DGKα: History
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Contributor:
José Carlos Bozelli
,
Richard M. Epand

Cancer immunotherapy has revolutionized the oncology field. Despite the success, new molecular targets are needed to increase the percentage of patients that benefits from this therapy. Diacylglycerol kinase α (DGKα) has gathered great attention as a potential molecular target in immunotherapy because of its role in cancer proliferation and immunosuppression. DGKα catalyzes the ATP-dependent phosphorylation of diacylglycerol (DAG) to produce phosphatidic acid (PA). Since both lipids are potent signaling messengers, DGKα acts as a switch between different signaling pathways. Its role in cancer and immunosuppression has long been ascribed to the regulation of DAG/PA levels. However, this paradigm has been challenged with the identification of DGKα substrate acyl chain specificity, which suggests its role in signaling could be specific to DAG/PA molecular species. In several biological processes where DGKα plays a role, large membrane morphological changes take place. DGKα substrate specificity depends on the shape of the membrane that the enzyme binds to. 

  • cancer
  • immune surveillance
  • immunotherapy
  • diacylglycerol kinase α

1. Introduction

Cancer continues to be one of the major health problems globally, bringing extreme physical, emotional, and financial strain on individuals, communities, and healthcare systems. However, in the past ten years, there has been a tremendous improvement in the survival and quality of life of cancer patients, especially terminal ones. This was brought by a change in the landscape of cancer treatment from previous standard of care (e.g., chemo- and radiotherapy) to the new era of immunotherapy [1][2][3]. In cancer immunotherapy, the idea is to resume the ability of the immune system, e.g., T-cells, to recognize and fight tumor cells. Cancer immunotherapy has revolutionized the oncology field and it has now established itself as a pillar of cancer treatment. One of the strategies in cancer immunotherapy is the immune checkpoint blockade, which relies on blocking negative regulators of T cells and, therefore, incites powerful T cell responses [4][5][6]. Immune checkpoint inhibitors often target receptors on the surface of immune cells, which in a tumor microenvironment are acting to silence the activation of T cells. Despite the increased success of immune checkpoint blockade against a range of cancers, currently only a small percentage of patients show significant improvement in health outcomes. Hence, currently, there is a need to find new molecular targets aiming at improving efficacy and broaden the use of immune checkpoint blockade.
Recently, there has been an increased interest in diacylglycerol kinase α (DGKα), an intracellular lipid kinase, as a potential target in immune checkpoint blockade [7][8][9]. This is because the silencing of DGKα activity provides a two-pronged attack on cancer cells. On the one hand, DGKα has been found to increase the survival, proliferation, migration, and invasion of some cancers and, therefore, its inhibition has been shown to prevent tumorigenesis [9][10][11][12][13][14]. On the other hand, DGKα has been shown to decrease activation and increase anergy, a hypo-responsive state, in T cells and, therefore, its inhibition has been shown to incite powerful T cell responses leading to greater immune clearance of cancer cells [15][16][17][18]. DGKα is an interfacial enzyme that utilizes ATP to phosphorylate diacylglycerol (DAG) at the membrane-water interface, yielding phosphatidic acid (PA). Both DAG and PA are potent lipid signaling molecules. It has been proposed that the DGKα role in cancer and immune cell biology is due to its ability to regulate the levels of these two lipid messengers. However, an idea that started to emerge is that it is not only the levels of DAG and PA, but also the nature of their molecular species that plays a role in the biological processes controlled by DGKα. This is based on the finding that DGKα exhibits substrate acyl chain specificity for its DAG substrate, a property that has been shown to be modulated by the shape of the membrane the enzymes bind to [19][20][21][22][23].

2. DGKα

2.1. Enzymatic Reaction and Substrate Acyl Chain Specificity

DGKα is a member of the family of lipid kinases namely diacylglycerol kinases (DGK). In humans, ten paralogues of DGK have been identified. The presence of gene splice variants increases the number of members of this family, highlighting the importance of the enzymatic reaction carried out by this family of enzymes. All DGK catalyze the same biochemical reaction, i.e., the ATP-dependent phosphorylation of DAG to produce PA. However, the different expression profile, subcellular location, structure, regulatory motifs, and substrate specificity suggest that each DGK paralog bears different biological functions, albeit catalyzing the same biochemical reaction. While for a long time it was believed that DGKε was the only paralog with substrate acyl chain specificity, recently it was shown that DGKα also presents substrate acyl chain specificity [20][24]. In celulla, it has been shown that DGKα bears specificity for DAG molecular species containing saturated/monounsaturated acyl chains [19][21][22]. This same acyl chain specificity was corroborated by a systematic in vitro study with purified DGKα and model membranes of variable physicochemical properties [23].

