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. Author manuscript; available in PMC: 2012 Dec 15.
Published in final edited form as: FEBS Lett. 2011 Nov 19;585(24):4025–4028. doi: 10.1016/j.febslet.2011.11.016

Substrate Specificity of Diacylglycerol kinase-epsilon and the Phosphatidylinositol Cycle

Yulia V Shulga 1, Matthew K Topham 2, Richard M Epand 1
PMCID: PMC3237191  NIHMSID: NIHMS341570  PMID: 22108654

Abstract

We show that diacylglycerol kinase-ε (DGKε) has less preference for the acyl chain at the sn-1 position of diacylglycerol (DAG) than the one at the sn-2 position. Although DGKε discriminates between 1-stearoyl-2-arachidonoyl-DAG and 1-palmitoyl-2-arachidonoyl-DAG, it has similar substrate preference for 1-stearoyl-2-arachidonoyl-DAG and 1,2-diarachidonoyl-DAG. We suggest that in addition to binding to the enzyme, the acyl chain at the sn-1 position may contribute to the depth of insertion of the DAG into the membrane. Thus, the DAG intermediate of the PI-cycle, 1-stearoyl-2-arachidonoyl-DAG, is not the only DAG that is a good substrate for DGKε, the DGK isoform involved in PI-cycling.

Keywords: Diacylglycerol kinase, diacylglycerol, polyunsaturated acyl chain, phosphatidylinositol cycling, acyl chain specificity

1. Introduction

Diacylglycerol kinases (DGKs) phosphorylate diacylglycerol (DAG), a second messenger involved in cell signalling, to produce phosphatidic acid (PA), which has signalling roles as well [1]. It is now widely accepted that conversion of DAG to PA by DGKs is the major pathway to remove the potent signaling molecule DAG.

It appears that the physiologically relevant DAGs are those containing a polyunsaturated acyl chain in the sn-2 position. DGKε is the only DGK isoform that shows substrate specificity in vitro for DAG with an arachidonoyl acyl chain at the sn-2 position [2,3]. Phosphorylation of 1-stearoyl-2-arachidonoyl-DAG (18:0/20:4-DAG), catalyzed by DGK, is the first step in the resynthesis of phosphatidylinositol (PI). It was shown that among all the isoforms of DGK, DGKε appears to be the most important for catalyzing this step in the PI cycle, since deletion of this enzyme significantly decreases the amounts of both PI and PA in the plasma membrane of the cells [4]. 1-stearoyl-2-arachidonoyl-DAG is formed as a result of phosphatidylinositol-4,5-bisphosphate-specific phospholipase C catalyzed hydrolysis of phosphatidylinositol-4,5-bisphosphate that itself is highly enriched in arachidonic acid at the same position. Thus, DGKε may be responsible for down-regulating the DAG signalling resulting from phosphatidylinositol cycling.

The product of the reaction catalyzed by DGK, PA, is also involved in the regulation of a wide variety of cellular events, including cell survival, cytoskeletal rearrangement and proliferation [5]. It is believed that each PA species can differentially activate proteins depending on the saturation and length of the acyl chains [6]. It was shown that the PA produced by different DGKs can fulfill different roles in the cell. Thus, PA produced by DGKε is enriched in polyunsaturated fatty acids, particularly arachidonate, and it is involved in the PI cycle. PA produced by DGKα is necessary to progress to S phase of the cell cycle in stimulated T lymphocytes [7] and PA produced by DGKζ is involved in the initialization of the cascade to cause actin rearrangements [8]. Therefore, DGK substrate specificity is crucial for the regulation of many cellular processes. In the present work we studied the substrate specificity of DGKε and for the first time showed that the DAG intermediate of the PI cycle, 1-stearoyl-2-arachidonoyl-DAG (18:0/20:4-DAG), is not the only preferred substrate for DGKε, but that this enzyme exhibits similar preference towards 1,2-diarachidonoyl-DAG (20:4/20:4-DAG).

2. Materials and methods

2.1. Preparation of Sf21 cells overexpressing DGKε and DGKζ

Baculovirus-infected Sf21 cells overexpressing either human DGKε with a C-terminal hexahistidine (DGKε-His6) or DGKζ with a C-terminal FLAG epitope (DGKζ-FLAG) were prepared as previously described [9].

