Diacylglycerol Kinase Delta in Growth and Development

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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.