2.2. Structural Properties

DGKα is an 80 kDa cytosolic DGK that in healthy humans has enhanced expression in esophagus, skin, and lymphoid tissue (www.proteinatlas.org (accessed on 9 August 2022)) [25]. DGKα is one of three type I DGK, which are characterized by bearing EF hand motifs. The domain architecture of DGKα involves the presence of two EF hand motifs in the N-terminus followed by two C1 domains, the catalytic domain, and an accessory domain close to the enzyme C-terminus. The EF hand motifs are regulatory motifs that bind Ca2+, while the C1 motifs have been shown to mediate DAG acyl chain specificity [22][26]. The catalytic domain is a conserved domain of lipid kinases responsible for carrying out the enzymatic reaction and the accessory domain, believed to assist with catalysis. The tridimensional high-resolution structure of full length DGKα has not been solved experimentally yet. However, recent advancements in the prediction of the structure of proteins by artificial intelligence has shed some light on its structure [27][28]. The Alphafold predicted structure of DGKα yields a globular protein where most of its domains are alpha-helical folded and the accessory domain exhibit mixtures of alpha-helix and beta-sheet secondary structures.
A pocket between the catalytic and accessory domains is believed to be the putative active site of the enzyme. This region in DGKα exhibits a high degree of conserved residues when comparing the sequence of the ten human DGK paralogues. This putative active site has a positively charged electrostatic surface and ATP, which is negatively charged, docks into this pocket. Indeed, recently it has been shown by in silico studies that this region is a conserved ATP-binding pocket in all ten human DGK paralogues [29]. Of the three type I DGKs, DGKα is the only one that is sensitive to changes in physiological levels of Ca2+, the other two paralogues have a higher affinity for Ca2+ and always have Ca2+ bound in vivo. It has been shown that the binding of Ca2+ to the EF hand motifs in DGKα triggers a conformational change in the enzyme N-terminus, which exposes active and hydrophobic sites promoting membrane binding [30]. The tridimensional high-resolution structure of the isolated EF hand motifs of DGKα bound to Ca2+ has been solved experimentally by X-ray crystallography [31]. When the structure of the isolated domains is overlapped with the predicted structure of the full-length enzyme, there is high degree of structural homology. In addition, the EF hand motifs are located away from the putative active site suggesting that the predicted structure could be the active form of the enzyme.

2.3. Subcellular Localization

DAG and PA are both lipid molecules, which have low water solubility and are mainly found embedded into the membrane. Hence, membrane interaction is a requirement for DGKα enzymatic catalysis. In resting cells, DGKα is found mainly in the cytosol [32]. However, upon cell stimulus DGKα has been described to show a rapid and transient partition to different biological membranes. For instance, activation of T-cell antigen receptor (TCR) or tyrosine kinase receptors triggers binding of DGKα to the plasma membrane (PM), which is dependent on both calcium binding to the EF hand domain of DGKα and Src (proto-oncogene tyrosine protein kinase)/Lck (lymphocyte-specific protein tyrosine kinase)-dependent phosphorylation of Tyr335 [12][17][26][33][34][35][36][37][38][39]. In lymphocytes, recruitment of DGKα to PM has also been shown to occur in response to an increase in the levels of phosphatidylinositol-3-kinase (PI3K) lipid products, i.e., phosphatidylinositol-3,4,5-triphosphate (PI-3,4,5-P3) and phosphatidylinositol-3,4-bisphosphate (PI-3,4-P2) [38]. In addition, upon TCR activation, DGKα has also been found associated with intracellular membranes involved in the secretory vesicle pathway, e.g., trans-Golgi network (TGN), endosomes, multivesicular bodies (MVB), and exosomes [40][41][42]. Furthermore, interleukin 2 (IL2) stimulation of T cells, which promotes proliferation of T cells, triggers DGKα translocation to the perinuclear region [32]. It, thus, seems that DGKα is recruited to different membranes in a stimulus-specific context. Therefore, DGKα could access different pools of DAG, which, in turn, would lead to its role in different biological processes.

