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Logo of jbcThe Journal of Biological Chemistry
 
J Biol Chem. 2016 April 8; 291(15): 8150–8161.
Published online 2016 February 17. doi:  10.1074/jbc.M115.648717
PMCID: PMC4825017

The Pleckstrin Homology Domain of Diacylglycerol Kinase η Strongly and Selectively Binds to Phosphatidylinositol 4,5-Bisphosphate*

Abstract

Type II diacylglycerol kinase (DGK) isozymes (δ, η, and κ) have a pleckstrin homology domain (PH) at their N termini. Here, we investigated the lipid binding properties of the PHs of type II DGK isozymes using protein-lipid overlay and liposome binding assays. The PH of DGKη showed the most pronounced binding activity to phosphatidylinositol (PI) 4,5-bisphosphate (PI(4,5)P2) among the various glycero- and sphingolipids including PI 3,4,5-trisphosphate, PI 3,4-bisphosphate, PI 3-phosphate, PI 4-phosphate, and PI 5-phosphate. Moreover, the PI(4,5)P2 binding activity of the DGKη-PH was significantly stronger than that of other type II DGK isozymes. Notably, compared with the PH of phospholipase C (PLC) δ1, which is generally utilized as a cellular PI(4,5)P2- probe, the DGKη-PH is equal to or superior than the PLCδ1-PH in terms of affinity and selectivity for PI(4,5)P2. Furthermore, in COS-7 cells, GFP-fused wild-type DGKη1 and its PH partly translocated from the cytoplasm to the plasma membrane where the PLCδ1-PH was co-localized in response to hyperosmotic stress in an inositol 5-phosphatase-sensitive manner, whereas a PH deletion mutant did not. Moreover, K74A and R85A mutants of DGKη-PH, which lack the conserved basic amino acids thought to ligate PI(4,5)P2, were indeed unable to bind to PI(4,5)P2 and co-localize with the PLCδ1-PH even in osmotically shocked cells. Overexpression of wild-type DGKη1 enhanced EGF-dependent phosphorylation of ERK, whereas either K74A or R85A mutant did not. Taken together, these results indicate that the DGKη-PH preferentially interacts with PI(4,5)P2 and has crucial roles in regulating the subcellular localization and physiological function of DGKη. Moreover, the DGKη-PH could serve as an excellent cellular sensor for PI(4,5)P2.

Keywords: cancer, diacylglycerol, extracellular signal-regulated kinase (ERK), inositol phospholipid, lipid-binding protein, phosphatidylinositol signaling, phosphatidylinositol 4,5-bisphosphate, pleckstrin homology domain, diacylglycerol kinase

Introduction

Diacylglycerol kinase (DGK)2 phosphorylates diacylglycerol to produce phosphatidic acid (1,5). Diacylglycerol (6,8) and phosphatidic acid (PA) (9, 10) are well recognized as lipid second messengers. DGK appears to participate in various physiological events by modulating the balance between the two bioactive lipids diacylglycerol and phosphatidic acid (1,5). Ten mammalian DGK isozymes (α, β, γ, δ, ϵ, ζ, η, θ, ι, and κ) containing two or three characteristic zinc finger-like C1 domains and a common catalytic region are divided into five groups (types I–V) according to their structural features (1,5).

Type II DGKs (11) consist of DGKδ (12), -η (13), and -κ (14). Moreover, alternative splicing products of DGKδ (δ1 and δ2) (15) and -η (η1 and η2) (16) have been identified. Type II DGKs have a pleckstrin homology domain (PH) in common at their N termini and a catalytic domain that is divided into two subdomains (catalytic subdomains a and b). DGKs δ1, δ2, and η2, but not DGKs η1 and κ, contain a sterile α-motif domain at their C termini. It has been demonstrated that DGKs δ1, δ2, and η2 form oligomers through interactions between their sterile α-motif domains and that this oligomer formation regulates the activities and subcellular localization of these DGK isoforms (15,19).

It has recently been demonstrated, using DGKδ knock-out mice and RNA interference, that DGKδ regulates the epidermal growth factor (EGF) receptor pathway in lung and skin epithelial cells (20) and insulin receptor signaling in skeletal muscle cells (21, 22) by modulating PKC activity. Moreover, a female patient with a disrupted DGKδ gene who exhibits seizures and a psychiatric disorder was found (23).

We recently reported that DGKη is required for the Ras/B-Raf/C-Raf/MEK/ERK signaling cascade to be activated by EGF in HeLa cells, which are derived from cervical cancer (24). Importantly, DGKη regulates the recruitment of B-Raf and C-Raf from the cytosol to membranes and controls their heterodimerization. Moreover, the study demonstrated that DGKη activates C-Raf, but not B-Raf, in an EGF-dependent manner. The data show that DGKη is a novel key regulator of the Ras/B-Raf/C-Raf/MEK/ERK signaling pathway. In addition, Nakano et al. (25) reported that depleting DGKη in lung cancer cell lines harboring a mutant EGF receptor reduced their growth on plastic and in soft agar and augmented the effects of afatinib, an EGF receptor inhibitor. In addition to cancer cells, DGKη is also highly expressed in the brain (13, 16, 26). It is interesting to note that a genome-wide association study recently indicated that the gene encoding DGKη is implicated in the etiology of bipolar disorder (27, 28). Moreover, it was reported that DGKη was highly expressed in the brain of bipolar disorder patients (29).

