Encyclopedia of Signaling Molecules

2018 Edition
| Editors: Sangdun Choi

Diacylglycerol Kinase

Reference work entry
DOI: https://doi.org/10.1007/978-3-319-67199-4_101585

Synonyms

Historical Background

Diacylglycerol kinase (DGK) is the enzyme which phosphorylates diacylglycerol (DG) to produce phosphatidic acid (PA) (Goto et al. 2007). Its enzymatic activity was first described in cabbage a half century ago by Hokin and Hokin (1959). Since their report, DGK activity has been found widely in many animal species. The substrate of DGK, DG, is a lipid derived from various sources, including phosphatidylinositol 4, 5-bisphosphate by the action of phospholipase C (PLC), phosphatidylcholine by phospholipase D (PLD), monoacylglycerol (MG) by acyltransferase, and triglyceride (TG) by TG lipase. DGs from different sources have distinct acyl chain composition. At least 50 distinct molecular species of DG have been identified. In the mid-1980s, the biological significance of sn-1,2-DG was highlighted: It is an allosteric activator of protein kinase C (PKC). The PKC family comprises of three classes (conventional, novel, and atypical). Of those, the activity of novel PKC is dependent solely on DG whereas that of conventional PKC depends upon both DG and Ca2+. Along with accumulating reports of widely various PKC functions such as proliferation and differentiation, DGK has attracted much attention as a physiological regulator of PKC activity (Fig. 1). Because of technical difficulties, however, the purification of DGK had to wait until 1983, followed by molecular cloning of 80 kDa DGK from porcine brain in 1990. The significance of DGK in various cellular functions has been inferred because RasGRP, a canonical transient receptor potential channels, Unc-13, and protein kinase D were found to be activated by DG. Furthermore, PA, the product of DGK, is shown to work as a second messenger to activate signaling molecules such as mammalian target of Rapamycin (mTOR), hypoxia-inducible factor 1α (HIF1α), and PKCζ. To date, DGK cDNAs have been cloned in widely various species including Homo sapiens, Mus musculus, Rattus norvegicus, Escherichia coli, Drosophila melanogaster, Dictyostelium discoideum, Streptococcus mutans, and Arabidopsis thaliana.
Diacylglycerol Kinase, Fig. 1

DG pool and DGK. DG pool contains a couple of DGs with different acyl chain positions, which are produced from various sources including phosphatidylinositol 4, 5-bisphosphate (PIP2), monoacylglycerol (MG), and triglyceride (TG). Of these, sn-1,2-DG activates cPKC, nPKC, RasGRP, and TRPCs. DGK phosphorylates sn-1,2-DG into phosphatidic acid (PA). PA activates PKCζ, mTOR, and HIF1α

Distinct Molecular Structure of DGK Isozyme

Ten mammalian DGK isozymes have been reported. They are classified into classes I–V, based upon their distinct structures (Fig. 2). DGKα, β, γ are grouped as class I; δ, η, and κ as class II; ε as class III; ζ and ι as class IV; and θ as class V. All isozymes share a conserved catalytic domain in the C-terminal region and at least two zinc fingers (C1 domains). In addition to these domains, class I isozymes have two EF-hand motifs that bind Ca2+, whereas class II isozymes contain a pleckstrin homology (PH) and separated catalytic domains. DGKδ and η are characterized by a sterile α-motif (SAM) domain for oligomer formation at the C-terminus. DGKκ has a postsynaptic density protein-95/discs large/zona occludens-1 (PDZ)-binding domain instead of the SAM domain. This isozyme contains a Glu-Pro-Ala-Pro (EPAP) repeat at the N-terminus. DGKε solely constitutes class III DGK, which is the simplest, but uniquely has substrate specificity towards 2-arachidonoyl-DG. Class IV DGKζ and ι contain ankyrin-like repeat domain at the C-terminus and nuclear localization signal (NLS) overlapped with myristoylated alanine-rich C kinase substrate (MARCKS). Class V DGKθ has three zinc fingers, proline/glycine rich (PR) and Ras-associating (RA) domains.
Diacylglycerol Kinase, Fig. 2

Schematic representation of mammalian DGK isozymes. Ten DGK isozymes are grouped into classes I–V based on distinct molecular structures. PH pleckstrin homology domain, SAM sterile α-motif, EPAP Glu-Pro-Ala-Pro repeat, PDZ binding postsynaptic density protein-95/discs large/zona occludens-1 binding domain, MARCKS myristoylated alanine-rich C kinase substrate, NLS nuclear localization signal, PR proline/glycine rich domain, RA Ras-associating domain