2.4. Modulation of DGKα by Membrane Physicochemical Properties

The description at the molecular level on how DGKα interacts with the membrane is not fully understood yet. However, it has been shown that membrane binding in intact cells is dependent on the C1 domains and the Pro-rich segment on the enzyme C-terminus [36]. Indeed, positioning of the Alphafold predicted tridimensional structure of DGKα on a model of mammalian PM shows that the energetically optimal spatial position involves interaction of DGKα C1 domains and Pro-rich segment at the C-terminus as well as the accessory domain with the PM. The predicted interaction between DGKα and the membrane is energetically favored (ΔGwater→membrane ≈ −13 kcal/mol) and the enzyme has a shallow insertion (~7 Å) into the membrane hydrophobic core. In this spatial arrangement, the surfaces of the catalytic and C1 domains laying juxtaposed to the membrane is positively charged. This would favor an electrostatic interaction with negatively charged lipids, which might approximate the catalytic site to the membrane interface, which, in turn, would promote enzymatic catalysis. This agrees with the fact that negatively charged lipids activate the enzyme (see below).
DGKα enzymatic properties are highly sensitive to the physicochemical properties of the membrane the enzyme binds to. It has been shown that major lipid components of the PM, one of the main biological membranes where DGKα exerts its biological function (see above), could either inhibit or activate DGKα enzymatic activity. For instance, sphingomyelin has been shown to inhibit DGKα in the presence of Ca2+ [43]. In the absence of Ca2+, DGKα can be activated by phosphatidylethanolamine (PE) and cholesterol [43]. In addition to these zwitterionic/neutral lipids, an anionic lipid enriched in the PM, i.e., phosphatidylserine (PS), has also been shown to activate DGKα [26][44]. It has been proposed that PE and cholesterol might induce a conformational change on the enzyme N-terminus, which, in turn, would expose the enzyme active site in a similar fashion to the one induced by Ca2+-binding to EF hand motifs [43]. On the other hand, PS activation has been proposed to be due to an interaction with the catalytic domain, which might serve to orient the catalytic site [44]. In addition to major lipids, potent lipid second messengers that are often present in low amounts at the membrane have been reported to regulate DGKα. It has been shown that PI-3,4-P2, PI-3,4,5-P3, and sphingosine activate DGKα [38][45][46][47]. Interestingly, while these PIPn are highly negatively charged, sphingosine is a positively charged lipid. Therefore, it seems that the modulations of DGKα by these lipid second messengers are occurring by interaction with different regions of the enzyme.
The activity of DGKα is sensitive to the lipid composition of the membrane the enzyme binds to. However, lipid composition does not seem to affect the DAG acyl chain specificity of the enzyme [48][49]. On the other hand, DGKα DAG acyl chain specificity seems to be modulated by membrane physical properties, i.e., to the shape of the membrane the enzyme binds to [23]. It has been shown that DGKα exhibits no preference for the molecular species of DAG when assayed in detergent micelles or in flat planar bilayer membranes. For instance, when the enzymatic activity was assayed in liposomes using four different DAG molecular species of DAG (i.e., 18:0/20:4-DAG, 18:1/18:1-DAG, 16:0/18:1-DAG, and 16:0/16:0-DAG), DKGα exhibited similar affinity as well as catalytic turn-over for all four DAG molecular species, indicating the enzyme bears no DAG acyl chain specificity in a flat planar bilayer. However, when the membrane phase is changed to increase the physical curvature of the interface, there is a preference for DAG species with saturated/monounsaturated acyl chains [23]. For instance, the enzymatic activity in curved membranes was found to be ca. 10-fold higher in comparison to flat membranes if the DAG species were either 16:0/18:1-DAG or 16:0/16:0-DAG, while the activity only increased ca. 2-fold if the DAG species were either 18:0/20:4-DAG or 18:1/18:1-DAG. These are the same molecular species found to be specific for DGKα activity in cells [19][21][22]. Thus, the substrate specificity depends on the shape of the membrane to which the DGKα is bound. This phenomenon is independent of membrane curvature bending strain but is dependent on membrane shape and possibly on the presence of Gaussian curvature [23]. An analogous effect of membrane shape on substrate preference was initially shown for DGKε [50]. Although it has been appreciated for many years that some biological processes are sensitive to curvature at a large length scale where curvature strain would not be present, it had not been demonstrated at the level of molecular events [51]. This has changed since the reports that DGKα/ε substrate acyl chain specificity is modulated by membrane shape and not curvature strain [23][50]. Hence, membrane shape modulation of enzymatic properties is a modern concept, which is proposed to be broadly found in different biological processes.