DGKκ is abundantly expressed in the testis (14, 30). A genome-wide association study indicated a potential relationship between DGKκ and hypospadias (31).

As described above, type II DGKs are physiologically and pathologically important. However, the binding targets and functions of their PHs are still poorly understood. In this study, we investigated the lipid binding properties of the PHs of DGKδ, -η, and -κ using protein-lipid overlay and liposome binding assays. We revealed that the PH of DGKη strongly and highly selectively binds to phosphatidylinositol (PI) 4,5-bisphosphate (PI(4,5)P2). The DGKδ-PH also, but to a lesser extent, selectively associated with PI(4,5)P2. However, the PH of DGKκ showed only weak binding activity to PI(4,5)P2.

Experimental Procedures

Materials

Monoclonal anti-glutathione S-transferase (GST) (B-14) and anti-GFP antibodies were purchased from Santa Cruz Biotechnology. Polyclonal anti-RFP antibody was purchased from Medical and Biological Laboratories. Monoclonal anti-FLAG M2 antibody was purchased from Sigma-Aldrich. Anti-ERK monoclonal antibody and anti-phospho-ERK antibody were obtained from BD Transduction Laboratories and Cell Signaling Technology, respectively. Peroxidase-conjugated goat anti-mouse IgG antibody was acquired from Bethyl Laboratories. Peroxidase-conjugated goat anti-rabbit IgG antibody was obtained from Jackson ImmunoResearch Laboratories. Phosphatidylserine (PS) from bovine brain, dioleoyl-PI 3-phosphate (PI(3)P), dioleoyl-PI(4)P, dioleoyl-PI(5)P, dioleoyl-PI 3,4-bisphosphate (PI(3,4)P2), 1-stearoyl-2-arachidonoyl-PI(4,5)P2 (18:0/20:4-PI(4,5)P2), dioleoyl-PI(4,5)P2 (18:1/18:1-PI(4,5)P2), phosphatidylglycerol (PG) from chicken egg yolk, 1-stearoyl-2-arachidonoyl-dioleoyl-phosphatidic acid (18:0/20:4-PA), and 1,2-distearoyl-PA (18:0/18:0-PA) were purchased from Avanti Polar Lipids. 1-Stearoyl-2-arachidonoyl-phosphoinositol 3,4,5-trisphosphate (PI(3,4,5)P3) was purchased from Echelon Biosciences. All other lipids, dioleoyl-PA (18:1/18:1-PA), phosphatidylcholine (PC) from chicken egg yolk, phosphatidylethanolamine (PE) from chicken egg yolk, PI from bovine liver, cardiolipin (CL) from bovine heart, sphingomyelin (SM) from chicken egg yolk, sphingosine (Sph), ceramide 1-phosphate (C1P) from bovine brain, and dipalmitoyl-PI(4,5)P2 (16:0/16:0-PI(4,5)P2) were purchased from Sigma-Aldrich. Myc/FLAG (DDK)-tagged human synaptojanin 1 cDNA (pCMV6-Entry-human synaptojanin 1) was purchased from OriGene.

Plasmid Constructs

pGEX-6P-1-DGKδ-PH, p3xFLAG-DGKη1 (splice variant 1), p3xFLAG-DGKη1-ΔPH, pAcGFP-DGKη1, pAcGFP-DGKη-PH, pAcGFP-DGKη-C1D, and pAcGFP-DGKη-PH+C1D were generated previously (32). To express GST-tagged DGKη-PH and DGKκ-PH, the cDNAs encoding these domains were generated from human DGKη1 (16) or DGKκ (14) cDNA clones by PCR and subcloned into the pGEX-6P-1 vector (GE Healthcare). The DGKη-PH cDNA was inserted into pDsRed monomer C1 (Takara Bio-Clontech). Human phospholipase C (PLC) δ1-PH cDNA (amino acids 21–130) (33) was amplified by reverse transcription-PCR with the following primers: forward, 5′-ATCTCGAGCGCTGCTGAAGGGCAGCCAGCT-3′; reverse, 5′-ATGGATCCTGAGTGGTGGATGATCTTGTGC-3′. It was then inserted into pDsRed-monomer C1.

DsRed monomer-DGKη-PH(K74A) and -(R85A) mutants contain substitutions of Ala for Lys-74 and Arg-85, respectively. All mutants were made with the in vitro oligonucleotide mutagenesis system (Takara Bio-Clontech).

Expression and Purification of GST Fusion Proteins

BL21 cells were transformed with pGEX-6P-1 constructs. GST alone and GST fusion proteins were expressed and purified according to the manufacturer's protocol (GE Healthcare). The expression of fusion proteins was induced with 0.1 mm isopropyl 1-thio-β-d-galactopyranoside (Wako Pure Chemical Industries) at 37 °C for 3 h. The cells were then lysed by sonication in 50 mm Tris-HCl, pH 7.4, 0.25 m sucrose, 1% (v/v) Triton X-100 (Nacalai Tesque), 1 mm EDTA (Dojindo), 1 mm dithiothreitol, 20 μg/ml aprotinin (Wako Pure Chemical Industries), 20 μg/ml leupeptin (Nacalai Tesque), 20 μg/ml pepstatin (Nacalai Tesque), 20 μg/ml soybean trypsin inhibitor (Wako Pure Chemical Industries), and 1 mm phenylmethylsulfonyl fluoride (Wako Pure Chemical Industries). The insoluble material was removed by centrifugation. The supernatants were purified by affinity chromatography on a glutathione-Sepharose 4B column (GE Healthcare) at 4 °C. The purified proteins were dialyzed in 10 mm Tris-HCl, pH 7.4.

