Aurora B inhibits MCAK activity through a phosphoconformational switch that reduces microtubule association.
Aurora B inhibits MCAK activity through a phosphoconformational switch that reduces microtubule association.
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DOI：
10.1016/j.cub.2013.10.054
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发表时间：
2013-12-16
期刊：
影响因子：
9.2
通讯作者：
Walczak, Claire E.
中科院分区：
文献类型：
--
作者：
Ems-McClung, Stephanie C.;Hainline, Sarah G.;Devare, Jenna;Zong, Hailing;Cai, Shang;Carnes, Stephanie K.;Shaw, Sidney L.;Walczak, Claire E.
正确的纺锤体组装和染色体分离依赖于精确的微管动力学，这在一定程度上是由驱动蛋白-13 MCAK控制的。MCAK微管解聚活性受到极光b依赖性磷酸化的抑制，但这种抑制的机制尚不清楚。在这里，我们开发了第一个基于fret的MCAK生物传感器，并表明MCAK在溶液中以c端结构域（CT）和颈部之间相互作用介导的封闭构象存在。利用荧光寿命成像（FLIM），我们发现与微管末端结合的MCAK相对于与微管晶格相关的MCAK是封闭的。颈部S196处的Aurora B磷酸化打开了MCAK构象，减少了CT和颈部之间的相互作用。通过FLIM和TIRF成像，我们发现MCAK构象的变化与MCAK对微管亲和力的降低有关。与运动蛋白不同，运动蛋白在做功时是开放的，而微管解聚运动蛋白的高亲和力结合状态是封闭的。磷酸化会改变MCAK的构象，从而抑制其与微管相互作用的能力，降低其微管解聚活性。这项工作表明，提出的调节激酶活性的构象模型并不普遍，微管解聚激酶利用独特的构象模式来调节对微管的亲和力，从而控制其催化效率。此外，我们的工作提供了一种机制，通过这种机制，可以快速调节Kinesin-13s的强大的微管解聚活性来控制细胞微管动力学。
Proper spindle assembly and chromosome segregation relies on precise microtubule dynamics, which are governed in part by the Kinesin-13 MCAK. MCAK microtubule depolymerization activity is inhibited by Aurora B-dependent phosphorylation, but the mechanism of this inhibition is not understood. Here we develop the first FRET-based biosensor for MCAK and show that MCAK in solution exists in a closed conformation mediated by an interaction between the C-terminal domain (CT) and the neck. Using fluorescence lifetime imaging (FLIM) we show that MCAK bound to microtubule ends is closed relative to MCAK associated with the microtubule lattice. Aurora B phosphorylation at S196 in the neck opens MCAK conformation and diminishes the interaction between the CT and the neck. Using FLIM and TIRF imaging we found that changes in MCAK conformation are associated with a decrease in MCAK affinity for the microtubule. Unlike motile kinesins, which are open when doing work, the high affinity binding state for microtubule depolymerizing kinesins is in a closed conformation. Phosphorylation switches MCAK conformation, which inhibits its ability to interact with microtubules and reduces its microtubule depolymerization activity. This work shows that the conformational model proposed for regulating kinesin activity is not universal and that microtubule depolymerizing kinesins utilize a distinct conformational mode to regulate affinity for the microtubule, thus controlling their catalytic efficiency. Furthermore, our work provides a mechanism by which the robust microtubule depolymerization activity of Kinesin-13s can be rapidly modulated to control cellular microtubule dynamics.
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