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• W3204188369 abstract "Apoptosis is a programmed cell death that efficiently removes damaged cells to maintain tissue homeostasis. Defect in apoptotic machinery can lead to tumor development, progression, and resistance to chemotherapy. PUMA (p53 upregulated modulator of apoptosis) and BAX (BCL2-associated X protein) are among the most well-known inducers of apoptosis. It has been reported that expression levels of BAX and PUMA are controlled at the posttranslational level by phosphorylation. However, the posttranslational regulation of these proapoptotic proteins remains largely unexplored. In this study, using biochemical, molecular biology, flow cytometric, and immunohistochemistry techniques, we show that PUMA and BAX are the direct target of the F-box protein FBXL20, which restricts their cellular levels. FBXL20 directs the proteasomal degradation of PUMA and BAX in a protein kinase AKT1-dependent manner to promote cancer cell proliferation and tumor growth. Interestingly, inactivation of AKT1 results in activation of another protein kinase GSK3α/β, which facilitates the proteasomal degradation of FBXL20 by another F-box protein, FBXO31. Thus, a switch between two signaling kinases AKT1 and GSK3α/β modulates the functional activity of these proapoptotic regulators, thereby determining cell survival or death. RNAi-mediated ablation of FBXL20 results in increased levels of PUMA as well as BAX, which further enhances the sensitivity of cancer cells to chemotherapeutic drugs. We showed that high level expression of FBXL20 in cancer cells reduces therapeutic drug-induced apoptosis and promotes chemoresistance. Overall, this study highlights the importance of targeting FBXL20 in cancers in conjunction with chemotherapy and may represent a promising anticancer strategy to overcome chemoresistance. Apoptosis is a programmed cell death that efficiently removes damaged cells to maintain tissue homeostasis. Defect in apoptotic machinery can lead to tumor development, progression, and resistance to chemotherapy. PUMA (p53 upregulated modulator of apoptosis) and BAX (BCL2-associated X protein) are among the most well-known inducers of apoptosis. It has been reported that expression levels of BAX and PUMA are controlled at the posttranslational level by phosphorylation. However, the posttranslational regulation of these proapoptotic proteins remains largely unexplored. In this study, using biochemical, molecular biology, flow cytometric, and immunohistochemistry techniques, we show that PUMA and BAX are the direct target of the F-box protein FBXL20, which restricts their cellular levels. FBXL20 directs the proteasomal degradation of PUMA and BAX in a protein kinase AKT1-dependent manner to promote cancer cell proliferation and tumor growth. Interestingly, inactivation of AKT1 results in activation of another protein kinase GSK3α/β, which facilitates the proteasomal degradation of FBXL20 by another F-box protein, FBXO31. Thus, a switch between two signaling kinases AKT1 and GSK3α/β modulates the functional activity of these proapoptotic regulators, thereby determining cell survival or death. RNAi-mediated ablation of FBXL20 results in increased levels of PUMA as well as BAX, which further enhances the sensitivity of cancer cells to chemotherapeutic drugs. We showed that high level expression of FBXL20 in cancer cells reduces therapeutic drug-induced apoptosis and promotes chemoresistance. Overall, this study highlights the importance of targeting FBXL20 in cancers in conjunction with chemotherapy and may represent a promising anticancer strategy to overcome chemoresistance. The phenomenon of programmed cell death (apoptosis) is a conserved cellular mechanism to maintain homeostasis during development and aging (1Elmore S. Apoptosis: A review of programmed cell death.Toxicol. Pathol. 