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, PHD2 and VHL form a complex. (A) RHOBTB3 interacts with endogenous PHD2 and VHL. Protein extracts of WT MEFs and RHOBTB3−/− MEFs (control) were immunoprecipitated with antibody against RHOBTB3 or control IgG, and analyzed by immunoblotting with antibodies indicated. (B) Ectopically expressed RHOBTB3 interacts simultaneously with PHD2 and VHL. HEK293T cells were transfected with different combinations of HA-RHOBTB3, MYC-VHL and FLAG-PHD2. At 16 h post-infection, cells were lysed and the protein extracts were immunoprecipitated with antibody against HA, and the IP product was analyzed by western blotting. (C) RHOBTB3, VHL and PHD2 form a complex. HEK293T cells were transfected with HA-RHOBTB3, FLAG-PHD2 and MYC-VHL. After 16 h, cells were harvested. Two-step co-IP was performed by first using anti-FLAG antibody, followed by elution with the FLAG peptide. The eluates were subjected to a second round of IP with anti-HA or control IgG, and the final precipitated proteins were analyzed by immunoblotting. (D) The N-terminal region of RHOBTB3 (aa 1-204) does not have a role in the degradation of HIF1α. RHOBTB3−/− MEFs were infected with lentiviruses expressing HA-RHOBTB3 or HA-RHOBTB3 (aa 1-201). At 36 h post-infection, cells were maintained in normoxia or exposed to hypoxia for 8 h, and analyzed after lysis by immunoblotting. (E) RHOBTB3 strengthens the interaction between PHD2 and VHL. RHOBTB3−/− MEFs and WT MEFs were lysed and the endogenous VHL was immunoprecipitated. The IP product was analyzed by immunoblotting. (F) RHOBTB3 promotes the PHD2-VHL interaction in vitro. In vitro translated RHOBTB3 and bacterially expressed GST-PHD2 were incubated with anti-MYC-conjugated resin-bound MYC-tagged VHL. The mixtures were then pulled down by centrifugation, and analyzed by immunoblotting. (G) RHOBTB3 promotes the interaction between PHD2 and HIF1α. HEK293T cells were transfected with different combinations of HIF1α, FLAG-PHD2, MYC-VHL and HA-RHOBTB3. At 8 h post-transfection, cells were treated with 10 μM MG-132 and maintained in normoxia or exposed to hypoxia for 10 h. The protein extracts were immunoprecipitated with antibody against MYC (for VHL), and precipitated proteins were analyzed by immunoblotting.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4559813&req=5

fig3: RHOBTB3, PHD2 and VHL form a complex. (A) RHOBTB3 interacts with endogenous PHD2 and VHL. Protein extracts of WT MEFs and RHOBTB3−/− MEFs (control) were immunoprecipitated with antibody against RHOBTB3 or control IgG, and analyzed by immunoblotting with antibodies indicated. (B) Ectopically expressed RHOBTB3 interacts simultaneously with PHD2 and VHL. HEK293T cells were transfected with different combinations of HA-RHOBTB3, MYC-VHL and FLAG-PHD2. At 16 h post-infection, cells were lysed and the protein extracts were immunoprecipitated with antibody against HA, and the IP product was analyzed by western blotting. (C) RHOBTB3, VHL and PHD2 form a complex. HEK293T cells were transfected with HA-RHOBTB3, FLAG-PHD2 and MYC-VHL. After 16 h, cells were harvested. Two-step co-IP was performed by first using anti-FLAG antibody, followed by elution with the FLAG peptide. The eluates were subjected to a second round of IP with anti-HA or control IgG, and the final precipitated proteins were analyzed by immunoblotting. (D) The N-terminal region of RHOBTB3 (aa 1-204) does not have a role in the degradation of HIF1α. RHOBTB3−/− MEFs were infected with lentiviruses expressing HA-RHOBTB3 or HA-RHOBTB3 (aa 1-201). At 36 h post-infection, cells were maintained in normoxia or exposed to hypoxia for 8 h, and analyzed after lysis by immunoblotting. (E) RHOBTB3 strengthens the interaction between PHD2 and VHL. RHOBTB3−/− MEFs and WT MEFs were lysed and the endogenous VHL was immunoprecipitated. The IP product was analyzed by immunoblotting. (F) RHOBTB3 promotes the PHD2-VHL interaction in vitro. In vitro translated RHOBTB3 and bacterially expressed GST-PHD2 were incubated with anti-MYC-conjugated resin-bound MYC-tagged VHL. The mixtures were then pulled down by centrifugation, and analyzed by immunoblotting. (G) RHOBTB3 promotes the interaction between PHD2 and HIF1α. HEK293T cells were transfected with different combinations of HIF1α, FLAG-PHD2, MYC-VHL and HA-RHOBTB3. At 8 h post-transfection, cells were treated with 10 μM MG-132 and maintained in normoxia or exposed to hypoxia for 10 h. The protein extracts were immunoprecipitated with antibody against MYC (for VHL), and precipitated proteins were analyzed by immunoblotting.

