Trieselmann Nadia, Armstrong Sheri, Rauw Jennifer, Wilde Andrew
Department of Medical Genetics and Microbiology, University of Toronto, 1 King's College Circle, Toronto, Ontario, Canada, M5S 1A8.
J Cell Sci. 2003 Dec 1;116(Pt 23):4791-8. doi: 10.1242/jcs.00798.
Ran, a GTPase in the Ras superfamily, is proposed to be a spatial regulator of microtubule spindle assembly by maintaining key spindle assembly factors in an active state close to chromatin. RanGTP is hypothesized to maintain the spindle assembly factors in the active state by binding to importin beta, part of the nuclear transport receptor complex, thereby preventing the inhibitory binding of the nuclear transport receptors to spindle assembly factors. To directly test this hypothesis, two putative downstream targets of the Ran spindle assembly pathway, TPX2, a protein required for correct spindle assembly and Kid, a chromokinesin involved in chromosome arm orientation on the spindle, were analyzed to determine if their direct binding to nuclear transport receptors inhibited their function. In the amino-terminal domain of TPX2 we identified nuclear targeting information, microtubule-binding and Aurora A binding activities. Nuclear transport receptor binding to TPX2 inhibited Aurora A binding activity but not the microtubule-binding activity of TPX2. Inhibition of the interaction between TPX2 and Aurora A prevented Aurora A activation and recruitment to microtubules. In addition we identified nuclear targeting information in both the amino-terminal microtubule-binding domain and the carboxy-terminal DNA binding domain of Kid. However, the binding of nuclear transport receptors to Kid only inhibited the microtubule-binding activity of Kid. Therefore, by regulating a subset of TPX2 and Kid activities, Ran modulates at least two processes involved in spindle assembly.
Ran是Ras超家族中的一种GTP酶，它被认为是微管纺锤体组装的空间调节因子，通过使关键的纺锤体组装因子保持在靠近染色质的活跃状态来实现这一功能。据推测，RanGTP通过与核转运受体复合物的一部分importin beta结合，使纺锤体组装因子维持在活跃状态，从而防止核转运受体与纺锤体组装因子的抑制性结合。为了直接验证这一假设，对Ran纺锤体组装途径的两个假定下游靶点进行了分析，这两个靶点分别是TPX2（正确纺锤体组装所需的一种蛋白质）和Kid（一种参与染色体臂在纺锤体上定向的染色体驱动蛋白），以确定它们与核转运受体的直接结合是否会抑制其功能。在TPX2的氨基末端结构域中，我们鉴定出了核靶向信息、微管结合活性和极光激酶A结合活性。核转运受体与TPX2的结合抑制了极光激酶A的结合活性，但没有抑制TPX2的微管结合活性。TPX2与极光激酶A之间相互作用的抑制阻止了极光激酶A的激活和向微管的募集。此外，我们在Kid的氨基末端微管结合结构域和羧基末端DNA结合结构域中都鉴定出了核靶向信息。然而，核转运受体与Kid的结合仅抑制了Kid的微管结合活性。因此，通过调节TPX2和Kid活性的一个子集，Ran至少调节了纺锤体组装过程中的两个过程。