2007; 35: 495-516Crossref PubMed Scopus (7813) Google Scholar). Cells undergo apoptosis by two distinct pathways, namely intrinsic and extrinsic pathway. BCL2 family proteins (BCL-2 homology domain containing proteins) play important roles in determining the cell fate by regulating the mitochondrial membrane potential in the intrinsic pathway of apoptosis. BCL-2 family proteins are classified into antiapoptotic and proapoptotic proteins. PUMA (p53 upregulated modulator of apoptosis) and BAX (BCL2-associated X protein) are most well-known apoptotic inducers. Under normal condition, apoptotic activity of BAX is impaired by inhibitory interactions with antiapoptotic BCL-2 proteins (2Shimizu S. Narita M. Tsujimoto Y. Bcl-2 family proteins regulate the release of apoptogenic cytochrome c by the mitochondrial channel VDAC.Nature. 1999; 399: 483-487Crossref PubMed Scopus (1892) Google Scholar, 3Gross A. McDonnell J.M. Korsmeyer S.J. BCL-2 family members and the mitochondria in apoptosis.Genes Dev. 1999; 13: 1899-1911Crossref PubMed Scopus (3212) Google Scholar, 4Cory S. Adams J.M. The Bcl2 family: Regulators of the cellular life-or-death switch.Nat. Rev. Cancer. 2002; 2: 647-656Crossref PubMed Scopus (3233) Google Scholar). Upon death stimuli, PUMA contributes to the induction of apoptosis by promoting the disruption of BAX-BCL2 interaction and thereby release BAX from BAX-BCL2 complex (5Yu J. Zhang L. PUMA, a potent killer with or without p53.Oncogene. 2008; 27: S71-S83Crossref PubMed Scopus (417) Google Scholar, 6Willis S.N. Adams J.M. Life in the balance: How BH3-only proteins induce apoptosis.Curr. Opin. Cell Biol. 2005; 17: 617-625Crossref PubMed Scopus (634) Google Scholar). Released BAX is then oligomerized on the mitochondrial membrane to lower the membrane potential, resulting in the release of cytochrome c and induction of apoptosis (7Jürgensmeier J.M. Xie Z. Deveraux Q. Ellerby L. Bredesen D. Reed J.C. Bax directly induces release of cytochrome c from isolated mitochondria.Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 4997-5002Crossref PubMed Scopus (1363) Google Scholar, 8Zhang M. Zheng J. Nussinov R. Ma B. Release of cytochrome C from Bax pores at the mitochondrial membrane.Sci. Rep. 2017; 7: 2635Crossref PubMed Scopus (65) Google Scholar, 9Narita M. Shimizu S. Ito T. Chittenden T. Lutz R.J. Matsuda H. Tsujimoto Y. Bax interacts with the permeability transition pore to induce permeability transition and cytochrome c release in isolated mitochondria.Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14681-14686Crossref PubMed Scopus (860) Google Scholar). The expression of PUMA and BAX is largely regulated at the transcriptional level by tumor suppressor p53 (10Yu J. Zhang L. Hwang P.M. Kinzler K.W. Vogelstein B. PUMA induces the rapid apoptosis of colorectal cancer cells.Mol. Cell. 2001; 7: 673-682Abstract Full Text Full Text PDF PubMed Scopus (1072) Google Scholar, 11Nakano K. Vousden K.H. PUMA, a novel pro-apoptotic gene, is induced by p53.Mol. Cell. 2001; 7: 683-694Abstract Full Text Full Text PDF PubMed Scopus (1827) Google Scholar, 12Chipuk J.E. Kuwana T. Bouchier-Hayes L. Droin N.M. Newmeyer D.D. Schuler M. Green D.R. Direct activation of Bax by p53 mediates mitochondrial membrane permeabilization and apoptosis.Science. 2004; 303: 1010-1014Crossref PubMed Scopus (1615) Google Scholar). Therefore, presence of p53 activity in tumor increases the potency of chemotherapeutic drugs through facilitating the induction of apoptosis. PUMA is also been shown to be regulated by other transcription factors such as p73, E2F1, and FOXO3a (13Melino G. Bernassola F. Ranalli M. Yee K. Zong W.X. Corazzari M. Knight R.A. Green D.R. Thompson C. Vousden K.H. p73 Induces apoptosis via PUMA transactivation and Bax mitochondrial translocation.J. Biol. Chem. 2004; 279: 8076-8083Abstract Full Text Full Text PDF PubMed Scopus (309) Google Scholar, 14Ming L. Sakaida T. Yue W. Jha A. Zhang L. Yu J. Sp1 and p73 activate PUMA following serum starvation.Carcinogenesis. 