br Conflicts of interest br
Conflicts of interest
Introduction Diacylglycerols (DAGs) and phosphatidic Heparin sodium (PA) play fundamental roles in biology as basic components of membranes, intermediates in lipid metabolism, and secondary messengers in cellular signaling (Carrasco and Merida, 2007, Fang et al., 2001). Cells regulate intracellular DAG and PA levels through metabolic networks that utilize distinct enzymes to produce or consume these secondary messengers/metabolites (Brown et al., 2017, Carrasco and Merida, 2007, Hsu et al., 2012, Shulga et al., 2011). One such enzymatic pathway that is central to signal transduction is ATP-dependent phosphorylation of DAGs to biosynthesize PA (Figure 1A) by a set of lipid kinases collectively known as diacylglycerol kinases (DGKs) (Shulga et al., 2011). DAG and PA are important lipid messengers that alter localization (Takai et al., 1979), activation (Newton and Koshland, 1989), and protein-protein interactions (Fang et al., 2001) of distinct sets of receptor proteins. Consequently, disruption of the same DGK protein in different cell types can result in opposing effects that can be leveraged, for example, in cancer to simultaneously block tumor growth and activate antitumor immunity (Merida et al., 2017, Sakane et al., 2016). Since DAG and PA serve as key intermediates in lipid metabolism, DGKs are uniquely positioned as key regulators of the structural, bio-energetic, and signaling demands of cells. Ten mammalian DGKs have been identified and classified into five subtypes based on structural features elucidated from primary sequence analysis (Figure 1B). At the N terminus, DGKs contain at least two cysteine-rich zinc-finger-like motifs similar to C1 domains found in protein kinase C (PKC) (Carrasco and Merida, 2007). DGKs contain a C-terminal catalytic domain composed of a conserved catalytic region (SMART domain [Schultz et al., 1998] DAGKc, SM000046), which is present in other eukaryotic lipid kinases and DGKs from Gram-positive bacteria (Adams et al., 2016), followed by an accessory subdomain (DAGKa, SM000045) of unknown function (Merida et al., 2017). While DGKs share the same basic domain organization, individual subtypes differ widely in regulatory domains proposed to mediate metal binding (EF hand motifs), oligomerization (SAM domain), membrane association (PH domain), subcellular localization (MARCKS domain), or protein-protein interactions (ankyrin repeats, PDZ domain) (Shulga et al., 2011). Given the enormous chemical diversity of DAG and PA lipids (Yetukuri et al., 2008), understanding the crosstalk between regulatory and catalytic domains of DGKs will be critical for assigning metabolic and signaling functions to individual isoforms. Attempts to define the function of individual DGK domains have resulted in inconclusive results. ATP binding motifs corresponding to the glycine-rich loops found in protein kinases (GxGxxG consensus sequence; Hanks et al., 1988, Hemmer et al., 1997) were identified in the first C1 and catalytic domains of DGKs (Sakane et al., 1990, Schaap et al., 1994). Mutation of lysines in these motifs, which abolishes ATP binding and protein kinase activity, did not affect catalytic function of DGKs and led others to hypothesize the existence of a DGK-specific ATP binding motif that remains to be defined (Sakane et al., 1996, Schaap et al., 1994). The role of C1 domains in DGK function is also enigmatic. With the exception of gamma and beta isoforms (Shindo et al., 2003), the C1 domains of DGKs lack conserved residues identified as being required for DAG binding in other proteins including PKC (Hurley and Misra, 2000). In vitro biochemical studies measuring the activity of C1 truncation mutants have produced conflicting reports with regard to whether C1 motifs are required (Abe et al., 2003, Houssa et al., 1997, Santos et al., 2002) or dispensable (Merino et al., 2007, Sakane et al., 1996) for maximal DGK catalytic activity.