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RHOBTB3 promotes proteasomal degradation of HIFα through facilitating hydroxylation and suppresses the Warburg effect.

Zhang CS, Liu Q, Li M, Lin SY, Peng Y, Wu D, Li TY, Fu Q, Jia W, Wang X, Ma T, Zong Y, Cui J, Pu C, Lian G, Guo H, Ye Z, Lin SC - Cell Res. (2015)

Bottom Line: Remarkably, RHOBTB3 dimerizes with LIMD1, and constructs a RHOBTB3/LIMD1-PHD2-VHL-HIFα complex to effect the maximal degradation of HIFα.Hypoxia reduces the RHOBTB3-centered complex formation, resulting in an accumulation of HIFα.Importantly, the expression level of RHOBTB3 is greatly reduced in human renal carcinomas, and RHOBTB3 deficiency significantly elevates the Warburg effect and accelerates xenograft growth.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Cellular Stress Biology, Innovation Center for Cell Signaling Network, School of Life Sciences, Xiamen University, Xiamen, Fujian 361005, China.

ABSTRACT
Hypoxia-inducible factors (HIFs) are master regulators of adaptive responses to low oxygen, and their α-subunits are rapidly degraded through the ubiquitination-dependent proteasomal pathway after hydroxylation. Aberrant accumulation or activation of HIFs is closely linked to many types of cancer. However, how hydroxylation of HIFα and its delivery to the ubiquitination machinery are regulated remains unclear. Here we show that Rho-related BTB domain-containing protein 3 (RHOBTB3) directly interacts with the hydroxylase PHD2 to promote HIFα hydroxylation. RHOBTB3 also directly interacts with the von Hippel-Lindau (VHL) protein, a component of the E3 ubiquitin ligase complex, facilitating ubiquitination of HIFα. Remarkably, RHOBTB3 dimerizes with LIMD1, and constructs a RHOBTB3/LIMD1-PHD2-VHL-HIFα complex to effect the maximal degradation of HIFα. Hypoxia reduces the RHOBTB3-centered complex formation, resulting in an accumulation of HIFα. Importantly, the expression level of RHOBTB3 is greatly reduced in human renal carcinomas, and RHOBTB3 deficiency significantly elevates the Warburg effect and accelerates xenograft growth. Our work thus reveals that RHOBTB3 serves as a scaffold to organize a multi-subunit complex that promotes the hydroxylation, ubiquitination and degradation of HIFα.

No MeSH data available.


