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Regulation of cullin RING E3 ubiquitin ligases by CAND1 in vivo.

Chua YS, Boh BK, Ponyeam W, Hagen T - PLoS ONE (2011)

Bottom Line: CAND1, which binds to cullins that are not conjugated with Nedd8 and not associated with substrate receptors, has been shown to function as a positive regulator of Cullin ligases in vivo.Furthermore, preventing binding of F-box substrate receptors to Cul1 does not increase CAND1 binding.In conclusion, our study suggests that CAND1 does not function by sequestering cullins in vivo to prevent substrate receptor autoubiquitination and is likely to regulate cullin RING ligase activity via alternative mechanisms.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore.

ABSTRACT
Cullin RING ligases are multi-subunit complexes consisting of a cullin protein which forms a scaffold onto which the RING protein Rbx1/2 and substrate receptor subunits assemble. CAND1, which binds to cullins that are not conjugated with Nedd8 and not associated with substrate receptors, has been shown to function as a positive regulator of Cullin ligases in vivo. Two models have been proposed to explain this requirement: (i) CAND1 sequesters cullin proteins and thus prevents autoubiquitination of substrate receptors, and (ii) CAND1 is required to promote the exchange of bound substrate receptors. Using mammalian cells, we show that CAND1 is predominantly cytoplasmically localized and that cullins are the major CAND1 interacting proteins. However, only small amounts of CAND1 bind to Cul1 in cells, despite low basal levels of Cul1 neddylation and approximately equal cytoplasmic endogenous protein concentrations of CAND1 and Cul1. Compared to F-box protein substrate receptors, binding of CAND1 to Cul1 in vivo is weak. Furthermore, preventing binding of F-box substrate receptors to Cul1 does not increase CAND1 binding. In conclusion, our study suggests that CAND1 does not function by sequestering cullins in vivo to prevent substrate receptor autoubiquitination and is likely to regulate cullin RING ligase activity via alternative mechanisms.

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Neddylation regulates the interaction between Cul1 and CAND1 in vivo.(a) HEK293 cells were transfected with wild type or K472E/R473E mutant Cul1-V5, followed by Western blotting with V5 antibody. (b–d) Lysates from cells transfected with wild type or mutant (K472E/R473E) Cul1-V5 were subjected to immunoprecipitation using V5 antibody followed by Western blotting of lysates and immunoprecipitates with the indicated antibodies. In (d) dnUbc12 tet-on cells were used for transfection and induced with 1 µg/ml tetracycline for 24 hours prior to cell lysis in order to block Cul1 neddylation. (e) Cells were transfected in 60 mm dishes with 1 µg wild type or K472E/R473E mutant Cul1-V5 and 1.5 µg dnCul1-V5 or empty vector, as indicated. After two days, all plates were transfected with 10 µg of recombinant GST-CAND1 in the presence or absence of TurboFect protein transfection reagent. One hour after protein transfection, cells were rinsed and lysed and cell lysates subjected to V5 immunoprecipitation. The immunoprecioitates were analyzed by Western blotting with GST and V5 antibodies. As expected, no GST-CAND1 was observed in cell lysate and V5 immunoprecipitates when no protein transfection agent was included (see lanes 5 and 10). When GST was transfected into cells as a negative control, no binding to Cul1 could be observed (not shown).
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pone-0016071-g002: Neddylation regulates the interaction between Cul1 and CAND1 in vivo.(a) HEK293 cells were transfected with wild type or K472E/R473E mutant Cul1-V5, followed by Western blotting with V5 antibody. (b–d) Lysates from cells transfected with wild type or mutant (K472E/R473E) Cul1-V5 were subjected to immunoprecipitation using V5 antibody followed by Western blotting of lysates and immunoprecipitates with the indicated antibodies. In (d) dnUbc12 tet-on cells were used for transfection and induced with 1 µg/ml tetracycline for 24 hours prior to cell lysis in order to block Cul1 neddylation. (e) Cells were transfected in 60 mm dishes with 1 µg wild type or K472E/R473E mutant Cul1-V5 and 1.5 µg dnCul1-V5 or empty vector, as indicated. After two days, all plates were transfected with 10 µg of recombinant GST-CAND1 in the presence or absence of TurboFect protein transfection reagent. One hour after protein transfection, cells were rinsed and lysed and cell lysates subjected to V5 immunoprecipitation. The immunoprecioitates were analyzed by Western blotting with GST and V5 antibodies. As expected, no GST-CAND1 was observed in cell lysate and V5 immunoprecipitates when no protein transfection agent was included (see lanes 5 and 10). When GST was transfected into cells as a negative control, no binding to Cul1 could be observed (not shown).

Mentions: CAND1 is known to bind to unneddylated cullin proteins [5], [6], [18]. To confirm that neddylation indeed regulates binding of CAND1 in vivo, we generated a hyperneddylation mutant of mouse Cul1 by mutating both Lys-472 and Arg-473 to Glu [5], [19]–[21]. K472E/R473E mutant Cul1 displayed a significantly increased level of Nedd8 modification (Fig. 2a). We hypothesized that the increased neddylation of K472E/R473E mutant Cul1 is due to reduced binding of the deneddylating CSN complex. We therefore measured the binding of wild type and K472E/R473E mutant Cul1 to the CSN5 subunit of the COP9 signalosome (CSN). As shown in Fig. 2b, in contrast to wild type Cul1, no interaction of CSN5 with K472E/R473E Cul1 could be detected, suggesting that the increased neddylation of the Cul1 mutant is due to reduced binding of CSN.


