FIGURE 6.

Human Mps1 contains a single destruction D-box that confers instability. A, D-box sequence (RNSLRQTN) located at the N terminus of human Mps1 is conserved in most mammalian species. Conserved amino acids are indicated in black bars. B, schematic representation of full-length human Mps1 wild type denoting its putative D-box (RNSL) versus a Mps1 D-box mutant (ANSA) created by site-directed mutagenesis. C, mutation of the D-box confers resistance to Mps1 degradation at anaphase in human 293T cells. GFP-Mps1 (WT) and (D-box mutant) DNA constructs were transiently transfected into 293T cells and arrested at prometaphase with nocodazole (40 ng/ml) treatment for 16 h. Cells were synchronously released into anaphase by washing twice in fresh media minus nocodazole. At the indicated times, cells were collected for Western analysis of recombinant Mps1 levels using a GFP antibody. Cyclin B degradation was monitored as a marker for anaphase. Actin was used as a loading control. Mitotic synchrony by nocodazole and exit into G₁ phase were monitored in parallel by flow cytometry. Results shown are representative of three independent experiments.