Limits...
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 and LIMD1 cooperatively regulate HIF1α. (A) RHOBTB3 and LIMD1 cooperatively suppress the protein level of HIF1α. HEK293T cells were infected with lentiviruses expressing siRNA targeting GFP, RHOBTB3 and/or LIMD1. At 16 h post-infection, cells were maintained in normoxia or exposed to hypoxia for 8 h before lysis and immunoblotting with antibodies indicated. (B) RHOBTB3 and LIMD1 cooperatively suppress the transcriptional activities of HIF1α. HEK293T cells were infected with different combinations of lentiviruses as indicated. Transcriptional activities of HIF1α were measured using a dual luciferase assay system as described in Figure 2E. Data are presented as mean ± SEM, n = 3 for each group, *P < 0.05, ***P < 0.001 (ANOVA followed by Tukey). (C) Knockdown of LIMD1 in RHOBTB3−/− MEFs further increases the protein levels of HIF1α. RHOBTB3−/− MEFs were infected with lentiviruses expressing siRNA targeting GFP or LIMD1. At 36 h post-infection, cells were maintained in normoxia or exposed to hypoxia for 8 h, before the western blot analysis. (D) RHOBTB3 and LIMD1 cooperatively promote the ubiquitination of HIF1α. HEK293T cells were transfected with different combinations of MYC-HIF1α, HA-RHOBTB3, HA-LIMD1 and FLAG-UB (ubiquitin). After treatment with 10 μM MG-132 for 10 h, the cells were lysed, and the lysates were subjected to IP with antibody against MYC (for HIF1α). The IP product was analyzed by western blotting to determine the ubiquitination levels of HIF1α. (E) Knockdown of RHOBTB3 and/or LIMD1 decreases PHD2-VHL interaction. HEK293T cells were infected with lentiviruses expressing siRNA targeting GFP, RHOBTB3 and/or LIMD1. At 16 h post-infection, cells were lysed and the endogenous VHL was immunoprecipitated, and the IP product was analyzed by immunoblotting. (F) Ectopically expressed RHOBTB3 and LIMD1 cooperatively promote PHD2-VHL interaction. HEK293T cells were transfected with different combinations of MYC-VHL, HA-RHOBTB3, HA-LIMD1 and FLAG-PHD2. Protein extracts from the transfected cells were subjected to IP with antibody against FLAG and analyzed by immunoblotting with antibodies indicated.
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fig4: RHOBTB3 and LIMD1 cooperatively regulate HIF1α. (A) RHOBTB3 and LIMD1 cooperatively suppress the protein level of HIF1α. HEK293T cells were infected with lentiviruses expressing siRNA targeting GFP, RHOBTB3 and/or LIMD1. At 16 h post-infection, cells were maintained in normoxia or exposed to hypoxia for 8 h before lysis and immunoblotting with antibodies indicated. (B) RHOBTB3 and LIMD1 cooperatively suppress the transcriptional activities of HIF1α. HEK293T cells were infected with different combinations of lentiviruses as indicated. Transcriptional activities of HIF1α were measured using a dual luciferase assay system as described in Figure 2E. Data are presented as mean ± SEM, n = 3 for each group, *P < 0.05, ***P < 0.001 (ANOVA followed by Tukey). (C) Knockdown of LIMD1 in RHOBTB3−/− MEFs further increases the protein levels of HIF1α. RHOBTB3−/− MEFs were infected with lentiviruses expressing siRNA targeting GFP or LIMD1. At 36 h post-infection, cells were maintained in normoxia or exposed to hypoxia for 8 h, before the western blot analysis. (D) RHOBTB3 and LIMD1 cooperatively promote the ubiquitination of HIF1α. HEK293T cells were transfected with different combinations of MYC-HIF1α, HA-RHOBTB3, HA-LIMD1 and FLAG-UB (ubiquitin). After treatment with 10 μM MG-132 for 10 h, the cells were lysed, and the lysates were subjected to IP with antibody against MYC (for HIF1α). The IP product was analyzed by western blotting to determine the ubiquitination levels of HIF1α. (E) Knockdown of RHOBTB3 and/or LIMD1 decreases PHD2-VHL interaction. HEK293T cells were infected with lentiviruses expressing siRNA targeting GFP, RHOBTB3 and/or LIMD1. At 16 h post-infection, cells were lysed and the endogenous VHL was immunoprecipitated, and the IP product was analyzed by immunoblotting. (F) Ectopically expressed RHOBTB3 and LIMD1 cooperatively promote PHD2-VHL interaction. HEK293T cells were transfected with different combinations of MYC-VHL, HA-RHOBTB3, HA-LIMD1 and FLAG-PHD2. Protein extracts from the transfected cells were subjected to IP with antibody against FLAG and analyzed by immunoblotting with antibodies indicated.

