While a complete review of the molecular mechanisms regulati

While a complete review of the molecular mechanisms regulating mammalian DGK activities is beyond the scope of this review, other reviews have covered much of this aspect (Shulga et al., 2011a; Topham and Epand, 2009; Tu-Sekine and Raben, 2011). In general, aside from enzyme kainate receptors levels, this regulation may involve one of more of the following: interactions with specific ions, protein-protein or protein-lipid interactions, post-translational modifications, membrane localization/substrate availability. By way of illustration, some isoforms (Class I DGKs) are sensitive to calcium levels, and responses to specific phospholipids varies among the various isoforms (Shulga et al., 2011b; Topham and Prescott, 2002). Additionally, phosphorylation may modulate activities of some DGK isoforms. For example, membrane association of DGK-δ (Imai et al., 2002) and DGK-θ (van et al., 2005), the nuclear localization of DGK-ζ (Topham et al., 1998), and intrinsic activity of DGK-α (Baldanzi et al., 2008) may involve phosphorylations. DGK-δ may also be regulated by oligomerization (Knight et al., 2010). In contrast, our understanding of protein modulators of DGK activities is less clear. DGK-ζ is activated by the hypo-phosphorylated Rb protein (pRb) as well as two related pocket proteins p107 and p130 (Los et al., 2006), while DGK-θ is inhibited by RhoA (Houssa et al., 1999), and is activated by proteins containing polybasic rich regions, termed polybasic activators (PBAs) (Tu-Sekine et al., 2013; Tu-Sekine and Raben, 2012). Importantly, how these mechanisms alter DGK conformations to affect activity remains unresolved. It is tempting to speculate that a hinge region similar to that seen in DGKB may be involved in modulating DGK structures but this appears to be unclear for SK1 (Wang et al., 2013). Clearly, there is a critical need for information regarding the three-dimensional structure of mammalian DGKs which will help elucidate the catalytic mechanisms. Solving these structures by rather conventional approaches, such as x-ray crystallography or NMR, have been hampered largely by the inability to obtain sufficient quantities of highly purified, monodispersed enzymes. The rapid development of single-particle cryo-electron microscopy (cryo-EM) has made it the most compelling technique for solving these challenging protein structures. Indeed, cryo-EM has recently been used to successfully solve the structure of a complex containing a lipid kinase, PtdIns4KIIIα, and two regulatory subunits designated TT7 and FAM126 (Lees et al., 2017). Importantly, besides solving large symmetric proteins, cryo-EM has been used to solve the structure of proteins size around 100 kD. Such small proteins were once thought to be beyond the resolution limit of cryo-EM but recent have advances have overcome the original limitations. For example, cryo-EM has been used to determine the structures of isocitrate dehydrogenase (IDH, 93 kD) and lactate dehydrogenase (LDH, 145 kD) at near atomic resolution (Merk et al., 2016). It is hoped that new approaches such as cryo-EM will finally allow us to obtain high resolution structural data of mammalian DGKs.