Subcellular Localization and Functional Significance

DGK isozymes catalyze DG species irrespective of acyl chain composition except arachidonoyl-specific DGKε. What is the significance of the structural diversity of the DGK family? DG is a minor component of the plasma membrane, but it is also distributed to other intracellular compartments including the nucleus, endoplasmic reticulum (ER), and Golgi apparatus. It is apparently reasonable that DGK isozymes are targeted to distinct subcellular regions to handle DG in a unique manner. Transfection of cDNA and immunocytochemistry using specific antibodies have revealed detailed subcellular localization and the site-specific function of DGK isozymes (Fig. 3).
Diacylglycerol Kinase, Fig. 3

Subcellular localization and translocation of DGK isozymes. Each isozyme shows unique subcellular localization and distinct translocation pattern including nucleus-cytoplasm, cytoplasm-plasma membrane, and cytoplasm-endosome. DGKκ and ι stably localize to the plasma membrane and cytoplasm, respectively

Cytosolic DGKα Translocates to the Plasma Membrane in Response to Extracellular Stimuli

Cytoplasmic localization of DGKα has been reported in various cell types including T cells, canine kidney cell line MDCK cells, melanoma cell lines, rat aortic smooth muscle cells, rat hepatocytes, and rat adrenal medullary chromaffin cells. The functional significance of DGKα has been investigated intensely in T cells, in which DGKα is expressed abundantly. DGKα is reported to regulate interleukin-2 (IL-2)-induced cell proliferation positively through PA production. In this case, DGKα translocates to the plasma membrane in response to the stimuli. T cell receptor (TCR) stimulation also moves DGKα to the plasma membrane. However, DGKα negatively regulates TCR stimulation by inhibiting RasGRP activity. This seems to be the opposite effect to that of IL-2-induced cell proliferation. However, these contradictory actions may be explained by the fact that distinct downstream signaling cascades of TCR are differently regulated (Sakane et al. 2007).

DGKβ, Associated with Actin Cytoskeleton, Facilitates Its Remodeling in a Kinase-Dependent Manner

DGKβ is colocalized with actin cytoskeleton in transfected COS7 cells. DGKβ transfection alters actin stress fiber arrangement, whereas kinase-dead mutant abolishes the association with stress fibers (Kobayashi et al. 2007). Furthermore, detailed immunoelectron microscopy reveals that DGKβ localizes to the perisynaptic membrane on dendritic spines of neurons (Hozumi et al. 2008). Overexpression of DGKβ into hippocampal neurons induces dendritic outgrowth and spine maturation depending on its enzymatic activity. In microglia, transfected DGKβ localizes to the plasma membrane before stimulation and moves to the phagocytic cup to engulf IgG-opsonized glass beads (Ueyama et al. 2004). Since both spine formation and phagocytic processes are regulated by actin rearrangement, DGKβ may play a role in actin cytoskeletal reorganization.

Cell Type-Dependent Expression Pattern and Function of DGKγ

Cell type-dependent subcellular localization and function are reported on DGKγ. DGKγ localizes to the Golgi apparatus in transfected COS7 cells (Kobayashi et al. 2007) and immunostained rat aortic endothelial cells (Nakano 2015). The Golgi apparatus is the organelle for posttranslational modification and sorting of proteins synthesized in the ER, suggesting involvement of DGKγ in these functions. However, some reports have described that DGKγ may play a role in cytoskeletal reorganization. In the process of differentiation of leukemic cell lines (HL-60 and U937 cells) into macrophages in response to phorbol ester (DG analogue serving as a strong PKC activator), DGKγ translocates from the cytoplasm to the F-actin-labeled cell margin. Upon stimulation with growth factors, DGKγ is shown to regulate cytoskeletal organization through Rac1 activity. In this regard, DGKγ interacts with β2-chimerin, a Rac-specific GAP. DGKγ may negatively regulate macrophage differentiation via its catalytic activity (Sakane et al. 2007).

