In contrast DAG phosphorylation to PA
In contrast, DAG phosphorylation to PA by diacylglycerol kinases (DGKs) represents a quantitatively minor metabolic pathway, but is generally regarded as a main disposal pathway for “signaling” DAG. PA produced by DGKs is an intermediate of the synthesis of CDP-DAG, cardiolipin and PI. However, PA is also a signaling lipid by itself, capable of regulating activity and localization of several proteins involved in signal transduction. Thus DGKs, downregulate a second messenger (DAG) and generate a new one in the process (PA) with different properties and effector functions (Mérida et al., 2008). These dual functions take place simultaneously, but the relative relevance of DAG depletion versus PA accumulation depends on cellular type and context. This is exemplified in T lymphocytes where (a) at first thymic selection critically requires DGKα and ζ produced PA (Guo et al., 2008), (b) DGKα and ζ act as negative regulators of TCR signaling in mature T N-Nonyldeoxynojirimycin australia by metabolizing DAG (Sanjuán et al., 2001, Zhong et al., 2002), and (c) IL-2 driven proliferation of activated T cells after TCR triggering involves PA generation via DGKα activity (Flores et al., 1996, Merida et al., 1993). Eukaryotic DGKs are a multigene family with ten isoforms characterized by a conserved catalytic domain preceded by two C1-like domains of unknown function. The different isoforms diverge in both the sequence of their N-terminal regulatory domains and in their pattern of tissue-specific expression (Sakane et al., 2007). In sharp contrast with the membrane DGK of E. coli, eukaryotic DGKs are DAG specific and thus selectively involved in DAG metabolism. Furthermore, eukaryotic DGKs are mainly soluble enzymes which must translocate from the cytosol to the plasma (Merino et al., 2007) or intracellular membranes such as Golgi (Nagaya et al., 2002) or nucleus (Goto et al., in press) under the control of extracellular and intracellular cues. The observation that localization and activity are controlled by the regulatory N-terminal domains which differentiate each isoform suggests that each DGK possesses distinct functions, possibly by metabolizing separate pools of DAG in response to different cues (Sakane et al., 2007). This idea is also supported by the observation that multiple isoforms are often co-expressed in the same cell, as observed in endothelial cells (Baldanzi et al., 2011a), hepatocytes (Baldanzi et al., 2010), or lymphocytes (Zhong et al., 2007). In fact, downregulation of different isoforms in the same cell gives rise to different phonotypes (Baldanzi et al., 2011a, Miele et al., 2007, Milne et al., 2008). One clear example of a DGK acting on a specific DAG pool for a dedicated purpose in metabolism is DGKɛ, which is unique in its selectivity for unsaturated substrates and contributes to selective metabolism of unsaturated DAG in the PI cycle (Lukiw et al., 2005, Nakano et al., 2009, Rodriguez de Turco et al., 2001). Among the different DAG metabolizing enzymes, DGKs seem to play a special role as terminators of the DAG pool involved in signaling. Indeed, in several instances DGK inhibition or silencing does not affect basal levels of DAG, but greatly potentiates agonist-induced DAG accumulation (de Chaffoy de Courcelles et al., 1985, Mizuno et al., 2012, Novotná et al., 2003, Rodriguez de Turco et al., 2001). This suggests a role for DGKs in establishing signaling thresholds, preventing inappropriate responses in low stimulatory conditions. Furthermore, several DGKs are targeted to specific membrane sub-compartments that contribute to localized DAG metabolism, as exemplified by DGKζ at the immune synapse (Gharbi et al., 2011) or DGKβ at dendritic spines (Hozumi and Goto, 2012). The role of DGKs as negative regulators of DAG signaling has been extensively investigated in T cells, in which the DGKα and ζ are the main isoforms expressed. Both isoforms are recruited to the TCR signalosome (Gharbi et al., 2011) and act in a synergistic manner to decrease TCR-induced DAG signaling to PKCθ and RAS-GRP1 both in vitro and in vivo (Jones et al., 2002, Olenchock et al., 2006, Sanjuán et al., 2001, Sanjuán et al., 2003, Zhong et al., 2002, Zhong et al., 2003). If one or both DAG isoforms are genetically or pharmacologically blocked, T lymphocytes show strongly enhanced DAG signaling and activation with higher sensitivity to TCR signaling and impaired ability to undergo anergy, a hyporesponsive state triggered by suboptimal stimulation (Zhong et al., 2007). In this scenario, basal signaling is unaffected and lymphocyte activation remains dependent on TCR triggering, suggesting a persistent requirement for upstream production of DAG through receptor-dependent PLC activity.