Cell Culture and cDNA Transfection

COS-7 cells were maintained in Dulbecco's modified Eagle's medium (DMEM) (Wako Pure Chemical Industries) supplemented with 10% fetal bovine serum (Biological Industries), 100 units/ml penicillin, and 100 μg/ml streptomycin (Wako Pure Chemical Industries) at 37 °C in an atmosphere containing 5% CO2. COS-7 cells were seeded in 60-mm dishes at a density of 2.5 × 105 cells/dish. cDNA was transfected into COS-7 cells by electroporation with a Gene Pulser XcellTM electroporation system (Bio-Rad) according to the manufacturer's instructions.

Western Blotting Analysis

COS-7 cells (~1 × 106 cells/60-mm dish) expressing AcGFP-tagged proteins or DsRed monomer-tagged proteins were lysed in 150 μl of 50 mm HEPES, pH 7.2, 150 mm NaCl, 5 mm MgCl2, 1 mm dithiothreitol, 1 mm phenylmethylsulfonyl fluoride, and Complete protease inhibitor mixture (Roche Applied Science). The mixture was centrifuged at 12,000 × g for 5 min at 4 °C. Cell lysates were separated using SDS-PAGE. The separated proteins were transferred to a polyvinylidene difluoride membrane (Bio-Rad) and blocked with 5% (w/w) skim milk. The membrane was incubated with polyclonal anti-RFP antibody (cross-reacts with DsRed monomer), monoclonal anti-FLAG M2 antibody, anti-ERK antibody, or anti-phospho-ERK antibody in 5% (w/v) skim milk for 1 h. The immunoreactive bands were visualized using peroxidase-conjugated goat anti-mouse IgG antibody or goat anti-rabbit IgG antibody and the ECL Western blotting detection system (GE Healthcare).

Protein-Lipid Overlay Assay

One hundred picomoles of the indicated lipids were spotted onto a nitrocellulose membrane (Bio-Rad). The membranes were subjected to blocking with 1% skim milk in Tris-buffered saline, pH 7.4, for 1 h at room temperature. After the blocking, 10 ml of 3% fatty acid-free bovine serum albumin in Tris-buffered saline, pH 7.4, containing lysates from cells expressing GST fusion protein of interest (final concentration, 20 nm) or 80 μl of the cell lysates containing DsRed monomer or 3xFLAG fusion protein were added to the membranes. The membranes were then incubated for 30 min at room temperature and then at 4 °C overnight. The membranes were incubated with an anti-GST antibody, anti-FLAG antibody, or anti-RFP antibody followed by incubation with peroxidase-conjugated goat anti-mouse IgG antibody or goat anti-rabbit IgG antibody. Finally, lipid-bound proteins were visualized using an ECL Western blotting detection system. Quantitative densitometry was performed using ImageJ software.

Liposome Binding Assay

The liposome preparation contained 1 mg/ml total lipid with the following composition: 100% (w/w) PC, 95% (w/w) PC and 5% (w/w) PS or 95% (w/w) PC and 5% (w/w) PA, and 95% (w/w) PC and 5% (w/w) PI(4,5)P2. The combined dried lipid mixture was resuspended in liposome buffer (100 mm NaCl, 1 mm dithiothreitol, and 20 mm HEPES, pH 7.4). Liposome formation was induced by 1-min sonication at 4 °C using a Branson Sonifier 450. For sedimentation assays, the cell lysates expressing GST alone and GST-DGKη-PH were ultracentrifuged at 100,000 × g for 30 min. One hundred microliters of the cell lysates (100,000 × g supernatant) were mixed with 100 μg of liposomes in 100 μl of the liposome buffer, incubated for 60 min at 4 °C, and ultracentrifuged at 100,000 × g for 60 min at 4 °C. The supernatant and pellet were analyzed by SDS-PAGE followed by immunoblotting. Quantitative densitometry was performed using ImageJ software.

DGK Activity Assay

The octyl glucoside mixed micellar assay for DGK activity was performed as described previously (12). In brief, the assay mixture (50 μl) contained 50 mm MOPS, pH 7.4, 50 mm octyl glucoside, 1 mm dithiothreitol, 100 mm NaCl, 20 mm NaF, 10 mm MgCl2, 1 mm EGTA, 5 mm phosphatidylserine, 1.5 mm diacylglycerol, and 1 mm [γ-32P]ATP (10,000 cpm/nmol; ICN Biomedicals). The reaction was initiated by adding cell lysates (5 μg of protein) and continued for 5 min at 30 °C. Lipids were extracted from the mixture, and phosphatidic acid separated by thin layer chromatography was scraped and counted by a liquid scintillation spectrophotometer.