2008; 29: 1878-1884Crossref PubMed Scopus (72) Google Scholar, 15Hershko T. Ginsberg D. Up-regulation of Bcl-2 homology 3 (BH3)-only proteins by E2F1 mediates apoptosis.J. Biol. Chem. 2004; 279: 8627-8634Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar, 16You H. Pellegrini M. Tsuchihara K. Yamamoto K. Hacker G. Erlacher M. Villunger A. Mak T.W. FOXO3a-dependent regulation of Puma in response to cytokine/growth factor withdrawal.J. Exp. Med. 2006; 203: 1657-1663Crossref PubMed Scopus (333) Google Scholar). Recent studies have demonstrated that both PUMA and BAX could be regulated at the proteasomal level in a phosphorylation-dependent manner (17Fricker M. O'Prey J. Tolkovsky A.M. Ryan K.M. Phosphorylation of Puma modulates its apoptotic function by regulating protein stability.Cell Death Dis. 2010; 1e59Crossref PubMed Scopus (53) Google Scholar, 18Sandow J.J. Jabbour A.M. Condina M.R. Daunt C.P. Stomski F.C. Green B.D. Riffkin C.D. Hoffmann P. Guthridge M.A. Silke J. Lopez A.F. Ekert P.G. Cytokine receptor signaling activates an IKK-dependent phosphorylation of PUMA to prevent cell death.Cell Death Differ. 2012; 19: 633-641Crossref PubMed Scopus (27) Google Scholar, 19Proto-Siqueira R. Panepucci R.A. Careta F.P. Lee A. Clear A. Morris K. Owen C. Rizzatti E.G. Silva Jr., W.A. Falcão R.P. Zago M.A. Gribben J.G. SAGE analysis demonstrates increased expression of TOSO contributing to Fas-mediated resistance in CLL.Blood. 2008; 112: 394-397Crossref PubMed Scopus (36) Google Scholar, 20Li B. Dou Q.P. Bax degradation by the ubiquitin/proteasome-dependent pathway: Involvement in tumor survival and progression.Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 3850-3855Crossref PubMed Scopus (408) Google Scholar), and this partly attributes to survival mechanisms of cancer cells (21Lee J.U. Hosotani R. Wada M. Doi R. Kosiba T. Fujimoto K. Miyamoto Y. Tsuji S. Nakajima S. Nishimura Y. Imamura M. Role of Bcl-2 family proteins (Bax, Bcl-2 and Bcl-X) on cellular susceptibility to radiation in pancreatic cancer cells.Eur. J. Cancer. 1999; 35: 1374-1380Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). However, mechanistic basis of proteasomal regulation of PUMA and BAX by E3 ubiquitin ligases is largely unknown. Human genome encodes for approximately 600 E3 ubiquitin ligases among which RING finger SCF (SKP1-Cullin1-F-box) E3 ubiquitin ligases are well studied and best known for targeting phosphorylated proteins for promoting their proteasomal degradation. SCF E3 ubiquitin ligases comprise of three invariable components (SKP1, Cullin1, and RBX1) and a variable component - F-box proteins. F-box proteins are associated with SKP1 through their conserved F-box motif to form SCF complex and function as substrate receptor (22Nakayama K.I. Nakayama K. Ubiquitin ligases: Cell-cycle control and cancer.Nat. Rev. Cancer. 2006; 6: 369-381Crossref PubMed Scopus (1106) Google Scholar, 23Jin J. Cardozo T. Lovering R.C. Elledge S.J. Pagano M. Harper J.W. Systematic analysis and nomenclature of mammalian F-box proteins.Genes Dev. 2004; 8: 2573-2580Crossref Scopus (523) Google Scholar). Typically, F-box proteins recognize phosphorylated substrates and promote their polyubiquitination. Previous reports showed that phosphorylated form of PUMA and BAX is less stable than nonphosphorylated form, indicating that SCF E3 ubiquitin ligase(s) might be involved in destabilization of PUMA and BAX (17Fricker M. O'Prey J. Tolkovsky A.M. Ryan K.M. Phosphorylation of Puma modulates its apoptotic function by regulating protein stability.Cell Death Dis. 2010; 1e59Crossref PubMed Scopus (53) Google Scholar, 18Sandow J.J. Jabbour A.M. Condina M.R. Daunt C.P. Stomski F.C. Green B.D. Riffkin C.D. Hoffmann P. Guthridge M.A. Silke J. Lopez A.F. Ekert P.G. Cytokine receptor signaling activates an IKK-dependent phosphorylation of PUMA to prevent cell death.Cell Death Differ. 