Human Aurora2 was originally identi®ed by its close homology to yeast IPL1 and¯y aurora, which are key regulators of chromosome segregation and a family of serine/threonine kinases. Here we demonstrate that the Aurora2 protein is degraded rapidly after G2/M phase release in mammalian cells. Aurora2 protein has a rapid turnover rate with a half-life of approximately 2 h. In eukaryotic cells, the ubiquitin-proteasome pathway is the major mechanism for the targeted degradation of unstable proteins. The treatment of mammalian cells with proteasome inhibitors blocks Aurora2 degradation. Furthermore, Aurora2 is polyubiquitinated in vivo and in vitro using anaphase-promoting complex (APC). These results demonstrate that Aurora2 protein is turned over through the APC-ubiquitin-proteasome pathway.
To accomplish the selective labeling of a specific protein in complicated biological systems, a peptide tag incorporated into the protein and a complementary small molecular probe are required. Although a variety of peptide tag/probe pairs have been developed as molecular tools for protein analyses, the availability of pairs suitable for real-time imaging of proteins is still limited. We now report a new peptide tag/artificial probe pair composed of a genetically encodable oligo-aspartate sequence (D4 tag, (D4)n, n = 1-3) and the corresponding multinuclear Zn(II) complexes (Zn(II)-DpaTyrs). The strong binding affinity of the Zn(II)-DpaTyr probes with the D4 tag was a result of the multiple coordination bonds and the multivalent effect. It was measured quantitatively by isothermal titration calorimetry. The high affinity between the tag and the probe, indispensable for the selective protein labeling, enabled the pair to be used for the labeling and fluorescence imaging of a membrane-bound receptor protein tethering a triply repeated D4 tag ((D4)3) in an intact cell configuration without significantly affecting the receptor signal transduction.
The human serine/threonine kinase Aurora-B is structurally related to the protein kinase Ipl1p from S cerevisiae and aurora from Drosophila melanogaster, which are key regulators of mitosis. The present study shows that human Aurora-B is activated by okadaic acid and forms complexes with the protein serine/threonine phosphatase type 1 (PP1) or PP2A, but not with PP5. These data identi®ed Aurora-B associated protein phosphatases as negative regulators of kinase activation. We then used a series of substrates based on a histone H3 phosphorylation site (residues 5 ± 15) to determine the substrate speci®city of human Aurora-B. We found that this enzyme is an arginine-directed kinase that can phosphorylate histone H3 at serines 10 and 28 in vitro, suggesting that human Aurora-B is a mitotic histone H3 kinase.
Human Aurora-A is related to a protein kinase originally identified by its close homology to Ipl1p from Saccharomyces cerevisiae and aurora from Drosophila melanogaster, which are key regulators of the structure and function of the mitotic spindle. We previously showed that human Aurora-A is turned over through the anaphase promoting complex/ cyclosome (APC/C)^ubiquitin^proteasome pathway. The association of two distinct WD40 repeat proteins known as Cdc20 and Cdh1, respectively, sequentially activates the APC/C. The present study shows that Aurora-A degradation is dependent on hCdh1 in vivo, not on hCdc20, and that Aurora-A is targeted for proteolysis through distinct structural features of the destruction box, the KEN box motifs and its kinase activity. ß
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