The Mechanism of Kinesin Self-Regulation

描述(申请人提供)：截断的动蛋白二聚体水解三磷酸腺苷并沿微管单向移动的机制是众所周知的。目前尚不清楚具有两条重链和两条轻链的全动蛋白四聚体是如何被调节和激活以进行货物运输的。在这项工作中，我们将检验这一假设，即当尾巴直接与头部结合时，Kinesin受到调控，以防止ADP释放或微管结合。激动素可能会受到轻链和微管之间电荷碰撞的进一步调节，当磷酸化的轻链与头部的尾巴竞争时，动蛋白可能会重新激活。我们将在四个具体目标上检验这些假设。前两个目的是利用全长Kinesin重链进行调节，后两个目的是探索轻链在调节和激活中的作用。在目标1中，我们将确定尾巴是否直接结合在微管结合部位或与头部的核苷酸传感元件结合，或者它是否变构影响头部的核苷酸或微管结合区。在目标1的结果指导下，目标2的实验将确定头部的哪个区域绑定尾巴，并将识别特定的头尾相互作用。在体内和体外进行的实验表明，轻链可能在调节Kinesin方面具有重要作用，这将在Aim#3中进行评估。最后，我们将在Aim#4中确定Kinesin轻链的磷酸化是否可以直接激活Kinesin。总之，这些实验将扩大我们对支配Kinesin活性的相互作用和构象变化的理解。此外，在这项工作中发现的调控相互作用可能揭示了几种动蛋白相似的抑制机制。这可能会导致更快地发现专门针对肌动蛋白的药物。

DESCRIPTION (provided by applicant): The mechanism by which truncated kinesin dimers hydrolyze ATP and move unidirectionally along microtubules is well understood. It is far less clear how the full kinesin heterotetramer, which has two heavy chains and two light chains, is regulated and activated for cargo transport. In this work, we will test the hypothesis that kinesin is regulated when the tails directly bind the heads to prevent ADP release or microtubule binding. Kinesin may be further regulated by a charge clash between its light chains and microtubules, and kinesin may be re-activated when phosphorylated light chains compete the tails away from the heads. We will test these hypotheses in four Specific Aims. The first two Aims address regulation using the full-length kinesin heavy chain, and the second two Aims explore the role of the light chains in regulation and activation. In Aim #1, we will determine whether the tail binds directly in the microtuble-binding site or to the nucleotide-sensing elements in the head, or whether it allosterically affects the nucleotide- or microtubule-binding regions of the head. The experiments of Aim #2, guided by the results of Aim #1, will determine what region of the head binds the tail and will identify specific head-tail interactions. Experiments performed both in vivo and in vitro indicate that the light chains may have a significant role in regulating kinesin, which will be assessed in Aim #3. Lastly, we will determine whether phosphorylation of kinesin light chains can directly activate kinesin in Aim #4. Together, these experiments will extend our understanding of the interactions and conformational changes that govern kinesin activity. Furthermore, the regulatory interactions that are found in this work may reveal inhibitory mechanisms that are similar in several kinesins. This may lead to quicker discovery of drugs that specifically target kinesins.