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

描述(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，该结构包括交错的肌动蛋白和肌球蛋白细丝、细丝锚定结构和一系列调节蛋白。在过去的几十年里，模式生物线虫的先进分子遗传学和细胞生物学为鉴定和研究对横纹肌细胞发育和功能至关重要的蛋白质提供了重要的发现工具。我们已经确定线虫肌肉影响基因UNC-82编码与人类蛋白ARK5(NUAK1)和SNARK(NUAK2)同源的丝氨酸/苏氨酸激酶。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都是因为它们的序列与AMPK激酶相似而命名的，AMPK激酶被应激激活，包括葡萄糖饥饿和由此导致的细胞内高水平的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中酶活性的激活以不同的机制发生。在葡萄糖饥饿的情况下，ARK5被Akt酶在特定调节部位的磷酸化激活，而SNARK不包含Akt酶识别的基序。有趣的是，通过选择性剪接，单个线虫基因UNC-82也产生不同于Akt调节磷酸化位点存在或不存在的蛋白质亚型。尽管UNC-82的某些异构体可能会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组织M-线的粗丝和膜远端成分所需的信号通路的一部分。这项提案中概述的实验旨在测试磷酸化在UNC-82在肌肉发育早期激活中的作用。此外，我们希望利用线虫系统的高级分子遗传学来确定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在横纹肌中的表达贯穿一生，这表明可能在心肌和骨骼肌的肌肉细胞发育、结构维护甚至收缩功能中扮演着不同的角色。大多数已发表的关于ARK5和SNARK的工作涉及这些蛋白在癌症中的作用，特别是在调节肿瘤细胞侵袭性方面。ARK5或SNARK激活后肿瘤细胞行为的改变可能暗示这些酶通过调节细胞骨架来影响侵袭性。如果是这样的话，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。 公共卫生相关性：我们已经确定了一种新的酶，UNC-82，它对于组织模式生物线虫的横纹肌细胞收缩装置的细丝很重要。有趣的是，这种酶的人类同源物已经被证明在人类癌症的转移中起着重要作用。UNC-82的人类同源物在心脏和骨骼肌中表达。了解UNC-82在线虫中的作用将有助于阐明人类酶在正常肌肉发育和功能中的作用。

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.