Regulation of macromolecular interactions by phosphorylation is crucial in signaling networks. In the spindle assembly checkpoint (SAC), which enables errorless chromosome segregation, phosphorylation promotes recruitment of SAC proteins to tensionless kinetochores. The SAC kinase Mps1 phosphorylates multiple Met-Glu-Leu-Thr (MELT) motifs on the kinetochore subunit Spc105/Knl1. The phosphorylated MELT motifs (MELTP) then promote recruitment of downstream signaling components. How MELTP motifs are recognized is unclear. In this study, we report that Bub3, a 7-bladed β-propeller, is the MELTP reader. It contains an exceptionally well-conserved interface that docks the MELTP sequence on the side of the β-propeller in a previously unknown binding mode. Mutations targeting the Bub3 interface prevent kinetochore recruitment of the SAC kinase Bub1. Crucially, they also cause a checkpoint defect, showing that recognition of phosphorylated targets by Bub3 is required for checkpoint signaling. Our data provide the first detailed mechanistic insight into how phosphorylation promotes recruitment of checkpoint proteins to kinetochores.
The cell cycle is the process by which a cell divides to produce two near-identical daughter cells. Two crucial parts of the cell cycle are the duplication of the chromosomes in the original cell, and the segregation of these chromosomes between the two daughter cells. These and other parts of the cell cycle are strictly regulated to prevent errors, which can lead to cancer and other diseases.
After chromosome duplication has taken place, the pairs of identical chromosomes, known as sister chromatids, remain tightly bound to each other. These sister chromatids line up in the middle of the cell, with protein filaments called microtubules connecting them to a bipolar structure called the spindle. For the cell to divide correctly, the sister chromatids in each pair must be connected to opposite poles of the spindle. A signalling network known as the spindle assembly checkpoint (SAC) ensures that the sister chromatids have enough time to line up correctly and to correct possible problems. Once everything is in place, the SAC releases its ‘break’, and the microtubules then pull the sister chromatids away from each other. This way, each daughter cell receives the same complement of chromosomes that was present in the mother cell.
The microtubules are not directly attached to the sister chromatids but to protein complexes called kinetochores that assemble on each sister chromatid. In particular, each microtubule binds to a very large protein complex called the KMN network. Knl1, which is part of this network, recruits two SAC proteins–Bub1 and Bub3–to the kinetochore. It is known that a phosphate group is added to Knl1 when the SAC is active, and that Knl1 can only recruit Bub1 and Bub3 after it has been phosphorylated. However, the details of the interactions between Knl1, Bub1 and Bub3 are not understood, and it is not clear whether these interactions are essential for the SAC.
Now Primorac et al. have shown that Bub3 binds directly to Knl1 through a region that contains multiple MELT motifs (where M, E, L and T are all amino acids), and that this interaction only happens if these ‘MELT repeats’ have been phosphorylated. Moreover, once bound to the Knl1, Bub3 then recruits Bub1 to the kinetochore. By showing that the recognition of phosphorylated Knl1 by the Bub1-Bub3 complex has a central role in the spindle assembly checkpoint, these results highlight the importance of phosphorylation as a way of regulating the timing of events during the cell cycle.