2.2. Enzyme Preparations for Enzymatic Activity Assay

Prior to assay, baculovirus-infected Sf21 cells overexpressing either human DGKε-His6 or DGKζ-FLAG were resuspended in ice-cold cell lysis buffer (1% (v/v) (octylphenoxy)polyethoxyethanol (Nonidet P-40), 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM activated sodium orthovanadate, and 1:100 protease inhibitor cocktail for use with mammalian cells and tissue (Sigma-Aldrich)), allowed to lyse for 10 minutes on ice, sonicated for 5 minutes and then centrifuged at 100,000 g, 30 min at 4 °C. The supernatants were used in the assay of DGK activity.

2.3. Quantification of Phosphatidic Acid

The concentration of all PA stocks used in this study was determined experimentally based on their phosphate content, as described previously [10].

2.4. Detergent-Phospholipid-Mixed Micelle-based DGK Enzymatic Activity Assay

DGK was assayed for enzymatic activity using a detergent-phospholipid-mixed micelle-based protocol described by Walsh et al. [2] as previously employed in our laboratory [11]. Lipid films composed of the substrate (DAG) and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC, for DGKε) or 1,2-dioleoyl-sn-glycero-3-[phospho-L-serine] (DOPS, for DGKζ)) were prepared. Enzymatic activity was measured with 15 mM Triton X-100, 0.1 mM [γ-32P]-ATP, 1.52 mol % DAG and 22.5 mol % DOPC or 22.5 mol% DOPS. The assays were performed in triplicate and the results are presented as the mean ± S.D.

2.5. Kinetic Analysis of the Micelle-Based Assay of DGK Activity

The Michaelis-Menten constants, Vmax and Km, were evaluated by a nonlinear regression analysis (initial velocity (v0) versus substrate concentration ([S])), as well as by using Hanes plots ([S]/v0 versus [S]). Origin (version 7.5) software was used to determine Vmax and Km parameters. Inhibition by PA was observed to be competitive, in agreement with previous observations [12]. Ki constants were evaluated by a nonlinear regression analysis for a competitive type of enzyme inhibition, using the GraphPad Prism software program (version 5.04).

3. Results and discussion

It has been recognized earlier that DGKε exhibits specificity for arachidonoyl-containing forms of DAG [13]. It has more recently been established that this isoform of DGK has a particularly important role in catalyzing one of the steps of the PI-cycle [3,14]. This finding correlated well with the known arachidonoyl specificity, since the predominant acyl chain in the sn-2 position of lipid intermediates of the PI-cycle is arachidonic acid. It is also established that these PI-cycle lipid intermediates contain predominantly stearoyl chains at the sn-1 position. We have shown that among saturated acyl chains, the stearoyl (18:0) chain is the most favoured for substrates of DGKε [12]. Furthermore, there is a decrease in 18:0 chains in PIs species in mouse embryo fibroblasts that have been knocked out for DGKε [12]. Thus the best substrate that we found for DGKε was 18:0/20:4-DAG, the form of DAG that is a precursor for the synthesis of PIs. The result of the present study, that 20:4/20:4-DAG has a similar activity to 18:0/20:4-DAG (Fig. 1, Table 1) was surprising. We therefore studied in more detail the acyl chain requirements for the substrates of DGKε.

Figure 1.

Figure 1

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Comparison of the enzyme activities for DGKε with 18:0/20:4-DAG, 20:4/20:4-DAG, 18:0/18:2-DAG and 18:2/18:2-DAG as substrates. Negative control (EV) is performed with the lysates from mock baculovirus-infected Sf21 cells.

Table 1.

Summary of the kinetic parameters for DGKε with 18:0/20:4-DAG, 20:4/20:4-DAG and 18:2/18:2-DAG as substrates. Results are presented as the mean ± S.D. Values of Vmax are relative values since the absolute amount of enzyme in the cell preparations is not known.