Related in: MedlinePlus

RHOBTB3 promotes HIFα hydroxylation and ubiquitination in a PHD2- and VHL-dependent manner. (A) RHOBTB3 promotes hydroxylation of HIF1α in MEFs. RHOBTB3−/− MEFs and WT MEFs were maintained in normoxia or exposed to hypoxia for 8 h. Cells were then lysed and the hydroxylation on proline-564 of HIF1α (OH-P564) was analyzed by immunoblotting. As a consequence of sustained accumulation of HIF1α in RHOBTB3- MEFs, the protein levels of PHD2 were increased. In contrast, the relatively short-term, 8-h hypoxic exposure did not change the protein levels of PHD2. (B) Ectopically expressed RHOBTB3 promotes hydroxylation of HIF1α in vitro. RHOBTB3−/− MEFs and WT MEFs were infected with blank lentiviruses or lentiviruses expressing FLAG-RHOBTB3. Following lysis, the cell lysates were incubated with nickel affinity resin-bound bacterially expressed His-HIF1α (aa 401-603) or the P564A mutant for 90 min at 30 °C. The mixtures were diluted twofold in a 2× SDS buffer, and analyzed by western blotting using antibodies indicated. (C)In vitro translated RHOBTB3 promotes hydroxylation of HIF1α. In vitro translated RHOBTB3 and His-HIF1α (aa 401-603) or the P564A mutant were separately added to cell lysates of RHOBTB3−/− MEFs, and the mixtures were incubated at 30 °C for 90 min. The mixtures were then analyzed for levels of HIF1α hydroxylation as in B. (D) Knockdown of PHD2 impairs RHOBTB3-induced degradation of HIF1α. HEK293T cells were infected by lentiviruses expressing control siRNA (GFP), or siRNA targeting RHOBTB3 or PHD2 or both. At 16 h post-infection, cells were exposed to hypoxia for 4 h, then lysed and analyzed by immunoblotting with antibodies indicated. (E) Double knockdown of RHOBTB3 and PHD2 does not significantly increase transcriptional activity of HIF1α in single knockdown of PHD2. HEK293T cells were infected with lentiviruses expressing different siRNAs as indicated. After 12 h, cells were treated with 200 μM CoCl2 for another 8 h and then lysed. The firefly luciferase reporter carrying HRE was measured and normalized against the Renilla luciferase activity in a dual luciferase assay system. Data are presented as mean ± SEM; n = 3 for each group; *P< 0.05 (ANOVA followed by Tukey); N.S., not significant. (F) RHOBTB3 promotes the interaction between HIF1α and VHL. HEK293T cells were transfected with different combinations of HIF1α, MYC-VHL and HA-RHOBTB3. At 16 h post-transfection, cells were treated with 10 μM MG-132 and maintained in normoxia or exposed to hypoxia for another 10 h, and were lysed. The protein extracts were immunoprecipitated with antibody against MYC for VHL, and were subjected to western blot analysis. TCL, total cell lysate. (G) Knockdown of PHD2 attenuates RHOBTB3-induced ubiquitination of HIF1α. HEK293T cells infected with lentiviruses expressing siRNA targeting GFP (control) or PHD2 were transfected with different combinations of MYC-HIF1α, HA-RHOBTB3 and FLAG-UB. At 16 h post-transfection, cells were treated with 10 μM MG-132 for another 10 h, and were then lysed with RIPA buffer containing 1% SDS and boiled. The protein extracts were diluted in RIPA buffer without SDS to a final concentration of 0.2% SDS, and were subjected to IP with antibody against MYC for HIF1α. The IP product was analyzed by immunoblotting. (H) Knockdown of VHL impairs RHOBTB3 deficiency-induced HIF1α accumulation. HEK293T cells were infected by lentiviruses expressing siRNA targeting GFP (control), RHOBTB3 and/or VHL. At 16 h post-infection, cells were exposed to hypoxia for 4 h, and analyzed by immunoblotting to determine HIF1α protein levels. Probably owing to the low expression levels of its 24 kDa isoform in HEK293T as described previously80 and the preferential affinity of antibody, only the 19 kDa isoform of VHL (VHL (p19)) could be detected and is shown here.
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fig2: RHOBTB3 promotes HIFα hydroxylation and ubiquitination in a PHD2- and VHL-dependent manner. (A) RHOBTB3 promotes hydroxylation of HIF1α in MEFs. RHOBTB3−/− MEFs and WT MEFs were maintained in normoxia or exposed to hypoxia for 8 h. Cells were then lysed and the hydroxylation on proline-564 of HIF1α (OH-P564) was analyzed by immunoblotting. As a consequence of sustained accumulation of HIF1α in RHOBTB3- MEFs, the protein levels of PHD2 were increased. In contrast, the relatively short-term, 8-h hypoxic exposure did not change the protein levels of PHD2. (B) Ectopically expressed RHOBTB3 promotes hydroxylation of HIF1α in vitro. RHOBTB3−/− MEFs and WT MEFs were infected with blank lentiviruses or lentiviruses expressing FLAG-RHOBTB3. Following lysis, the cell lysates were incubated with nickel affinity resin-bound bacterially expressed His-HIF1α (aa 401-603) or the P564A mutant for 90 min at 30 °C. The mixtures were diluted twofold in a 2× SDS buffer, and analyzed by western blotting using antibodies indicated. (C)In vitro translated RHOBTB3 promotes hydroxylation of HIF1α. In vitro translated RHOBTB3 and His-HIF1α (aa 401-603) or the P564A mutant were separately added to cell lysates of RHOBTB3−/− MEFs, and the mixtures were incubated at 30 °C for 90 min. The mixtures were then analyzed for levels of HIF1α hydroxylation as in B. (D) Knockdown of PHD2 impairs RHOBTB3-induced degradation of HIF1α. HEK293T cells were infected by lentiviruses expressing control siRNA (GFP), or siRNA targeting RHOBTB3 or PHD2 or both. At 16 h post-infection, cells were exposed to hypoxia for 4 h, then lysed and analyzed by immunoblotting with antibodies indicated. (E) Double knockdown of RHOBTB3 and PHD2 does not significantly increase transcriptional activity of HIF1α in single knockdown of PHD2. HEK293T cells were infected with lentiviruses expressing different siRNAs as indicated. After 12 h, cells were treated with 200 μM CoCl2 for another 8 h and then lysed. The firefly luciferase reporter carrying HRE was measured and normalized against the Renilla luciferase activity in a dual luciferase assay system. Data are presented as mean ± SEM; n = 3 for each group; *P< 0.05 (ANOVA followed by Tukey); N.S., not significant. (F) RHOBTB3 promotes the interaction between HIF1α and VHL. HEK293T cells were transfected with different combinations of HIF1α, MYC-VHL and HA-RHOBTB3. At 16 h post-transfection, cells were treated with 10 μM MG-132 and maintained in normoxia or exposed to hypoxia for another 10 h, and were lysed. The protein extracts were immunoprecipitated with antibody against MYC for VHL, and were subjected to western blot analysis. TCL, total cell lysate. (G) Knockdown of PHD2 attenuates RHOBTB3-induced ubiquitination of HIF1α. HEK293T cells infected with lentiviruses expressing siRNA targeting GFP (control) or PHD2 were transfected with different combinations of MYC-HIF1α, HA-RHOBTB3 and FLAG-UB. At 16 h post-transfection, cells were treated with 10 μM MG-132 for another 10 h, and were then lysed with RIPA buffer containing 1% SDS and boiled. The protein extracts were diluted in RIPA buffer without SDS to a final concentration of 0.2% SDS, and were subjected to IP with antibody against MYC for HIF1α. The IP product was analyzed by immunoblotting. (H) Knockdown of VHL impairs RHOBTB3 deficiency-induced HIF1α accumulation. HEK293T cells were infected by lentiviruses expressing siRNA targeting GFP (control), RHOBTB3 and/or VHL. At 16 h post-infection, cells were exposed to hypoxia for 4 h, and analyzed by immunoblotting to determine HIF1α protein levels. Probably owing to the low expression levels of its 24 kDa isoform in HEK293T as described previously80 and the preferential affinity of antibody, only the 19 kDa isoform of VHL (VHL (p19)) could be detected and is shown here.

