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，使其在感觉细胞中发挥关键作用。肌球蛋白IIIA基因的破坏会导致脊椎动物耳聋，以及无脊椎动物的视网膜退化和光传导缺陷。肌球蛋白III是一种以肌动蛋白为基础的分子马达，它包含肌球蛋白超家族特有的保守运动域，但也包含一个N-末端的激酶域。我们认为，激酶活性是通过激活域激活环的磷酸化来激活的，这会刺激运动域中环2的自动磷酸化，从而导致运动活性的下调。磷酸酶活性使马达返回到其活动状态，并使激活域失活。我们将研究肌球蛋白IIIA结构域激活/失活以及运动域激活/失活的酶学、运动性和结构特性。肌球蛋白IIIA与肌球蛋白IIIB的不同之处在于它的C末端含有一个肌动蛋白结合基序。我们认为，尾巴上额外的肌动蛋白结合位点允许肌球蛋白IIIA使肌动蛋白细丝交联，协同激活运动酶循环，并通过增加肌球蛋白IIIA对肌动蛋白的整体亲和力来增强其运动特性。我们将通过研究全长肌球蛋白IIIB和肌球蛋白IIIA的酶活性、运动性和交联性来直接确定尾部肌动蛋白结合基序的作用，无论是否有功能的尾部肌动蛋白结合基序。我们还将研究尾肌动蛋白结合基序与肌动蛋白细丝相互作用的结构基础。肌球蛋白IIIA沿肌动蛋白束的运动将用单分子运动性分析来检测。这些具有生物化学特征的构建体将在培养的HeLa/COS细胞、光感受器和内耳毛细胞中进行检测，以确定肌球蛋白III的活性如何介导其细胞定位、肌动蛋白动力学和体内运动。总体而言，在体外和细胞生物学实验中研究肌球蛋白IIIA和IIIB的酶、运动和调节特性将确定其在感觉细胞中的生理作用。此外，这些研究很好地为了解肌球蛋白IIIA基因的破坏如何导致耳聋和视网膜退化提供了基础。

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 critical role in sensory cells. Disruption of the myosin IIIA gene results in deafness in vertebrates as well as retinal degeneneration and phototransduction defects in invertebrates. Myosin III is an actin-based molecular motor that contains a conserved motor domain characteristic of the myosin superfamily, but also contains an N-terminal kinase domain. We propose that kinase activity is activated by phosphorylation of the kinase domain activation loop which stimulates autophosphorylation of loop 2 in the motor domain and results in downregulation of motor activity. Phosphatase activity returns the motor to its active state and inactivates the kinase domain. We will examine the enzymatic, motile, and structural properties of myosin IIIA constructs with the kinase domain activated/inactivated, as well motor domain activated/inactivated. Myosin IIIA is different from myosin IIIB in that it contains an actin-binding motif in its C-terminal tail. We propose that the additional actin binding site in the tail allows myosin IIIA to cross-link actin filaments, cooperatively activate the motor enzymatic cycle, and enhances its motile properties by increasing the overall affinity of myosin IIIA for actin. We will directly determine the role of the tail actin binding motif by examining the enzymatic, motile, and cross-linking properties of full length myosin IIIB and myosin IIIA, with and without a functional tail actin binding motif. We will also examine the structural basis for the interaction of the tail actin binding motif with actin filaments. The movement of myosin IIIA along actin bundles will be examined with single molecule motility assays. The biochemically characterized constructs will be examined in cultured HeLa/COS cells, photoreceptors, and inner ear hair cells to determine how the activity of myosin III mediates its cellular localization, actin dynamics, and in vivo motility. Overall, studying the enzymatic, motor, and regulatory properties of myosin IIIA and IIIB in vitro and in cell biology experiments will identify its physiological role in sensory cells. In addition, these studies well provide the basis for understanding how disruption of the myosin IIIA gene leads to deafness and retinal degeneration.