L02 and HepG2 cells after MG132 treatments and found that HepG2 cells underwent more robustly ubiquitination of Mst1/2 than L02 cells (Fig. 5C). Likewise, Western blotting analyses indicated that knockdown or overexpression of Smurf1 significantly increased or decreased the Mst1/2 protein levels in HepG2 cells, respectively (Fig. 5, D and E), whereas overexpression of WT Smurf1 but not its inactive mutant (CA) robustly enhanced the ubiquitination of Mst1/2 in HepG2 cells (Fig. 5F). Thus, Smurf1 induces the polyubiquitination and degradation of Mst1/2 in HepG2 HCC cells either. To determine whether Smurf1 has a critical role in the regulation of proliferation, apoptosis, and tumorigenesis of HCCs, we generated stably Smurf1- or Smurf1-shRNA-overexpressing HepG2 cells and performed Cell Counting Kit-8 (Yeasen), flow cytometry, and colony formation assays. Smurf1 knockdown or overexpression significantly increased or decreased the cell apoptosis, respectively, whereas Smurf1 knockdown or overexpression robustly decreased or increased the proliferation and colony formation of HepG2 cells, respectively (Fig. 5, G–I). To explore the in vivo effect of Smurf1 in the tumorigenesis of HCC, we generated Smurf1-shRNA- or Smurf1-expressing HCC xenografts in nude mice and assessed their volumes on day 14 postinoculation. Knockdown or overexpression of Smurf1 significantly decreased or increased the volumes of HCC xenografts, respectively (Fig. 5J). However, knockdown or overexpression of Smurf1 in xenografts significantly induced or negated Mst1/2 protein levels in HCC xenografts, respectively, as demonstrated by Western blotting and immunohistochemistry analyses (Fig. 5, K and L). Thus, Smurf1 polyubiquitinates and degrades Mst1/2 to attenuate their tumor-suppressor functions. In order to investigate further the role of ubiquitination on K285/K282 of Mst1/2 in the regulation of tumorigenesis of HCC, we generated HepG2 cells stably expressing WT Mst1/2 or deubiquitinated variants of Mst1/2, Mst1/2(K285R/K282R). Overexpression of K285R/K282R variants of Mst1/2 consistently increased and decreased the cytosolic and nuclear YAP/TAZ levels, as compared with overexpression of WT Mst1/2, respectively (Fig. 6, A and B). Likewise, Cell Counting Kit-8 and colony formation assays revealed that overexpression of K285R/K282R variants of Mst1/2 significantly decreased the cell proliferation at 3 days postculture and colony formation at 14 days postculture, as compared with overexpression of WT Mst1/2, respectively (Fig. 6, C–F). Whereas overexpression of K285R/K282R variants of Mst1/2 significantly increased the cell apoptosis at 3 days postculture, as compared with overexpression of WT Mst1/2, respectively (Fig. 6, G and H). Thus, deubiquitination of Mst1/2 on K285/K282 stabilizes Mst1/2 to enhance their tumor-suppressor functions. To confirm further the role of ubiquitination on K285/K282 of Mst1/2 in the regulation of their tumor-suppressor functions in vivo, we generated stably vector-, Mst1/2(WT)-, or Mst1/2(K285R/K282R)-expressing HepG2 cell xenografts in nude mice. The volumes of HepG2 cell xenografts expressing vector, WT, or K285R/K282R were time-dependently increased within 38-day postinoculation (Fig. 7, A and B). From day 10, the volumes of WT-expressing xenografts were significantly smaller than those of vector-expressing xenografts, whereas the volumes of K285R/K282R-expressing xenografts were much smaller than those of WT-expressing xenografts (Fig. 7, A and B). On day 38, the volumes from WT-expressing xenografts were approximately 60% of those from vector-expressing xenografts; however, the volumes of K285R/K282R-expressing xenografts were 50% and 40% of those from WT-expressing xenografts, respectively (Fig. 7, C–F). Moreover, analyses of mRNA levels revealed that WT-expressing xenografts significantly decreased the Ctgf and Cyr61 mRNA levels, as compared with vector-expressing xenografts, and K285R/K282R-expressing xenografts further decreased these mRNA levels, as compared with WT-expressing xenografts (Fig. 7, G and H). The cell proliferation and apoptosis in vector-, WT-, and K285R/K282R-expressing xenografts at 38 day postinoculation were assessed by Ki-67 and TUNEL staining analyses. The TUNEL-positive cells in xenografts expressing Mst1/2(WT) were significantly more than those in xenografts expressing vector, whereas the TUNEL-positive cells in xenografts expressing Mst1/2(K285R/K282R) were much more than those in xenografts expressing Mst1/2(WT) (Fig. 7I). In contrast, Ki-67-positive cells in xenografts expressing Mst1/2(WT) were significantly less than those in xenografts expressing vector, and the Ki-67-positive cells in xenografts expressing Mst1/2(K285R/K282R) were much less than those in xenografts expressing Mst1/2(WT) (Fig. 7J). Taken together, Smurf1-mediated ubiquitination on K285/K282 of Mst1/2 attenuates their tumor-suppressor functions in HepG2 cell xenografts. By biochemical, genetic, and xenograft approaches, the present study, to the best of our knowledge, is the first work uncovering that Smurf1-mediated ubiquitination on K285/K282 of Mst1/2 destabilizes Mst1/2 to attenuate their tumor-suppressor functions. We have previously demonstrated that Smurf1 ubiquitinates and destabilizes Lats1/2 to induce the transcriptional output of Hippo signaling (21Qu M. Gong Y. Jin Y. Gao R. He Q. Xu Y. et al.HSP90beta chaperoning SMURF1-mediated LATS proteasomal degradation in the regulation of bone formation.Cell. Signal. 2023; 102110523Crossref PubMed Scopus (1) Google Scholar). Thus, Smurf1 targets multiple acceptor sites of Hippo signaling pathway to attenuate the tumor-suppressor functions (Fig. 8). Our present study is consistent with previous studies that E3 Ub ligases, carboxy terminus of Hsp70-interacting protein and tumor necrosis factor receptor–associated factor 6, negatively regulate Mst1 stability, and that SCFβTrCP negatively regulates Mst2 stability through ubiquitination degradation in mammalian cells (28Li J.A. Kuang T. Pu N. Fang Y. Han X. Zhang L. et al.TRAF6 regula
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