Diacylglycerol kinase epsilon is selective for both acyl chains of phosphatidic acid or diacylglycerol - PubMed (original) (raw)

Diacylglycerol kinase epsilon is selective for both acyl chains of phosphatidic acid or diacylglycerol

Michael Lung et al. J Biol Chem. 2009.

Abstract

The phosphatidylinositol (PI) cycle mediates many cellular events by controlling the metabolism of many lipid second messengers. Diacylglycerol kinase epsilon (DGK epsilon) has an important role in this cycle. DGK epsilon is the only DGK isoform to show inhibition by its product phosphatidic acid (PA) as well as substrate specificity for sn-2 arachidonoyl-diacylglycerol (DAG). Here, we show that this inhibition and substrate specificity are both determined by selectivity for a combination of the sn-1 and sn-2 acyl chains of PA or DAG, respectively, preferring the most prevalent acyl chain composition of lipids involved specifically in the PI cycle, 1-stearoyl-2-arachidonoyl. Although the difference in rate for closely related lipid species is small, there is a significant enrichment of 1-stearoyl-2-arachidonoyl PI because of the cyclical nature of PI turnover. We also show that the inhibition of DGK epsilon by PA is competitive and that the deletion of the hydrophobic segment and cationic cluster of DGK epsilon does not affect its selectivity for the acyl chains of PA or DAG. Thus, this active site not only recognizes the lipid headgroup but also a combination of the two acyl chains in PA or DAG. We propose a mechanism of DGK epsilon regulation where its dual acyl chain selectivity is used to negatively regulate its enzymatic activity in a manner that ensures DGK epsilon remains committed to the PI turnover cycle. This novel mechanism of enzyme regulation within a signaling pathway could serve as a template for the regulation of enzymes in other pathways in the cell.

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Figures

FIGURE 1.

Overview of DGKϵ, DGKϵΔ58, and DGKζ structure. The various domains/motifs in each of these proteins are presented here. CRD, cysteine-rich domain; extCRD, extension of the cysteine-rich domains; MARCKS, myristoylated alanine-rich protein kinase C substrate; NLS, nuclear localization sequence; PDZ, PSD95/DlgA/zo-1. The structures presented here are not drawn to exact scale.

FIGURE 2.

DGKϵ inhibition by PA is dependent upon both its _sn_-1 and _sn_-2 acyl chains. A, enriched lysates from Sf21-overexpressing DGKϵ-His6 (+DGK_ϵ) or from mock baculovirus-infected Sf21 cells (−_ve control) were assayed for DGK enzymatic activity with 7.5 m

Triton X-100, 7.5 m

Triton X-114, 0.1 m

[γ-32P]ATP, and 1.5 mol % SAG in the presence of either 1.5 mol % SLPA, PAPA, SAPA, 1-arachidoyl-2-arachidonoyl phosphatidic acid (AAPA), 1,2-diarachidonoyl phosphatidic acid (DAPA), or SOPA. The enzymatic activity presented was normalized to DGKϵ enzymatic activity in the absence of PA, which was 0.932 ± 0.080 nmol of PA/min and defined as 100% enzymatic activity. B, enriched lysates from Sf21-overexpressing DGKϵ-His6 (+DGK_ϵ) or from mock baculovirus-infected Sf21 cells (−_ve control) were assayed for DGK enzymatic activity as in A except either 1.5 mol % 1-palmitoyl-2-oleoyl phosphatidic acid (POPA), PAPA, or SAPA was used instead of the PAs used in A. The enzymatic activity presented was normalized to DGKϵ enzymatic activity in the absence of PA, which was 0.873 ± 0.001 nmol of PA/min and defined as 100% enzymatic activity. C, enriched lysates from Sf21-overexpressing DGKϵ-His6 (+DGK_ϵ) or from mock baculovirus-infected Sf21 cells (−_ve control) were assayed for DGK enzymatic activity as in A except 3.0 mol % 1-stearoyl-2-linoleoyl-_sn_-glycerol (SLG) was used instead of SAG as the DAG lipid substrate, and 0.75 mol % of the PAs was used. The enzymatic activity presented was normalized to DGKϵ enzymatic activity in the absence of PA, which was 0.266 ± 0.005 nmol of PA/min and defined as 100% enzymatic activity.

FIGURE 3.

DGKζ is activated by PA in a nonacyl chain-specific manner. Enriched lysates from Sf21-overexpressing DGKζ-FLAG (+DGK_ζ) or from mock baculovirus-infected Sf21 cells (−_ve control) were assayed for DGK enzymatic activity with 7.5 m

Triton X-100, 7.5 m

Triton X-114, 0.1 m

[γ-32P]ATP, and 1.5 mol % DOG, in the presence of either 8.1 mol % SLPA, SOPA, or SAPA. The enzymatic activity presented was normalized to DGKζ enzymatic activity in the absence of PA, which was 0.252 ± 0.001 nmol of PA/min and defined as 100% enzymatic activity.

FIGURE 4.