3. DAG and PA: Intermediates of Lipid Metabolism, Membrane Curvature Generators and Signaling Molecules

DAG and PA are lipids present in low levels in biological membranes. However, upon cell stimulus, their levels are rapidly and transiently increased [52][53][54]. The levels of these lipids are highly regulated since both lipids are: (i) key intermediates in lipid metabolism, (ii) precursors for the biosynthesis of new lipids, (iii) important for the generation of membrane curvature, and (iv) potent signaling molecules. Hence, both lipids are critical for maintaining cellular homeostasis [53][54][55].
DAG and PA are key intermediates in the metabolism of glycerophospholipids (e.g., PA, PE, phosphatidylcholine—PC, phosphatidylinositol—PI, PS) and triacylglycerols [53]. DAG and PA also play a role in modulating the physicochemical properties of the membrane they are embedded into. DAG has a hydroxyl group as the headgroup and, therefore, it is a neutral lipid. On the other hand, PA has a phosphate group as a headgroup and, consequently, it is negatively charged. PA has a second pKa around physiological pH and, therefore, it can exist as an equilibrium between mono- or divalent anionic species [52]. Each of these lipids are present in cells as a variety of molecular species due to differences in length (i.e., 14–22 carbon atoms long) and unsaturation (i.e., 0–6 unsaturation) of the acyl chains. These differences in the chemistry of these lipids contribute to the different interactions with downstream signaling molecules. In addition, both lipids present a conical molecular shape, which makes them inducers/stabilizers of membrane negative curvature [56][57]. Their molecular shape enables the penetration of proteins into the membrane–water interface by decreasing the lipid packing at the headgroup region. Indeed, they are often found to regulate biological processes where large membrane morphological changes take place, where they facilitate vesicle budding and fusion [58].
DAG signaling is important for a variety of cellular processes including cell proliferation, survival, motility, and membrane trafficking and secretion [59]. DAG triggers downstream signaling by recruiting proteins containing DAG binding motifs, i.e., C1 domains, to the membrane. Proteins that are regulated by DAG include protein kinase C (PKC), protein kinase D (PKD), Ras guanyl nucleotide-releasing protein 1 (RasGRP1), and chimaerins [59]. Recently, it has been shown that PKC is sensitive to the nature of the DAG molecular species, i.e., different PKC bind different DAG molecular species in live cells [60]. Contrary to the well-established role of DAG in cellular signaling, PA signaling was long believed to occur, but the lack of the identification of PA-binding domains in proteins led to an elusive role. More recently this has changed, and it has been shown that PA can regulate a variety of biological processes due to the regulation of proteins involved in cell growth, differentiation, migration, and membrane trafficking [20][54]. Proteins regulated by PA include mammalian target of rapamycin (mTOR), PKCε/ζ, phospholipase C (PLC) β1/γ1, Ras GTPase activating protein (RasGAP), chimaerin, ADP-ribosylation factor 1 (Arf1), and Ras-related C3 botulinum toxin substrate 1 (Rac1) [20][54]. It also seems that some of these proteins present specificity for the molecular species of PA they bind to [20]. For a long time, DAG/PA cellular signaling was believed to be dependent on the regulation of their levels in different biological membranes. This paradigm seems to be challenged by the identification of proteins that have specificity for different molecular species DAG/PA. It thus seems reasonable to propose that contrary to the long-believed concept, it is currently thought that DAG/PA signaling is not solely a result of change in their concentrations, but also of the change in the concentration of specific molecular species of DAG/PA.