Confocal Laser Scanning Microscopy

Confocal laser scanning microscopy was carried out as described previously (34). Briefly, COS-7 cells were grown on poly-l-lysine-coated glass coverslips and transiently transfected with expression plasmids containing DGKη1 or its mutant cDNAs that were N-terminally fused with AcGFP and a DsRed monomer-tagged PLCδ1-PH (35). After 48 h, the cells were serum-starved with DMEM (0.1% fetal bovine serum) for 3 h and incubated with sorbitol in DMEM (final concentration, 500 mm) for 30 min to induce hyperosmotic stress. The cells were then fixed in 3.7% formaldehyde. For immunofluorescence microscopy, COS-7 cells were permeabilized with 0.1% Triton X-100 in phosphate-buffered saline for 10 min at room temperature. Cells were incubated in phosphate-buffered saline containing 1% bovine serum albumin for 10 min at room temperature as a blocking step. The cells were then incubated in 1% bovine serum albumin in phosphate-buffered saline containing anti-FLAG monoclonal antibody for 30 min at room temperature. After being washed twice with phosphate-buffered saline, cells were incubated with the Alexa Fluor-conjugated secondary antibodies (Invitrogen). The coverslips were mounted using Vectashield (Vector Laboratories). Fluorescence images were acquired using an Olympus FV1000-D confocal laser scanning microscope.

Cell Fractionation

COS-7 cells (~1 × 106 cells/60-mm dish) expressing AcGFP-tagged proteins were lysed in 150 μl of 50 mm HEPES, pH 7.2, 150 mm NaCl, 5 mm MgCl2, 1 mm dithiothreitol, 1 mm phenylmethylsulfonyl fluoride, and Complete protease inhibitor mixture. The mixture was centrifuged at 12,000 × g for 5 min at 4 °C and further centrifuged at 100,000 × g for 60 min at 4 °C to separate supernatant and precipitate (membrane) fractions. AcGFP-tagged proteins were detected by Western blotting with anti-GFP antibody.

Results

Lipid Binding Activity of the PHs of DGKη, -δ, and -κ

We expressed GST alone and the GST-fused PHs of DGKδ, DGKη, and DGKκ in Escherichia coli and highly purified them by affinity chromatography (Fig. 1). These proteins were soluble with expected molecular masses of 26, 38, 38, and 38 kDa, respectively (Fig. 1). We determined the lipid binding activities of the GST-fused PHs of DGKδ, DGKη, and DGKκ using a protein-lipid overlay assay. A nitrocellulose membrane was spotted with 100 pmol each of PI, PI(3)P, PI(4)P, PI(5)P, PI(3,4)P2, PI(4,5)P2, PI(3,4,5)P3, PC, PA, and PS as indicated (Fig. 2A). The PH of DGKη strongly bound to PI(4,5)P2 (Fig. 2, A and B). Compared with PI(4,5)P2, the PH was associated with PI(3,4)P2 and PI(3,4,5)P3 to a lesser extent. PI(3)P, PI(4)P, PI(5)P, and PA showed only weak binding activities. The binding activities of PI, PC, and PS were not detectable. The DGKδ-PH also strongly interacted with PI(4,5)P2 (Fig. 2A). However, compared with the PH of DGKη, the PH of DGKδ bound less strongly to PI(4,5)P2 (Fig. 2, A and B). The PH of DGKκ showed only very weak binding activity to PI(4,5)P2 (Fig. 2, A and B). Therefore, among the PHs of type II DGKs, the PH of DGKη most strongly and selectively binds to PI(4,5)P2.

FIGURE 1.
Expression and purification of GST fusion proteins. GST alone and GST-tagged PHs of DGKδ, -η, and -κ were expressed in BL21 E. coli cells and purified by affinity chromatography. These proteins were separated by SDS-PAGE and stained ...
FIGURE 2.
Lipid binding activities of GST-tagged PHs of DGKδ, -η, and -κ. A and C, equimolar amounts (100 pmol) of PI, PI(3)P, PI(4)P, PI(5)P, PC, PI(3,4)P2, PI(4,5)P2, PI(3,4,5)P3, PA, and PS (A) and PA, PS, CL, PC, SM, Sph, PG, PE, PI, ...

We examined the binding activities of the PHs of DGKη, -δ, and -κ compared with other glycerolipids and sphingolipids. In this experiment, their lipid binding activities were detected after longer exposure. Compared with PA, the DGKη-PH bound to CL to a lesser extent (Fig. 2, C and D). PG, PE, SM, Sph, and C1P exhibited either subtle or no detectable binding activities. In contrast, the PH of DGKδ more strongly bound to PG and CL than to PA and showed either subtle or no detectable binding activities to PE, SM, Sph, and C1P (Fig. 2, C and D). The binding activities of the PH of DGKκ to these lipids were not detectable (Fig. 2, C and D). Taken together, the PH of DGKη exhibited the most pronounced binding activity to PI(4,5)P2 among the various glycero- and sphingolipids. Although the binding activity is weaker than that of the DGKη-PH, the PH of DGKδ also selectively interacts with PI(4,5)P2.

Characterization of Selective Binding Activity of the DGKη-PH to PI(4,5)P2

Next, we more quantitatively determined the affinity of the PH of DGKη for PI(4,5)P2. Various concentrations of PI(4,5)P2, PI(3,4)P2, and PI(3,4,5)P3 were spotted on a nitrocellulose membrane. As shown in Fig. 3, the intensity of the spot of the DGKη-PH on 6.3 pmol of PI(4,5)P2 was almost equivalent to those of 100 pmol of PI(3,4)P2 and PI(3,4,5)P3. Therefore, the result indicates that the binding affinity of the DGKη-PH for PI(4,5)P2 is markedly stronger than for PI(3,4)P2 and PI(3,4,5)P3. Moreover, in the same experiment, the intensity of the spot of the DGKη-PH on 3.1 pmol of PI(4,5)P2 was almost equivalent to that of 100 pmol of PI(4,5)P2 of the DGKδ-PH (data not shown).

