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SAG/RBX2 is a novel substrate of NEDD4-1 E3 ubiquitin ligase and mediates NEDD4-1 induced chemosensitization.

Zhou W, Xu J, Zhao Y, Sun Y - Oncotarget (2014)

Bottom Line: Consistently, SAG protein half-life is shortened or extended by NEDD4-1 overexpression or silencing, respectively.Biologically, NEDD4-1 overexpression sensitizes cancer cells to etoposide-induced apoptosis by reducing SAG levels through targeted degradation.Thus, SAG is added to a growing list of NEDD4-1 substrates and mediates its biological function.

View Article: PubMed Central - PubMed

Affiliation: Division of Radiation and Cancer Biology, Department of Radiation Oncology, University of Michigan, Ann Arbor, MI.

ABSTRACT
Sensitive to apoptosis gene (SAG), also known as RBX2, ROC2, or RNF7, is a RING component of SCF E3 ubiquitin ligases, which regulates cellular functions through ubiquitylation and degradation of many protein substrates. Although our previous studies showed that SAG is transcriptionally induced by redox, mitogen and hypoxia via AP-1 and HIF-1, it is completely unknown whether and how SAG is ubiquitylated and degraded. Here we report that NEDD4-1, a HECT domain-containing E3 ubiquitin ligase, binds via its HECT domain directly with SAG's C-terminal RING domain and ubiquitylates SAG for proteasome-mediated degradation. Consistently, SAG protein half-life is shortened or extended by NEDD4-1 overexpression or silencing, respectively. We also found that SAG bridges NEDD4-1 via its C-terminus and CUL-5 via its N-terminus to form a NEDD4-1/SAG/CUL-5 tri-complex. Biologically, NEDD4-1 overexpression sensitizes cancer cells to etoposide-induced apoptosis by reducing SAG levels through targeted degradation. Thus, SAG is added to a growing list of NEDD4-1 substrates and mediates its biological function.

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Levels of SAG and NEDD4-1 in lung cancer cells and SAG-NEDD4-1 binding(A). Protein levels of NEDD4-1, SAG, and ROC1 in multiple lung cancer cell lines and a normal cell line, BEAS-2B. Cell lysates were prepared from indicated cell lines, followed by Western blotting with equal amount protein loaded, using indicated antibodies. (B). SAG immunoprecipitates endogenous NEDD4-1. 293 cells were transiently transfected with FLAG-SAG and FLAG-ROC1, along with pCDNA3.1 vector control. Cells were lysed and immunoprecipitated with FLAG antibody, followed by IB with indicated antibodies. (C&D). Binding of endogenous SAG and NEDD4-1. H1299 cells were left untreated or treated with 10 μM MG132 for 4 hrs prior to harvesting. Whole cell extracts were immunoprecipitated with antibodies against NEDD4-1 (C) or SAG (D), along with IgG control, followed by Western blotting with indicated antibodies.
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Figure 1: Levels of SAG and NEDD4-1 in lung cancer cells and SAG-NEDD4-1 binding(A). Protein levels of NEDD4-1, SAG, and ROC1 in multiple lung cancer cell lines and a normal cell line, BEAS-2B. Cell lysates were prepared from indicated cell lines, followed by Western blotting with equal amount protein loaded, using indicated antibodies. (B). SAG immunoprecipitates endogenous NEDD4-1. 293 cells were transiently transfected with FLAG-SAG and FLAG-ROC1, along with pCDNA3.1 vector control. Cells were lysed and immunoprecipitated with FLAG antibody, followed by IB with indicated antibodies. (C&D). Binding of endogenous SAG and NEDD4-1. H1299 cells were left untreated or treated with 10 μM MG132 for 4 hrs prior to harvesting. Whole cell extracts were immunoprecipitated with antibodies against NEDD4-1 (C) or SAG (D), along with IgG control, followed by Western blotting with indicated antibodies.

Mentions: In a recent large scale proteomic study, SAG was identified as a binding partner of human NEDD4-1, but not NEDD4-2 [27]. Since it is unknown how SAG is ubiquitylated and degraded, and by which E3 ligase, we hypothesized that NEDD4-1 may be an E3 ligase for SAG. We first determined potential correlation in the protein levels between SAG and NEDD4-1 in multiple human lung cancer cell lines, and found that the level of SAG, but not its family member RBX1, is largely correlated in a inverse manner with NEDD4-1 (Figure 1A). We next determined whether the two proteins would bind to each other by an IP pull-down assay. Indeed, ectopically expressed SAG, but to a lesser extent, ROC1, pulled down endogenous NEDD4-1 in 293 cells (Figure 1B). We further determined endogenous binding of two proteins and found that SAG was detected in NEDD4-1 immunoprecipitates, but not in the IgG control. Reciprocally, NEDD4-1 was detected in SAG immunoprecipitates (Figure 1C&D). Moreover, SAG-NEDD4-1 binding was significantly enhanced when protein degradation was inhibited in the presence of MG132 (Figure 1C&D). Thus, SAG binds to NEDD4-1 under the physiological conditions.


