Diacylglycerol Kinase Delta in Growth and Development

二酰基甘油激酶（DGKs）磷酸化二酰基甘油（DAG）生成磷脂酸（PA）。DAG和PA都具有信号特性，使dgk处于重要的生物学十字路口。已经鉴定出9种哺乳动物dgk，这表明它们不仅在生物学上很重要，而且每一种dgk都有不同的功能。由于细胞通常表达几种DGK抑制剂，而这些抑制剂只能抑制三种DGK，因此很难确定每种DGK同工酶的功能。先前确定其功能的努力依赖于过表达DGKisotype或失活突变体。这是一种简单而有效的方法，但也有缺点，因为在细胞中过度表达一种蛋白质会导致非特异性作用。如果没有适当的控制，结果可能会产生误导。克服这些技术困难的一个有效方法是，对小鼠进行定向删除感兴趣的基因的工程。因此，为了了解每种DGK同型的作用，我们启动了在小鼠中删除这些基因的项目。我们发现靶向缺失DGKdelta的小鼠的表型与靶向缺失表皮生长因子受体（EGFR）或肿瘤坏死因子- α转换酶（TACE）的小鼠非常相似。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dgk在EGFR信号传导中起作用。我们还观察到，靶向删除DGKepsilon或iota的小鼠没有这种表型，表明这是DGKdelta的独特特性。进一步的研究表明，删除DGKdelta并不影响EGFR下游的信号事件，而是表明DGK对于TACE的适当激活是必要的，TACE是一种跨膜酶，可以从细胞表面蛋白水解释放EGFR配体。事实上，DGKdelta与TACE共免疫预测，在已知激活TACE的条件下，它们的相关性得到增强。因此，TACE激活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的异常活动将导致不受控制的生长和发育。在本提案中，我们概述了通过剖析导致DGKdelta激活TACE的事件以及检查异常高DGKdelta活性的后果来验证假设的实验。

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.