Mentions: We then explored the mechanism by which RHOBTB3 downregulates the protein levels of HIFα. In the presence of lysosomal inhibitor chloroquine, RHOBTB3 could still suppress the protein levels of HIF1α, while addition of MG-132 strongly blocked RHOBTB3-mediated HIF1α degradation, suggesting that RHOBTB3 promotes HIF1α degradation in a proteasome-specific manner (Supplementary information, Figure S2A). We next explored the possibility that RHOBTB3 promotes HIFα hydroxylation and ubiquitination, two essential modifications prior to proteasomal degradation. While the total protein levels of HIF1α and its target gene, PHD1-3 was increased in RHOBTB3−/− MEFs, the level of its hydroxylation at proline-564 (OH-P564) was significantly reduced under normoxic or hypoxic conditions (Figure 2A and Supplementary information, Figure S2B), indicating that RHOBTB3 is important for the hydroxylation of HIF1α. Consistently, overexpression of RHOBTB3 effectively downregulated wild-type HIF1α, but had little effect on the hydroxylation-defective mutant P564A (Supplementary information, Figure S2C). To confirm the role of RHOBTB3 in HIF1α hydroxylation, we carried out in vitro hydroxylation assays. We mixed bacterially expressed ODD domain of HIF1α (aa 401-603 of human HIF1α) with different cell lysates, and found that lysate from RHOBTB3-overexpressing cells strongly stimulated the hydroxylation of HIF1α (OH-P564) (Figure 2B). Conversely, hydroxylation at P564 of HIF1α was significantly reduced when the cell lysate prepared from RHOBTB3−/− MEFs was added, even though higher protein levels of PHD2 hydroxylase were present in these cells, and the level of OH-P564 was increased by the lysate from RHOBTB3−/− MEFs supplemented with RHOBTB3 (Figure 2B). Moreover, adding in vitro translated RHOBTB3 protein into the lysates of RHOBTB3−/− MEFs markedly enhanced the OH-P564 levels of HIF1α (Figure 2C), excluding the possibility that RHOBTB3 regulates hydroxylation through affecting cellular concentrations of other factors such as co-factors for PHDs46,69. Depletion of PHD2 strongly impaired the RHOBTB3-induced hydroxylation and degradation of HIF1α in HEK293T cells (Supplementary information, Figure S2D and S2E). Moreover, double knockdown of RHOBTB3 and PHD2 did not significantly increase the protein levels or transcriptional activity of HIF1α compared with PHD2 single knockdown (Figure 2D and 2E), suggesting that RHOBTB3 and PHD2 function in the same pathway.


