NUCLEAR MYOSIN I

我们之前已经证明了细胞核中存在一种肌球蛋白I样蛋白。这种蛋白的表观分子量为120,000，它与K+-EDTA ATPase活性有关，它与钙调蛋白结合，它是用ATP标记的光亲和性，它在没有ATP的情况下与肌动蛋白结合，它能被从肾上腺提纯的肌球蛋白I的抗体识别。对120 kDa蛋白质的显微测序证实它是肌球蛋白超家族肌动蛋白分子马达中肌球蛋白Iβ亚家族的成员。我们已经利用微测序数据克隆了该蛋白的cDNA。显微测序和基因序列分析表明，120 kDa的蛋白质存在一个16个氨基酸的N-末端延伸。对肌球蛋白Iβ基因的分析发现了两个新的外显子的存在，并在核苷酸水平上证明了肌球蛋白Iβ在核和细胞质形式上的不同基础。有趣的是，核肌球蛋白I的一个标记表位位于细胞核，但当标记表位与细胞质肌球蛋白I结合时，仅在细胞质中发现。我们还产生了针对16个氨基酸的多肽的抗体，并表明它们对细胞核染色。综上所述，这些数据为肌球蛋白I在细胞核中的存在提供了强有力的支持，并表明核肌球蛋白I特有的16个氨基酸延伸负责该蛋白的核定位。我们现在提出三个特定的目标，它们具有定义核肌球蛋白I的生理功能的长期目标，它们将依次解决以下问题：(A)我们如何获得核肌球蛋白Iβ蛋白？(B)这种特定的肌球蛋白Iβ亚型是如何进入细胞核的？(C)它在细胞核中做什么？具体目标1是我们正在进行的肌球蛋白Iβ基因特征的继续。我们对该基因的分析表明，核肌球蛋白Iβ亚型和胞浆肌球蛋白Iβ亚型是由11号染色体上的单个基因衍生的多个mRNA物种的结果。特定目的1将检验这一假说。具体目标2的目标是揭示将核肌球蛋白I导向细胞核的机制，而不是肌球蛋白1β的其他异构体。它将检验16个氨基酸延伸将肌球蛋白I定位于细胞核的假设。具体目标3将通过测试核肌球蛋白I具有结构作用和/或活跃转录基因的功能的假设来研究肌球蛋白I在细胞核中的生理功能。解决这些特定的目标将帮助我们达到了解核肌球蛋白I的生理功能的最终目标。这项提议还将开发一些工具(例如：质粒、单抗和抗肽抗体、杆状病毒表达的蛋白质、敲除细胞)，这些工具将有助于实现我们的长期目标。

We have previously demonstrated the presence of a myosin I like protein in the nucleus. This protein has an apparent molecular weight of 120,000, it is associated with K+-EDTA ATPase activity, it binds calmodulin, it is photoaffinity labelled with ATP, it binds actin in the absence, but not the presence, of ATP and it is recognized by an antibody to myosin I purified from adrenal glands. Microsequencing of the 120 kDa protein has positively identified it as a member of the myosin I beta subfamily of the myosin superfamily of actin-based molecular motors. We have used the microsequencing data to clone the cDNA for this protein. The microsequencing and cDNA have independently shown the presence of a 16 amino acid N-terminal extension that is unique to the 120 kDa protein. Analysis of the myosin I beta gene has uncovered the presence of 2 new exons and demonstrated the basis, at the nucleotide level, for the differences in the nuclear and cytoplasmic forms of myosin I beta. Interestingly, a FLAG epitope attached to the cDNA for nuclear myosin I localizes in the nucleus but the FLAG epitope is found only in the cytoplasm when attached to the cytoplasmic myosin I. We have also produced antibodies to the 16 amino acid peptide and shown that they stain the nucleus. Taken together, these data provide strong support for the presence of myosin I in the nucleus and suggest that the 16 amino acid extension unique to nuclear myosin I is responsible for the nuclear localization of this protein. We now propose 3 Specific Aims that have the long range goal of defining the physiological functions of nuclear myosin I. They will, in order, address the following questions: (a) How do we get a nuclear myosin I beta protein? (b) How does this specific myosin I beta isoform get into the nucleus? (c) What does it do in the nucleus? Specific Aim 1 is a continuation of our ongoing characterization of the myosin I beta gene. Our analysis of this gene has suggested that the nuclear and cytoplasmic myosin I beta isoforms result from multiple mRNA species derived from the single gene on chromosome 11. Specific Aim 1 will test this hypothesis. The goal of Specific Aim 2 is to uncover the mechanisms that direct nuclear myosin I, but not other isoforms of myosin 1beta, to the nucleus. It will test the hypothesis that the 16 amino acid extension targets myosin I to the nucleus. Specific Aim 3 will investigate the physiological functions of myosin I in the nucleus by testing the hypothesis that nuclear myosin I has a structural role and/or the functioning of actively transcribing genes. Addressing these Specific Aims will help us to reach our ultimate goal of understanding the physiological functions of nuclear myosin I. This proposal will also develop a number of tools (eg: plasmids, monoclonal and anti-peptide antibodies, proteins expressed in baculovirus, knock-out cells) that will be instrumental in realizing our long range goal.