The Unique N Terminus of the UbcH10 E2 Enzyme Controls the Threshold for APC Activation and Enhances Checkpoint Regulation of the APC  Matthew K. Summers, Borlan Pan, Kiran Mukhyala, Peter K. Jackson  Molecular Cell  Volume 31, Issue 4, Pages 544-556 (August 2008) DOI: 10.1016/j.molcel.2008.07.014 Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 1 UbcH10 Is Sufficient and Required for APC-Mediated Destruction Events, and Its N Terminus Regulates Ubiquitination Activity (A) Comparison of UbcH10 and UbcH5 catalyzed APC ubiquitination of 35S-labeled cyclin B and Securin in vitro. Immunopurified APC was activated with Cdh1 and mixed with E1, Ub, substrate, an energy-regenerating mix, and E2s at room temperature for the indicated times. Reaction products were analyzed by SDS-PAGE and autoradiography. (B) Mitotic HeLa extracts were supplemented with an energy-regenerating system and nondegradeable cyclin B to maintain the mitotic state. Reactions were initiated by the addition of 35S-Securin. Two micromolars of E2s with or without 50 μM Ub were added as indicated. The stability of substrates over time was assessed by SDS-PAGE autoradiography. (C) Securin destruction assays were set up as in (A). Destruction was induced by addition of 10 μM p31Comet. Catalytically inactive E2s (2 μM) were added as indicated. (D) Comparison of the UbcH10 N terminus from multiple species. Residue conservation is indicated by shading. The sites of the N-terminal mutations generated are indicated above the sequences. (E and F) HeLa cells were transfected with FLAG-tagged proteins, and mitotic index, in the presence of Taxol, was determined after release from thymidine. (E) Representative images of UbcH10 and ΔN-expressing cells. (F) Cells were fixed at the indicated times and stained for FLAG expression and phosphohistone H3 (S10). The data from two experiments with duplicate samples ± SEM are shown. (G) Comparison of UbcH10, ΔN, and UbcH5 catalyzed APC ubiquitination of 35S-labeled cyclin A in vitro, as in (A). Molecular Cell 2008 31, 544-556DOI: (10.1016/j.molcel.2008.07.014) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 2 UbcH10-APC Specificity and Activity Are Determined by UbcH10core and the N-Terminal Extension, Respectively (A) Comparison of UbcH10, ΔN, and UbcH5 activity in the presence of APC11. Recombinant APC11 was mixed with E1, FLAG-Ub, substrate, ATP, and E2s at 30°C for 45 min. Reaction products were analyzed by SDS-PAGE and immunoblotting for the FLAG epitope. (B) Comparison of UbcH10, ΔN, and UbcH5 catalyzed APC2-APC11 ubiquitination of 35S-labeled Securin in vitro. Baculovirus-expressed APC proteins were mixed with E1, Ub, substrate, energy-regenerating mix, and E2s at 30°C for 45 min. Reaction products were analyzed by SDS-PAGE and autoradiography. (C) Activity of UbcH10 N-terminal mutants with the APC holoenzyme. E2s were added to mitotic HeLa extract, and Securin destruction was monitored as in Figure 1B. Lower, the same E2s were used in in vitro APC reactions, as in Figure 1A, with Securin as substrate. (D) UbcH10, UbcH5, or a Ubch10 N-term-UbcH5 chimera were used in in vitro APC reactions, as in Figure 1A, with Securin as substrate. Molecular Cell 2008 31, 544-556DOI: (10.1016/j.molcel.2008.07.014) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 3 The N Terminus of UbcH10 Restricts the Number of Substrate Lysines Targeted by the APC and Enhances D Box Selectivity (A) UbcH10 and ΔN were used in in vitro APC reactions, as in Figure 1A, utilizing wild-type (WT) or no lysine (NoK) Ub with Securin as substrate. The number of substrate lysines conjugated to Ub is indicated. (Right) Densitometry was used to calculate the percentage of ubiquitinated substrate represented by each species. (B) UbcH10 and N-terminal mutants were used in in vitro APC reactions, as in Figure 1A. WT D box mutant (Db−) and KEN box, D box mutant (Kb−Db−) Securin were used as substrates. (Left) Autorads. (Right) The fraction of ubiquitinated substrate was determined. For each E2, the activity toward WT substrate was set as maximal, and the percentage of this activity toward the mutant substrates was determined. (C) (Left) UbcH10 (WT) and ΔN were added to mitotic HeLa extract, and the destruction of WT D box (Db−) or KEN box, D box mutant (Kb−Db−, lower) Securin was monitored as in Figure 1B. (Left) Autorads. (Right) Graphical representation of destruction assays ± SEM of three independent experiments. Molecular Cell 2008 31, 544-556DOI: (10.1016/j.molcel.2008.07.014) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 4 Constraints Imposed by the N Terminus Confer Enhanced Regulation of APC Activity In Vitro (A) 1.7 μM UbcH10 and the ΔN mutant were used in in vitro APC reactions, as in Figure 1A, with 35S-cyclin B1 N terminus as substrate in the presence of MBP-Emi1. (Left) Autorads. (Right) Quantitation of results. Data is representative of three independent experiments. (B) Titration of UbcH10 and the ΔN mutant in mitotic HeLa extract. Securin stability was monitored as in Figure 1A. (Upper) Autorads. (Lower) Quantitation. The data is representative of four independent experiments. Molecular Cell 2008 31, 544-556DOI: (10.1016/j.molcel.2008.07.014) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 5 NMR Analysis of the UbcH10 N Terminus (A) 1H-15N HSQC spectra of UbcH10 (blue) and ΔN (red). The crosspeaks displaying chemical shift perturbations in the WT relative to ΔN are boxed. (B) Recombinant E2s were incubated with 35S-labeled APC2, captured by Ni-affinity resin, and bound protein detected by SDS-PAGE and autoradiography. (C) UbcH10, APC2, and APC11 were modeled onto the structure of Rbx1-Cul1. The catalytic cysteine of UbcH10 is represented as yellow spheres, and the chelated zinc in the APC11 RING domain are represented as purple spheres. Arrows indicate the junction of the N terminus and the core E2 domain. Molecular Cell 2008 31, 544-556DOI: (10.1016/j.molcel.2008.07.014) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 6 Model of the N-Terminal Regulation of UbcH10-APC Activity (Upper panel) Optimal engagement of the substrate allows the substrate lysines to efficiently accept Ub from UbcH10. (Lower left) In the absence of proper engagement, e.g., in the absence of a D box, Ub transfer to the substrate is inefficient, preventing substrate destruction. (Lower right) Under the same conditions, the increased ubiquitination activity of ΔN is able to transfer Ub to the substrate, promoting its destruction. Molecular Cell 2008 31, 544-556DOI: (10.1016/j.molcel.2008.07.014) Copyright © 2008 Elsevier Inc. Terms and Conditions