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: 6465329
• 负责人: MATTHEW KENT TOPHAM
• 金额: $ 26.7万
• 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/6465329
• 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.

二酰甘油激酶(DGKs)使二酰基甘油(DAG)磷酸化，生成磷脂酸(PA)。DAG和PA都具有信号特性，使DGK处于重要的生物十字路口。已鉴定出九种哺乳动物DGK，这表明它们不仅具有重要的生物学意义，而且每一种都有不同的功能。因为细胞通常表达几种DGK抑制剂--只能抑制三种DGK--所以很难确定每一种DGK同工酶的功能。以前确定其功能的努力依赖于过度表达DGKistype或不活跃的突变体。这是一种简单且往往有效的方法，但也有缺点，因为在细胞中过度表达蛋白质可能会导致非特异性效应。如果不包括适当的控制措施，结果可能会产生误导。绕过这些技术困难的一个有效方法是通过定向缺失目的基因来改造小鼠。因此，为了了解每种DGK亚型的作用，我们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转换酶(TACE)靶向缺失的小鼠非常相似的表型。这种相似性令人惊讶，因为之前没有证据表明DGK Delta或其他DGK在EGFR信号转导中发挥作用。我们还观察到，DGKepsilon或IOTA靶向缺失的小鼠没有这种表型，表明这是DGKDelta的一个独特属性。进一步的研究表明，删除DGK Delta不会影响EGFR下游的信号事件，相反，DGK对于TACE的正确激活是必要的，TACE是一种跨膜酶，可以蛋白质水解性地从细胞表面释放EGFR配体。事实上，DGKDelta与TACE共同免疫，在已知激活TACE的条件下，它们的关联性得到增强。因此，TACE激活中的一个关键事件似乎是它与DGKDelta的关联。然而，DGKDelta激活TACE的具体机制尚不清楚。我们假设DGKDelta直接或间接地与TACE结合，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的异常活动将导致放松对增长和发展的管制。在这项提案中，我们概述了通过解剖导致DGKDelta激活TACE的事件和检查异常高的DGKDelta活动的后果来检验假说的实验。