Mentions: LIMD1 has been reported to serve as an adaptor protein for PHD2 and VHL in the degradation of HIF1α, although if it can enhance HIFα hydroxylation has not been demonstrated58. We thus explored whether there is a functional linkage between RHOBTB3 and LIMD1. Consistent with previous reports, we found that knockdown of LIMD1, such as RHOBTB3, elevated the protein levels and transcriptional activities of HIF1α, and simultaneous knockdown of RHOBTB3 and LIMD1 led to a further increase in protein levels and transcriptional activity of HIF1α (Figure 4A and 4B). Likewise, knockdown of LIMD1 in RHOBTB3−/− MEFs resulted in an increase of HIF1α above the level observed in control RHOBTB3−/− MEFs (Figure 4C). Consistently, co-expressing RHOBTB3 and LIMD1 produced an additive effect on HIF1α ubiquitination (Figure 4D). Moreover, knockdown of RHOBTB3 or LIMD1 decreased the interaction between endogenous PHD2 and VHL and knockdown of both RHOBTB3 and LIMD1 further dampened this interaction (Figure 4E). Conversely, overexpression experiment showed that RHOBTB3 and LIMD1 cooperatively strengthened this interaction (Figure 4F). These observations together suggest that both RHOBTB3 and LIMD1 are required for the suppression of HIF1α.


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 and LIMD1 cooperatively regulate HIF1α. (A) RHOBTB3 and LIMD1 cooperatively suppress the protein level of HIF1α. HEK293T cells were infected with lentiviruses expressing siRNA targeting GFP, RHOBTB3 and/or LIMD1. At 16 h post-infection, cells were maintained in normoxia or exposed to hypoxia for 8 h before lysis and immunoblotting with antibodies indicated. (B) RHOBTB3 and LIMD1 cooperatively suppress the transcriptional activities of HIF1α. HEK293T cells were infected with different combinations of lentiviruses as indicated. Transcriptional activities of HIF1α were measured using a dual luciferase assay system as described in Figure 2E. Data are presented as mean ± SEM, n = 3 for each group, *P < 0.05, ***P < 0.001 (ANOVA followed by Tukey). (C) Knockdown of LIMD1 in RHOBTB3−/− MEFs further increases the protein levels of HIF1α. RHOBTB3−/− MEFs were infected with lentiviruses expressing siRNA targeting GFP or LIMD1. At 36 h post-infection, cells were maintained in normoxia or exposed to hypoxia for 8 h, before the western blot analysis. (D) RHOBTB3 and LIMD1 cooperatively promote the ubiquitination of HIF1α. HEK293T cells were transfected with different combinations of MYC-HIF1α, HA-RHOBTB3, HA-LIMD1 and FLAG-UB (ubiquitin). After treatment with 10 μM MG-132 for 10 h, the cells were lysed, and the lysates were subjected to IP with antibody against MYC (for HIF1α). The IP product was analyzed by western blotting to determine the ubiquitination levels of HIF1α. (E) Knockdown of RHOBTB3 and/or LIMD1 decreases PHD2-VHL interaction. HEK293T cells were infected with lentiviruses expressing siRNA targeting GFP, RHOBTB3 and/or LIMD1. At 16 h post-infection, cells were lysed and the endogenous VHL was immunoprecipitated, and the IP product was analyzed by immunoblotting. (F) Ectopically expressed RHOBTB3 and LIMD1 cooperatively promote PHD2-VHL interaction. HEK293T cells were transfected with different combinations of MYC-VHL, HA-RHOBTB3, HA-LIMD1 and FLAG-PHD2. Protein extracts from the transfected cells were subjected to IP with antibody against FLAG and analyzed by immunoblotting with antibodies indicated.