Cytoplasmic DGKδ Translocates to the Plasma Membrane in Response to Glucose Stimulation

Class II DGKs, DGKδ and η, localize to the cytoplasm but they behave differently after stimulation. Splicing variants of DGKδ (DGKδ1 and δ2) and DGKη reside in the cytoplasm of HEK293 and C2C12 myoblast cells (Takeuchi et al. 2012). Of those, only DGKδ1 translocates to the plasma membrane upon stimulation with high glucose. In this process, PH and C1 domains are responsible for this action. DGKδ2 and DGKη remain cytoplasmic after high-glucose stimulation, although DGKη translocates from the cytoplasm to endosomes by osmotic shock in transfected COS7 cells (Murakami et al. 2003).

DGKκ Stably Localizes to the Plasma Membrane

DGKκ stably localizes to the plasma membrane in HEK293 cells under basal and stimulated conditions (Imai et al. 2005), which contrasts sharply to other type II DGKs (δ and η). Functional significance of this isozyme remains unknown.

DGKε Resides in the ER and Is Implicated in Arachidonic Acid Metabolism

DGKε, which localizes to the ER in transfected COS7 and HeLa cells (Kobayashi et al. 2007), contains no specific ER retention signal such as the KDEL sequence, but the hydrophobic region in the N-terminus determines its ER localization (Matsui et al. 2014). The ER plays a pivotal role in protein and lipid synthesis and Ca2+ homeostasis. As described above, DGKε is a unique isozyme with respect to substrate specificity towards arachidonoyl-DG. Arachidonate is a component of phospholipids, especially in phosphoinositide (PI), of the plasma and ER membranes. Cyclooxygenase (COX), which plays a central role in arachidonate metabolism, also localizes to the ER. Under recurrent epileptic seizures, it is noteworthy that the COX-2 level is higher in the hippocampus of DGKε-knockout (KO) mice than the wild-type mice (Lukiw et al. 2005). DGKε may be intimately involved in the metabolism of PI together with COX-2.

DGKζ Shuttles Between the Nucleus and Cytoplasm

DGKζ, which is characterized by NLS, is well known as “nuclear DGK.” In both cDNA transfection and immunocytochemistry, DGKζ is detected in the nuclei of neurons, alveolar epithelial cells, macrophages, and aortic smooth muscle cells. Under conditions of transient ischemia and kainate-induced seizures, DGKζ translocates from the nucleus to cytoplasm. It never relocates to the nucleus in hippocampal neurons. In addition to NLS, the nuclear export signal (NES) is identified in DGKζ. Subcellular localization of DGKζ may be determined by the balance between the NLS/importin and NES/exportin systems (Goto et al. 2014).

DGKι Localizes to the Cytoplasm Irrespective of NLS

DGKι belongs to class IV DGK. This isozyme contains the NLS similar to DGKζ but localizes to the cytoplasm (Ito et al. 2004). Deletion of DGKι is shown to reduce Ras signaling through Rap1 activity (Regier et al. 2005), suggesting involvement in Ras signaling.

DGKθ Resides in the Nucleus and Cytoplasm and Translocates to the Plasma Membrane

DGKθ localizes mainly to the nucleus of MDA-MB-453, MCF-7, PC12, HeLa, and IIC9 cells. In H295 human adrenocortical cells, DGKθ is activated transcriptionally by a complex of nuclear steroidogenic factor-1 (SF-1) and sterol regulatory element binding protein 1 (SREBP1) in response to cAMP stimulation, thereby leading to glucocorticoid production (Cai and Sewer 2013). DGKθ responds to cAMP both in the cytoplasm and the nucleus, but nuclear DGKθ is more responsive to cAMP. In A431 and HEK293 cells, DGKθ translocates from the cytoplasm to plasma membrane in a PKC-dependent manner and regulates epidermal growth factor receptor (EGFR) signaling (van Baal et al. 2012).

DGKs in Health and Diseases

Numerous reports of investigations of human patients, animal models, and population studies suggest that DGKs are closely associated with disease pathogenesis (Nakano 2015).

Cancer

Cancer cells reach an abnormally hyperproliferative state. The primary function of DGK is an attenuator of PKC activity via DG metabolism. Therefore, DGK downregulation theoretically engenders prolonged activation of PKC, thereby stimulating cell proliferation, a characteristic feature of cancer pathogenesis. However, downregulation of DGKδ engenders activation of PKCα and subsequent PH domain leucine-rich repeat protein phosphatase 2 (PHLPP2), resulting in Akt dephosphorylation. DGKη can modulate EGFR signaling. Its downregulation reduces cellular proliferation in HeLa cells. In addition, DGKη downregulation reduces the proliferation of human lung cancer cells. DGK inhibitor R59949 strongly inhibits hepatocyte growth factor (HGF)-induced cellular proliferation in Kaposi’s sarcoma. Taken together, under certain conditions, DGK downregulation exerts an inhibitory, rather than stimulatory, effect on cellular proliferation. These findings suggest that DGK regulates not only DG-PKC pathway but also other signaling pathways.

