Activation of the APC/C ubiquitin ligase by enhanced E2 efficiency

Abstract

The anaphase-promoting complex/cyclosome (APC/C) is a protein-ubiquitin ligase (E3) that initiates the final events of mitosis by catalyzing the ubiquitination and proteasomal destruction of securin, cyclins, and other substrates [1, 2]. Like other members of the RING family of E3s [3, 4], the APC/C catalyzes direct ubiquitin transfer from an E2-ubiquitin conjugate (E2-Ub) to lysine residues on the protein substrate. The APC/C is activated at specific cell-cycle stages by association with an activator subunit, Cdc20 or Cdh1, which provides binding sites for specific substrate sequence motifs, or degrons. Activator might also stimulate catalytic activity [5, 6], but the underlying mechanisms are not known. Here, we dissected activator function using an artificial fusion substrate in which the N-terminal region of securin was linked to an APC/C core subunit. This fusion substrate bound tightly to the APC/C and was ubiquitinated at a low rate in the absence of activator. Ubiquitination of this substrate was stimulated by activator, due primarily to a dramatic stimulation of E2 sensitivity (Km) and catalytic rate (kcat), which together resulted in a 670-fold stimulation of kcat/Km. Thus, activator is not simply a substrate adaptor, but also enhances catalysis by promoting a more efficient interaction with the E2-Ub. Interestingly, full E2 stimulation required activator interaction with degron motifs on the substrate. We conclude that formation of a complete APC/C-activator-substrate complex leads to a major enhancement of E2 efficiency, providing an unusual substrate-assisted catalytic mechanism that limits efficient ubiquitin transfer to specific substrates.

Figure 1. Fusion Substrate Ubiquitination by the APC/C

(A) Schematic diagram of the securin-N-Apc10 and Clb2-N-Apc10 fusion substrates used in this study. Lysine residues in substrate regions are indicated by red bars. See also Figure S1B. (B) APC/C (~5 nM) was immunoprecipitated from strains lacking Apc10 and Cdh1 and incubated with the indicated ³⁵S-labeled substrates, translated in rabbit reticulocyte lysates. After washing, the APC/C-substrate complex was incubated with Cdh1 as indicated, and reactions were started by the addition of E1 (300 nM), E2 (50 μM Ubc4), and methyl-ubiquitin. Reaction products were analyzed by SDS-PAGE and autoradiography. Results are representative of two independent experiments. (C) APC/C reactions were performed as in (B) with the securin-N-Apc10 fusion substrate, using either Cdc20 or Cdh1 as the added activator. Results are representative of three independent experiments. The greater stimulation by Cdh1 was likely due to the fact that this activator is generally more stable in vitro than Cdc20. (D) APC/C reactions were performed with either the securin-N-Apc10 or Clb2-N-Apc10 fusion substrate, as in (B). Results are representative of three independent experiments. (E, F) APC/C reactions were performed as in (B) with the indicated ³⁵S-labeled fusion substrates, including mutant substrates in which KEN and/or D box residues were replaced with alanines (K-AAA and D-AAA mutants, respectively). Activity (bottom) reflects the total amount of ubiquitinated substrate, quantified with ImageQuant software and normalized relative to activity with wild-type fusion protein in the absence of activator (lanes 2). Results are representative of two independent experiments.

Figure 2. Activator Stimulatesk_cat/K_m of Ubc4 and Ubc1

(A, B) APC/C (~1 nM) reactions were performed as in Figure 1B with ³⁵S-labeled securin-NApc10 fusion substrate, in the absence (left) or presence (right) of wild-type Cdh1. Purified E2 was titrated into the reactions at the indicated concentrations. Reactions with Ubc4 (A) were incubated for 20 min in the absence of Cdh1 and 10 min in the presence of Cdh1, and reactions with Ubc1 (B) were incubated for 30 min. The rate of substrate turnover (k_obs) was determined by dividing the moles of total substrate modified per minute by the moles of APC/C. Values represent the means (+/− SEM) from three independent experiments. Data were analyzed using Prism and fit using Michaelis-Menten parameters to determine the apparent K_m and maximal k_obs (k_cat), which are summarized in Table S1. Insets show close-ups of activity at lower E2 concentrations. (C) APC/C reactions were performed at various Ubc4 concentrations as in (A), in the absence or presence of a mutant form of Cdh1 in which critical residues of the C-box were mutated (I57A, P58A). Data from three independent experiments (means +/− SEM) are presented, and the apparent K_m and maximal k_obs (k_cat) are summarized in Table S1.

Figure 3. Stimulation of E2 Depends on Substrate Binding

(A, B) APC/C (~1 nM) reactions were performed at various Ubc4 concentrations, as in Figure 2A, using ³⁵S-labeled securin-N-Apc10 fusion substrate in the absence (left) or presence (right) of Cdh1. The experiments in (A) were performed with a mutant fusion substrate from which both the 3-residue KEN box and nine-residue D box were deleted. The experiments in (B) were performed with a mutant form of Cdh1 in which a key residue involved in D-box binding was mutated (V279M). For both panels, data from three independent experiments (means +/− SEM) are presented, and the apparent K_m and maximal k_obs (k_cat) are summarized in Table S1.

Figure 4. Activator Stimulation of Ubc4 Depends on Apc10

(A) APC/C-Apc10Δ (~1 nM) reactions were performed as in Figure 2A with ³⁵S-labeled securin N-terminal fragment (residues 1-110) translated in reticulocyte lysate and purified. Purified Cdh1, also produced by translation in vitro, was added to all reactions. Recombinant Apc10 was added to a final concentration of 20 μM to half the reactions as indicated, and purified Ubc4 was titrated into the reactions. Data were analyzed using Prism and fit using Michaelis-Menten parameters to determine the half-maximal E2 concentration, or apparent K_m. Results are representative of two independent experiments. (B) APC/C reactions were performed with soluble securin fragment as in (A), using immunopurified wild-type APC/C (left panel) or APC/C carrying the Apc10-4A mutant subunit (right panel). Results are representative of two independent experiments. APC/C-Apc10-4A activity is more robust than the activity of APC/C lacking Apc10 (A) because deletion of Apc10 causes a nonspecific loss of activity [15].