Enzymatic and Motor Properties of Myosin III

描述（由申请人提供）：本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基因的破坏导致脊椎动物的耳聋以及无脊椎动物的视网膜变性和光转导缺陷。肌凝蛋白III是一种基于肌动蛋白的分子马达，包含肌凝蛋白超家族的保守马达结构域，但也包含n端激酶结构域。我们认为激酶活性是由激酶结构域激活环的磷酸化激活的，磷酸化刺激运动结构域环2的自磷酸化，从而导致运动活性的下调。磷酸酶活性使马达恢复其活性状态并使激酶结构域失活。我们将研究激酶结构域激活/失活以及运动结构域激活/失活的肌球蛋白IIIA结构体的酶学、运动性和结构特性。Myosin IIIA与Myosin IIIB的不同之处在于，它的c端尾部含有一个肌动蛋白结合基序。我们提出，尾部额外的肌动蛋白结合位点允许肌动蛋白IIIA交联肌动蛋白丝，协同激活运动酶循环，并通过增加肌动蛋白IIIA对肌动蛋白的整体亲和力来增强其运动特性。我们将通过检测全长myosin IIIB和myosin IIIA的酶促、运动和交联特性，直接确定尾部肌动蛋白结合基序的作用，无论是否具有功能性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丝相互作用的结构基础。肌凝蛋白IIIA沿着肌动蛋白束的运动将通过单分子运动测定来检测。我们将在培养的HeLa/COS细胞、光感受器和内耳毛细胞中检测这些生物化学特征结构，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性。总之，在体外和细胞生物学实验中研究肌球蛋白IIIA和IIIB的酶促、运动和调节特性将确定其在感觉细胞中的生理作用。此外，这些研究为理解肌球蛋白IIIA基因的破坏如何导致耳聋和视网膜变性提供了基础。

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.