RHOBTB3 promotes proteasomal degradation of HIFα through facilitating hydroxylation and suppresses the Warburg effect.

Zhang CS, Liu Q, Li M, Lin SY, Peng Y, Wu D, Li TY, Fu Q, Jia W, Wang X, Ma T, Zong Y, Cui J, Pu C, Lian G, Guo H, Ye Z, Lin SC - Cell Res. (2015)

RHOBTB3 promotes HIFα hydroxylation and ubiquitination in a PHD2- and VHL-dependent manner. (A) RHOBTB3 promotes hydroxylation of HIF1α in MEFs. RHOBTB3−/− MEFs and WT MEFs were maintained in normoxia or exposed to hypoxia for 8 h. Cells were then lysed and the hydroxylation on proline-564 of HIF1α (OH-P564) was analyzed by immunoblotting. As a consequence of sustained accumulation of HIF1α in RHOBTB3- MEFs, the protein levels of PHD2 were increased. In contrast, the relatively short-term, 8-h hypoxic exposure did not change the protein levels of PHD2. (B) Ectopically expressed RHOBTB3 promotes hydroxylation of HIF1α in vitro. RHOBTB3−/− MEFs and WT MEFs were infected with blank lentiviruses or lentiviruses expressing FLAG-RHOBTB3. Following lysis, the cell lysates were incubated with nickel affinity resin-bound bacterially expressed His-HIF1α (aa 401-603) or the P564A mutant for 90 min at 30 °C. The mixtures were diluted twofold in a 2× SDS buffer, and analyzed by western blotting using antibodies indicated. (C)In vitro translated RHOBTB3 promotes hydroxylation of HIF1α. In vitro translated RHOBTB3 and His-HIF1α (aa 401-603) or the P564A mutant were separately added to cell lysates of RHOBTB3−/− MEFs, and the mixtures were incubated at 30 °C for 90 min. The mixtures were then analyzed for levels of HIF1α hydroxylation as in B. (D) Knockdown of PHD2 impairs RHOBTB3-induced degradation of HIF1α. HEK293T cells were infected by lentiviruses expressing control siRNA (GFP), or siRNA targeting RHOBTB3 or PHD2 or both. At 16 h post-infection, cells were exposed to hypoxia for 4 h, then lysed and analyzed by immunoblotting with antibodies indicated. (E) Double knockdown of RHOBTB3 and PHD2 does not significantly increase transcriptional activity of HIF1α in single knockdown of PHD2. HEK293T cells were infected with lentiviruses expressing different siRNAs as indicated. After 12 h, cells were treated with 200 μM CoCl2 for another 8 h and then lysed. The firefly luciferase reporter carrying HRE was measured and normalized against the Renilla luciferase activity in a dual luciferase assay system. Data are presented as mean ± SEM; n = 3 for each group; *P< 0.05 (ANOVA followed by Tukey); N.S., not significant. (F) RHOBTB3 promotes the interaction between HIF1α and VHL. HEK293T cells were transfected with different combinations of HIF1α, MYC-VHL and HA-RHOBTB3. At 16 h post-transfection, cells were treated with 10 μM MG-132 and maintained in normoxia or exposed to hypoxia for another 10 h, and were lysed. The protein extracts were immunoprecipitated with antibody against MYC for VHL, and were subjected to western blot analysis. TCL, total cell lysate. (G) Knockdown of PHD2 attenuates RHOBTB3-induced ubiquitination of HIF1α. HEK293T cells infected with lentiviruses expressing siRNA targeting GFP (control) or PHD2 were transfected with different combinations of MYC-HIF1α, HA-RHOBTB3 and FLAG-UB. At 16 h post-transfection, cells were treated with 10 μM MG-132 for another 10 h, and were then lysed with RIPA buffer containing 1% SDS and boiled. The protein extracts were diluted in RIPA buffer without SDS to a final concentration of 0.2% SDS, and were subjected to IP with antibody against MYC for HIF1α. The IP product was analyzed by immunoblotting. (H) Knockdown of VHL impairs RHOBTB3 deficiency-induced HIF1α accumulation. HEK293T cells were infected by lentiviruses expressing siRNA targeting GFP (control), RHOBTB3 and/or VHL. At 16 h post-infection, cells were exposed to hypoxia for 4 h, and analyzed by immunoblotting to determine HIF1α protein levels. Probably owing to the low expression levels of its 24 kDa isoform in HEK293T as described previously80 and the preferential affinity of antibody, only the 19 kDa isoform of VHL (VHL (p19)) could be detected and is shown here.
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fig2: RHOBTB3 promotes HIFα hydroxylation and ubiquitination in a PHD2- and VHL-dependent manner. (A) RHOBTB3 promotes hydroxylation of HIF1α in MEFs. RHOBTB3−/− MEFs and WT MEFs were maintained in normoxia or exposed to hypoxia for 8 h. Cells were then lysed and the hydroxylation on proline-564 of HIF1α (OH-P564) was analyzed by immunoblotting. As a consequence of sustained accumulation of HIF1α in RHOBTB3- MEFs, the protein levels of PHD2 were increased. In contrast, the relatively short-term, 8-h hypoxic exposure did not change the protein levels of PHD2. (B) Ectopically expressed RHOBTB3 promotes hydroxylation of HIF1α in vitro. RHOBTB3−/− MEFs and WT MEFs were infected with blank lentiviruses or lentiviruses expressing FLAG-RHOBTB3. Following lysis, the cell lysates were incubated with nickel affinity resin-bound bacterially expressed His-HIF1α (aa 401-603) or the P564A mutant for 90 min at 30 °C. The mixtures were diluted twofold in a 2× SDS buffer, and analyzed by western blotting using antibodies indicated. (C)In vitro translated RHOBTB3 promotes hydroxylation