Mentions: We next asked whether RHOBTB3 forms a complex with VHL and PHD2, particularly since RHOBTB3 and VHL showed interaction in our yeast two-hybrid screen (Supplementary information, Figure S1A) and that RHOBTB3 promoted PHD2-mediated HIFα hydroxylation. We found that endogenous or ectopically expressed RHOBTB3 was co-immunoprecipitated with VHL or vice versa, but not with the control IgG (Figure 3A and Supplementary information, Figure S3A). Consistently, immunofluorescent staining revealed the co-localization of RHOBTB3 with VHL (Supplementary information, Figure S3B). Similarly, PHD2 was readily co-precipitated with RHOBTB3 (Figure 3A and Supplementary information, Figure S3C). We next asked whether RHOBTB3, PHD2 and VHL co-exist in the same complex. It was observed that RHOBTB3 interacted simultaneously with PHD2 and VHL (Figure 3B). We further employed a two-step co-immunoprecipitation (IP) assay, and found that all three components were detected in the final immunoprecipitates (Figure 3C), indicating their co-existence in the same complex. Of note, HSP90, reported as a chaperone of HIFs71,72 and PHD273, was co-precipitated with RHOBTB3 only when HIF1α was present, suggesting that the HSP90 does not directly interact with RHOBTB3 (Supplementary information, Figure S3D). Although HSP90 has been shown to interact with PHD273 and RHOBTB3 interacts with PHD2 as shown in the present study, PHD2 could co-precipitate HSP90 and RHOBTB3 only when HIF1α was present, suggesting the PHD2-HSP90 complex depends on HIF1α to form a co-complex with RHOBTB3 (Supplementary information, Figure S3D). In the presence of ectopic HIF1α, more HSP90 was co-precipitated with RHOBTB3 under normoxia (Supplementary information, Figure S3E), consistent with a stronger interaction of HIF1α with RHOBTB3 under this condition (Figure 2F). These data indicate that HSP90 does not directly interact with RHOBTB3.


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, PHD2 and VHL form a complex. (A) RHOBTB3 interacts with endogenous PHD2 and VHL. Protein extracts of WT MEFs and RHOBTB3−/− MEFs (control) were immunoprecipitated with antibody against RHOBTB3 or control IgG, and analyzed by immunoblotting with antibodies indicated. (B) Ectopically expressed RHOBTB3 interacts simultaneously with PHD2 and VHL. HEK293T cells were transfected with different combinations of HA-RHOBTB3, MYC-VHL and FLAG-PHD2. At 16 h post-infection, cells were lysed and the protein extracts were immunoprecipitated with antibody against HA, and the IP product was analyzed by western blotting. (C) RHOBTB3, VHL and PHD2 form a complex. HEK293T cells were transfected with HA-RHOBTB3, FLAG-PHD2 and MYC-VHL. After 16 h, cells were harvested. Two-step co-IP was performed by first using anti-FLAG antibody, followed by elution with the FLAG peptide. The eluates were subjected to a second round of IP with anti-HA or control IgG, and the final precipitated proteins were analyzed by immunoblotting. (D) The N-terminal region of RHOBTB3 (aa 1-204) does not have a role in the degradation of HIF1α. RHOBTB3−/− MEFs were infected with lentiviruses expressing HA-RHOBTB3 or HA-RHOBTB3 (aa 1-201). At 36 h post-infection, cells were maintained in normoxia or exposed to hypoxia for 8 h, and analyzed after lysis by immunoblotting. (E) RHOBTB3 strengthens the interaction between PHD2 and VHL. RHOBTB3−/− MEFs and WT MEFs were lysed and the endogenous VHL was immunoprecipitated. The IP product was analyzed by immunoblotting. (F) RHOBTB3 promotes the PHD2-VHL interaction in vitro. In vitro translated RHOBTB3 and bacterially expressed GST-PHD2 were incubated with anti-MYC-conjugated resin-bound MYC-tagged VHL. The mixtures were then pulled down by centrifugation, and analyzed by immunoblotting. (G) RHOBTB3 promotes the interaction between PHD2 and HIF1α. HEK293T cells were transfected with different combinations of HIF1α, FLAG-PHD2, MYC-VHL and HA-RHOBTB3. At 8 h post-transfection, cells were treated with 10 μM MG-132 and maintained in normoxia or exposed to hypoxia for 10 h. The protein extracts were immunoprecipitated with antibody against MYC (for VHL), and precipitated proteins were analyzed by immunoblotting.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4559813&req=5

