Klotzbucher A et al. (2002), A Method for Analyzing the Ubiquitination and D...

XB-ART-5334

Biol Proced Online 2002 Nov 11;4:62-69. doi: 10.1251/bpo35.

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A Method for Analyzing the Ubiquitination and Degradation of Aurora-A.

Klotzbucher A , Pascreau G , Prigent C , Arlot-Bonnemains Y .

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The cell cycle machinery consists of regulatory proteins that control the progression through the cell cycle ensuring that DNA replication alternates with DNA segregation in mitosis to maintain cell integrity. Some of these key regulators have to be degraded at each cell cycle to prevent cellular dysfunction. Mitotic exit requires the inactivation of cyclin dependent kinase1 (cdk1) and it is the degradation of the cyclin subunit that inactivates the kinase. Cyclin degradation has been well characterized and it was shown that it is ubiquitin proteasome pathway that leads to the elimination of cyclins. By now, many other regulatory proteins were shown to be degraded by the same pathway, among them members of the aurora kinase family, degraded many other regulatory proteins. Aurora kinases are involved in mitotic spindle formation as well as in cytokinesis. The abundance and activity of the kinase is precisely regulated during the cell cycle. To understand how proteolysis regulates transitions through the cell cycle we describe two assays for ubiquitination and degradation of xenopus aurora kinase A using extracts from xenopus eggs or somatic cell lines.

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Species referenced: Xenopus
Genes referenced: aurka ccnb1 ccnb2

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	Fig. 1. Cell cycle-dependent expression of aurora A protein. XL2 cells were blocked at various stages of the cell cycle by serum starvation or treatment with the different drugs as described in material and methods. After release from the block, cells were harvested at different stages of the cell cycle. The cell lysates prepared from 5x105 cells were separated by SDS PAGE and analyzed by Western blot with XlAurora A antibody and anti γ tubulin as a control. The appropriate portion of a blot (according to the molecular weight of proteins to be analyzed) was separately probed with anti X1Aurora-A (dilution: 1/500) and anti γ tubulin (dilution: 1/200) for the internal control of quantity of protein (Fig. 1A). The cell cycle distribution was analyzed by BrdU analysis. Percentage of cells in different phases of the cell cycle was calculated (Fig. 1B). The membrane has been scanned on a fluoroimager (Molecular Dynamics) and quantification of the protein has been realized with Image Quant.
	Fig. 2. Diagram to compare the amino acid sequences of the destruction boxes and flanking sequences of cyclin B1, cyclin B2 and aurora-A. The destruction boxes are marked by a rectangle and the conserved amino acids are indicated in bold.
	Fig. 3. Ubiquitination of p Aurora A in a xenopus cell extract. In vitro-translated pAurora A was incubated in Xenopus CSF arrested eggs extracts (35 µg of protein) in the presence of 1µM ubiquitin aldehyde and 1mg/ml of methylated ubiquitin (Lane 1). Lane 2: ubiquitin aldehyde and methylated ubiquitin were omitted from the reaction. Lane 3: without in vitro translation of pAurora-A. Lane 4: without CSF extract the reactions were stopped, analyzed on SDS-PAGE and transferred onto nitrocellulose membrane. Western blot analysis was performed with the monoclonal antibody ICI (dilution 1:500) in order to detect the pAurora-A-ubiquitin conjugates (Published in FEBS Lett, 508: 149-152, 2001 and used with permission).
	Fig. 4. In vitro degradation assay of the ubiquitinated protein pAuroraA. A specific amount of the recombinant protein (Lane 1) was analyzed on a 12.5% polyacrylamide gel and compared with the endogenous protein from a G1 cell extract (Lane 2). Western blot analysis was performed with the monoclonal antibody 1C1 (dilution 1:500) in order to detect the recombinant pAurora-A and the endogenous protein.Fig. 4B: XL2 cells were arrested in G1 phase by serum starvation. Cell extracts were prepared with lysis buffer complemented with an ATP regenerating system and incubated in the presence of pAurora A wild type or pAurora A mdb (mutated destruction box). Samples were analysed on a 12.5% SDS-PAGE followed by a western blot performed with the 1C1 monoclonal antibody. Degradation kinetics of the various recombinant aurora-A proteins containing potential destruction box deletions were performed in XL2 cell extracts prepared from G1 cells. The reaction was incubated at 30°C. The specificity of the degradation was controlled by addition of the APC/C inhibitor ALLN. Reaction products were separated by 12.5% SDS-polyacrylamide gel electrophoresis and analysed by western blot using the 1C1 monoclonal antibody (1:200). Aliquots of the reaction were taken at 0, 15, 30 and 120 minutes. Western blots were quantified with Imag Quant and the amount of protein detected at various time points was expressed as a percentage of the protein detected at t = 0 min (input).

References [+] :

Arlot-Bonnemains, Identification of a functional destruction box in the Xenopus laevis aurora-A kinase pEg2. 2001, Pubmed, Xenbase