1. A Role for the Fizzy/Cdc20 Family of Proteins in Activation of the APC/C Distinct from Substrate Recruitment 
Yuu Kimata, Joanne E. Baxter, Andrew M. Fry, Hiroyuki Yamano 
Molecular Cell 
Volume 32, Issue 4, Pages (November 2008)
DOI: /j.molcel
Copyright © 2008 Elsevier Inc. Terms and Conditions

2. Figure 1
The Destruction of Nek2A, but Not Canonical APC/C Substrates, Is Activated by the N-Terminal Fragments of Fizzy/Cdc20
(A) Different modes of substrate recognition. (Left) Nek2A directly binds the APC/C through its MR motif. (Right) Canonical substrates normally require both APC/C and Fizzy/Cdc20 for their efficient recruitment.
(B) Nek2A requires Fizzy/Cdc20 for its destruction in a cell-free destruction assay. Mock-depleted or Fizzy/Cdc20-depleted Xenopus egg extracts were used. As substrates, 35S-labeled in vitro-translated Nek2A, Cdc13 (fission yeast cyclin B), and cyclin A were used alongside a version of Cdc13 lacking the N-terminal 67 residues (Δ67, stable control). Samples were taken at indicated time points after addition of CaCl2.
(C) A schematic diagram of Fizzy/Cdc20 mutants used in this study.
(D) Addition of N-terminal 159 residues of Fizzy/Cdc20 (N159) is sufficient to trigger Nek2A destruction in Fizzy/Cdc20-depleted egg extracts, whereas the full-length Fizzy/Cdc20 is required for Cdc13 and Mes1 destruction. Endogenous Fizzy/Cdc20 was depleted from CSF extracts and the destruction of APC/C substrates was examined after in vitro-translated Fizzy/Cdc20 constructs were supplemented into the extracts. Samples were taken at the indicated time points after addition of CaCl2. Asterisk indicates N159 protein used for this assay.
(E) Bacterially purified N-Cdc20 initiates Nek2A destruction, but not Cdc13. GST- and MBP-fused Fizzy/Cdc20 N159 alongside in vitro-translated (IVT) full-length Fizzy and FzyN159 were used for destruction assay of Cdc13 and Nek2A in Fizzy/Cdc20-depleted extracts.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2008 Elsevier Inc. Terms and Conditions

3. Figure 2
The Conserved C Box of the Fizzy/Cdc20 Family of Proteins Is Required for Nek2A Destruction and APC/C Interaction
(A) Mock, GST-fused WT, or C box mutant (ΔC box) versions of N159 were added into Fizzy/Cdc20-depleted egg extracts and examined for activation of Nek2A and Cdc13 destruction.
(B) APC/C-binding assays with GST-N159. The indicated amounts of purified GST-N159 WT, C box mutant (ΔC), or GST alone (25 μg) were bound to GSH-Sepharose and incubated with CSF extracts at 23°C for 20 min. The amounts of bound APC/C were analyzed by immunoblotting with anti-Apc3 antibody.
(C) The C box-dependent activation of the APC/C is conserved. GST-fused N-terminal 186 residues of Xenopus Fizzy-related/Cdh1 (FzrN186) or N-terminal 151 residues of human Cdc20 (Cdc20N151) were added into Fizzy/Cdc20-depleted egg extracts and the destruction of Nek2A and Cdc13 examined. Adding back mock- and GST-N159 serves as a negative and a positive control, respectively.
(D) The C box is required for APC/C activation in not only mitosis but also interphase. Mock, GST-FzrN186 WT, or C box mutant (ΔC box) was added into Fizzy/Cdc20-depleted CSF or interphase extracts and the destruction of Nek2A and Cdc13 examined.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2008 Elsevier Inc. Terms and Conditions

