APC/Fizzy-Related targets Aurora-A kinase for proteolysis

Abstract

Aurora-A kinase is a mitotic spindle-pole-associated protein that has been implicated in duplication and separation of centrosomes and in spindle assembly. The proper timing and amplitude of Aurora-A expression seems to be important, as elevated levels of this protein have been associated with centrosome abnormalities and aneuploidy in mammalian cells. We show that Aurora-A increases at the G2-M transistion and disappears completely at G1 in XL2 cells. Using Xenopus oocyte extracts, we demonstrate that degradation of Aurora-A is mediated by the anaphase-promoting complex (APC) and is regulated by Fizzy-Related but not by Fizzy. Degradation of Aurora-A depends on a D-Box, but not on its KEN-Box motif, as mutation of its C-terminal D-Box sequence induces stabilization of the protein. Accordingly, addition into the extracts of a cyclin B-type D-Box-motif-containing peptide completely suppresses its degradation. Furthermore, APC/Fizzy-Related ubiquitylates the wild type but not a D-Box mutant form of Aurora-A in vitro. Consistent with these data, ectopic expression of Fizzy-Related in Xenopus oocytes induces complete degradation of endogenous Aurora-A. Aurora-A is thus the first protein, at least in our assay system, that undergoes a D-Box-dependent degradation mediated by APC/Fizzy-Related but not by APC/Fizzy.

Fig. 1. Fizzy-Related but not Fizzy is required for Aurora-A degradation. (A) CSF extract (20 µl) was supplemented with 0.5 mM CaCl₂ where indicated (CSF+Ca²⁺). Samples (2 µl) were taken at different times, and endogenous Aurora-A and cyclin B2 levels were analysed by western blotting. The asterisk represents an unspecific band recognized by the anti-cyclin B2 antibodies in the CSF extracts. (B) Metaphase-II-arrested oocytes were activated by the calcium ionophore A32187 (Activated Oocytes), homogenized individually and analysed at various times for the degradation of endogenous Aurora-A and cyclin B2. (C) Fizzy-Related mRNA was added to interphase extracts (50 µl) as indicated (Interphase Extract +Fzr). One hour later, 1 µl of either in vitro translated ³⁵S-labelled Aurora-A or the same amount of ³⁵S-labelled cyclin B was added. Samples (3 µl) were taken at different times and analysed by autoradiography. Fizzy-Related translation was verified by western blotting (Interphase Extract + Fzr and Fzr). (D) Interphase extracts (50 µl) were supplemented with Fizzy-Related mRNA as indicated (Interphase Extracts +Fzr). Endogenous Aurora-A and Fizzy degradation were analysed by western blotting. To verify Fizzy-Related translation, Aurora-A and Fizzy-Related were analysed on the same nitrocellulose membrane (Interphase Extract +Fzr, Aurora-A/Fzr).

Fig. 2. The degradation of Aurora-A is mediated by the APC/Fizzy-Related complex and is blocked by the addition of a cyclin B D-Box-motif-containing peptide. (A) An interphase extract (50 µl) was first depleted with either α-CDC27 antibodies (α-CDC27 IP) or non-specific antibodies (Control IP) and then complemented with Fizzy-Related mRNA. A sample of 2 µl was then taken at different times, and endogenous Aurora-A levels were analysed by western blotting. The initial interphase extract (CDC27, Int) and the supernatants of α-CDC27 (CDC27, SN Cdc 27) and non-specific (CDC27, SN Ct) immunoprecipitations were analysed by western blotting with α-CDC27 antibodies to verify CDC27 depletion. (B) Fizzy-Related mRNA was added to interphase extracts (50 µl); 20 min later, we added a purified recombinant cyclin A protein (2 ng/µl, +Cyclin A), the cyclin B D-Box-containing peptide (7 µg/µl, +DBox Peptide) or buffer (CT). Endogenous levels of Aurora-A were then analysed by western blotting at different times.

Fig. 3. Aurora-A targeting by the APC/Fizzy-Related complex requires a D-Box. (A) Schematic drawing of Xenopus Aurora-A depicting the presence of all the putative KEN-Box and D-Box motifs. (B) ³⁵S-radiolabelled KEN-Box mutant of Aurora-A was added to interphase extracts containing Fizzy-Related mRNA. The degradation of this protein was then analysed by autoradiography. (C) Similar to (B), except for the addition of D-Box 1 (ΔDBox1), 2 (ΔDBox2) or 3 (ΔDBox3) mutants instead of KEN-Box mutant. (D) ³⁵S-labelled wild type (WT and WT + Fzr) and D-Box 3 mutant (D-Box + Fzr) were added to an immunopurified APC in the presence (WT + Fzr) or absence (WT) of in vitro translated Fizzy-Related and assayed for ubiquitylation.

Fig. 4. APC/Fizzy-Related induces in vivo degradation of endogenous Aurora-A and Fizzy. Oocytes were microinjected with Fizzy-Related mRNA. Homogenates were prepared from individual oocytes hourly and analysed by coupled western blotting in the same nitrocellulose membrane for endogenous Aurora-A and Fizzy-Related. Fizzy degradation was followed by western blotting on a separate membrane.