Multimodal activation of the ubiquitin ligase SCF by Nedd8 conjugation - PubMed (original) (raw)

Multimodal activation of the ubiquitin ligase SCF by Nedd8 conjugation

Anjanabha Saha et al. Mol Cell. 2008.

Abstract

Conjugation of ubiquitin-like protein Nedd8 to cullins (neddylation) is essential for the function of cullin-RING ubiquitin ligases (CRLs). Here, we show that neddylation stimulates CRL activity by multiple mechanisms. For the initiator ubiquitin, the major effect is to bridge the approximately 50 A gap between naked substrate and E2 approximately Ub bound to SCF. The gap between the acceptor lysine of ubiquitinated substrate and E2 approximately Ub is much smaller, and, consequentially, the impact of neddylation on transfer of subsequent ubiquitins by Cdc34 arises primarily from improved E2 recruitment and enhanced amide bond formation in the E2 active site. The combined effects of neddylation greatly enhance the probability that a substrate molecule acquires >or= 4 ubiquitins in a single encounter with a CRL. The surprisingly diverse effects of Nedd8 conjugation underscore the complexity of CRL regulation and suggest that modification of other ubiquitin ligases with ubiquitin or ubiquitin-like proteins may likewise have major functional consequences.

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Figures

Figure 1. Neddylation enhances Cdc34 binding to Cul1-Rbx1

(A) Fluorescence emission spectra of buffer, 40 nM Cul1–Rbx1, 300 nM Cdc34, and Cdc34–Cul1–Rbx1 complex following excitation at 430 nm. (B, C) Equilibrium binding titrations of Cdc34 (B) and Cdc34~Ub (C) with Cul1–Rbx1 and Nedd8 Cul1–Rbx1. FRET efficiency was plotted as a function of Cdc34 concentration. (D) Quantification of dissociation constant (mean ± SD).

Figure 2. Cul1 neddylation enhances E2– SCFβ-TrCP interaction and the rate of ubiquitination of β-catenin and monoubiquitinated β-catenin substrates

(A) 32P-labeled β-catenin (5 µM) was incubated with E1, ATP, ubiquitin, Cdc34 (indicated amounts), and 300 nM SCFβ-TrCP for 75 min (lanes 1 through 10) or 300 nM Nedd8 SCFβ-TrCP for 60 min (lanes 11 through 20). Substrate ubiquitination per min was normalized to the amount of SCF and rate of formation of modified β-catenin was plotted as a function of Cdc34 concentration. (B) Same as in panel (A) but using UbcH5c as the E2 and 300 nM SCFβ-TrCP for 10 min (lanes 1 through 11) or 120 nM Nedd8 SCFβ-TrCP for 6 min (lanes 12 through 22). (C) 32P-labeled monoubiquitinated β-catenin (5 µM) was incubated with E1, ATP, ubiquitin, Cdc34 (indicated amounts), and 120 nM SCFβ-TrCP for 12 min (lanes 1 through 10) or neddylated SCFβ-TrCP for 6 min (lanes 11 through 20). Rate of substrate ubiquitination was plotted as a function of Cdc34 concentration. (D) Same as in panel (A) but using UbcH5c as the E2, and 300 nM SCFβ-TrCP for 40 min (lanes 1 through 11) or 120 nM neddylated SCFβ-TrCP for 6 min (lanes 12 through 22). Shown is a representative experiment.

Figure 3. Neddylation increases the rate of ubiquitin transfer

(A) Phosphorylated p27 (200 nM) in complex with Cdk2–Cyc E was ubiquitinated under single-turnover conditions in the presence of E1, ATP, ubiquitin, 40 µM Cdc34, and 600 nM SCFSkp2 (lanes 1 through 7) or Nedd8 SCFSkp2 (lanes 8 through 14). These reactions were carried out at both 23°C (shown above) and at 5°C to accurately estimate substrate turnover (Figure S11). The ubiquitin transfer rates at 23°C are reported. (B) Same as in panel (A) but using 40 µM UbcH5c as the E2. (C) 32P-labeled β-catenin (100 nM) was ubiquitinated under single-turnover conditions in the presence of E1, ATP, ubiquitin, 40 µM Cdc34, and 300 nM SCFβ-TrCP (lanes 1 through 7) or Nedd8 SCFβ-TrCP (lanes 8 through 14). The ubiquitin transfer rates at 23°C are reported. (D) Same as in panel (C) but using 40 µM UbcH5c as the E2. Shown is a representative experiment.

Figure 4. Neddylation enables crosslinking of β-catenin substrate to the UbcH5c active site cysteine and increases the rate of Cdc34~Ub decay to form Ub-NHOH

(A) β-catenin peptide (100 nM) coupled to a crosslinker containing maleimide group was incubated with premixed UbcH5c (30 µM) and 300 nM SCFβ-TrCP or subcomplexes thereof. Reaction products were analyzed by immunobloting with β-catenin antibody. Blots were also stained with Ponceau S to detect UbcH5c (bottom panel) or probed with Cul1 and Skp1 antibodies to confirm equal loading (data not shown). (B) Cdc34 (40 µM) was pre-incubated with 80 µM 32P-labeled K48R ubiquitin in the presence of 2 µM E1 and ATP for 10 min at 23°C, followed by 5 min incubation with apyrase and no Cul1–Rbx1 (lanes 1 through 6), 400 nM Cul1–Rbx1 (lanes 7 through 12), or 400 nM Nedd8 Cul1–Rbx1 (lanes 13 through 18). Discharge of Cdc34~Ub was initiated by adding 5 mM hydroxylamine, aliquots were removed at indicated times and quenched with non-reducing SDS-PAGE sample buffer with 5 mM NEM. Relative discharge rate of Cdc34~Ub to Ub-NHOH was estimated (Figure S12) and reported (mean ± SD).

Figure 5. Neddylation increases the fraction of substrate that acquires a long ubiquitin chain in a single E3 binding event

(A) 32P-labeled β-catenin (100 nM) was incubated under single-turnover conditions with 40 µM Cdc34 and 300 nM SCFβ-TrCP using three different order-of-addition schemes. In scheme 1 (lanes 2–4) reaction was initiated by mixing E1, E2 and Ub with E3 and labeled substrate. In scheme 2 (lanes 5–7) reaction was initiated by mixing E1, E2 and Ub with E3, labeled substrate and 100 µM cold substrate. In scheme 3 (lanes 8–10) reaction was initiated by mixing E1, E2, Ub and cold substrate with E3 and labeled substrate. (B) Same as in panel (A) but using 300 nM Nedd8 SCFβ-TrCP. (C) Phosphoimager analysis of the results shown in (A) and (B). The number of ubiquitins conjugated is shown only for the modified substrates in a single binding event. (D) Same as in panel (A) but using 40 µM UbcH5c. (E) Same as in panel (D) but using 300 nM Nedd8 SCFβ-TrCP. (F) Phosphoimager analysis of the results shown in (D) and (E).

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Multimodal activation of the ubiquitin ligase SCF by Nedd8 conjugation - PubMed (original) (raw)