br Regulation of DGK activity Activation of

Regulation of DGK activity Activation of the DGKs is complex and unique for each DGK isotype. In most cases, DGKs must translocate to a membrane compartment to access DAG. However, translocation does not necessarily activate the enzyme [57]. In addition, DGK activity can be modified by other cofactors such as lipids and calcium, and several DAG kinases are also regulated by post-translational modifications. Finally, tissue-specific alternative splicing of DGKs β, γ, δ, ζ, and η, and probably other isotypes, allows for additional regulation [31], [34], [35], [58], [59]. This complexity permits cell- or tissue-specific regulation of each DGK isotype depending on the availability of cofactors and the type of stimulus that the cell receives. DGKα is perhaps the best example of the contextually dependent, differential regulation of DGKs. DGKα translocated to at least two different membrane compartments in T lymphocytes depending upon the agonist used to activate the cells: from the cytosol to a perinuclear region in T sphingosine kinase stimulated with IL-2 [55], [60], and to the plasma membrane upon activation of the T cell antigen receptor [20]. Once at a membrane compartment, the DAG kinase activity of DGKα can be modified by the availability of several cofactors. Calcium is known to bind to EF hand structures and stimulated DGKα activity in vitro [32]. Sanjuan et al. [20] demonstrated that deleting the EF hand motifs of DGKα caused it to associate with the plasma membrane and significantly increased its DAG kinase activity. This observation led to the hypothesis that in the absence of calcium, the EF hand structures of DGKα inhibit DAG kinase activity—possibly by masking a motif necessary for catalytic activity—and somehow reduce membrane association. Binding calcium releases the inhibition and causes translocation to the membrane, allowing maximal DAG kinase activity. When associated with the membrane, the activity of DGKα is further modified by binding to lipid components: phosphatidylserine, sphingosine [61], [62], and the phosphatidylinositol (PI) 3-kinase lipid products, PI-3,4-P2 and PI-3,4,5-P3[63], activated DGKα in vitro and likely in vivo as well. Finally, DGKα can be phosphorylated by several protein kinases including some PKC isoforms [64], [65] and Src kinase [66], which may further enhance its DAG kinase activity. Thus, numerous events are required to fully activate DGKα, combinations of which can fine-tune its activity to the appropriate level. Similar to DGKα, other DGK isotypes appear to be sensitively regulated by a number of factors. For example, type II DGKs have a PH domain that may affect intracellular localization by interacting with either phosphatidylinositols or with other proteins. Indeed, the PH domain of the type II DGKδ could bind inositol phosphates [33]. However, the binding was nonselective and weaker than a typical high affinity protein–lipid interaction, suggesting that it may not be a physiological interaction. Its DAG kinase activity was not affected by PIP2[23]. In contrast, the activity of DGK types III and IV can be modified by phosphatidylinositols and phosphatidylserine in opposing ways. DGKε, the type III enzyme, was inhibited by both PIP2 and phosphatidylserine, whereas DGKζ was activated by both lipids [67]. Like DGKα, the subcellular localization of type IV DGKs is exquisitely regulated. These enzymes have a nuclear localization signal that can be regulated by PKC phosphorylation [22], [39]. Additionally, members of the syntrophin family of scaffolding proteins further regulate the subcellular location of DGKζ by associating with its carboxy terminal PDZ-binding domain, anchoring the protein in the cytoplasm [42]. Further, Davidson et al. [68] recently demonstrated that activating the type I gonadotropin-releasing hormone receptor caused DGKζ to associate with active Src kinase and translocate to the plasma membrane. Association with Src significantly enhanced its DAG kinase activity. Finally, DGKθ, a type V DGK, can be regulated through its association with active RhoA: binding this GTPase abolished its DAG kinase activity [44]. Thus, depending on the context of activation, the availability of cofactors, and the activation state of protein kinases, DGKs can be differentially regulated.