of HIF1α. In vitro translated RHOBTB3 and His-HIF1α (aa 401-603) or the P564A mutant were separately added to cell lysates of RHOBTB3−/− MEFs, and the mixtures were incubated at 30 °C for 90 min. The mixtures were then analyzed for levels of HIF1α hydroxylation as in B. (D) Knockdown of PHD2 impairs RHOBTB3-induced degradation of HIF1α. HEK293T cells were infected by lentiviruses expressing control siRNA (GFP), or siRNA targeting RHOBTB3 or PHD2 or both. At 16 h post-infection, cells were exposed to hypoxia for 4 h, then lysed and analyzed by immunoblotting with antibodies indicated. (E) Double knockdown of RHOBTB3 and PHD2 does not significantly increase transcriptional activity of HIF1α in single knockdown of PHD2. HEK293T cells were infected with lentiviruses expressing different siRNAs as indicated. After 12 h, cells were treated with 200 μM CoCl2 for another 8 h and then lysed. The firefly luciferase reporter carrying HRE was measured and normalized against the Renilla luciferase activity in a dual luciferase assay system. Data are presented as mean ± SEM; n = 3 for each group; *P< 0.05 (ANOVA followed by Tukey); N.S., not significant. (F) RHOBTB3 promotes the interaction between HIF1α and VHL. HEK293T cells were transfected with different combinations of HIF1α, MYC-VHL and HA-RHOBTB3. At 16 h post-transfection, cells were treated with 10 μM MG-132 and maintained in normoxia or exposed to hypoxia for another 10 h, and were lysed. The protein extracts were immunoprecipitated with antibody against MYC for VHL, and were subjected to western blot analysis. TCL, total cell lysate. (G) Knockdown of PHD2 attenuates RHOBTB3-induced ubiquitination of HIF1α. HEK293T cells infected with lentiviruses expressing siRNA targeting GFP (control) or PHD2 were transfected with different combinations of MYC-HIF1α, HA-RHOBTB3 and FLAG-UB. At 16 h post-transfection, cells were treated with 10 μM MG-132 for another 10 h, and were then lysed with RIPA buffer containing 1% SDS and boiled. The protein extracts were diluted in RIPA buffer without SDS to a final concentration of 0.2% SDS, and were subjected to IP with antibody against MYC for HIF1α. The IP product was analyzed by immunoblotting. (H) Knockdown of VHL impairs RHOBTB3 deficiency-induced HIF1α accumulation. HEK293T cells were infected by lentiviruses expressing siRNA targeting GFP (control), RHOBTB3 and/or VHL. At 16 h post-infection, cells were exposed to hypoxia for 4 h, and analyzed by immunoblotting to determine HIF1α protein levels. Probably owing to the low expression levels of its 24 kDa isoform in HEK293T as described previously80 and the preferential affinity of antibody, only the 19 kDa isoform of VHL (VHL (p19)) could be detected and is shown here.
Mentions: We then explored the mechanism by which RHOBTB3 downregulates the protein levels of HIFα. In the presence of lysosomal inhibitor chloroquine, RHOBTB3 could still suppress the protein levels of HIF1α, while addition of MG-132 strongly blocked RHOBTB3-mediated HIF1α degradation, suggesting that RHOBTB3 promotes HIF1α degradation in a proteasome-specific manner (Supplementary information, Figure S2A). We next explored the possibility that RHOBTB3 promotes HIFα hydroxylation and ubiquitination, two essential modifications prior to proteasomal degradation. While the total protein levels of HIF1α and its target gene, PHD1-3 was increased in RHOBTB3−/− MEFs, the level of its hydroxylation at proline-564 (OH-P564) was significantly reduced under normoxic or hypoxic conditions (Figure 2A and Supplementary information, Figure S2B), indicating that RHOBTB3 is important for the hydroxylation of HIF1α. Consistently, overexpression of RHOBTB3 effectively downregulated wild-type HIF1α, but had little effect on the hydroxylation-defective mutant P564A (Supplementary information, Figure S2C). To confirm the role of RHOBTB3 in HIF1α hydroxylation, we carried out in vitro hydroxylation assays. We mixed bacterially expressed ODD domain of HIF1α (aa 401-603 of human HIF1α) with different cell lysates, and found that lysate from RHOBTB3-overexpressing cells strongly stimulated the hydroxylation of HIF1α (OH-P564) (Figure 2B). Conversely, hydroxylation at P564 of HIF1α was significantly reduced when the cell lysate prepared from RHOBTB3−/− MEFs was added, even though higher protein levels of PHD2 hydroxylase were present in these cells, and the level of OH-P564 was increased by the lysate from RHOBTB3−/− MEFs supplemented with RHOBTB3 (Figure 2B). Moreover, adding in vitro translated RHOBTB3 protein into the lysates of RHOBTB3−/− MEFs markedly enhanced the OH-P564 levels of HIF1α (Figure 2C), excluding the possibility that RHOBTB3 regulates hydroxylation through affecting cellular concentrations of other factors such as co-factors for PHDs46,69. Depletion of PHD2 strongly impaired the RHOBTB3-induced hydroxylation and degradation of HIF1α in HEK293T cells (Supplementary information, Figure S2D and S2E). Moreover, double knockdown of RHOBTB3 and PHD2 did not significantly increase the protein levels or transcriptional activity of HIF1α compared with PHD2 single knockdown (Figure 2D and 2E), suggesting that RHOBTB3 and PHD2 function in the same pathway.

