Fraschini R, Beretta A, Sironi L, Musacchio A, Lucchini G, Piatti S
Dipartimento di Biotecnologie e Bioscienze, Piazza della Scienza 2, 20126 Milano, Italy.
EMBO J. 2001 Dec 3;20(23):6648-59. doi: 10.1093/emboj/20.23.6648.
The kinetochore checkpoint pathway, involving the Mad1, Mad2, Mad3, Bub1, Bub3 and Mps1 proteins, prevents anaphase entry and mitotic exit by inhibiting the anaphase promoting complex activator Cdc20 in response to monopolar attachment of sister kinetochores to spindle fibres. We show here that Cdc20, which had previously been shown to interact physically with Mad2 and Mad3, associates also with Bub3 and association is up-regulated upon checkpoint activation. Moreover, co-fractionation experiments suggest that Mad2, Mad3 and Bub3 may be concomitantly present in protein complexes with Cdc20. Formation of the Bub3-Cdc20 complex requires all kinetochore checkpoint proteins but, surprisingly, not intact kinetochores. Conversely, point mutations altering the conserved WD40 motifs of Bub3, which might be involved in the formation of a beta-propeller fold devoted to protein-protein interactions, disrupt its association with Mad2, Mad3 and Cdc20, as well as proper checkpoint response. We suggest that Bub3 could serve as a platform for interactions between kinetochore checkpoint proteins, and its association with Mad2, Mad3 and Cdc20 might be instrumental for checkpoint activation.
动粒检查点通路涉及Mad1、Mad2、Mad3、Bub1、Bub3和Mps1蛋白，通过抑制后期促进复合体激活因子Cdc20，防止后期进入和有丝分裂退出，该抑制作用是对姐妹动粒与纺锤体纤维单极附着的响应。我们在此表明，先前已证明与Mad2和Mad3发生物理相互作用的Cdc20，也与Bub3相关联，并且在检查点激活时这种关联上调。此外，共分级分离实验表明，Mad2、Mad3和Bub3可能同时存在于与Cdc20的蛋白复合物中。Bub3-Cdc20复合物的形成需要所有动粒检查点蛋白，但令人惊讶的是，并不需要完整的动粒。相反，改变Bub3保守WD40基序的点突变可能参与形成致力于蛋白质-蛋白质相互作用的β-螺旋桨折叠，破坏其与Mad2、Mad3和Cdc20的关联以及适当的检查点反应。我们认为，Bub3可能作为动粒检查点蛋白之间相互作用的平台，其与Mad2、Mad3和Cdc20的关联可能对检查点激活至关重要。