Regulation of cullin RING E3 ubiquitin ligases by CAND1 in vivo.

Chua YS, Boh BK, Ponyeam W, Hagen T - PLoS ONE (2011)

Neddylation regulates the interaction between Cul1 and CAND1 in vivo.(a) HEK293 cells were transfected with wild type or K472E/R473E mutant Cul1-V5, followed by Western blotting with V5 antibody. (b–d) Lysates from cells transfected with wild type or mutant (K472E/R473E) Cul1-V5 were subjected to immunoprecipitation using V5 antibody followed by Western blotting of lysates and immunoprecipitates with the indicated antibodies. In (d) dnUbc12 tet-on cells were used for transfection and induced with 1 µg/ml tetracycline for 24 hours prior to cell lysis in order to block Cul1 neddylation. (e) Cells were transfected in 60 mm dishes with 1 µg wild type or K472E/R473E mutant Cul1-V5 and 1.5 µg dnCul1-V5 or empty vector, as indicated. After two days, all plates were transfected with 10 µg of recombinant GST-CAND1 in the presence or absence of TurboFect protein transfection reagent. One hour after protein transfection, cells were rinsed and lysed and cell lysates subjected to V5 immunoprecipitation. The immunoprecioitates were analyzed by Western blotting with GST and V5 antibodies. As expected, no GST-CAND1 was observed in cell lysate and V5 immunoprecipitates when no protein transfection agent was included (see lanes 5 and 10). When GST was transfected into cells as a negative control, no binding to Cul1 could be observed (not shown).
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Related In: Results  -  Collection

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pone-0016071-g002: Neddylation regulates the interaction between Cul1 and CAND1 in vivo.(a) HEK293 cells were transfected with wild type or K472E/R473E mutant Cul1-V5, followed by Western blotting with V5 antibody. (b–d) Lysates from cells transfected with wild type or mutant (K472E/R473E) Cul1-V5 were subjected to immunoprecipitation using V5 antibody followed by Western blotting of lysates and immunoprecipitates with the indicated antibodies. In (d) dnUbc12 tet-on cells were used for transfection and induced with 1 µg/ml tetracycline for 24 hours prior to cell lysis in order to block Cul1 neddylation. (e) Cells were transfected in 60 mm dishes with 1 µg wild type or K472E/R473E mutant Cul1-V5 and 1.5 µg dnCul1-V5 or empty vector, as indicated. After two days, all plates were transfected with 10 µg of recombinant GST-CAND1 in the presence or absence of TurboFect protein transfection reagent. One hour after protein transfection, cells were rinsed and lysed and cell lysates subjected to V5 immunoprecipitation. The immunoprecioitates were analyzed by Western blotting with GST and V5 antibodies. As expected, no GST-CAND1 was observed in cell lysate and V5 immunoprecipitates when no protein transfection agent was included (see lanes 5 and 10). When GST was transfected into cells as a negative control, no binding to Cul1 could be observed (not shown).
Mentions: CAND1 is known to bind to unneddylated cullin proteins [5], [6], [18]. To confirm that neddylation indeed regulates binding of CAND1 in vivo, we generated a hyperneddylation mutant of mouse Cul1 by mutating both Lys-472 and Arg-473 to Glu [5], [19]–[21]. K472E/R473E mutant Cul1 displayed a significantly increased level of Nedd8 modification (Fig. 2a). We hypothesized that the increased neddylation of K472E/R473E mutant Cul1 is due to reduced binding of the deneddylating CSN complex. We therefore measured the binding of wild type and K472E/R473E mutant Cul1 to the CSN5 subunit of the COP9 signalosome (CSN). As shown in Fig. 2b, in contrast to wild type Cul1, no interaction of CSN5 with K472E/R473E Cul1 could be detected, suggesting that the increased neddylation of the Cul1 mutant is due to reduced binding of CSN.

Bottom Line: CAND1, which binds to cullins that are not conjugated with Nedd8 and not associated with substrate receptors, has been shown to function as a positive regulator of Cullin ligases in vivo.Furthermore, preventing binding of F-box substrate receptors to Cul1 does not increase CAND1 binding.In conclusion, our study suggests that CAND1 does not function by sequestering cullins in vivo to prevent substrate receptor autoubiquitination and is likely to regulate cullin RING ligase activity via alternative mechanisms.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore.

ABSTRACT
Cullin RING ligases are multi-subunit complexes consisting of a cullin protein which forms a scaffold onto which the RING protein Rbx1/2 and substrate receptor subunits assemble. CAND1, which binds to cullins that are not conjugated with Nedd8 and not associated with substrate receptors, has been shown to function as a positive regulator of Cullin ligases in vivo. Two models have been proposed to explain this requirement: (i) CAND1 sequesters cullin proteins and thus prevents autoubiquitination of substrate receptors, and (ii) CAND1 is required to promote the exchange of bound substrate receptors. Using mammalian cells, we show that CAND1 is predominantly cytoplasmically localized and that cullins are the major CAND1 interacting proteins. However, only small amounts of CAND1 bind to Cul1 in cells, despite low basal levels of Cul1 neddylation and approximately equal cytoplasmic endogenous protein concentrations of CAND1 and Cul1. Compared to F-box protein substrate receptors, binding of CAND1 to Cul1 in vivo is weak. Furthermore, preventing binding of F-box substrate receptors to Cul1 does not increase CAND1 binding. In conclusion, our study suggests that CAND1 does not function by sequestering cullins in vivo to prevent substrate receptor autoubiquitination and is likely to regulate cullin RING ligase activity via alternative mechanisms.

Show MeSH
Related in: MedlinePlus