NUCLEAR MYOSIN I

核肌球蛋白 I

• 批准号: 6525417
• 负责人: PRIMAL DE LANEROLLE
• 金额: $ 29.49万
• 依托单位: UNIVERSITY OF ILLINOIS AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-08-01 至 2004-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6525417
• 关键词: Baculoviridae; antisense nucleic acid; cell nucleus; chromosomes; embryonic stem cell; gene targeting; genetic transcription; genetic translation; image processing; immunoelectron microscopy; immunoprecipitation; intracellular transport; laboratory mouse; laboratory rabbit; messenger RNA; monoclonal antibody; myosins; nucleic acid structure; nucleoproteins; oligonucleotides; plasmids; protein isoforms; protein structure function; tissue /cell culture; transfection /expression vector

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.

我们之前已经证明了在细胞核中存在肌凝蛋白I类蛋白。这种蛋白的表观分子量为120000，它与K+-EDTA ATP酶活性有关，它与钙调蛋白结合，它和光亲和标记有ATP，它在没有ATP的情况下与肌动蛋白结合，但不存在ATP，它被从肾上腺中纯化的肌球蛋白I抗体识别。120 kDa蛋白的微测序结果表明，该蛋白是肌动蛋白超家族肌球蛋白I β亚家族的成员。我们利用微测序数据克隆了该蛋白的cDNA。微测序和cDNA独立显示存在一个16个氨基酸的n端延伸，这是120 kDa蛋白所特有的。对myosin I β基因的分析发现了2个新的外显子的存在，并在核苷酸水平上证明了myosin I β核和细胞质形式差异的基础。有趣的是，核肌球蛋白I cDNA上的FLAG表位定位在细胞核中，但当与细胞质肌球蛋白I连接时，FLAG表位仅在细胞质中发现。我们还制备了针对16个氨基酸肽的抗体，并证明它们可以染色细胞核。综上所述，这些数据为肌凝蛋白I在细胞核中的存在提供了强有力的支持，并表明核肌凝蛋白I特有的16个氨基酸延伸负责该蛋白的核定位。我们现在提出了3个特定目标，这些目标具有定义核肌球蛋白I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I β蛋白？(b)这种特殊的肌凝蛋白I β同型体是如何进入细胞核的？(c)它在细胞核中起什么作用？特异性目标1是我们正在进行的肌球蛋白I β基因表征的延续。我们对该基因的分析表明，核和细胞质肌球蛋白I β异构体是由11号染色体上的单个基因衍生的多个mRNA物种产生的。具体目标1将检验这一假设。特异性目标2的目标是揭示指导核肌凝蛋白1的机制，而不是其他肌凝蛋白1 β的同工型。它将验证16个氨基酸延伸将肌球蛋白I靶向到细胞核的假设。特异性目的3将通过验证核肌球蛋白I具有结构作用和/或活性转录基因功能的假设来研究肌球蛋白I在细胞核中的生理功能。解决这些具体目标将有助于我们达到了解核肌球蛋白i的生理功能的最终目标。该建议还将开发一些工具（例如：质粒，单克隆和抗肽抗体，杆状病毒表达的蛋白质，敲除细胞），这将有助于实现我们的长期目标。