fig3: RHOBTB3, PHD2 and VHL form a complex. (A) RHOBTB3 interacts with endogenous PHD2 and VHL. Protein extracts of WT MEFs and RHOBTB3−/− MEFs (control) were immunoprecipitated with antibody against RHOBTB3 or control IgG, and analyzed by immunoblotting with antibodies indicated. (B) Ectopically expressed RHOBTB3 interacts simultaneously with PHD2 and VHL. HEK293T cells were transfected with different combinations of HA-RHOBTB3, MYC-VHL and FLAG-PHD2. At 16 h post-infection, cells were lysed and the protein extracts were immunoprecipitated with antibody against HA, and the IP product was analyzed by western blotting. (C) RHOBTB3, VHL and PHD2 form a complex. HEK293T cells were transfected with HA-RHOBTB3, FLAG-PHD2 and MYC-VHL. After 16 h, cells were harvested. Two-step co-IP was performed by first using anti-FLAG antibody, followed by elution with the FLAG peptide. The eluates were subjected to a second round of IP with anti-HA or control IgG, and the final precipitated proteins were analyzed by immunoblotting. (D) The N-terminal region of RHOBTB3 (aa 1-204) does not have a role in the degradation of HIF1α. RHOBTB3−/− MEFs were infected with lentiviruses expressing HA-RHOBTB3 or HA-RHOBTB3 (aa 1-201). At 36 h post-infection, cells were maintained in normoxia or exposed to hypoxia for 8 h, and analyzed after lysis by immunoblotting. (E) RHOBTB3 strengthens the interaction between PHD2 and VHL. RHOBTB3−/− MEFs and WT MEFs were lysed and the endogenous VHL was immunoprecipitated. The IP product was analyzed by immunoblotting. (F) RHOBTB3 promotes the PHD2-VHL interaction in vitro. In vitro translated RHOBTB3 and bacterially expressed GST-PHD2 were incubated with anti-MYC-conjugated resin-bound MYC-tagged VHL. The mixtures were then pulled down by centrifugation, and analyzed by immunoblotting. (G) RHOBTB3 promotes the interaction between PHD2 and HIF1α. HEK293T cells were transfected with different combinations of HIF1α, FLAG-PHD2, MYC-VHL and HA-RHOBTB3. At 8 h post-transfection, cells were treated with 10 μM MG-132 and maintained in normoxia or exposed to hypoxia for 10 h. The protein extracts were immunoprecipitated with antibody against MYC (for VHL), and precipitated proteins were analyzed by immunoblotting.
Mentions: We next asked whether RHOBTB3 forms a complex with VHL and PHD2, particularly since RHOBTB3 and VHL showed interaction in our yeast two-hybrid screen (Supplementary information, Figure S1A) and that RHOBTB3 promoted PHD2-mediated HIFα hydroxylation. We found that endogenous or ectopically expressed RHOBTB3 was co-immunoprecipitated with VHL or vice versa, but not with the control IgG (Figure 3A and Supplementary information, Figure S3A). Consistently, immunofluorescent staining revealed the co-localization of RHOBTB3 with VHL (Supplementary information, Figure S3B). Similarly, PHD2 was readily co-precipitated with RHOBTB3 (Figure 3A and Supplementary information, Figure S3C). We next asked whether RHOBTB3, PHD2 and VHL co-exist in the same complex. It was observed that RHOBTB3 interacted simultaneously with PHD2 and VHL (Figure 3B). We further employed a two-step co-immunoprecipitation (IP) assay, and found that all three components were detected in the final immunoprecipitates (Figure 3C), indicating their co-existence in the same complex. Of note, HSP90, reported as a chaperone of HIFs71,72 and PHD273, was co-precipitated with RHOBTB3 only when HIF1α was present, suggesting that the HSP90 does not directly interact with RHOBTB3 (Supplementary information, Figure S3D). Although HSP90 has been shown to interact with PHD273 and RHOBTB3 interacts with PHD2 as shown in the present study, PHD2 could co-precipitate HSP90 and RHOBTB3 only when HIF1α was present, suggesting the PHD2-HSP90 complex depends on HIF1α to form a co-complex with RHOBTB3 (Supplementary information, Figure S3D). In the presence of ectopic HIF1α, more HSP90 was co-precipitated with RHOBTB3 under normoxia (Supplementary information, Figure S3E), consistent with a stronger interaction of HIF1α with RHOBTB3 under this condition (Figure 2F). These data indicate that HSP90 does not directly interact with RHOBTB3.

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