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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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Cul1 binds preferentially to substrate receptors.HEK293 cells were cotransfected with the indicated plasmids. Cell lysates were used for immunoprecipitation with FLAG antibody followed by Western blotting.
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pone-0016071-g006: Cul1 binds preferentially to substrate receptors.HEK293 cells were cotransfected with the indicated plasmids. Cell lysates were used for immunoprecipitation with FLAG antibody followed by Western blotting.

Mentions: Cul1 can bind to Skp1 adaptor and substrate receptor subunits and to CAND1 in a mutually exclusive manner [5]–[8]. To determine with which interaction partner Cul1 associates preferentially in vivo, we transfected cells with a plasmid encoding Cul1 carrying an N-terminal FLAG and a C-terminal V5 tag. The cells were co-transfected with V5-tagged CAND1 and β-TrCP or Skp2 substrate receptors. FLAG antibody was then used to immunoprecipiate Cul1 protein complexes. The FLAG-Cul1-V5 immunoprecipitates were analyzed in Western blots with V5 antibody to directly compare the amounts of Cul1, CAND1, β-TrCP and Skp2 in the Cul1 complex. Although CAND1 and β-TrCP were expressed at approximately equal amounts in the cell lysate, much more binding of β-TrCP to Cul1 was observed compared to CAND1 (see lane 4 of the total cell lysates and lane 4 of the FLAG immunoprecipitates in Fig. 6). Transfected Skp2 was expressed at higher concentrations. When comparing the ratio of Skp2 in the FLAG-immunopreciptates to that in the lysate, a marked enrichment of Skp2 protein was seen in the immunoprecipitates compared to CAND1 (compare the rations of Skp2 and CAND1 between lane 2 of the FLAG immunoprecipitates and lane 3 of the lysates). The enrichment of Skp2 in the immunoprecipiates compared to lysate was smaller than that observed for β-TrCP, which is likely due to the higher basal expression of Skp2 and saturation of Cul1 binding sites. When CAND1, β-TrCP and Skp2 were transfected in the absence of Cul1, none of the proteins was detected in the FLAG immunoprecipitates, confirming the specificity of the assay (see lane 2 of the lysates and lane 3 of the FLAG immunoprecipitates). Given the predominant localization of CAND1 in the cytoplasm, we also performed analogous experiments using cytoplasmic cellular fractions. Similarly to the total cell lysate, strong binding of both β-TrCP and Skp2 to Cul1 was observed while specific binding of CAND1 to Cul1 was low or undetectable (data not shown). Taken together, the results in Fig. 6 suggest that under in vivo conditions the substrate receptor proteins bind much stronger to Cul1 compared to CAND1. Our results are consistent with data published by Bornstein et al. [23], who showed that Skp2–Skp1 promotes the dissociation of CAND1 from Cul1 in vitro.


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

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

Cul1 binds preferentially to substrate receptors.HEK293 cells were cotransfected with the indicated plasmids. Cell lysates were used for immunoprecipitation with FLAG antibody followed by Western blotting.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3020946&req=5

pone-0016071-g006: Cul1 binds preferentially to substrate receptors.HEK293 cells were cotransfected with the indicated plasmids. Cell lysates were used for immunoprecipitation with FLAG antibody followed by Western blotting.
Mentions: Cul1 can bind to Skp1 adaptor and substrate receptor subunits and to CAND1 in a mutually exclusive manner [5]–[8]. To determine with which interaction partner Cul1 associates preferentially in vivo, we transfected cells with a plasmid encoding Cul1 carrying an N-terminal FLAG and a C-terminal V5 tag. The cells were co-transfected with V5-tagged CAND1 and β-TrCP or Skp2 substrate receptors. FLAG antibody was then used to immunoprecipiate Cul1 protein complexes. The FLAG-Cul1-V5 immunoprecipitates were analyzed in Western blots with V5 antibody to directly compare the amounts of Cul1, CAND1, β-TrCP and Skp2 in the Cul1 complex. Although CAND1 and β-TrCP were expressed at approximately equal amounts in the cell lysate, much more binding of β-TrCP to Cul1 was observed compared to CAND1 (see lane 4 of the total cell lysates and lane 4 of the FLAG immunoprecipitates in Fig. 6). Transfected Skp2 was expressed at higher concentrations. When comparing the ratio of Skp2 in the FLAG-immunopreciptates to that in the lysate, a marked enrichment of Skp2 protein was seen in the immunoprecipitates compared to CAND1 (compare the rations of Skp2 and CAND1 between lane 2 of the FLAG immunoprecipitates and lane 3 of the lysates). The enrichment of Skp2 in the immunoprecipiates compared to lysate was smaller than that observed for β-TrCP, which is likely due to the higher basal expression of Skp2 and saturation of Cul1 binding sites. When CAND1, β-TrCP and Skp2 were transfected in the absence of Cul1, none of the proteins was detected in the FLAG immunoprecipitates, confirming the specificity of the assay (see lane 2 of the lysates and lane 3 of the FLAG immunoprecipitates). Given the predominant localization of CAND1 in the cytoplasm, we also performed analogous experiments using cytoplasmic cellular fractions. Similarly to the total cell lysate, strong binding of both β-TrCP and Skp2 to Cul1 was observed while specific binding of CAND1 to Cul1 was low or undetectable (data not shown). Taken together, the results in Fig. 6 suggest that under in vivo conditions the substrate receptor proteins bind much stronger to Cul1 compared to CAND1. Our results are consistent with data published by Bornstein et al. [23], who showed that Skp2–Skp1 promotes the dissociation of CAND1 from Cul1 in vitro.

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