Characterization of the role of COP9 signalosome in regulating cullin E3 ubiquitin ligase activity - PubMed (original) (raw)

Characterization of the role of COP9 signalosome in regulating cullin E3 ubiquitin ligase activity

Yin Yin Choo et al. Mol Biol Cell. 2011 Dec.

Abstract

Cullin RING ligases (CRLs) are the largest family of cellular E3 ubiquitin ligases and mediate polyubiquitination of a number of cellular substrates. CRLs are activated via the covalent modification of the cullin protein with the ubiquitin-like protein Nedd8. This results in a conformational change in the cullin carboxy terminus that facilitates the ubiquitin transfer onto the substrate. COP9 signalosome (CSN)-mediated cullin deneddylation is essential for CRL activity in vivo. However, the mechanism through which CSN promotes CRL activity in vivo is currently unclear. In this paper, we provide evidence that cullin deneddylation is not intrinsically coupled to substrate polyubiquitination as part of the CRL activation cycle. Furthermore, inhibiting substrate-receptor autoubiquitination is unlikely to account for the major mechanism through which CSN regulates CRL activity. CSN also did not affect recruitment of the substrate-receptor SPOP to Cul3, suggesting it may not function to facilitate the exchange of Cul3 substrate receptors. Our results indicate that CSN binds preferentially to CRLs in the neddylation-induced, active conformation. Binding of the CSN complex to active CRLs may recruit CSN-associated proteins important for CRL regulation. The deneddylating activity of CSN would subsequently promote its own dissociation to allow progression through the CRL activation cycle.

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Figures

FIGURE 1:

Role of the conserved proline residue in determining F-box protein binding to Cul1 and F-box protein stability. (A) HEK293T cells were transfected with the wild-type or proline mutant V5-Skp2 or V5-Fbxo4. Cell lysates were subjected to V5 immunoprecipitation and immunoprecipitates were analyzed by Western blotting with Cul1 and V5 antibodies. An asterisk (*) denotes a nonspecific band (heavy chain). (B) Cells were transfected with wild-type or P101A V5-Skp2 and subjected to chase analysis with 40 μM cycloheximide in which cells were lysed at the indicated time points after addition of the protein synthesis inhibitor. The relative amounts of wild-type and P101A V5-Skp2, determined by densitometry, are shown in the graph at the right. (C) Cells were transfected with negative control or CSN5 siRNA for 3 d and with wild-type or mutant V5-Skp2 or V5-Fbxo4 for the last 2 d. Cycloheximide (40 μM) was added where indicated 8 h before cell lysis and cells lysates were analyzed by Western blotting with the indicated antibodies. Bottom, quantification of the V5-Skp2 and V5-Fbxo4 abundance by densitometry. The results represent the average of two (V5-Skp2) or three (V5-Fbxo4) independent experiments.

FIGURE 2:

In vivo cullin deneddylation rates in the presence and absence of ongoing substrate ubiquitination. (A) HEK293 cells were grown in 12-well plates and 3 μM MLN4924 was added at time zero. Cells were lysed at the indicated time points and cell lysates were analyzed by Western blotting with Cul1 and Cul2 antibodies. (B) Cells were transfected with negative control or CAND1 siRNA for 3 d. The Cul1 deneddylation rate was determined as in (A). NS, nonspecific band that served as a loading control. (C) Cells were cotreated with 1 μM myxothiazol and 2.5 mM iodoacetate to rapidly deplete cellular ATP concentrations. ATP concentrations were measured as described in Materials and Methods. (D) To measure the Cul1 deneddylation rate in the presence and absence of ongoing substrate ubiquitination, HEK293, HCT116, and HeLa cells were treated with either MLN-4924 (3 μM) or myxothiazol (1 μM) and iodoacetate (2.5 mM) at time zero. Cells were lysed at the indicated time points and cell lysates were analyzed by Western blotting with Cul1 antibody. The Western blots shown are representative of at least three independent experiments in each cell line and did not show any consistent difference in the rate at which neddylation occurs. (E) The relative expression levels of CSN and Cul1 in HEK293, HCT116, and HeLa cells was compared by Western blotting of equal protein amounts of cell lysate with the indicated antibodies.

FIGURE 3:

Role of CSN-mediated cullin deneddylation in facilitating substrate-receptor exchange. Cells grown in 60-mm dishes were transfected with negative control or CAND1 siRNA, which was followed after 1 d by transfection of Cul3-V5 and FLAG-SPOP (in a tetracycline-inducible plasmid). Three days after siRNA transfection, 1 μg/ml tetracycline was added at time zero and cells were lysed at the indicated times; this was followed by V5 immunoprecipitation and Western blotting of immunoprecipitates and cell lysates with the indicated antibodies. (B) The experiment was performed as in (A), except that cells were transfected with negative control or CSN5 siRNA. NS, nonspecific band.

FIGURE 4:

Role of CSN in preventing CAND1-mediated CRL disassembly. Cells were transfected with negative control or CSN5 siRNA, which was followed after 1 d by transfection of Cul1-V5. Cells were lysed 3 d after siRNA transfection, and cell lysates were subjected to V5 immunoprecipitation and Western blotting with the indicated antibodies.

FIGURE 5:

Preferential binding of CSN5 to neddylated Cul1. (A) Cells with stable expression of FLAG-CSN5 were transfected with Cul1-V5 and treated with 3 μM MLN-4924 for 3 h, as indicated. Cell lysates were used for V5 immunoprecipitation, which was followed by Western blotting. (B) Cells with stable expression of FLAG-CSN5 were transfected with wild-type or K720R mutant Cul1-V5 and subjected to 30 min of myxothiazol (1 μM) and iodoacetate (2.5 mM) treatment, indicated as ATP depletion, prior to cell lysis. Each condition in this experiment was performed in duplicate to ensure consistency of the results. (C) Cells were transfected with wild-type or D151N mutant FLAG-CSN5, which was followed by FLAG immunoprecipitation and Western blotting of immunoprecipitates and cell lysates with Cul1 and FLAG antibodies.

FIGURE 6:

CSN5 binds preferentially to active CRLs. (A and B) HEK293T cells were transfected in 60-mm cell culture plates for 2 d with expression constructs for the full-length or extreme C-terminal deletion mutants of Cul2 and Cul3, as indicated at the top of each panel. The SFE mutant of Cul3 corresponds to the S53A/F54A/E55A mutant of Cul3, which is unable to bind to substrate-receptor subunits. The cells were lysed, and the lysates were subjected to V5 immunoprecipitation (IP), as described in Materials and Methods. Immunoprecipitates and aliquots of the cell lysates were analyzed by Western blotting with the indicated antibodies.

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Characterization of the role of COP9 signalosome in regulating cullin E3 ubiquitin ligase activity - PubMed (original) (raw)