2012; 19: 633-641Crossref PubMed Scopus (27) Google Scholar, 19Proto-Siqueira R. Panepucci R.A. Careta F.P. Lee A. Clear A. Morris K. Owen C. Rizzatti E.G. Silva Jr., W.A. Falcão R.P. Zago M.A. Gribben J.G. SAGE analysis demonstrates increased expression of TOSO contributing to Fas-mediated resistance in CLL.Blood. 2008; 112: 394-397Crossref PubMed Scopus (36) Google Scholar, 20Li B. Dou Q.P. Bax degradation by the ubiquitin/proteasome-dependent pathway: Involvement in tumor survival and progression.Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 3850-3855Crossref PubMed Scopus (408) Google Scholar). In this study, we have identified FBXL20 as a potential regulator of proapoptotic proteins PUMA and BAX. We show that FBXL20 regulates PUMA and BAX at the posttranslational level by promoting their degradation through 26S proteasome. FBXL20-mediated degradation of PUMA and BAX reduces the potency of chemotherapeutic drug-induced apoptosis and thereby promotes tumor growth. Our study revealed that inactivation of AKT1 results in the stabilization of BAX and PUMA due to the proteasomal degradation of FBXL20 by SCFFBXO31 E3 ubiquitin ligase in GSK3α/β-dependent manner. Thus, our study delineates an important cellular signaling pathway required to maintain the basal levels of proapoptotic regulators. Overall, we propose that targeting FBXL20 in cancers in conjunction with chemotherapy may be a promising anticancer strategy. Proapoptotic genes, PUMA and BAX, are expressed at low levels in unstressed proliferating cells and rapidly induced in response to wide range of cellular stresses (5Yu J. Zhang L. PUMA, a potent killer with or without p53.Oncogene. 2008; 27: S71-S83Crossref PubMed Scopus (417) Google Scholar, 21Lee J.U. Hosotani R. Wada M. Doi R. Kosiba T. Fujimoto K. Miyamoto Y. Tsuji S. Nakajima S. Nishimura Y. Imamura M. Role of Bcl-2 family proteins (Bax, Bcl-2 and Bcl-X) on cellular susceptibility to radiation in pancreatic cancer cells.Eur. J. Cancer. 1999; 35: 1374-1380Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). In general, induction of PUMA and BAX is considered to occur at the transcriptional level (7Jürgensmeier J.M. Xie Z. Deveraux Q. Ellerby L. Bredesen D. Reed J.C. Bax directly induces release of cytochrome c from isolated mitochondria.Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 4997-5002Crossref PubMed Scopus (1363) Google Scholar, 8Zhang M. Zheng J. Nussinov R. Ma B. Release of cytochrome C from Bax pores at the mitochondrial membrane.Sci. Rep. 2017; 7: 2635Crossref PubMed Scopus (65) Google Scholar, 9Narita M. Shimizu S. Ito T. Chittenden T. Lutz R.J. Matsuda H. Tsujimoto Y. Bax interacts with the permeability transition pore to induce permeability transition and cytochrome c release in isolated mitochondria.Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14681-14686Crossref PubMed Scopus (860) Google Scholar, 10Yu J. Zhang L. Hwang P.M. Kinzler K.W. Vogelstein B. PUMA induces the rapid apoptosis of colorectal cancer cells.Mol. Cell. 2001; 7: 673-682Abstract Full Text Full Text PDF PubMed Scopus (1072) Google Scholar, 11Nakano K. Vousden K.H. PUMA, a novel pro-apoptotic gene, is induced by p53.Mol. Cell. 2001; 7: 683-694Abstract Full Text Full Text PDF PubMed Scopus (1827) Google Scholar, 12Chipuk J.E. Kuwana T. Bouchier-Hayes L. Droin N.M. Newmeyer D.D. Schuler M. Green D.R. Direct activation of Bax by p53 mediates mitochondrial membrane permeabilization and apoptosis.Science. 