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Fig. 1. Destruction of human Aur-A protein in G1-phase extracts of HeLa cells requires the D box and the A box. (A) HeLa cells were synchronized by a double thymidine block, released into nocodazole to collect cells in M phase and subsequently released into G1 phase. Extracts were prepared from M-phase and G1-phase populations, and supplemented with ubiquitin and UbcH10. IVT human cyclin B1 or non-degradable cyclin B1 D1-90 (A) or variants of human Aur-A were then added, with the proteasome inhibitor MG-115 where indicated. Samples were taken at the times and processed for SDS-PAGE followed by autoradiography. (B) Schematic of potential destruction signals and catalytic residues in human Aur-A. The sequence alignment compares the A box in human (h) and Xenopus (x) Aur-A with the corresponding region in Aur-B. Residues conserved with human Aur-A are shaded. The solid bar indicates the conserved QRxL motif. The circled P indicates the serine phosphorylation site (S51 in human Aur-A). (C) IVT mutants of human Aur-A were assayed for destruction in G1-phase HeLa-cell lysates as described in A.
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