Enzymatic and motor properties of myosin III

描述(申请人提供)：该研究项目的长期目标是了解肌球蛋白III的酶和运动特性，使其在光感受器的光传导过程中发挥直接作用。肌球蛋白III是一种以肌动蛋白为基础的分子马达，含有肌球蛋白超家族特有的保守运动结构域，但在其C末端尾部还含有一个N-末端的激酶结构域和一个额外的肌动蛋白结合基序。我们认为，肌球蛋白结构域上自动磷酸化位置的能力降低了肌球蛋白III的酶和运动特性，但当肌球蛋白结构域失活或与另一底物结合时，肌球蛋白III马达被激活。我们将研究肌球蛋白III结构的酶学和运动学性质，其中肌球蛋白III结构域被删除或失活，以及在有细胞底物存在的情况下。肌球蛋白III的激酶调节可能提供了一种机制，通过这种机制，肌球蛋白III可以被激活/抑制以响应特定的细胞信号。我们认为，尾巴上额外的肌动蛋白结合位点允许肌球蛋白III使肌动蛋白细丝交联，协同激活肌球蛋白III的酶循环，并通过增加肌球蛋白对肌动蛋白的整体亲和力来增强肌球蛋白III的运动特性。我们将直接检测尾部肌动蛋白结合基序的肌动蛋白结合亲和力，并确定它在调节肌球蛋白III的酶和运动特性中所起的作用。因此，额外的肌球蛋白结合位点可能允许肌球蛋白III定位并帮助稳定肌动蛋白细丝网络，这是组装参与光传导信号复合体的蛋白质所必需的。总之，研究肌球蛋白III的酶、运动和调节特性可能揭示光转导信号复合体如何在光感受器中形成和稳定的潜在机制。

DESCRIPTION (provided by applicant): The long-term goal of this research project is to understand the enzymatic and motor properties of myosin III that allow it to play a direct role in the phototransduction process in photoreceptors. Myosin III is an actinbased molecular motor that contains a conserved motor domain characteristic of the myosin superfamily, but also contains an N-terminal kinase domain as well as an additional actin-binding motif in its C-terminal tail. We propose that the ability of the kinase domain to autophosphorylate sites on the myosin domain reduces the enzymatic and motile properties of myosin III, but when the kinase domain is inactivated or bound to another substrate the myosin III motor is activated. We will examine the enzymatic and motile properties of myosin III constructs with the kinase domain deleted or inactivated, as well as in the presence of cellular substrates for the kinase domain. The kinase regulation of myosin III may provide a mechanism whereby myosin III can be activated/inhibited in response to specific cellular signals. We propose that the additional actin binding site in the tail allows myosin III to cross-link actin filaments, cooperatively activate the myosin III enzymatic cycle, and enhance the motile properties of myosin III by increasing the overall affinity of myosin for actin. We will directly examine the actin binding affinity of the tail actin-binding motif, and determine the role that it plays in mediating the enzymatic and motile properties of myosin III. Thus, the additional actin-binding site may allow myosin III localize to and help stabilize actin filament networks, which are necessary for assembling the proteins involved in the phototransduction signaling complex. Overall, studying the enzymatic, motor, and regulatory properties of myosin III may reveal potential mechanisms of how the formation and stabilization of the phototransduction signaling complex occurs in photoreceptors.