Identification of a new APC/C recognition domain, the A box, which is required for the Cdh1-dependent destruction of the kinase Aurora-A during mitotic exit.
The mitotic kinase Aurora A (Aur-A) is required for formation of a bipolar mitotic spindle and accurate chromosome segregation. In somatic cells, Aur-A protein and kinase activity levels peak during mitosis, and Aur-A is degraded during mitotic exit. Here, we investigated how Aur-A protein and kinase activity levels are regulated, taking advantage of the rapid synchronous cell division cycles of Xenopus eggs and cell-free systems derived from them. Aur-A kinase activity oscillates in the early embryonic cell cycles, just as in somatic cells, but Aur-A protein levels are constant, indicating that regulated activation and inactivation, instead of periodic proteolysis, is the dominant mode of Aur-A regulation in these cell cycles. Cdh1, the APC/C activator that targets many mitotic proteins for ubiquitin-dependent proteolysis during late mitosis and G1 in somatic cells, is missing in Xenopus eggs and early embryos. We find that addition of Cdh1 to egg extracts undergoing M phase exit is sufficient to induce rapid degradation of Aur-A. Aur-A contains both of the two known APC/C recognition signals, (1) a C-terminal D box similar to those required for ubiquitin-dependent destruction of cyclin B and several other mitotic proteins, and (2) an N-terminal KEN box similar to that found on cdc20, which is ubiquitinated in response to APC/C(Cdh1). The D box is required for Cdh1-induced destruction of Aur-A but the KEN box is not. Destruction also requires a short region in the N terminus, which contains a newly identified recognition signal, the A box. The A box is conserved in vertebrate Aur-As and contains serine 53, which is phosphorylated during M phase. Mutation of serine 53 to aspartic acid, which can mimic the effect of phosphorylation, completely blocks Cdh1-dependent destruction of Aur-A. These results suggest that dephosphorylation of serine 53 during mitotic exit could control the timing of Aur-A destruction, allowing recognition of both the A box and D box by Cdh1-activated APC/C.
Littlepage
Laurie E
LE
Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115, USA.
Ruderman
Joan V
JV
D016475:3T3 Cells; D000595:Amino Acid Sequence; D064173:Anaphase-Promoting Complex-Cyclosome; D000818:Animals; D064096:Aurora Kinase A; D064127:Aurora Kinases; D001665:Binding Sites; D018797:Cell Cycle Proteins; D017124:Conserved Sequence; D005260:Female; D005306:Fertilization / Q000502:physiology; D066298:In Vitro Techniques; D008025:Ligases / Q000378:metabolism*; D008297:Male; D051379:Mice; D008938:Mitosis / Q000502:physiology*; D008969:Molecular Sequence Data; D009154:Mutation; D009865:Oocytes / Q000166:cytology / Q000378:metabolism; D011494:Protein Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases; D017434:Protein Structure, Tertiary; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D043743:Ubiquitin-Protein Ligase Complexes; D014981:Xenopus; D029867:Xenopus Proteins
D018797:Cell Cycle Proteins; D029867:Xenopus Proteins; D043743:Ubiquitin-Protein Ligase Complexes; D064173:Anaphase-Promoting Complex-Cyclosome; D011494:Protein Kinases; C112134:AURKA protein, Xenopus; C576918:Aurka protein, mouse; D064096:Aurora Kinase A; D064127:Aurora Kinases; D017346:Protein Serine-Threonine Kinases; D008025:Ligases