2004; 303: 1010-1014Crossref PubMed Scopus (1615) Google Scholar, 13Melino G. Bernassola F. Ranalli M. Yee K. Zong W.X. Corazzari M. Knight R.A. Green D.R. Thompson C. Vousden K.H. p73 Induces apoptosis via PUMA transactivation and Bax mitochondrial translocation.J. Biol. Chem. 2004; 279: 8076-8083Abstract Full Text Full Text PDF PubMed Scopus (309) Google Scholar, 14Ming L. Sakaida T. Yue W. Jha A. Zhang L. Yu J. Sp1 and p73 activate PUMA following serum starvation.Carcinogenesis. 2008; 29: 1878-1884Crossref PubMed Scopus (72) Google Scholar, 15Hershko T. Ginsberg D. Up-regulation of Bcl-2 homology 3 (BH3)-only proteins by E2F1 mediates apoptosis.J. Biol. Chem. 2004; 279: 8627-8634Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar, 16You H. Pellegrini M. Tsuchihara K. Yamamoto K. Hacker G. Erlacher M. Villunger A. Mak T.W. FOXO3a-dependent regulation of Puma in response to cytokine/growth factor withdrawal.J. Exp. Med. 2006; 203: 1657-1663Crossref PubMed Scopus (333) Google Scholar). However, recent studies indicated that stability of PUMA and BAX is also regulated at the posttranslational level (17Fricker M. O'Prey J. Tolkovsky A.M. Ryan K.M. Phosphorylation of Puma modulates its apoptotic function by regulating protein stability.Cell Death Dis. 2010; 1e59Crossref PubMed Scopus (53) Google Scholar, 18Sandow J.J. Jabbour A.M. Condina M.R. Daunt C.P. Stomski F.C. Green B.D. Riffkin C.D. Hoffmann P. Guthridge M.A. Silke J. Lopez A.F. Ekert P.G. Cytokine receptor signaling activates an IKK-dependent phosphorylation of PUMA to prevent cell death.Cell Death Differ. 2012; 19: 633-641Crossref PubMed Scopus (27) Google Scholar, 20Li B. Dou Q.P. Bax degradation by the ubiquitin/proteasome-dependent pathway: Involvement in tumor survival and progression.Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 3850-3855Crossref PubMed Scopus (408) Google Scholar). We therefore presumed that PUMA and BAX might be regulated at the proteasomal level and performed a screen wherein we ectopically expressed ORFs of 54 SCF-F-box proteins to identify putative F-box protein(s) that may regulate PUMA. From this screen, F box protein FBXL20 emerged as the potential candidate that reduced PUMA levels upon ectopic expression (Figs. 1A and S1A). Interestingly, we found that ectopically expressed FBXL20 also reduced BAX levels (Figs. 1A and S1A). To determine whether FBXL20 maintains physiological levels of PUMA and BAX, we generated FBXL20 stable knockdown (FBXL20-KD) MCF7 cells using two unrelated lentiviral shRNAs. As compared with the cells expressing nonsilencing shRNA (NS), the levels of PUMA and BAX were significantly increased in FBXL20-KD cells (Figs. 1B and S1B), while the levels of BIM and BCL2 were not affected by FBXL20 knockdown. However, the transcript levels of both PUMA and BAX were not altered in FBXL20-depleted cells (Fig. S1C). An increase in PUMA and BAX levels upon FBXL20 knockdown in MCF7 cells indicated that FBXL20-depleted cells could be more sensitive to chemotherapeutic drugs than wild-type NS cells. We therefore performed a series of experiments to examine the sensitivity of FBXL20 knockdown cells to Doxorubicin (DOXO) and Camptothecin (CPT). First, we performed MTT assay in MCF7 cells expressing NS and FBXL20 shRNA with varying concentrations of either DOXO or CPT and evaluated 50% growth inhibitory concentration (IC50) of these drugs. As shown in Figure 1C, the IC50 values of DOXO were ∼2.7 fold less in FBXL20 knockdown cells (IC50 0.18 ± 0.05 μM) than that in NS cells (IC50 0.5 ± 0.09 μM). Consistent with this result, the IC50 values of CPT were also approximately threefold lower in FBXl20-KD cells (IC50 1.0 ± 0.25 μM) as compared with