权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

Molecular Genetic Analysis of UNC-82 Kinase Function in C. Elegans Muscle

线虫肌肉 UNC-82 激酶功能的分子遗传学分析

基本信息

批准号：
7778424
负责人：
PAMELA E HOPPE
金额：
$ 18.56万
依托单位：
WESTERN MICHIGAN UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-03-01 至 2015-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7778424
关键词：
AMP-activated protein kinase kinase Actins Affect Alternative Splicing Animal Model Biochemical Caenorhabditis elegans Cancer cell line Cell Shape Cellular biology Cytoskeleton Data Defect Development Distal Embryo Enzymes Excision Filament Genes Genetic Techniques Glucose Heart Homologous Gene Human Human Cell Line Knowledge Life Light Maintenance Malignant Neoplasms Membrane Molecular Genetics Muscle Muscle Cells Muscle Contraction Muscle Development Myeloma Proteins Myosin ATPase Myosin Heavy Chains Names Neoplasm Metastasis Paratropomyosin Pathway interactions Phenotype Phosphorylation Phosphorylation Site Phosphotransferases Physiology Play Protein Isoforms Protein-Serine-Threonine Kinases Proteins Publishing RNA Interference RNA Splicing Regulation Role Signal Pathway Signal Transduction Site Skeletal Muscle Starvation Stress Striated Muscles Structure System Testing Thick Filament Variant Visual Work biological adaptation to stress cell behavior cell type design enzyme activity genetic analysis genetic regulatory protein human ARK5 protein in vivo knock-down member mutant neoplastic cell public health relevance research study response tool

项目摘要

DESCRIPTION (provided by applicant): Striated muscle contraction is accomplished by a highly ordered cytoskeletal structure consisting of interdigitating actin and myosin filaments, the filament anchoring structures, and a host of regulatory proteins. Over the past several decades, the advanced molecular genetics and cell biology of the model organism C. elegans have provided an important discovery tool for identifying and studying proteins critical for striated muscle cell development and function. We have determined that the C. elegans muscle-affecting gene unc-82 encodes a serine/threonine kinase that is orthologous to human proteins ARK5 (NUAK1) and SNARK (NUAK2). Both ARK5 and SNARK are expressed in striated muscle, but the role that either plays in muscle development or physiology is unknown. Both ARK5 and SNARK were named for their sequence similarity to AMPK kinases, which are activated by stress, including glucose starvation and the resulting high levels of cellular AMP. However, sequence comparisons and biochemical data from human cell lines suggest that the activation of enzyme activity occurs by different mechanisms in ARK5 and SNARK. Whereas ARK5 is activated by phosphorylation at a particular regulatory site by the kinase Akt in conditions of glucose starvation, SNARK does not contain the motif recognized by the Akt enzyme. Interestingly, through alternative splicing, the single C. elegans gene unc-82 also produces protein isoforms that differ in the presence or absence of the Akt regulatory phosphorylation site. Although some isoforms of UNC-82 may be activated in response to stress, we hypothesize that in embryonic muscle UNC-82 is part of a signaling pathway that is required for the organization of thick filaments and membrane-distal components of the M-line in response to changes in cell shape. Experiments outlined in this proposal are designed to test the role of phosphorylation in the activation of UNC-82 in early muscle development. In addition, we hope to exploit the advanced molecular genetics of the C. elegans system to identify other members of the UNC-82 signaling pathway, including UNC-82 substrates and potential kinase partners in a signaling cascade. UNC-82 and the human proteins ARK5 and SNARK are expressed in striated muscle throughout life, indicating perhaps diverse roles in muscle cell development, structural maintenance, or even contractile function in both heart and skeletal muscle. The majority of the published work on ARK5 and SNARK concerns the role of these proteins in cancer, and particularly in regulation of the invasiveness of tumors cells. The change in tumor cell behavior in response to activation of ARK5 or SNARK may imply that the enzymes are affecting invasiveness through regulation of the cytoskeleton. If so, the knowledge gained about the role of UNC-82 in normal muscle development may promote understanding of the role of ARK5 and SNARK in promotion metastasis. PUBLIC HEALTH RELEVANCE: We have identified a new enzyme, UNC-82, that is important for organizing the filaments of the striated muscle cell contractile apparatus in the model organism C. elegans. Interestingly, the human homologs of this enzyme have been shown to be important in metastasis of human cancers. The human homologs of UNC-82 are expressed in heart and skeletal muscle. Understanding the role of UNC-82 in C. elegans will shed light on the role of the human enzymes in normal muscle development and function.

描述（由申请人提供）：横纹肌收缩是由高度有序的细胞骨架结构完成的，该结构由交错的肌动蛋白和肌球蛋白丝、丝锚定结构和一系列调节蛋白组成。在过去的几十年里，模式生物秀丽隐杆线虫的先进分子遗传学和细胞生物学为鉴定和研究横纹肌细胞发育和功能的关键蛋白质提供了重要的发现工具。我们已经确定秀丽隐杆线虫肌肉影响基因unc-82编码一种丝氨酸/苏氨酸激酶，该激酶与人类蛋白ARK5 （NUAK1）和SNARK （NUAK2）同源。ARK5和SNARK都在横纹肌中表达，但在肌肉发育或生理中所起的作用尚不清楚。ARK5和SNARK因其序列与AMPK激酶相似而命名，AMPK激酶可被应激激活，包括葡萄糖饥饿和由此产生的高水平细胞AMP。然而，序列比较和来自人类细胞系的生化数据表明，ARK5和SNARK中酶活性的激活是通过不同的机制发生的。在葡萄糖饥饿的情况下，ARK5在一个特定的调控位点被Akt激酶磷酸化激活，而SNARK不包含Akt酶识别的基序。有趣的是，通过选择性剪接，秀丽隐杆线虫的单一基因unc-82也会产生不同的蛋白异构体，这种异构体在存在或不存在Akt调控磷酸化位点时是不同的。尽管UNC-82的一些同工型可能在应激反应中被激活，但我们假设在胚胎肌肉中，UNC-82是响应细胞形状变化而形成粗丝和m系远端膜组分所需的信号通路的一部分。本提案中概述的实验旨在测试磷酸化在早期肌肉发育中活化UNC-82的作用。此外，我们希望利用秀丽隐杆线虫系统的先进分子遗传学来鉴定UNC-82信号通路的其他成员，包括UNC-82底物和信号级联中的潜在激酶伙伴。UNC-82和人类蛋白ARK5和SNARK在横纹肌中表达，这表明可能在心脏和骨骼肌的肌肉细胞发育、结构维持甚至收缩功能中发挥不同的作用。已发表的关于ARK5和SNARK的大部分工作都涉及这些蛋白在癌症中的作用，特别是在调节肿瘤细胞的侵袭性方面。ARK5或SNARK激活后肿瘤细胞行为的变化可能暗示这些酶通过调节细胞骨架影响侵袭性。如果是这样，对UNC-82在正常肌肉发育中的作用的了解可能会促进对ARK5和SNARK在促进转移中的作用的理解。