4. Figure 3
The N-Terminal Domain of Fizzy/Cdc20 Is Required for APC/C Substrate Ubiquitylation
(A) APC/C was purified from Xenopus mitotic extracts from which Fizzy/Cdc20 had been predepleted and was used for in vitro ubiquitylation assays with buffer (mock), IVT-full-length Fizzy (FzyWT), purified recombinant GST-FzyN159, or GST-FzyN159-ΔC box. Nek2A and Cdc13 were used as substrates. Samples were taken at the indicated time points and analyzed by SDS-PAGE and fluorography.
(B) As for (A), except that Xenopus securin was used as a substrate.
(C) The APC/C-bound fraction of Nek2A is efficiently ubiquitylated by addition of FzyN159. In vitro-translated Nek2A was incubated in CSF extracts in the presence of the proteasome inhibitor MG-132. Nek2A-bound APC/C was immunopurified with anti-Apc3 antibody and used for ubiquitylation assay with buffer (+mock) or GST-FzyN159.
(D) The WD40 repeat domain is dispensable if FzyN159 is fused to substrates. (Left) Schematic diagram of FzyN159 and the fusion constructs. Filled box in Mes1 indicates mutations in the D box and KEN box. (Right) Ubiquitylation assay was performed using APC/C purified from Fizzy/Cdc20-depleted mitotic extracts. FzyN159 or the fusion constructs were added as substrates and activators to initiate the reaction. Samples were taken at the indicated time points and analyzed by SDS-PAGE and fluorography.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2008 Elsevier Inc. Terms and Conditions

5. Figure 4
The N-Terminal Domain of Cdc20 Also Regulates Nek2A Destruction In Vivo
(A) Human U2OS:EGFP-Nek2A cells were induced with 1 μg/ml doxycycline and treated with 100 nM siRNA oligonucleotides directed against Cdc20 or GAPDH. After 36 hr, cells were treated for a further 12 hr with 500 ng/ml nocodazole before mitotic cells were collected by shake-off. Extracts were prepared using RIPA buffer and analyzed by immunoblotting with antibodies against Cdc20, GFP, or α-tubulin.
(B) U2OS:EGFP-Nek2A cells were induced and depleted of Cdc20 as described above but without nocodazole treatment. After 24 hr, cells were either untransfected or transfected with Flag-tagged N-terminal 151 residues of human Cdc20 (+N-Cdc20) or the same construct with C box mutation (+ N-Cdc20 ΔC box) for a further 24 hr. Cell extracts were prepared as above and analyzed by immunoblotting with indicated antibodies.
(C) Cells treated as in (B) were processed for immunofluorescence microscopy using antibodies against Flag (Flag-Cdc20) or GFP (GFP-Nek2A). Images of mitotic cells are shown indicating that both Flag-tagged N-Cdc20 proteins localize predominantly to the cytoplasm. The WT N-Cdc20 appears more intense than the N-Cdc20-ΔC box, as the cell is more rounded up. Merge images include Flag-Cdc20 (red), GFP-Nek2A (green), and DNA stained with Hoechst (blue). Scale bar, 10 μm.
(D) The histogram indicates the percentage of mitotic cells, treated as described in (B), that stained positively for GFP-Nek2A. Fifty cells were counted in three independent experiments, and error bars show standard error.
(E) A model for activation of the APC/C by the C box of the Fizzy family of activators. In the absence of activators, the APC/C is inactive and cannot ubiquitylate any substrates (indicated by the color blue). A substrate (e.g., Nek2A) that directly binds the APC/C only requires the N-terminal region (N-Cdc20), whereas canonical substrates (e.g., cyclin B and securin) need both the N- and C-terminal regions (+Full Cdc20) for their ubiquitylation. The C-terminal WD40 repeat domain contributes to recruitment of substrates to the APC/C; however, the N-terminal domain (including the C box) seems to activate the APC/C by an as-yet-unknown mechanism (here indicated by a conformational and color change). When a substrate is directly fused to the N-Cdc20, despite D box or KEN box mutations, the fusion substrate (N-Cdc20-DK) can be ubiquitylated, indicating that the WD40 domain is dispensable once substrates are recruited to the APC/C.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2008 Elsevier Inc. Terms and Conditions