Substrate Km (mol%) Vmax (nmol PA min−1) Vmax/Km (mol%−1sec−1)
18:0/20:4 DAG 2.0 ± 0.7 1.7 ± 0.3 0.8 ± 0.3
20:4/20:4 DAG 2.0 ± 0.7 1.6 ± 0.2 0.8 ± 0.3
18:2/18:2 DAG 3.5 ± 0.4 0.89 ± 0.06 0.26 ± 0.03
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Maintaining 18:0 as the sn-1 acyl chain, we confirmed that a linoleoyl chain (18:2) at the sn-2 position is also a substrate for DGKε, but one that is poorer than 18:0/20:4-DAG (Fig. 1). Although 18:0 at sn-1 of DAG makes a better DGKε substrate than 16:0, the difference is not very great [12]. However, 16:0/16:0-DAG is a poor substrate for DGK [15,16]. We showed that 16:0/18:1-DAG and 18:1/18:1-DAG are also poor substrates (Fig. 2). DGKε is very abundant in the brain and retina, suggesting an important physiological role of this enzyme in CNS and visual function. At the same time, docosahexaenoic acid (DHA, 22:6-fatty acid) is the most abundant omega-3 fatty acid in the brain and retina, comprising 40% of the polyunsaturated fatty acids in the brain and 60% in the retina. Despite these facts, 18:0/22:6-DAG is not a substrate for DGKε (Fig. 3A). This is in contrast with the behaviour of another DGK isoform, DGKζ, that does not discriminate among DAGs with different acyl chains (Fig. 3B). Thus, for DGKε there is a very high specificity for the acyl chain at the sn-2 position with only two acyl groups, arachidonoyl or linoleoyl, showing any substantial activity. Interestingly, these two acyl chains are also the only two that are recognized by lipoxygenases. Generally mammalian lipoxygenases are more specific for arachidonic acid, while the homologous enzymes from plants have greater specificity for linoleic acid. Recognition of polyunsaturated acyl chains by both DGKε and by lipoxygenases is due in part to a common amino acid motif in a segment of both proteins [17].

Figure 2.

Figure 2

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A. Comparison of the enzyme activities for DGKε with 18:0/20:4-DAG, 18:1/18:1-DAG and 16:0/18:1-DAG as substrates. Negative control (EV) is performed with the lysates from mock baculovirus-infected Sf21 cells.

Figure 3.

Figure 3

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A. Comparison of the enzyme activities for DGKε with 18:0/20:4-DAG and 18:0/22:6-DAG as substrates. Negative control (EV) is performed with the lysates from mock baculovirus-infected Sf21 cells. B. Comparison of the enzyme activities for DGKζ with 18:0/20:4-DAG, 18:1/18:1-DAG, 18:0/22:6-DAG and 20:4/20:4-DAG as substrates. Negative control (EV) is performed with the lysates from mock baculovirus-infected Sf21 cells.

The requirements for the acyl chain of DAG at the sn-1 position are much more flexible as shown by the finding that 20:4/20:4-DAG has similar activity to 18:0/20:4-DAG (Fig. 1, Table 1). Although, with 20:4 in the sn-2 position, 18:0 was the best acyl chain for sn-1 among saturated acyl chains and had a higher affinity with DGKε than either 16:0/20:4-DAG or 20:0/20:4-DAG [12], the 18:0 acyl chain at sn-1 can be replaced by 20:4 with almost complete retention of activity. Furthermore, 18:2/18:2-DAG has a higher activity than even 18:0/18:2-DAG (Fig. 1). 18:2/18:2-DAG is also the precursor for PA with this acyl chain composition. 18:2/18:2-PA is a potent inhibitor of insulin receptor signalling [18].

Studies of the crystal structure of different fatty acids bound to autotaxin has shown that acyl chains containing unsaturation turn sharply at the unsaturated bonds, allowing longer lipid tails to be accommodated in a hydrophobic pocket [19]. A similar phenomenon can explain the finding that longer acyl chains can be incorporated into the sn-1 position of DAG and still be a good substrate for DGKε, provided that the longer chains have unsaturation.

