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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: 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.

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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Related in: MedlinePlus

SAG bridges NEDD4-1 and CUL-5 to form a NEDD4-1/SAG/CUL-5 tri-complex(A&B). NEDD4-1, SAG, and CUL-5 bind to each other under physiological condition. 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 (A) or SAG (B), along with IgG control, followed by Western blotting with indicated antibodies.
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Figure 3: SAG bridges NEDD4-1 and CUL-5 to form a NEDD4-1/SAG/CUL-5 tri-complex(A&B). NEDD4-1, SAG, and CUL-5 bind to each other under physiological condition. 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 (A) or SAG (B), along with IgG control, followed by Western blotting with indicated antibodies.

Mentions: It has been previously shown that a) under physiological conditions, SAG preferentially binds with CUL-5 [30], and b) SAG, through its N-terminal domain (NTD) binds to the C-terminal domain (CTD) of CUL-5 [31, 32]. We, therefore, tested our hypothesis that NEDD4-1/SAG/CUL-5 might form a tri-complex under physiological conditions by a pull-down assay. Indeed, both SAG and CUL-5 were detected in NEDD4-1 immunoprecipitates (Figure 3A). Likewise, both NEDD4-1 and CUL-5 were detected in SAG immunoprecipitates (Figure 3B). Furthermore, treatment with proteasome inhibitor MG132 increased the SAG levels as well as the binding among the three components (Figure 3A&B). Taken together, our results indicated that SAG binds to CUL-5 via its NTD [31, 32] and NEDD4-1 via its CTD to form a NEDD4-1/SAG/CUL-5 tri-complex under 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)

SAG bridges NEDD4-1 and CUL-5 to form a NEDD4-1/SAG/CUL-5 tri-complex(A&B). NEDD4-1, SAG, and CUL-5 bind to each other under physiological condition. 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 (A) or SAG (B), 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 3: SAG bridges NEDD4-1 and CUL-5 to form a NEDD4-1/SAG/CUL-5 tri-complex(A&B). NEDD4-1, SAG, and CUL-5 bind to each other under physiological condition. 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 (A) or SAG (B), along with IgG control, followed by Western blotting with indicated antibodies.
Mentions: It has been previously shown that a) under physiological conditions, SAG preferentially binds with CUL-5 [30], and b) SAG, through its N-terminal domain (NTD) binds to the C-terminal domain (CTD) of CUL-5 [31, 32]. We, therefore, tested our hypothesis that NEDD4-1/SAG/CUL-5 might form a tri-complex under physiological conditions by a pull-down assay. Indeed, both SAG and CUL-5 were detected in NEDD4-1 immunoprecipitates (Figure 3A). Likewise, both NEDD4-1 and CUL-5 were detected in SAG immunoprecipitates (Figure 3B). Furthermore, treatment with proteasome inhibitor MG132 increased the SAG levels as well as the binding among the three components (Figure 3A&B). Taken together, our results indicated that SAG binds to CUL-5 via its NTD [31, 32] and NEDD4-1 via its CTD to form a NEDD4-1/SAG/CUL-5 tri-complex under physiological conditions.

Bottom Line: 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.

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