FIGURE 3.
Dose-dependent binding activities of DGKη-PH to PI(3,4)P2, PI(4,5)P2, PI(3,4,5)P2, PA, PC, and PS. Indicated amounts (1.6, 3.1, 6.3, 13, 25, 50, and 100 pmol) of PI(3,4)P2, PI(4,5)P2, PI(3,4,5)P3, PA, PC, and PS were spotted onto a nitrocellulose ...

The effects of the tag and expression system were examined next. The DGKη-PH was tagged with DsRed monomer protein instead of GST and expressed in mammalian COS-7 cells instead of E. coli (Fig. 4A). It was confirmed that DsRed monomer-DGKη-PH was expressed with the expected molecular mass (38 kDa). As shown in Fig. 4, B and C, the DsRed monomer-DGKη-PH expressed in COS-7 cells strongly bound to PI(4,5)P2. Similar to the GST-DGKη-PH, the binding affinity for PI(4,5)P2 was markedly stronger than those for PI(3,4,5)P3, PI(3,4)P2, PI(3)P, PI(4)P, PI(5)P, and PI.

FIGURE 4.
Lipid binding activities of DsRed monomer-tagged DGKη-PH and PLCδ1-PH expressed in COS-7 cells. A, expression of DsRed monomer alone, DsRed monomer-DGKη-PH, and DsRed monomer-PLCδ1-PH was confirmed by Western blotting. ...

The PH of PLCδ1 is a widely used PI(4,5)P2-binding probe (35, 36). The binding selectivity and affinity of the PLCδ1-PH for PI(4,5)P2 was compared with those of DGKη. The DsRed monomer-tagged PLCδ1-PH was also expressed with the expected molecular mass (40 kDa) in COS-7 cells (Fig. 4A). The PLCδ1-PH strongly bound to PI(4,5)P2 and, to a lesser extent, to PI(3)P, PI(4)P, PI(5)P, PI(3,4)P2, and PI(3,4,5)P3 (Fig. 4, B and C). The result is essentially the same as reported previously (37). Intriguingly, the intensity of the spot of the DGKη-PH on PI(4,5)P2 was equal to or greater than that of the PLCδ-PH (Fig. 4, B and C). Moreover, the selectivity of the DGKη-PH for PI(4,5)P2 compared with PI(3,4)P2 and PI(3,4,5)P3 was also equal to or better than that of the PLCδ1-PH (Fig. 4, B and C).

In addition to the protein-lipid overlay assay, we confirmed the binding of the DGKη-PH to PI(4,5)P2 using a liposome binding assay. GST alone was not recovered in the precipitate fraction of PI(4,5)P2 liposome, indicating that GST alone was not associated with PI(4,5)P2 (Fig. 5, A and B). However, more than 60% of the GST-DGKη-PH was recovered in the precipitate fraction of PI(4,5)P2 liposome (Fig. 5, A and B). In contrast, only ~10, 20, and 40% of the DGKη-PH was detected in PC, PS, and PA liposomes, respectively. Similar to the lipid-protein overlay assay, these results indicate that the DGKη-PH more strongly binds to PI(4,5)P2 than to PC, PS, and PA.

FIGURE 5.
Binding of DGKη-PH to PI(4,5)P2-containing liposomes. A, GST-tagged DGKη-PH and GST alone were incubated with liposomes containing PC alone and a 9.5:0.5 molar ratio of PC to PS, PA, and PI(4,5)P2 as indicated. Liposome-binding proteins ...

PI(4,5)P2 Binding Activity of Full-length DGKη via Its PH

We next determined whether full-length DGKη indeed binds to PI(4,5)P2. A lipid-protein overlay assay was performed using 3xFLAG-tagged full-length DGKη1 (splice variant 1) expressed in COS-7 cells (Fig. 6A). Similar to the PH alone (Figs. 2 and and4),4), 3xFLAG-full-length DGKη most strongly interacted with PI(4,5)P2 and, to a lesser extent, with PI(3)P, PI(4)P, PI(5)P, PI(3,4)P2, and PI(3,4,5)P3 (Fig. 6, B and C). A DGKη1 mutant lacking a PH exhibited much less binding activity for PI(4,5)P2 (Fig. 6, B and C). Therefore, these results indicate that the full-length DGKη1 strongly and selectively interacts with PI(4,5)P2 through its PH.

FIGURE 6.
PI(4,5)P2 binding of full-length DGKη and its PH-deleted mutant. A, 3xFLAG-tagged full-length DGKη1 (splice variant 1) and DGKη1-ΔPH (amino acids 164–1164) were expressed in COS-7 cells. The Western blotting analysis ...

We also examined whether PI(4,5)P2 can activate DGKη. Although 1.0 mol % PI(4,5)P2 was added to the assay mixture, the activity of DGKη1 was not increased (data not shown). Thus, it seems likely that PI(4,5)P2 is not directly involved in the activation mechanism of DGKη in vitro.