Bottom Line: Remarkably, RHOBTB3 dimerizes with LIMD1, and constructs a RHOBTB3/LIMD1-PHD2-VHL-HIFα complex to effect the maximal degradation of HIFα.Hypoxia reduces the RHOBTB3-centered complex formation, resulting in an accumulation of HIFα.Importantly, the expression level of RHOBTB3 is greatly reduced in human renal carcinomas, and RHOBTB3 deficiency significantly elevates the Warburg effect and accelerates xenograft growth.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Cellular Stress Biology, Innovation Center for Cell Signaling Network, School of Life Sciences, Xiamen University, Xiamen, Fujian 361005, China.

ABSTRACT
Hypoxia-inducible factors (HIFs) are master regulators of adaptive responses to low oxygen, and their α-subunits are rapidly degraded through the ubiquitination-dependent proteasomal pathway after hydroxylation. Aberrant accumulation or activation of HIFs is closely linked to many types of cancer. However, how hydroxylation of HIFα and its delivery to the ubiquitination machinery are regulated remains unclear. Here we show that Rho-related BTB domain-containing protein 3 (RHOBTB3) directly interacts with the hydroxylase PHD2 to promote HIFα hydroxylation. RHOBTB3 also directly interacts with the von Hippel-Lindau (VHL) protein, a component of the E3 ubiquitin ligase complex, facilitating ubiquitination of HIFα. Remarkably, RHOBTB3 dimerizes with LIMD1, and constructs a RHOBTB3/LIMD1-PHD2-VHL-HIFα complex to effect the maximal degradation of HIFα. Hypoxia reduces the RHOBTB3-centered complex formation, resulting in an accumulation of HIFα. Importantly, the expression level of RHOBTB3 is greatly reduced in human renal carcinomas, and RHOBTB3 deficiency significantly elevates the Warburg effect and accelerates xenograft growth. Our work thus reveals that RHOBTB3 serves as a scaffold to organize a multi-subunit complex that promotes the hydroxylation, ubiquitination and degradation of HIFα.

No MeSH data available.


Related in: MedlinePlus