DGKϵ substrate specificity is dependent upon both the _sn_-1 and _sn_-2 acyl chains of DAG. Enriched lysates from Sf21-overexpressing DGKϵ-His6 (+DGK_ϵ) or from mock baculovirus-infected Sf21 cells (−_ve control) were assayed for DGK enzymatic activity with 7.5 m

Triton X-100, 7.5 m

Triton X-114, and 0.1 m

[γ-32P]ATP, using either 0.38 mol % PAG, SAG, AAG, or DOG as a DAG lipid substrate. The enzymatic activity presented was normalized to DGKϵ enzymatic activity using SAG as a DAG lipid substrate, which was 0.234 ± 0.002 nmol of PA/min and defined as 100% enzymatic activity.

FIGURE 5.

DGKζ does not exhibit an acyl chain-dependent substrate specificity. Enriched lysates from Sf21-overexpressing DGKζ-FLAG (+DGK_ζ) or from mock baculovirus-infected Sf21 cells (−_ve control) were assayed for DGK enzymatic activity with 7.5 m

Triton X-100, 7.5 m

Triton X-114, and 0.1 m

[γ-32P]ATP, using either 0.38 mol % PAG, SAG, AAG, or DOG as a DAG lipid substrate. The enzymatic activity presented was normalized to DGKζ enzymatic activity using AAG as a DAG lipid substrate, which was 0.169 ± 0.025 nmol of PA/min and defined as 100% enzymatic activity.

FIGURE 6.

Lipidomic analysis of DGKϵ KO and WT MEFs. Phospholipids in DGKϵ KO and WT MEFs were determined by direct infusion mass spectrometry and identified by liquid chromatography-tandem mass spectrometry. Different phospholipid classes (PA, phosphatidic acids; PC, phosphatidylcholines; PE, phosphatidylethanolamines; PG, phosphatidylglycerols; PI, phosphatidylinositols; PS, phosphatidylserines) within a specific group of phospholipids with the same acyl chain composition were compared. Fold differences in the KO samples compared with the WT samples are presented as well as the standard deviation from three independent experiments (each in triplicate). A, 18:0-containing phospholipids; B, 16:0-containing phospholipids; C, 36:2 phospholipids.

FIGURE 7.

DGKϵ PA or DAG acyl chain selectivity is retained after deletion of the cationic cluster and hydrophobic segment of DGKϵ. COS-7 cells overexpressing 3XFLAG-DGKϵFL (full-length) or 3XFLAG-DGKϵΔ58 (Δ58) protein or transfected with empty vector (−ve control) were resuspended in lysis buffer containing 1% Nonidet P-40 and centrifuged at 100 000 × g for 30 min. A, resultant supernatants (enriched lysates) were assayed for DGK enzymatic activity with 7.5 m

Triton X-100, 7.5 m

Triton X-114, 0.1 m

[γ-32P]ATP, and 3.0 mol % SAG, in the presence of either 1.5 mol % SAPA or SLPA. These enzymatic activity values are presented as a percentage of 3XFLAG-DGKϵΔ58 enzymatic activity in the absence of PA, which was 0.038 ± 0.003 nmol of PA/min. B, enriched lysates were also assayed for DGK enzymatic activity with 75 m

OG and 0.5 m

[γ-32P]ATP, using either 0.30 mol % SAG or DOG as a DAG lipid substrate. These enzymatic activity values are presented as a percentage of 3XFLAG-DGKϵΔ58 enzymatic activity using SAG as a lipid substrate, which was 0.0054 ± 0.0011 nmol of PA/min. An immunoblot was also performed on the enriched lysates using a 1:2500 dilution of mouse anti-FLAG M2 primary, 1:2500 dilution of goat anti-mouse horseradish peroxidase secondary antibody (C) or a 1:500 dilution of goat anti-actin primary, 1:2500 dilution of donkey anti-goat horseradish peroxidase secondary antibody (D).

FIGURE 8.

DGKϵ acyl chain-specific inhibition by PA is detectable using OG/DOPC-based mixed micelles. Enriched lysates from Sf21-overexpressing DGKϵ-His6 (+DGK_ϵ) or from mock baculovirus-infected Sf21 cells (−_ve control) were assayed for DGK enzymatic activity with 75 m

OG, 0.1 m

[γ-32P]ATP, and 2.0 mol % SAG, in the presence of either 2.0 mol % SLPA, SAPA, or SOPA. The enzymatic activity presented was normalized to DGKϵ enzymatic activity in the absence of PA, which was 1.056 ± 0.043 nmol of PA/min and defined as 100% enzymatic activity.

FIGURE 9.

Cornish-Bowden plot for DGKϵ phosphorylation of SAG in the presence of different concentrations of SAPA indicates that the inhibition of DGKϵ by PA is competitive. Enriched lysates from Sf21-overexpressing DGKϵ-His6 were assayed for DGK enzymatic activity assayed for enzymatic activity with 75 m

OG, 0.1 m

[γ-32P]ATP, either 0.125, 0.250, 0.500, 1.00, or 2.00 mol % SAG, and either 0, 0.630, 2.00, 2.50, or 3.40 mol % SAPA.

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Diacylglycerol kinase epsilon is selective for both acyl chains of phosphatidic acid or diacylglycerol - PubMed (original) (raw)