Full-length DGKη1 and Its PH Co-localize with PLCδ1-PH in Osmotically Shocked Cells

Because the PH of PLCδ1 recognizes and co-localizes with cellular PI(4,5)P2 (35, 36), it is generally used as a cellular PI(4,5)P2 probe. Moreover, osmotic shock increases the amount of PI(4,5)P2 in the plasma membrane (38, 39). We next observed the co-localization of AcGFP-tagged full-length DGKη1 with the DsRed monomer-tagged PLCδ1-PH in osmotically shocked cells. Indeed, the PLCδ1-PH was partly localized to the plasma membrane in osmotically shocked cells (Fig. 7). As reported previously (16, 32), full-length DGKη1 was translocated to punctate vesicles in response to osmotic shock (Fig. 7B). Interestingly, full-length DGKη1 was partly co-localized with the PLCδ1-PH at the punctate regions close to the plasma membrane (Fig. 7B). However, AcGFP alone did not exhibit such co-localization (Fig. 7A). These results indicate that in addition to punctate vesicles in the cytoplasm full-length DGKη1 was translocated to subcellular compartments enriched with PI(4,5)P2 in the plasma membrane.

FIGURE 7.
Subcellular localization of full-length DGKη and its mutants (DGKη-PH+C1D, DGKη-PH, and DGKη-C1D) in COS-7 cells treated and untreated with hyperosmotic shock. AcGFP alone (A), AcGFP-tagged full-length DGKη1 (splice ...

An AcGFP-tagged DGKη mutant containing the PH and the C1 domains (C1Ds) was co-localized with the DsRed monomer-tagged PLCδ-PH at punctate vesicles close to the plasma membrane in osmotically shocked cells (Fig. 7C). However, the C1Ds alone lacking a PH were not co-localized with the PLCδ1-PH (Fig. 7E). In contrast, the AcGFP-tagged DGKη-PH alone was translocated to punctate regions in the plasma membrane and markedly co-localized with the DsRed monomer-tagged PLCδ1-PH there in response to osmotic shock (Fig. 7D). We performed the same experiments using DsRed monomer-tagged DGKη-PH and AcGFP-tagged PLCδ-PH and obtained essentially the same results (Fig. 7G). Taken together, these results indicate that the PH of DGKη plays an important role in recognition and co-localization with PI(4,5)P2-containing subcellular compartments.

To further evaluate the co-localization of DGKη-PH with PI(4,5)P2-containing subcellular compartments, we next examined the effect of overexpression of the PI(4,5)P2 phosphatase synaptojanin. Synaptojanin dephosphorylates the D-5 position phosphate from PI(4,5)P2 (40). As shown in Fig. 8, AcGFP-DGKη-PH was markedly translocated from the cytoplasm to the plasma membrane in response to osmotic shock. However, synaptojanin significantly inhibited the osmotic shock-induced plasma membrane translocation of AcGFP-DGKη-PH. The result further indicates that DGKη-PH interacted and co-localized with PI(4,5)P2 in an osmotic shock-dependent manner.

FIGURE 8.
Subcellular localization of DGKη-PH in COS-7 cells treated with hyperosmotic shock in the presence of synaptojanin. A, AcGFP-tagged DGKη-PH was co-expressed with FLAG-tagged synaptojanin in COS-7 cells. After 48 h of transfection, the ...

To confirm the subcellular translocation of AcGFP-DGKη-PH, we performed cell fractionation. As shown in Fig. 9, the DGKη-PH was translocated to the 100,000 × g precipitate (membrane) fraction in osmotically shocked cells, whereas AcGFP alone was not. It was also confirmed that the PLCδ1-PH was translocated to the membrane fraction in response to hyperosmotic shock (data not shown).

FIGURE 9.
Subcellular fractionation of DGKη-PH in COS-7 cells treated and untreated with hyperosmotic shock. COS-7 cells expressing AcGFP alone and AcGFP-tagged DGKη-PH were lysed. The mixture was centrifuged at 12,000 × g for 5 min at 4 ...

Ferguson et al. (41) reported that Lys-30, Lys-32, Arg-40, Ser-55, Arg-56, and Lys-57 in the PLCδ1-PH play important roles in strong PI(4,5)P2-selective binding activity. The four amino acid residues Lys-30, Arg-40, Ser-55, and Arg-56 in the PLCδ1-PH are conserved in the DGKη-PH (Lys-74, Arg-85, Ser-100, and Lys-101, respectively) (Fig. 10). Therefore, we made DsRed monomer-DGKη-PH(K74A) and -(R85A). It was confirmed that both DsRed monomer-DGKη-PH(K74A) and -(R85A) were expressed with the expected molecular mass (38 kDa) (Fig. 11A) and that these mutants failed to bind to PI(4,5)P2 in vitro (Fig. 11B). These results indicate that Lys-74 and Arg-85 in DGKη-PH are also critical residues for PI(4,5)P2 binding activity. We confirmed that DsRed monomer-DGKη-PH(K74A) and -(R85A) were unable to translocate to the plasma membrane and co-localize with the PLCδ1-PH even in osmotically shocked cells (Fig. 11, C and D), suggesting that Lys-74 and Arg-85 are important to detect cellular PI(4,5)P2 and further indicating that the PH of DGKη plays an important role in recognition and co-localization with PI(4,5)P2-containing subcellular compartments.