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fig4: RHOBTB3 and LIMD1 cooperatively regulate HIF1α. (A) RHOBTB3 and LIMD1 cooperatively suppress the protein level of HIF1α. HEK293T cells were infected with lentiviruses expressing siRNA targeting GFP, RHOBTB3 and/or LIMD1. At 16 h post-infection, cells were maintained in normoxia or exposed to hypoxia for 8 h before lysis and immunoblotting with antibodies indicated. (B) RHOBTB3 and LIMD1 cooperatively suppress the transcriptional activities of HIF1α. HEK293T cells were infected with different combinations of lentiviruses as indicated. Transcriptional activities of HIF1α were measured using a dual luciferase assay system as described in Figure 2E. Data are presented as mean ± SEM, n = 3 for each group, *P < 0.05, ***P < 0.001 (ANOVA followed by Tukey). (C) Knockdown of LIMD1 in RHOBTB3−/− MEFs further increases the protein levels of HIF1α. RHOBTB3−/− MEFs were infected with lentiviruses expressing siRNA targeting GFP or LIMD1. At 36 h post-infection, cells were maintained in normoxia or exposed to hypoxia for 8 h, before the western blot analysis. (D) RHOBTB3 and LIMD1 cooperatively promote the ubiquitination of HIF1α. HEK293T cells were transfected with different combinations of MYC-HIF1α, HA-RHOBTB3, HA-LIMD1 and FLAG-UB (ubiquitin). After treatment with 10 μM MG-132 for 10 h, the cells were lysed, and the lysates were subjected to IP with antibody against MYC (for HIF1α). The IP product was analyzed by western blotting to determine the ubiquitination levels of HIF1α. (E) Knockdown of RHOBTB3 and/or LIMD1 decreases PHD2-VHL interaction. HEK293T cells were infected with lentiviruses expressing siRNA targeting GFP, RHOBTB3 and/or LIMD1. At 16 h post-infection, cells were lysed and the endogenous VHL was immunoprecipitated, and the IP product was analyzed by immunoblotting. (F) Ectopically expressed RHOBTB3 and LIMD1 cooperatively promote PHD2-VHL interaction. HEK293T cells were transfected with different combinations of MYC-VHL, HA-RHOBTB3, HA-LIMD1 and FLAG-PHD2. Protein extracts from the transfected cells were subjected to IP with antibody against FLAG and analyzed by immunoblotting with antibodies indicated.
Mentions: LIMD1 has been reported to serve as an adaptor protein for PHD2 and VHL in the degradation of HIF1α, although if it can enhance HIFα hydroxylation has not been demonstrated58. We thus explored whether there is a functional linkage between RHOBTB3 and LIMD1. Consistent with previous reports, we found that knockdown of LIMD1, such as RHOBTB3, elevated the protein levels and transcriptional activities of HIF1α, and simultaneous knockdown of RHOBTB3 and LIMD1 led to a further increase in protein levels and transcriptional activity of HIF1α (Figure 4A and 4B). Likewise, knockdown of LIMD1 in RHOBTB3−/− MEFs resulted in an increase of HIF1α above the level observed in control RHOBTB3−/− MEFs (Figure 4C). Consistently, co-expressing RHOBTB3 and LIMD1 produced an additive effect on HIF1α ubiquitination (Figure 4D). Moreover, knockdown of RHOBTB3 or LIMD1 decreased the interaction between endogenous PHD2 and VHL and knockdown of both RHOBTB3 and LIMD1 further dampened this interaction (Figure 4E). Conversely, overexpression experiment showed that RHOBTB3 and LIMD1 cooperatively strengthened this interaction (Figure 4F). These observations together suggest that both RHOBTB3 and LIMD1 are required for the suppression of HIF1α.

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