NS cells (IC50 3.0 ± 0.1 μM) (Fig. 1C). These results demonstrated that FBXL20 depletion increased the sensitivity to Doxorubicin or Camptothecin. We then performed clonogenic survival assay and results showed that FBXL20-KD cells showed an increased sensitivity to DOXO or CPT as compared with the NS cells treated with DMSO (Figs. 1, D and E and S1D). In contrast, knockdown of PUMA in FBXL20-KD cells resulted in formation of increased number of colonies as compared with FBXL20-KD cells treated with either DOXO or CPT indicating that increased levels of PUMA in FBXL20-KD increased sensitivity to chemotherapeutic agents, at least in part (Fig. 1, D and E). Next, we determined the apoptotic index of MCF7 cells expressing either NS shRNA or FBXL20 shRNA, or coexpressing FBXL20 and PUMA shRNAs or coexpressing FBXL20 shRNA and BAX shRNA following treatment of DOXO. FACS analysis of Annexin-V/7-AAD stained cells revealed that percentage of apoptotic cells was increased in FBXL20-KD cells as compared with NS cells, which further increased following treatment with DOXO or CPT (Figs. 1F and S1E). However, codepletion of PUMA or BAX in FBXL20 knockdown cells decreased the percentage of apoptotic cells as compared with the FBXL20-KD cells alone (Figs. 1F and S1E). We next performed comet assay to determine whether increased level of apoptosis of DOXO/CPT treated FBXL20-KD cells was due to induction of DNA fragmentation. The results of comet assay demonstrated that FBXL20-KD cells were more susceptible to DNA fragmentation (as evident by the appearance of higher percentage of apoptotic comets and DNA in tail) as compared with the NS cells following treatment with either DOXO or CPT (Fig. 1, G and H). However, the extent of DNA tailing was significantly reduced upon knocked down of PUMA in FBXL20-KD cells, indicating that accumulated PUMA in FBXL20-KD cells could be responsible for induction of severe DNA damage (Fig. 1H). Conversely, ectopic expression of FBXL20 decreased DNA fragmentation and DNA tailing following treatment with either DOXO or CPT (Fig. S1, F and G). In addition to this, an ectopic expression of FBXL20 also inhibited the cleavage of caspase 9 and PARP1 induced by DOXO treatment, suggesting the role of FBXL20 in counteracting apoptosis (Fig. S1H). Mitochondrial membrane potential alteration is one of the markers routinely measured to detect apoptotic cells. We then monitored the mitochondrial membrane potential using JC1 dye, which exhibits potential dependent fluorescence emission shift from red to green upon induction of intrinsic apoptosis. FACS analysis of cells expressing either vector or FBXL20, treated with CPT, and stained with JC1 dye showed that FBXL20 expressing cells display substantially less emission shift as compared with the cells expressing vector (Fig. 1I). BAX oligomerization on mitochondrial outer membrane leads to membrane polarization, release of cytochrome c, and induction of apoptosis (9Narita M. Shimizu S. Ito T. Chittenden T. Lutz R.J. Matsuda H. Tsujimoto Y. Bax interacts with the permeability transition pore to induce permeability transition and cytochrome c release in isolated mitochondria.Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14681-14686Crossref PubMed Scopus (860) Google Scholar, 10Yu J. Zhang L. Hwang P.M. Kinzler K.W. Vogelstein B. PUMA induces the rapid apoptosis of colorectal cancer cells.Mol. Cell. 2001; 7: 673-682Abstract Full Text Full Text PDF PubMed Scopus (1072) Google Scholar). We found that FBXL20 also negatively regulates BAX (Fig. 1, A and B); we therefore, assessed the extent of BAX oligomerization in FBXL20-KD cells following treatment with Doxorubicin. Immunoblotting data showed that knockdown of FBXL20 resulted in an elevated level of BAX oligomers, which was further