The D-Box-activating domain (DAD) is a new proteolysis signal that stimulates the silent D-Box sequence of Aurora-A

Abstract

We have demonstrated previously that Xenopus Aurora-A is degraded at late mitosis by the APC/Fizzy-Related in a D-Box-dependent manner. Here we demonstrate that, although Aurora-B possesses the same D-Box as Aurora-A, Aurora-B is not degraded by this ubiquitin ligase. We have constructed a chimera Aurora-A/B with the N-terminus of Aurora-A and the C-terminus of Aurora-B and we have examined its degradation by APC/Fizzy-Related. We demonstrate that the N-terminus of Aurora-A confers degradation capacity on the C-terminus of Aurora-B and that this feature is blocked by mutation of the conserved D-Box sequence. We characterize the minimal degradation signal at the N-terminus of Aurora-A and demonstrate that its deletion blocks the degradation of this protein by APC/Fizzy-Related. Thus, we conclude that two different degradation signals are required for proteolysis of Aurora-A. The first one, which we designated D-Box-activating domain, within the N-terminal domain of Aurora-A confers the functionality to the second, a silent D-Box, present within the C-terminus of the kinase.

Figure 1

Xenopus Aurora-B is not degraded by the APC/Fizzy-Related complex. (A) CSF extract (20 μl) was supplemented with 2 μl of in vitro translated ³⁵S-labelled Aurora-B and the same volume of ³⁵S-labelled cyclin B. Subsequently, a final concentration of 0.5 mM CaCl₂ was added where indicated (CSF+Ca²⁺). Samples (2 μl) were taken at different times and analysed by autoradiography. (B) Endogenous Aurora-B and cyclin B2 levels were analysed in a similar experiment by western blotting. ^* Represents unspecific bands recognized by the anti-cyclin B2 antibodies in the CSF extracts. (C) Fizzy-Related mRNA was added to interphase extracts (20 μl) as indicated (INT+Fzr). One hour later 2 μl of ³⁵S-labelled Aurora-A or the same amount of ³⁵S-labelled Aurora-B were added. Samples (2 μl) were taken at different times and analysed by autoradiography. Fizzy-Related translation was verified by western blotting (lower panel, Fzr). The arrow at 60 min in the α-Fizzy-Related western blot indicates the time-point at which radiolabelled Aurora-A or B was added to the extract. (D) Interphase extracts (20 μl) were supplemented with Fizzy-Related mRNA and endogenous protein levels of Aurora-A and Aurora-B were analysed by western blotting. (E) Cell cycle expression of Xenopus Aurora-A and Aurora-B. XL2 cells were synchronized at different phases of the cell cycle as described in Uzbekov et al. (1998). Protein levels were then analysed by western blotting with α-Aurora-A and α-Aurora-B antibodies (1/1000), and α-β-tubulin antibodies (1/1500).

Figure 2

The N-terminal domain of Aurora-A confers degradation capacity on the C-terminal domain of Aurora-B. (A) Schematic domain structure of Aurora-A, Aurora-B and Aurora-A/B chimera. (B) An interphase extract (20 μl) was complemented (INT+Fzr) or not (INT-Fzr) with Fizzy-Related mRNA. One hour later both extracts were supplemented with 2 μl of either in vitro translated ³⁵S-labelled A/B chimera or the Dbox mutated form of this chimerical protein. Samples of 2 μl were then taken at different times and the two chimeras were analysed by autoradiography.

Figure 3

Schematic representation of the sequence of Aurora-A/B chimera and the different mutants; N, N-terminus. Where internal sequences were deleted, they are depicted by shaded rectangles. The numbers in brackets indicate the deleted amino acids. All mutants of Aurora-A/B chimera were in vitro translated and incubated in Fizzy-Related-containing extracts to measure protein stability.

Figure 4

The DAD is required for the physiological degradation of Aurora-A. (A) An interphase extract (20 μl) was complemented with Fizzy-Related mRNA. One hour later the extract was supplemented with 2 μl of either in vitro translated ³⁵S-labelled wild type Aurora-A or a mutated form of this kinase in which amino acids 44–55 were deleted. Samples of 2 μl were then taken at different times and the two proteins were analysed by autoradiography. (B) Interphase extracts containing Fizzy-Related were supplemented with all the different radiolabelled punctual mutated forms of Aurora-A resulting from the substitution to alanine of every one of the amino acids from position 44–55. Samples of 2 μl were then taken at 0, 60 and 120 min and Aurora-A protein levels were analysed by autoradiography. (C) Alignment of the DAD of Xenopus (Xl), mouse (Mm), rat (Rn) and human (Hs) sequences of Aurora-A. Amino acids included in the DAD minimal sequence are underlined.