FIGURE 10.
Alignment of the PHs of PLCδ1, DGKη, DGKδ, and DGKκ. Lys-30, Lys-32, Arg-40, Ser-55, Arg-56, and Lys-57 in the PLCδ1-PH indicated by asterisks are important for strong PI(4,5)P2-selective binding activity. Lys-74 ...
FIGURE 11.
Lipid binding activities and subcellular localization of DsRed monomer-tagged DGKη-PH(K74A) and DGKη-PH(R85A) expressed in COS-7 cells. A, expression of DsRed monomer alone, DsRed monomer-DGKη-PH(K74A), and DsRed monomer-DGKη-PH(R85A) ...

Effects of DGKη1(K74A) and DGKη1(R85A) on EGF-dependent Activation of ERK

DGKη1 is a positive regulator of the EGF receptor/Ras/B-Raf/C-Raf/MEK/ERK signaling pathway (24). To address physiological significance of PI(4,5)P2 binding activity of DGKη1, we tested the effects of overexpression of wild-type DGKη1 and the PI(4,5)P2 binding-negative (full-length) mutants DGKη1(K74A) and DGKη1(R85A) on EGF-induced ERK phosphorylation. As shown in Fig. 12, wild-type DGKη1 markedly increased EGF-dependent phosphorylation levels of ERK. However, neither DGKη1(K74A) nor DGKη1(R85A) enhanced the ERK phosphorylation even though their protein expression levels were similar to that of wild-type DGKη1. These results suggest that PI(4,5)P2 binding activity of DGKη1 plays an important role in regulating the EGF receptor/ERK signal transduction pathway.

FIGURE 12.
Effects of DGKη1(K74A) and DGKη1(R85A) on EGF-dependent activation of ERK. COS-7 cells were transfected with pAcGFP vector, pAcGFP-DGKη1, pAcGFP-DGKη1(K74A), or pAcGFP-DGKη1(R85A). After 24 h, the cells were serum-starved ...

Discussion

The lipid binding properties of the PH of DGKη have not been clear. In this study, we demonstrated that the DGKη-PH is a PI(4,5)P2-selective binding domain with high affinity (Figs. 2 and and4).4). It was also confirmed that the full-length DGKη1 strongly and selectively interacts with PI(4,5)P2 through its PH (Fig. 6). Affinities for various phosphoinositides and phospholipids were as follows: PI(4,5)P2 [dbl greater-than sign] PI(3,4)P2 ≈ PI(3,4,5)P3 > PA ≈ PI(3)P ≈ PI(4)P ≈ PI(5)P [dbl greater-than sign] CL [dbl greater-than sign] PG ≈ PE ≈ PS ≈ PC > PI ≈ SM ≈ Sph ≈ C1P (Figs. 2 and and3).3). Therefore, these results indicate that the DGKη-PH is highly selective for PI(4,5)P2.

Compared with the PHs of DGKδ and DGKκ, the affinity of the PH of DGKη for PI(4,5)P2 is markedly strong (DGKη-PH [dbl greater-than sign] DGKδ-PH [dbl greater-than sign] DGKκ-PH) (Fig. 2). Partial formation of the proper conformation may cause low affinity for PI(4,5)P2. However, high concentrations of the PHs of DGKδ and DGKκ, which probably increase the number of the domains that have the proper conformation, showed no effect on their binding affinities for PI(4,5)P2, indicating that the different affinities are intrinsic properties.

It was reported that Lys-30, Lys-32, Arg-40, Ser-55, Arg-56, and Lys-57 in the PLCδ1-PH are important for strong PI(4,5)P2-selective binding activity (41). The four amino acid residues Lys-30, Arg-40, Ser-55, and Arg-56 in the PLCδ1-PH are conserved in the DGKη-PH (Lys-74, Arg-85, Ser-100, and Lys-101, respectively) (Fig. 10). We confirmed that K74A and R85A in DGKη-PH are indeed critical residues for PI(4,5)P2 binding activity (Fig. 11B). In the DGKδ-PH, the three amino acid residues Lys-62, Arg-73, and Lys-89, corresponding to Lys-30, Arg-40, and Arg-56 in the PLCδ1-PH, are conserved. However, in the DGKκ-PH, only two amino acid residues, Lys-225 and Arg-236, are conserved. Therefore, it is possible that the differences in the sequences of these PHs cause the distinct affinities for PI(4,5)P2 among them (DGKη-PH > DGKδ-PH > DGKκ-PH; see Fig. 2). The presence of Gly at the end of the β1 strand of PH is important to confer PI(3,4,5)P3 binding activity (42, 43). Because the amino acid residue at the end of the β1 strand of the DGKη-PH is Asn, it is reasonable that it does not preferably bind to PI(3,4,5)P3 (Fig. 2). However, the determination of the three-dimensional structure of the PHs of DGKη, -δ, and -κ is required to analyze their different PI(4,5)P2 binding properties in more detail.

A previous study demonstrated that the PH of DGKδ non-selectively interacted with PI(4,5)P2 because it also strongly bound to PS (37). However, high amounts (~20 times higher) of lipids were used in the overlay assay in the previous report (37). Therefore, it is likely that the selectivity of the PH of DGKδ for PI(4,5)P2 was detected in the present study (Fig. 2), which used relatively low amounts of phospholipids. The PH of DGKδ is important for its phorbol ester-dependent plasma membrane localization (15, 44). However, it is still unclear whether PI(4,5)P2 is involved in the translocation.