SAG/RBX2 is a novel substrate of NEDD4-1 E3 ubiquitin ligase and mediates NEDD4-1 induced chemosensitization.

Zhou W, Xu J, Zhao Y, Sun Y - Oncotarget (2014)

Levels of SAG and NEDD4-1 in lung cancer cells and SAG-NEDD4-1 binding(A). Protein levels of NEDD4-1, SAG, and ROC1 in multiple lung cancer cell lines and a normal cell line, BEAS-2B. Cell lysates were prepared from indicated cell lines, followed by Western blotting with equal amount protein loaded, using indicated antibodies. (B). SAG immunoprecipitates endogenous NEDD4-1. 293 cells were transiently transfected with FLAG-SAG and FLAG-ROC1, along with pCDNA3.1 vector control. Cells were lysed and immunoprecipitated with FLAG antibody, followed by IB with indicated antibodies. (C&D). Binding of endogenous SAG and NEDD4-1. H1299 cells were left untreated or treated with 10 μM MG132 for 4 hrs prior to harvesting. Whole cell extracts were immunoprecipitated with antibodies against NEDD4-1 (C) or SAG (D), along with IgG control, followed by Western blotting with indicated antibodies.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4196160&req=5

Figure 1: Levels of SAG and NEDD4-1 in lung cancer cells and SAG-NEDD4-1 binding(A). Protein levels of NEDD4-1, SAG, and ROC1 in multiple lung cancer cell lines and a normal cell line, BEAS-2B. Cell lysates were prepared from indicated cell lines, followed by Western blotting with equal amount protein loaded, using indicated antibodies. (B). SAG immunoprecipitates endogenous NEDD4-1. 293 cells were transiently transfected with FLAG-SAG and FLAG-ROC1, along with pCDNA3.1 vector control. Cells were lysed and immunoprecipitated with FLAG antibody, followed by IB with indicated antibodies. (C&D). Binding of endogenous SAG and NEDD4-1. H1299 cells were left untreated or treated with 10 μM MG132 for 4 hrs prior to harvesting. Whole cell extracts were immunoprecipitated with antibodies against NEDD4-1 (C) or SAG (D), along with IgG control, followed by Western blotting with indicated antibodies.
Mentions: In a recent large scale proteomic study, SAG was identified as a binding partner of human NEDD4-1, but not NEDD4-2 [27]. Since it is unknown how SAG is ubiquitylated and degraded, and by which E3 ligase, we hypothesized that NEDD4-1 may be an E3 ligase for SAG. We first determined potential correlation in the protein levels between SAG and NEDD4-1 in multiple human lung cancer cell lines, and found that the level of SAG, but not its family member RBX1, is largely correlated in a inverse manner with NEDD4-1 (Figure 1A). We next determined whether the two proteins would bind to each other by an IP pull-down assay. Indeed, ectopically expressed SAG, but to a lesser extent, ROC1, pulled down endogenous NEDD4-1 in 293 cells (Figure 1B). We further determined endogenous binding of two proteins and found that SAG was detected in NEDD4-1 immunoprecipitates, but not in the IgG control. Reciprocally, NEDD4-1 was detected in SAG immunoprecipitates (Figure 1C&D). Moreover, SAG-NEDD4-1 binding was significantly enhanced when protein degradation was inhibited in the presence of MG132 (Figure 1C&D). Thus, SAG binds to NEDD4-1 under the physiological conditions.

Bottom Line: Consistently, SAG protein half-life is shortened or extended by NEDD4-1 overexpression or silencing, respectively.Biologically, NEDD4-1 overexpression sensitizes cancer cells to etoposide-induced apoptosis by reducing SAG levels through targeted degradation.Thus, SAG is added to a growing list of NEDD4-1 substrates and mediates its biological function.

View Article: PubMed Central - PubMed

Affiliation: Division of Radiation and Cancer Biology, Department of Radiation Oncology, University of Michigan, Ann Arbor, MI.

ABSTRACT
Sensitive to apoptosis gene (SAG), also known as RBX2, ROC2, or RNF7, is a RING component of SCF E3 ubiquitin ligases, which regulates cellular functions through ubiquitylation and degradation of many protein substrates. Although our previous studies showed that SAG is transcriptionally induced by redox, mitogen and hypoxia via AP-1 and HIF-1, it is completely unknown whether and how SAG is ubiquitylated and degraded. Here we report that NEDD4-1, a HECT domain-containing E3 ubiquitin ligase, binds via its HECT domain directly with SAG's C-terminal RING domain and ubiquitylates SAG for proteasome-mediated degradation. Consistently, SAG protein half-life is shortened or extended by NEDD4-1 overexpression or silencing, respectively. We also found that SAG bridges NEDD4-1 via its C-terminus and CUL-5 via its N-terminus to form a NEDD4-1/SAG/CUL-5 tri-complex. Biologically, NEDD4-1 overexpression sensitizes cancer cells to etoposide-induced apoptosis by reducing SAG levels through targeted degradation. Thus, SAG is added to a growing list of NEDD4-1 substrates and mediates its biological function.

Show MeSH
Related in: MedlinePlus