Arguably, our lack of knowledge about the three dimensional structure of mammalian DGKs remains as one of our most difficult challenges. Currently, our knowledge of the structural features of DGKs is essentially limited to the primary sequence of these enzymes (Figure 1). As noted in many excellent reviews (for example, see van Blitterswijk and Houssa, 2000; Kanoh et al., 2002; Mérida et al., 2008; Shulga et al., 2011) DGK-θ's major distinguishing feature in this regard is that it possess three, instead of two, C1 domains. Like other DGKs, the C1 domains of DGK-θ is homologous to PKC C1A and C1C domains with an extended region closest to the presumed catalytic domain near the C terminus. Despite the temptation to assume these domains bind DAG, their precise role remains unclear. Such caution is supported by the observation that the C1 domains of DGK-θ does not bind phorbol esters (Shindo et al., 2003). Further, the structural studies of Hurley and colleagues showed clear differences between these domains and those found in PKCs (Hurley et al., 1997). As suggested previously (Shulga et al., 2011), these data suggest that these DGK-θ domains may bind other lipids or participate in protein-protein interactions. Such a notion is supported by the evidence showing the C1 domain of another DGK, DGKζ, mediates the interactions with β-arrestins (Nelson et al., 2007) and Rac1 (Yakubchyk et al., 2005). We should note, that there is some evidence that this domain may be involved in membrane association in response to activation of some G-protein coupled receptors (van Baal et al., 2005).

Another feature of DGK-θ is the presence of a sequence with homology to Ras binding domains which is termed the Ras-Association (RA) domain (Houssa et al., 1997). It is important to note, however, that an in silico analyses based on binding energies predicts that the RA domain of DGK-θ probably does not bind Ras (Kiel et al., 2005). It is interesting, in this regard, that DGK-θ has been shown to bind a different small GTPase, active (GTP-bound) RhoA, and this association inhibits the kinase activity (Houssa et al., 1997). As with the C1 domains, however, it is not clear that RhoA binds specifically to this domain either and it is equally unclear as to whether RhoA is a regulator under various physiological conditions (see Regulation below).

Another major distinguishing feature of DGK-θ is the presence of a proline/glycine-rich region near its N-terminus. It is interesting to speculate that this region, may bind to proteins containing an SH3 domain given the presence of a pXPXXP motif (Yu et al., 1994). Further, a splice form of DGKδ, DGKδ2, also contains a proline-rich domain which is believed to play a role in regulating its membrane association in response to certain stimuli (Takeuchi et al., 2012). While these data are interesting, the precise role for the proline/glycine-rich domain in DGK-θ has not been fully examined.

The above clearly highlights the need for a greater understanding of the structural features of DGK-θ as well as other DGKs. Much of our structural understanding of these enzymes comes from studies of two prokaryotic DGKs. The first is a DGK found in the gram-negative organism Escherichia coli. This enzyme is different from the mammalian enzymes in that in contains three membrane spanning domains and functions as a trimer (see Van Horn and Sanders, 2012). Another DGK, found in gram-positive organisms such as Staphylococcus aureus, is a soluble enzyme with 15–18% homology to human DGKs (Miller et al., 2008). The structural studies of this enzyme showed the active site appears to form at a homodimer interface. Further, the data suggest a putative DAG substrate recognition loop that may provide an important difference between DGKs and related enzymes such as YegS and NAD kinases that are not bona fide DGKs (Bakali et al., 2007; Nichols et al., 2007; see Miller et al., 2008). Importantly, there is a clear need for clear structural studies on eukaryotic enzymes to determine not only the catalytic site, but also the structure of likely regulatory domains, such as the N-terminus, which are not present in the prokaryotic enzymes.