increased upon Doxorubicin treatment (Fig. 1J). DNA damaged-induced increased oligomerization of BAX in FBXL20-KD cells could be due to either the increased expression levels of BAX or the increased disruption of BAX-BCL2 interaction resulting from the increased levels of PUMA. To address these possibilities, we performed coimmunoprecipitation assay to examine BAX and BCL2 interaction in MCF7 cells expressing NS and FBXL20 shRNA treated with and without Doxorubicin. Immunoblotting results of immunoprecipitates showed that BAX and BCL2 interaction was disrupted in NS and FBXL20-KD cells treated with Doxorubicin (Fig. S1I). Therefore, increased oligomerization of BAX in FBXL20-KD cells could be due to both increase levels of BAX as well as disruption of BCL2-BAX interaction. Collectively, these results suggest that FBXL20 inhibits the induction of apoptosis by reducing the expression levels of proapoptotic proteins PUMA and BAX. We next sought to understand the mechanism(s) by which FBXL20 regulates PUMA and BAX. To understand how FBXL20 reduces the levels of PUMA and BAX, we first examined mRNA level of PUMA and BAX upon ectopic expression of FBXL20. Real-time RT-PCR results showed that ectopic expression FBXL20 in MCF7 cells did not affect the mRNA levels of PUMA and BAX (Fig. 2, A and B). In contrast, protein levels of PUMA and BAX are significantly reduced by FBXL20 (Figs. 1A and S1, A and B). Generally, F-box proteins function by modulating activity of their substrates at the proteasomal level. We then examined whether PUMA and BAX are substrates of FBXL20 and are regulated at the proteasomal level. Consistent with this idea, treatment of cells with proteasomal inhibitor, MG132 inhibited the ability of FBXL20 to reduce expression levels of PUMA and BAX (Fig. 2C). This suggested that FBXL20-mediated reduction of PUMA and BAX protein level is a posttranscriptional event. Preceding results demonstrated that PUMA and BAX protein levels are increased upon FBXL20 knockdown (Fig. 1B). We therefore monitored PUMA and BAX protein turnover by cycloheximide chase assay. As shown in Figure 2, D–F, the turnover of PUMA and BAX decreased in FBXL20-KD cells (Fig. 2, D–F). PUMA and BAX are known to be regulated at the transcriptional level predominantly by p53 (11Nakano K. Vousden K.H. PUMA, a novel pro-apoptotic gene, is induced by p53.Mol. Cell. 2001; 7: 683-694Abstract Full Text Full Text PDF PubMed Scopus (1827) Google Scholar, 12Chipuk J.E. Kuwana T. Bouchier-Hayes L. Droin N.M. Newmeyer D.D. Schuler M. Green D.R. Direct activation of Bax by p53 mediates mitochondrial membrane permeabilization and apoptosis.Science. 2004; 303: 1010-1014Crossref PubMed Scopus (1615) Google Scholar, 24Yu J. Wang Z. Kinzler K.W. Vogelstein B. Zhang L. PUMA mediates the apoptotic response to p53 in colorectal cancer cells.Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 1931-1936Crossref PubMed Scopus (508) Google Scholar). We therefore sought to examine whether FBXL20-mediated degradation of PUMA or BAX was p53-dependent. Immunoblot data showed that ectopically expressed FBXL20 reduced the expression levels of BAX and PUMA in both p53-wild-type and p53-deficient cells, indicating that FBXL20-mediated degradation of PUMA and BAX was p53-independent (Fig. 2G) Typically, F-box proteins associate with SCF complex through their F-box motif to promote substrate polyubiquitylation. To determine the involvement of F-box motif of FBXL20 in PUMA or BAX degradation, we generated F-box motif deletion mutant of FBXL20 (DDK-ΔF-FBXL20). Unlike wild type, ectopically expressed DDK-ΔF-FBXL20 failed to degrade both PUMA and BAX (Fig. 2, H and I), indicating that FBXL20 promotes the degradation of both PUMA and BAX via SCF complex. F-box protein-mediated degradation of their substrates