BIOCHEMISTRY AND BIOLOGY OF A MYOSIN BINDING PROTEIN

非肌肉细胞调节重要动作的方式 肌球蛋白介导的形状变化负责细胞分裂， 伪足延伸、内吞作用和许多其他形式的 运动还没有完全理解。 大量的知识 存在着关于各种肌动蛋白结合的生物化学和生物学 调节细胞肌动蛋白装配的蛋白质。 另 一方面，虽然对肌球蛋白的生物化学有很多了解， 通过磷酸化调节，关于细胞如何 改变肌球蛋白在细胞内的组装状态和定位 运动 肌细胞有许多肌球蛋白相关的 包括轻链和重链激酶的蛋白质，其介导 肌球蛋白组装，以及作为部分的星云蛋白、肌联蛋白和C蛋白 的粗灯丝。 然而，人们对非肌肉组织知之甚少， 细胞肌球蛋白相关蛋白的生物化学以外， 激酶。 有趣的是，肌球蛋白表现出非常不寻常的低离子 在高速细胞提取物中的强度溶解度，条件 当它纯净时，它会组装成双极细丝， 与肌动蛋白及其相关蛋白质结合，并在肌球蛋白中收缩， 依赖的方式。 我们鉴定了一个新的53 kD蛋白质 亚基分子量为海胆卵提取物， 直接介导这种通常的低离子强度溶解度误差 以肌球蛋白的低离子强度影响肌球蛋白的溶解度 直接作用于肌球蛋白以影响肌球蛋白的低离子强度溶解度。 我们也有初步证据表明53 kD分子与头部结合- 肌球蛋白的杆状连接，并表现出钙调素非依赖性 肌球蛋白轻链激酶活性。 在本提案中，我们打算 进一步表征这种蛋白质在其方面： 其他轻链激酶，功能结构域，精确结合位点 对肌球蛋白的影响，磷酸化调节，对肌球蛋白的影响 ATP酶和装配，以及普遍性。 一系列生物学 将通过使用中询问关于53 kD功能的问题 体外肌球蛋白介导的运动试验，通过研究 肌球蛋白被53 kD结合或磷酸化对运动性的影响。 53 kD在体内的作用将通过分析效应来研究 显微注射53 kD的抑制性单克隆抗体 对受精海胆卵卵裂的活性和通过定位 53 kD在整个细胞周期中，在这个和其他细胞。 这些 对一种新蛋白质的研究应该有助于弥合我们之间的差距， 肌球蛋白活性调节的分子知识， 细胞控制肌球蛋白的动态组装和定位， 运动

The way in which non-muscle cells regulate the important acto- myosin mediated shape changes responsible for cell division, pseudopod extension, endocytosis, and many other forms of movement are not fully understood. A great deal of knowledge exists about the biochemistry and biology of various actin binding proteins which regulate cellular actin assemblies. On the other hand, while a good deal is known about the biochemistry of myosin regulation by phosphorylation, little is known about how the cell changes the assembly state and localization of myosin during cell movement. Muscle cells have a number of myosin-associated proteins including light and heavy chain kinases, which mediate myosin assembly, and nebulin, titin, and C-protein which are part of the thick filament. However, little is known about non-muscle cell myosin-associated proteins other than the biochemistry of the kinases. Interestingly, myosin exhibits very unusual low ionic strength solubility in high speed cellular extracts, conditions where when pure it assembles into bipolar filament, which can gel with actin and its associated proteins and contract in a myosin- dependent manner. We have identified a new protein of 53 kD subunit molecular weight is sea urchin egg extracts which mediates this ususual low ionic strength solubility error directly to myosin to affect myosin low ionic strength solubility by binding directly to myosin to affect myosin low ionic strength solubility. We also have preliminary evidence that 53 kD binds to the head- rod junction of myosin and exhibits calmodulin-independent myosin light chain kinase activity. In this proposal we intend to further characterize this protein in terms of its: relatedness to other light chain kinases, functional domains, exact binding site on myosin, regulation by phosphorylation, effects on myosin ATPase and assembly, and ubiquity. A series of biological questions will be asked about 53 kD function through the use of in vitro myosin-mediated motility assays by studying the effects of myosin being bound by, or phosphorylated by, 53 kD on motility. The role of 53 kD in vivo will be studied by analyzing the effects of microinjected inhibitory monoclonal antibodies to 53 kD activity on cleavage of fertilized sea urchin eggs and by localizing 53 kD throughout the cell cycle in this and in other cells. These studies on a novel protein should help bridge the gap between our molecular knowledge of the regulation of myosin activity and how the cell controls myosin's dynamic assembly and localization for movement.