Progress in understanding the structure of eukaryotic DGKs including DGK-θ has been hampered by our inability to purify a sufficient amount of enzyme in a uniform, monodisperse solution that would be amenable to classic methodologies such as NMR and X-ray crystallography. So far, only the SAM (sterile alpha motif) domain of DGKδ1 has been determined by x-ray crystallography at a resolution of 2.9 angstrom (Harada et al., 2008). This study indicated that oligomerization of the SAM domain in a head to tail orientation leads to the cytosolic sequestration, and inhibition, of this enzyme. In addition to this structure, a solution structure of the C1 domain of DGKδ1, and although a structure of DGKα has been deposited in a structure database, it has not yet been published. We are clearly in need of new and creative approaches to determine the structure of DGK-θ and other DGK isoforms.

DGK-θ belongs to a class of enzymes referred to as interfacial enzymes. These enzymes are generally soluble and have at least one insoluble substrate that is present in membranes or lipid aggregates. As a result, the enzymology of these enzymes, including DGK-θ, cannot be approached as is done for soluble enzymes with soluble substrates. Binding to membranes is often independent of catalysis and enzyme and substrate approach each other via diffusion within a membrane or lipid aggregate. Further complicating the analyses is the amount of time the enzyme resides on the interface, i.e., the enzyme's processivity, which may vary with the composition or architecture of the interface. We recently published a protocol (Tu-Sekine and Raben, submitted) that outlines an approach to the enzymology of these enzymes.

Analyses of DGK-θ present in cytosolic lysates illuminated two interesting aspects of its regulation (Tu-Sekine et al., 2006, 2007; Tu-Sekine and Raben, 2010). First, the enzyme is sensitive to both the bulk as well as surface concentration of the DAG substrate (Tu-Sekine et al., 2007). Second, the apparent Km (Km(app)) of the enzyme in these lysates was dependent on prior treatment of the cells. When DGK-θ was overexpressed in fibroblasts, the Km(app) of the enzyme for diacylglycerol (DAG) was increased in lysates obtained from mitogen-induced cells. The Km(app) then decreased with increasing bulk concentration of DAG. Additionally, the apparent Vmax (Vmax(app)) was increased in response to increases in the product, PtdOH (Tu-Sekine et al., 2007). It is noteworthy that the activation of DGK-θ in response to PtdOH occurs at 6-fold lower surface and bulk concentrations of DAG than observed when the activation occurs in response to phosphatidyserine (PtdSer). Importantly, the significant changes in the K_M and V_max values were observed only when physiological levels of both PtdOH and PtdSer were reached (Tu-Sekine et al., 2007).

To fully understand the enzymology, studies must be conducted on purified enzymes. One of the surprising discoveries when isolated DGK-θ was analyzed was that the enzyme is not fully active when purified. Full activity was observed when the purified enzyme was assayed in the presence of proteins rich in polybasic residues. In the presence of such polybasic cofactors, which directly interact with the enzyme, the apparent K(m) of the enzyme for DAG at the membrane surface (K(m)((surf))) was decreased These cofactors also decreased the Km for ATP. Furthermore, the cofactors synergistically increase the activity 10- to 30-fold in the presence of acidic phospholipids which were known to enhance enzyme activity (below).

There has been considerable interest in gaining a complete understanding of the enzymology and catalytic mechanism of DGK-θ and related kinases. In 2002, an interesting review appeared that highlighted the possibility that the similarity in phosphate-donor-binding sites may lead to a similar phosphorylation mechanism for four enzymes; DGKs, sphingosine kinases, NAD kinases, and phosphofructo-6-kinase (Labesse et al., 2002). Clearly, a confirmation of this hypothesisfor DGKs requires a comprehensive understanding of their structures. For example, it is possible that DGK-θ and other DGKs, act as a dimer similar to that suggested for DGKB in Staphylococcus aureus (Miller et al., 2008). Indeed, this may be a common paradigm for lipid kinases as another lipid kinase, sphingosine kinase 1, has also been suggested to function as a dimer (see Adams et al., 2016). As with enzymes in general, a complete understanding of DGK-θ's enzymology and catalytic mechanism will also provide critical information for the design of specific DGK-θ inhibitors.

