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First published online 9 November 2004
doi: 10.1242/jcs.01418

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Requirements for the destruction of human Aurora-A

Department of Cell Biology, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115, USA

View larger version (38K): [in a new window]   Fig. 1. Destruction of human Aur-A protein in G1-phase extracts of HeLa cells requires the D box and the A box. (A) HeLa cells were synchronized by a double thymidine block, released into nocodazole to collect cells in M phase and subsequently released into G1 phase. Extracts were prepared from M-phase and G1-phase populations, and supplemented with ubiquitin and UbcH10. IVT human cyclin B1 or non-degradable cyclin B1 D1-90 (A) or variants of human Aur-A were then added, with the proteasome inhibitor MG-115 where indicated. Samples were taken at the times and processed for SDS-PAGE followed by autoradiography. (B) Schematic of potential destruction signals and catalytic residues in human Aur-A. The sequence alignment compares the A box in human (h) and Xenopus (x) Aur-A with the corresponding region in Aur-B. Residues conserved with human Aur-A are shaded. The solid bar indicates the conserved QRxL motif. The circled P indicates the serine phosphorylation site (S51 in human Aur-A). (C) IVT mutants of human Aur-A were assayed for destruction in G1-phase HeLa-cell lysates as described in A.

View larger version (27K): [in a new window]   Fig. 2. Cdh1 is required for the destruction of Aur-A in HeLa cells. (A) HeLa cells were infected with retrovirus encoding His6-tagged N-terminal dominant negative fragment of Cdh1 [Cdh1(1-125)] or an empty retrovirus control. 24 hours after infection, cells were lysed and processed for western blotting with antibodies against His6 or Aur-A, as indicated, or for propidium iodide staining and flow cytometry. (B) HeLa cells were transfected with or without pcDNA3-FLAG-CUL1(1-452). 48 hours after transfection, cells were lysed and processed for western blotting with antibodies against FLAG, p27 or Aur-A, as indicated. In both A and B, the Aur-A blot was stripped and reprobed with anti-Cdc2 antibody as a loading control. (C) IVT Myc tag, Myc-Cdh1 or Myc-Cdc20 were incubated with anti-Myc beads and washed extensively. Beads were then incubated with [35S]-labeled IVT GFP or GFP/Aur-A variants, as indicated, followed by washes and elution into SDS sample buffer. Samples were analysed by PAGE and autoradiography alongside 10% of the input IVT product, and bands quantified by phosphorimager analysis.

View larger version (40K): [in a new window]   Fig. 3. Human Aur-A IVT product is destroyed in egg extract without additional Cdh1. (A,B) Xenopus egg CSF extract was incubated for 5 minutes with the APC/C activators Cdc20 or Cdh1. Radiolabeled IVT substrates were added as indicated and incubated for a further 10 minutes. Calcium was added to trigger exit from meiosis-I metaphase arrest and entry into the first cell cycle. Samples were taken at the indicated times and analysed by PAGE and autoradiography. (C) Radiolabeled IVT products were prepared, using 40 µg µl-1 DNA template or 20 µg ml-1 IVT and purified mRNA template. Templates were: (1) Xenopus Aur-A DNA; (2) human Aur-A DNA; (3) Xenopus Aur-A mRNA; (4) human Aur-A mRNA; (5) human Aur-A D-box-mutant DNA; (6) human Aur-A (66-403) DNA; and (7) human Aur-A DNA in the presence of 10 ng µl-1 Cdh1(1-125) protein. (D) Following coupled in vitro transcription-translation of the indicated templates for 90 minutes in rabbit reticulocyte lysate, 100 µg ml-1 cycloheximide (CHX) was added to inhibit further protein synthesis. Reaction mixes were shifted to 23°C and samples were taken at subsequent times for analysis by SDS-PAGE followed by autoradiography. Where indicated, the proteasome inhibitor MG-115 (1.6 µM) was added 10 minutes before CHX.

View larger version (34K): [in a new window]   Fig. 4. Reticulocyte lysate contains a Cdh1-dependent destruction activity. (A) Destruction of Aur-A in reticulocyte lysate was analysed as follows. Following coupled in vitro transcription-translation of the indicated templates for 90 minutes in rabbit reticulocyte lysate, 100 µg ml-1 cycloheximide (CHX) was added to inhibit further protein synthesis. Reaction mixes were shifted to 23°C and samples were taken at subsequent times for analysis by SDS-PAGE followed by autoradiography. Five minutes prior to cycloheximide (CHX) addition, 10 ng µl-1 Cdh1(1-125) protein was added as indicated. (B) Xenopus egg CSF extract was incubated for 5 minutes with Cdh1 protein, reticulocyte lysate (RL), or reticulocyte lysate and Cdh1(1-125) protein. Calcium was added to trigger exit from meiosis I. Samples were taken at the indicated times and analysed by PAGE and western blot for endogenous Xenopus Aur-A.

View larger version (60K): [in a new window]   Fig. 5. Epitope tags reduce the destruction of Aur-A in vitro. (A) Radiolabeled IVT products were prepared for the indicated proteins using 0.2 µg DNA templates: (1) Xenopus Aur-A; (2) N-terminal AU1-tagged Xenopus Aur-A; (3) human Aur-A; (4) N-terminal AU1-tagged human Aur-A; (5) N-terminal GFP-tagged human Aur-A; and (6) C-terminal GFP-tagged human Aur-A. (B) IVT products were incubated with G1-phase HeLa-cell lysates. Samples were taken at the indicated times and monitored by SDS-PAGE followed by autoradiography. (C) Destruction assays were performed in Xenopus egg extracts as described in Fig. 3A.

View larger version (17K): [in a new window]   Fig. 6. Destruction of purified, recombinant human Aur-A protein in Xenopus egg extract requires the D box and sequences within the A box. Destruction assays were performed in Xenopus egg extracts as follows. Xenopus egg CSF extract was incubated for 5 minutes with the APC/C activators Cdc20 or Cdh1. His6-tagged human Aur-A wild-type (A) or mutant (B) protein substrates were added at a final concentration of 220 nM and incubated for a further 10 minutes. Calcium was added to trigger exit from meiosis-II metaphase arrest and entry into the first cell cycle. Samples were taken at the indicated times and analysed SDS-PAGE and western blotted with anti-Aur-A antibodies.