Another factor that may affect the efficiency of phosphorylation of different species of DAG is the extent to which the substrate penetrates into the membrane. It is suggestive that this may be a factor, although at the present time the evidence is incomplete and indirect. There is however evidence from neutron diffraction studies showing that the position of tocopherol in a lipid environment is very similar for tocopherol embedded into 20:4/20:4-PC as it is when embedded in 16:0/20:4-PC and different from results with other forms of PC not containing polyunsaturated acyl chains [20]. Thus, replacement of 16:0 in the sn-1 position with a 20:4 chain does not alter the depth of burial of tocopherol and thus this change of acyl group would also not likely effect the depth of burial of DAG, despite the large difference in structure and properties of these acyl groups. The similar location of 18:0/20:4-DAG and 20:4/20:4-DAG in the membrane can contribute to their similar location with respect to the enzyme active site, resulting in similar activities.

Previously we showed that 18:0/20:4-PA is the best inhibitor of DGKε [12]. We tested if DGKε inhibition by this PA depends on the acyl chains of the substrate. Our results showed that 18:0/20:4-PA is still the most potent inhibitor of DGKε with three different substrates 18:0/20:4-DAG, 20:4/20:4-DAG and 18:1/18:1-DAG (Fig. 4). Further, we determined the inhibition constants Ki for 20:4/20:4-PA as an inhibitor of DGKε activity with 18:0/20:4-DAG and 20:4/20:4-DAG as substrates (Table 2). Comparison of Ki suggests that 20:4/20:4-PA binds and inhibits DGKε activity somewhat to a greater extend with 20:4/20:4-DAG as a substrate than 18:0/20:4-DAG. PA is a competitive inhibitor of DAG phosphorylation [12]. Thus, the potency of inhibition of different species of PA depends on how well that PA binds to the active site of DGKε. In contrast, to be a good substrate, the DAG must not only bind to the active site, but must also be at an optimal location with regard to the catalytic groups of the enzyme to undergo efficient catalysis. We suggest that 20:4/20:4-DAG is at the optimal location for catalysis because its depth of insertion into the membrane is optimal and therefore it is a good substrate, even though it does not bind optimally to the enzyme. Furthermore, with 20:4/20:4-PA, inhibition is slightly stronger because of lower binding of the substrate to the enzyme.

Figure 4.

Figure 4

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Comparison of inhibition of DGKε by PAs in presence of different substrates. DGKε enzymatic activity was measured with 15 mM Triton X-100, 0.1 mM [γ-32P]-ATP, 30 mol % DOPC, and 1.34 mol % either 18:0/20:4-DAG, 20:4/20:4-DAG and 18:1/18:1-DAG (shown as black bars) or with the addition of either 0.67 mol % 18:0/20:4-PA (grey bars), 20:4/20:4-PA (light grey bars) or 18:1/18:1-PA (white bars).

Table 2.

Summary of the inhibition constants Ki for DGKε with 18:0/20:4-DAG and 20:4/20:4-DAG as substrates and 20:4/20:4-PA as inhibitor. Results are presented as the mean ± S.D.

Substrate Ki, mol%
18:0/20:4-DAG 4.4 ± 1.0
20:4/20:4-DAG 2.6 ± 0.3
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In summary, our results demonstrate that 18:0/20:4-DAG is not unique in being a good substrate for DGKε and that there is a qualitative difference between the nature and extent of acyl chain specificity of DGKε for the sn-1 and sn-2 positions of the substrate.

Highlights.

  • Several different acyl chains can occupy the sn-1 position of good DAG substrates

  • 18:2 and 20:4 are only chains possible at sn-2 of good DAG substrates

  • The best substrate is the PI-cycle intermediate 18:0/20:4-DAG

  • 20:4/20:4-DAG surprisingly has equivalent activity to 18:0/20:4-DAG

Acknowledgments

This work was supported in part by a grant from the Natural Sciences and Engineering Research Council of Canada, grant 9848 (to R.M.E.) and from the National Institutes of Health Grant R01CA095463 (to M.K.T.). We wish to thank Dr. Stephen Wassall for providing us with the neutron diffraction results prior to publication.

Abbreviations used

DGK

diacylglycerol kinase

DAG

diacylglycerol

PA

phosphatidic acid

PI

phosphatidylinositol

DAG

diacylglycerol

18:0/20:4-DAG

1-stearoyl-2-arachidonoyl-DAG

20:4/20:4-DAG

1,2-diarachidonoyl-DAG

DHA

docosahexaenoic acid

Footnotes

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