Lee S E, Jensen S, Frenz L M, Johnson A L, Fesquet D, Johnston L H
Division of Yeast Genetics, National Institute for Medical Research, The Ridgeway, Mill Hill, London, NW7 1AA, UK.
J Cell Sci. 2001 Jun;114(Pt 12):2345-54. doi: 10.1242/jcs.114.12.2345.
In eukaryotes an abnormal spindle activates a conserved checkpoint consisting of the MAD and BUB genes that results in mitotic arrest at metaphase. Recently, we and others identified a novel Bub2-dependent branch to this checkpoint that blocks mitotic exit. This cell-cycle arrest depends upon inhibition of the G-protein Tem1 that appears to be regulated by Bfa1/Bub2, a two-component GTPase-activating protein, and the exchange factor Lte1. Here, we find that Bub2 and Bfa1 physically associate across the entire cell cycle and bind to Tem1 during mitosis and early G1. Bfa1 is multiply phosphorylated in a cell-cycle-dependent manner with the major phosphorylation occurring in mitosis. This Bfa1 phosphorylation is Bub2-dependent. Cdc5, but not Cdc15 or Dbf2, partly controls the phosphorylation of Bfa1 and also Lte1. Following spindle checkpoint activation, the cell cycle phosphorylation of Bfa1 and Lte1 is protracted and some species are accentuated. Thus, the Bub2-dependent pathway is active every cell cycle and the effect of spindle damage is simply to protract its normal function. Indeed, function of the Bub2 pathway is also prolonged during metaphase arrests imposed by means other than checkpoint activation. In metaphase cells Bub2 is crucial to restrain downstream events such as actin ring formation, emphasising the importance of the Bub2 pathway in the regulation of cytokinesis. Our data is consistent with Bub2/Bfa1 being a rate-limiting negative regulator of downstream events during metaphase.
在真核生物中,异常纺锤体激活一个由MAD和BUB基因组成的保守检查点,导致有丝分裂在中期停滞。最近,我们和其他人发现了该检查点一个新的依赖Bub2的分支,它会阻止有丝分裂退出。这种细胞周期停滞依赖于对G蛋白Tem1的抑制,而Tem1似乎受双组分GTP酶激活蛋白Bfa1/Bub2和交换因子Lte1的调控。在这里,我们发现Bub2和Bfa1在整个细胞周期中都存在物理关联,并在有丝分裂和G1早期与Tem1结合。Bfa1以细胞周期依赖性方式发生多重磷酸化,主要磷酸化发生在有丝分裂期。这种Bfa1磷酸化依赖于Bub2。Cdc5部分控制Bfa1以及Lte1的磷酸化,但Cdc15或Dbf2则不然。纺锤体检查点激活后,Bfa1和Lte1的细胞周期磷酸化会延长,并且某些磷酸化形式会增强。因此,依赖Bub2的途径在每个细胞周期都处于活跃状态,纺锤体损伤的作用仅仅是延长其正常功能。实际上,在通过检查点激活以外的其他方式导致的中期停滞期间,Bub2途径的功能也会延长。在中期细胞中,Bub2对于抑制诸如肌动蛋白环形成等下游事件至关重要,这强调了Bub2途径在胞质分裂调控中的重要性。我们的数据与Bub2/Bfa1作为中期下游事件的限速负调节因子一致。