As noted above, DGK-θ is very sensitive to the bulk and surface concentration of the DAG substrate. This provides a mechanism for regulating the enzyme in response to increases in DAG that accompany many signaling pathways. Additionally, as noted, DGK-θ activity is increased in response to PtdSer as well as PtdOH. Taken together, the data suggest that when DAG is generated in a signaling pathway, the Km(app) decreases and as the DAG concentration and PtdOH concentrations increase, the enzyme becomes more efficient. This could help explain the very transient nature of DAG increases in signaling pathways.

In addition to variations in membrane lipids, there are strong data supporting a role for protein-protein interactions that regulate DGK-θ activity. In 1999, Houssa et al. reported that activated (GTP-bound) RhoA associated with DGK-θ and inhibited its activity. Consistent with these data, Nurrish and colleagues have provided some fascinating data showing the homolog of DGK-θ in C. elegans, DGK-1, is regulated by RhoA and to modulate acetylcholine release at neuromuscular junctions (Nurrish et al., 1999; McMullan et al., 2006). As indicated above, there is a Ras-associating (RA) domain within the PH domain of DGK-θ and it has been tempting to speculate that this region was responsible for RhoA binding. Los et al., however, showed that RhoA bound to the C-terminal portion of the catalytic domain although they did not completely rule out another interaction with the RA domain (Los et al., 2004). While the precise RhoA binding regions have not been fully identified, a potential important role for these interactions is given support from the observations that other DGKs may show similar associations. For example, Tolias et al. presented evidence that a DGK associated with Rac1 (Tolias et al., 1998). This enzyme, however, is unlikely to be DGK-θ as another study showed DGK-θ does not associate with Rac1 or cdc42 (Houssa et al., 1999).

The role of phosphorylation in regulating DGK-θ is unclear. Using lysates of Cos-7 cells, PKC-ε has been shown to phosphorylate DGK-θ in vitro and studies using A431 cells suggested that this phosphorylation may promote membrane association (van Baal et al., 2005). Akt has also been shown to associate with and stimulate DGK-θ catalytic activity. Consistent with this, PI-3 kinase regulates the translocation and activation of DGK-θ in rat mesenteric arteries although, importantly, the mechanism of this regulation is unclear and may not involve DGK-θ phosphorylation (Walker et al., 2001; Clarke et al., 2007). Additional studies are clearly required to define the precise role of DGK-θ phosphorylation in membrane association and catalytic activation.

As indicated above, full activation of DGK-θ requires an acidic phospholipid and a polybasic cofactor. Additional analyses showed that while the acidic phospholipids recruit polybasic cofactors to the surface of artificial membranes, they do not affect the membrane association of DGK-θ suggesting interfacial association and catalytic activity are independently regulated. The data provide support for a model (Figure 2) in which DGK-θ exists as an auto-inhibited enzyme and that full interfacial activity requires a triad of enzyme, acidic lipid, and basic protein (Tu-Sekine et al., 2007). The enzyme is also likely to be modulated by the presence of RhoA or another small GTPase under physiological conditions. Importantly, the polybasic protein that represents the physiological regulator of DGK-θ in various tissues has/have not been identified and represents an important challenge in the field.

While we have learned quite a bit about DGK-θ, there is clearly much more to be discovered. Understanding the structure of DGK-θ will provide essential insights into its catalytic mechanism and regulation. Developing new strategies to elucidate the structure of this enzyme, and identification of physiologically relevant polybasic regulators will be key in our understanding of this enzyme. Such data will be critical in our efforts to develop specific inhibitors for this isozyme which are at present not available. The importance of such inhibitors will be highlighted as we learn more about the various physiological roles of DGK-θ. For example, if DGK-θ is indeed critical during intense neuronal stimulation, a specific inhibitor may be useful in the treatment pathophysiological conditions in which such stimulation plays an important role (e.g., epilepsy). Conversely, the enzyme may play a role under physiological conditions where rapid SV cycling is essential such as ribbon synapses of some sensory neurons. The future of research on DGK-θ promises to be exciting and impactful.

All authors listed, have made substantial, direct and intellectual contribution to the work, and approved it for publication.

DR is funded by the National Institutes of Health RO1NS077923.