4. DGKα in Cell Biology

4.1. T Cells

Both DAG and PA are potent lipid signaling molecules. Therefore, most of what is known about the role of DGK in cell biology is a consequence of either attenuation of DAG and/or intensification of PA signaling pathways. Since DGKα is highly expressed in T cells, its roles in T cell biological processes have been best characterized [61]. For instance, DGKα controls T cell polarity at the immunological synapse (IS), i.e., at the interface between antigen-presenting cell (APC) and T cells [62]. Upon TCR activation, a signaling platform is formed in response to membrane remodeling and organelle assembly (e.g., Golgi apparatus, secretory lysosomes, mitochondria) at the inner leaflet of the PM of T cells around the IS [62]. Polarization of the microtubule organizing center (MTOC) of T cells towards the IS allows directionality in the secretory pathway towards the APC. In this process, it has been shown that DGKα controls MTOC polarity by limiting the diffusion of DAG (through its conversion into PA) from the periphery of the IS, where it is preferentially localized likely via a specific interaction with PIP3 [63]. DGKα has also been shown to play a role in T cell proliferation, whereupon T cell stimulation with IL2 DGKα translocates from the cytosol to the perinuclear region, where the production of PA by DGKα has been associated with its mitogenic properties [32]. In addition to these positive effects of DGKα in T cell biology, DGKα has also been shown to exhibit negative effects on T cells, often through its increased expression and, consequently, decrease in DAG signaling. For instance, in the absence of costimulatory signals or weak activation of TCR, DGKα expression is increased and, consequently, there is an attenuation of DAG signaling at the PM [15][17]. This leads to a hyporesponsive state in T cells, namely anergy [64]. This is a consequence of decreased recruitment of RasGRP1 to the PM, which is dependent on DAG, and, therefore, inactivation of extracellular signal-regulated kinase (ERK) signaling pathway [16][18]. In addition, DGKα overexpression downregulates secretory vesicular trafficking at the IS in T cells. Upon TCR activation, T cells can secrete exosomes through a series of membrane fusion/fission events, which starts with vesicle budding at the TGN and ends with the fusion of MVB with the PM to secrete exosomes containing soluble factors towards the target cell. It has been shown that increased DGKα expression downregulates the formation of MVB and exosomes secretion [40][41][42]. It has been proposed that this is a consequence of DGKα kinase activity at the TGN, which, in turn, inhibits PKD, a key enzyme to control secretory vesicle budding in the TGN, and, therefore, the formation of MVB [40][41][42].
It is important to mention that DGKα is not the only paralogue that is highly expressed in T cells, DGKζ is also highly expressed and, therefore, DGKζ also plays a role in the biology of T cells by regulating DAG/PA levels. DGKα and DGKζ show redundant and specialized roles in T cell biology. For instance, both DGKα and DGKζ have been shown to modulate DAG levels at the IS, which is critical for proper organization of the signaling platform [63][65][66]. On the other hand, expansion of innate-like cytotoxic T lymphocytes mediated by cytokine and independent of antigen stimulation is predominantly limited by DGKζ activity [67]. The study of the molecular mechanisms governing the function of DGKα and DGKζ in T cell biology is a field of increased interest. However, in this entry, the primary focus is on DGKα and the reader is referred to excellent reviews addressing the topic [61][68][69]. Nevertheless, it will be of interest to evaluate the modulation of DGKζ enzymatic activity and/or DAG substrate acyl chain specificity by membrane shape and how this compares to DGKα. This might help to clarify the redundant/specialized roles of these enzymes in T cell biology.

4.2. Cancer Cells

DGKα is also highly expressed in various cancer cells, where it has been shown to be involved in cancer cell survival, proliferation, migration, and invasiveness [9][10][11][12][70]. While DAG attenuation has been associated with the majority of DGKα roles in T cell biology, it seems that the role of DGKα in cancer cell biology is mainly associated with PA production. In human hepatocarcinoma, it has been shown that DGKα is associated with tumor progression by activation of the mitogen-activated protein kinase (MAPK) pathway [13]. In melanoma, DGKα has been shown to inhibit apoptosis by activating tumor necrosis factor α (TNFα)-induced activation of nuclear factor kappa-light chain enhancer of activated B cells (NF-κB), which was proposed to be a result of DGKα-produced PA activating PKCζ-mediated phosphorylation of S311 of NF-κB p65 [10][71]. In glioblastoma cells, DGKα production of PA has been associated with inhibition of apoptosis and regulation of hypoxia-inducible factor 1-α (HIF1-α) and mammalian target of rapamycin (mTOR) oncogenic pathways [14][70]. In ovarian cancer, it has been shown that DGKα promotes platinum resistance by producing PA, which activates the transcription factor c-JUN and, consequently, enhancing the transcription of cell-cycle checkpoint regulator WEE1 gene [72]. In esophageal squamous cell carcinoma, PA production by DGKα has been associated with the activation of Akt/NF-κB signaling, which, in turn, reduced cAMP levels and PTEN activity leading to tumor progression [73]. DGKα has also been shown to promote tumor invasion and progression by generating PA at invasive pseudopods triggering the localization of atypical PKC, which control Rac-mediated protrusion elongation and Rab-coupling protein (RCP)-dependent integrin recycling [74][75]. In several cases, inhibition of DGKα in the cancer models described above has been associated with antitumor properties.

This entry is adapted from the peer-reviewed paper 10.3390/cancers14215259

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