Ischemia

In brain ischemia, DGKζ is deeply involved in the pathogenesis in both neurons and nonneuronal cells. Neurons are susceptible to cell damage by hypoxia because of their high metabolic rate. Transient loss of blood supply easily engenders ischemic injury. In rat forebrain ischemia, DGKζ disappears rapidly from the nucleus and translocates to the cytoplasm of hippocampal CA1 neurons. This phenomenon is recapitulated in an acute hippocampal slice culture model under oxygen-glucose deprivation (Goto et al. 2014). In an infarction model of rat brain, DGKζ-immunoreactivity disappears rapidly in cortical neurons. It is particularly interesting that the immunoreactivity reappears in endothelial cells and activated microglia.

Diabetes

Reduced expression of DGKδ in skeletal muscles of type II diabetes patients and diabetic rats has been reported. Skeletal muscles of DGKδ-KO mice are characterized by defects in glucose usage, together with reduced tyrosine phosphorylation of insulin receptor and insulin receptor substrate-1 (IRS-1), suggesting insulin-resistant states in DGKδ-deficient skeletal muscles. Decreased levels of DGKδ engender PKCδ activation, which results in serine phosphorylation of IRS-1 and subsequent insulin resistance. Another study reveals that DGKδ deficiency causes decreased activity of AMP-activated protein kinase (AMPK), which is known to be an energy sensor in skeletal muscles (Jiang et al. 2016). Consequently, modulating DG levels by DGK seems to play an important role in the regulation of glucose homeostasis. A single nucleotide polymorphism (SNP) study of human population has shown that DGKε polymorphism is associated with higher plasma TG levels. Taken together, these studies suggest that DGKs are deeply involved in both glucose and lipid metabolism. Further investigations are warranted to unveil precise mechanisms of DGK in the pathogenesis of metabolic syndromes such as diabetes and obesity.

Kidney Disease

DGKε is implicated in the field of kidney diseases such as hemolytic uremic syndrome. Mutations in DGKε gene cause thrombotic microangiopathy, characterized by endothelial injury of small vessels (Lemaire et al. 2013). In the pathogenesis of DGKε-KO mice, reduced prostaglandin production induces cell adhesion, causing endothelial cell injury in kidney glomeruli (Zhu et al. 2016).

Summary

DGK phosphorylates DG to produce PA. DG serves as an activator of conventional and novel classes of PKC, RasGRP, TRPCs, etc. PA also works as an activator of PKCζ, HIF1α, etc. These facts suggest that DGK acts as a hub between DG-mediated and PA-mediated signalings. To date, ten mammalian isozymes have been reported. These isozymes constitute the enzyme family, which comprises five subgroups (class I, α, β, γ; class II, δ, η, κ; class III, ε; class IV, ζ, ι; and class V, θ) depending upon their distinct molecular structures. Each DGK isozyme exhibits diverse subcellular localization and translocation patterns. DGKα localizes to the cytoplasm and translocates to the plasma membrane in response to extracellular stimuli. DGKβ is associated with actin cytoskeleton. DGKγ localizes to the Golgi apparatus and may be associated with cytoskeleton. DGKδ/η localizes mainly to the cytoplasm and translocates to the plasma membrane. DGKκ stably localizes to the plasma membrane. DGKε resides in the ER. DGKζ and θ are nuclear isozymes, but they translocate to the cytoplasm or the plasma membrane. DGKι is in the cytoplasm irrespective of the NLS. DG includes at least 50 molecular species, but only DGKε shows substrate specificity towards arachidonoyl-DG. DGKs are implicated in widely various diseases including cancer, diabetes, ischemia, and kidney diseases. The original idea on DGK function was based upon the regulation of PKC activity, the so-called DG-PKC pathway, but diverse effects on the other signaling pathways are emerging now.

See Also

References

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© Springer International Publishing AG 2018

Authors and Affiliations

  1. 1.Department of Anatomy and Cell BiologyYamagata University School of MedicineYamagata CityJapan