requires an interaction with the substrates. To ascertain whether FBXL20 interacts with PUMA and BAX, we performed a series of coimmunoprecipitation experiments. First, we ectopically expressed DDK-tagged FBXL20 and HA-tagged PUMA in MCF7 cells and whole cell lysates were immunoprecipitated with anti-DDK or anti-HA antibodies, respectively. Figure 3A showed the presence of HA-PUMA in DDK-FBXL20 immunoprecipitates. The reciprocal immunoprecipitation showed the presence of DDK-FBXL20 in HA-PUMA immunoprecipitates (Fig. 3A). Consistent with this observation, the interaction of FBXL20 with PUMA and BAX was also observed at the endogenous level (Fig. 3B). In addition, we also examined physical interaction of PUMA and BAX with FBXL20 by an in vitro by GST pull-down assay. As shown in Figure 3, C and D, in vitro interaction between purified GST-PUMA or GST-BAX and His-FBXL20 was detected. These results confirm that FBXL20 directly interacts with BAX and PUMA. Given that BH3 domain in PUMA is involved in protein–protein interaction, we presumed that the FBXL20 might interact with BH3 domain of PUMA. To investigate this possibility, we ectopically expressed DDK-FBXL20 and HA-PUMA or HA-ΔBH3-PUMA in MCF7 cells and immunoprecipitated with either anti-DDK or anti-HA antibodies, respectively. As shown in Figure 3E, DDK-FBXL20 interacted with HA-PUMA and not with HA-ΔBH3-PUMA indicating that FBXL20 interacts with BH3 domain of PUMA. In agreement with previous study, we found that BH3 domain of PUMA is important to interact with BAX. Interestingly, immunoblotting of FBXL20 immunoprecipitates revealed that interaction of FBXL20 with BAX is independent of PUMA (Fig. 3E). Typically, F-box proteins direct proteasomal degradation of their substrates by promoting their polyubiquitination (22Nakayama K.I. Nakayama K. Ubiquitin ligases: Cell-cycle control and cancer.Nat. Rev. Cancer. 2006; 6: 369-381Crossref PubMed Scopus (1106) Google Scholar, 25Ciechanover A. The ubiquitin-proteasome pathway: On protein death and cell life.EMBO J. 1998; 17: 7151-7160Crossref PubMed Scopus (1165) Google Scholar). We then asked whether FBXL20 promotes polyubiquitination of PUMA and BAX. Ubiquitin possesses seven lysine residues (K6, K11, K27, K29, K33, K48, and K63) that may be utilized to form polyubiquitin chains. Among these lysine residues, K48-linked polyubiquitin chains are generally marked for proteasomal degradation of target proteins (26Jacobson A.D. Zhang N.Y. Xu P. Han K.J. Noone S. Peng J. Liu C.W. The lysine 48 and lysine 63 ubiquitin conjugates are processed differently by the 26 s proteasome.J. Biol. Chem. 2009; 284: 35485-35494Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). We therefore examined whether FBXL20 promotes K48-linked polyubiquitination of BAX and PUMA. Immunoprecipitation of MCF7 cell extracts expressing either vector or DDK-FBXL20 or DDK-ΔF-FBXL20 with BAX and PUMA antibodies followed by immunoblotting with K48 linked Ub antibody showed that DDK-FBXL20 promoted K48-linked polyubiquitination of PUMA (Fig. 3F) and BAX (Fig. 3G). However, the knockdown of FBXL20 reduced K48-linked ubiquitination of PUMA (Fig. 3H) and BAX (Fig. 3I). Consistent with these results, in vitro ubiquitination assay also demonstrated that FBXL20 can promote the polyubiquitination of recombinant PUMA and BAX (Fig. 3, J and K). Collectively, these results suggested that FBXL20 interacts with PUMA and BAX and promotes polyubiquitina" @default.
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• W3204188369 date "2021-10-01" @default.
• W3204188369 modified "2023-10-02" @default.
• W3204188369 title "FBXL20 promotes breast cancer malignancy by inhibiting apoptosis through degradation of PUMA and BAX" @default.
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