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First published online 9 November 2004
doi: 10.1242/jcs.01418
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Research Article |
Department of Cell Biology, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115, USA
* Author for correspondence (e-mail: ruderman{at}hms.harvard.edu)
Accepted 26 July 2004
The mitotic kinase Aurora A (Aur-A) is overexpressed in a high proportion of human tumors, often in the absence of gene amplification. In somatic cells, Aur-A protein levels fall following mitosis or upon overexpression of Cdh1, an activator of the ubiquitin ligase APC/C. Thus, mutations that reduce or block the rate of Aur-A destruction might also be expected to contribute to its oncogenic potential. Previous work had defined two short sequences of Xenopus Aur-A that are required for its Cdh1-inducible destruction in extracts of Xenopus eggs, an N-terminal A box and a C-terminal D box, and a serine residue within the A box whose phosphorylation might inhibit destruction. Here, we show that these same sequences are required for the destruction of human Aur-A during mitotic exit and G1 in the somatic cell cycle. Expression of a dominant negative Cdh1 protein leads to accumulation of Aur-A, further indicating that the Cdh1-activated form of the APC/C is responsible for destruction of Aur-A during the somatic cell cycle in vivo. During the course of this work, we found some previously unsuspected problems in commonly used in vitro destruction assays, which can result in misleading results. Potentially confounding factors include: (i) the presence of D-box- and A-box-dependent destruction-promoting activities in the reticulocyte in vitro translation mix that is used to produce radiolabeled substrates for destruction assays; and (ii) the ability of green-fluorescent-protein tags to reduce the destruction rate of Aur-A substantially. These findings have direct relevance for studies of Aur-A destruction itself, and for broader approaches that use in vitro translation products in screens for additional APC/C targets.
Key words: Aurora kinases, Mitosis, Xenopus, Cdh1, A box, Ubiquitin-dependent proteolysis, Cell cycle
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