Diacylglycerol Kinase Delta in Growth and Development

二酰甘油激酶 Delta 在生长和发育中的作用

• 批准号: 6745620
• 负责人: MATTHEW KENT TOPHAM
• 金额: $ 26.61万
• 依托单位: UNIVERSITY OF UTAH
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-04-25 至 2007-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6745620
• 关键词: alcohol phosphotransferase; biological signal transduction; cell growth regulation; diacylglycerols; enzyme activity; enzyme inhibitors; enzyme linked immunosorbent assay; enzyme substrate; epidermal growth factor; gene deletion mutation; gene expression; genetically modified animals; growth factor receptors; human tissue; isozymes; laboratory mouse; metalloendopeptidases; mitogen activated protein kinase; phenotype; protein protein interaction; transfection /expression vector; transforming growth factors; tumor necrosis factor alpha

Diacylglycerol kinases (DGKs) phosphorylate diacylglycerol (DAG) to generate phosphatidic acid (PA). Both DAG and PA have signaling properties, placing DGKs at an important biological crossroads. Nine mammalian DGKs have been identified, suggesting not only they are biologically important, but also that each of them has a distinct function. Because cells often express several DGK inhibitors- which can inhibit only three DGKs- it has been very difficult to determine the function of each DGK isozyme. Previous efforts to determine their functions have relied on overexpressing a DGKisotype or an inactive mutant. This is a simple and often effective approach, but has drawbacks because overexpressing a protein in cells can lead to non-specific effects. If proper controls are not included, the results can be misleading. An effective approach to circumvent these technical difficulties, is to engineer mice with targeted deletion of the gene of interest. So, to understand the role of each DGK isotype, we have initiated projects to delete the genes in mice. We found that mice with targeted deletion of DGKdelta, have a phenotype very similar to mice with targeted deletion of either the epidermal growth factor receptor (EGFR) or tumor necrosis factor-alpha converting enzyme (TACE). This similarity was surprising because there is no previous evidence indicating a role for DGKdelta or other DGKs in EGFR signaling. We have also observed that mice with targeted deletion of either DGKepsilon or iota do not have this phenotype, indicating that this is a distinct property of DGKdelta. Further studies indicated that deleting DGKdelta did not affect signaling events downstream of the EGFR, but instead suggested that DGK was necessary for proper activation of TACE, a transmembrane enzyme that proteolytically releases EGFR ligands from the cell surface. Indeed, DGKdelta co-immunoprecited with TACE and their association was enhanced in conditions known to activate TACE. Thus, it appears that a crucial event in TACE activation is its association with DGKdelta. However, the specific mechanism by which DGKdelta activates TACE is not clear. We hypothesize that DGKdelta associates either directly or indirectly with TACE and then activates growth factor shedding through its DAG kinase activity. Additionally, we believe that abnormal activity of DGKdelta will result in deregulated growth and development. In this proposal, we outline experiments to test hypotheses by dissecting the events leading to DGKdelta's activation of TACE and by examining the consequences of abnormally high DGKdelta activity.

二酰基甘油激酶（DGK）磷酸化二酰基甘油（DAG）以产生磷脂酸（PA）。 DAG和PA都具有信号传导特性，将DGK置于重要的生物学十字路口。 已经鉴定出9种哺乳动物DGK，这表明它们不仅具有生物学重要性，而且它们中的每一种都具有独特的功能。 由于细胞通常表达几种DGK抑制剂-其只能抑制三种DGK-因此很难确定每种DGK同工酶的功能。 以前确定其功能的努力依赖于过表达DGK同种型或失活突变体。 这是一种简单且通常有效的方法，但有缺点，因为在细胞中过度表达蛋白质可能导致非特异性效应。如果不包括适当的控制，结果可能会产生误导。 规避这些技术困难的有效方法是用目标基因的靶向缺失来改造小鼠。 因此，为了了解每个DGK同种型的作用，我们已经启动了删除小鼠基因的项目。 我们发现DGKdelta靶向缺失的小鼠具有与表皮生长因子受体（EGFR）或肿瘤坏死因子-α转化酶（TACE）靶向缺失的小鼠非常相似的表型。 这种相似性是令人惊讶的，因为之前没有证据表明DGKdelta或其他DGKs在EGFR信号传导中的作用。 我们还观察到具有DGKdelta或iota的靶向缺失的小鼠不具有这种表型，表明这是DGKdelta的独特性质。 进一步的研究表明，删除DGKdelta并不影响EGFR下游的信号传导事件，而是表明DGK对于TACE的适当活化是必需的，TACE是一种从细胞表面蛋白水解释放EGFR配体的跨膜酶。 事实上，DGKdelta与TACE共免疫表达，并且它们的关联在已知激活TACE的条件下增强。 因此，TACE激活中的关键事件似乎是其与DGKdelta的相关性。然而，DGKdelta激活TACE的具体机制尚不清楚。 我们假设DGKdelta与TACE直接或间接相关，然后通过其DAG激酶活性激活生长因子脱落。 此外，我们认为DGKdelta的异常活性将导致生长和发育失调。 在这个建议中，我们概述了实验，通过解剖导致DGKdelta激活TACE的事件，并通过检查异常高的DGKdelta活性的后果来测试假设。