The EGF receptor signaling pathway, which is regulated by DGKη (24), augments PI(4,5)P2 (45). Osmotic shock also increases the amount of PI(4,5)P2 at the plasma membrane (38, 39). In this study, we also found that the PLCδ1-PH, which is known to be a PI(4,5)P2 sensor, was localized at the plasma membrane in response to osmotic shock (Fig. 7). DGKη-PH(K74A) and -(R85A), which lacked PI(4,5)P2 binding activities (Fig. 11B), failed to translocate to the plasma membrane and to co-localize with the PLCδ1-PH even in osmotically shocked cells (Fig. 11, C and D). These results strongly suggest that PI(4,5)P2 was increased in the plasma membrane. The PH of DGKη was also co-distributed with the PLCδ1-PH at the plasma membrane. Moreover, depletion of PI(4,5)P2 by the PI(4,5)P2 phosphatase synaptojanin significantly reduced the osmotic shock-dependent plasma membrane localization of DGKη-PH (Fig. 8). The result further indicates that DGKη-PH binds to and is co-localized with PI(4,5)P2 in cells. Therefore, the PH of DGKη could also function as a cellular PI(4,5)P2 sensor. Compared with the PH of PLCδ1, the PH of DGKη is equal to or superior to the PLCδ1-PH in terms of affinity and selectivity for PI(4,5)P2 (Fig. 4). The PH of PLCδ1 is widely used as a cellular PI(4,5)P2 detector. Therefore, the PH of DGKη could serve as an excellent PI(4,5)P2-selective probe with high affinity and selectivity like the PLCδ1-PH.

We have reported that DGKη1 was osmotic shock-dependently translocated to punctate vesicles and that the PH and C1Ds are important for the redistribution (16, 32). In this study, we observed that the PH alone was translocated to the punctate regions of the plasma membrane where the PLCδ1-PH was co-localized in response to osmotic shock (Fig. 7, D and G), whereas the C1 domains alone, which were distributed to punctate vesicles in the cytoplasm, were not co-localized with the PLCδ1-PH (Fig. 7E). In osmotically shocked cells, full-length DGKη and a DGKη mutant containing both the PH and the C1Ds were distributed to both PLCδ1-PH-co-localized punctate regions at the plasma membrane and punctate vesicles in the cytoplasm, which do not co-localize with the PLCδ1-PH (Fig. 7, B and C). These results suggest that the PH and C1Ds competitively recruit DGKη to subcellular compartments with and without PI(4,5)P2, respectively.

We did not detect obvious competition between the PLCδ1-PH and the DGKη-PH in both confocal microscopy and cell fractionation. Whether the PI(4,5)P2 binding competition between these PHs occurs or not is probably dependent on the amount of PI(4,5)P2 in the plasma membrane. The amount of PI(4,5)P2 produced in osmotically shocked cells may be greater than the sum of the amounts of the PLCδ1-PH and DGKη-PH that are able to access PI(4,5)P2 in the plasma membrane.

We reported previously that DGKη1 positively regulates the EGF receptor/Ras/B-Raf/C-Raf/MEK/ERK signaling pathway (24). Wild-type DGKη1 markedly increased EGF-dependent phosphorylation levels of ERK, whereas both DGKη1(K74A) and DGKη1(R85A) failed to show such effect (Fig. 12). Therefore, it is possible that PI(4,5)P2 binding activity of DGKη1 regulates its stimulation-dependent subcellular localization and plays an important role in regulating the signal transduction pathway.

DGKη was reported to be implicated in the etiology of bipolar disorder (27,29). In the brain, DGKη is enriched in the dentate gyrus of the hippocampus and the Purkinje cells of the cerebellum (26), which are known to be associated with bipolar disorder (46,48). A common treatment for bipolar disorder is a mood stabilizer, lithium, which attenuates PI turnover (49). Therefore, DGKη may regulate the pathogenesis of bipolar disorder through the PH-dependent binding to PI(4,5)P2 generated by PI turnover in the dentate gyrus and the Purkinje cells.

Among more than 300 PHs, only the PH of PLCδ1 is an established PI(4,5)P2-selective binding domain (50, 51). In this study, we added the DGKη-PH to the list as a new member. Although the affinity of the DGKδ-PH for PI(4,5)P2 is relatively lower than that of the DGKη-PH, the PH of DGKδ is an additional PI(4,5)P2-selective PH. The PH of DGKη could be an excellent cellular sensor for PI(4,5)P2 that is equal or superior to the PLCδ1-PH. DGKη1 has been reported to be involved in EGF-dependent cell proliferation (24) and in the pathogenesis of lung cancer (25) and bipolar disorder (27,29). It will be interesting to determine what role the DGKη isozyme, which has the highly PI(4,5)P2-selective binding motif, plays in modulating these physiologically and pathologically important events.

Author Contributions

F. S. conceived and coordinated the study and wrote the paper. A. K. designed, performed, and analyzed the experiments shown in Figs. 117 and and10.10. T. U. designed, performed, and analyzed the experiments shown in Figs. 2, ,7779, ,11,11, and and12.12. S. K. designed, performed, and analyzed the experiments shown in Figs. 779, ,11,11, and and12.12. K. K. designed, performed, and analyzed the experiments shown in Figs. 8, ,11,11, and and12.12. E. T. designed, performed, and analyzed the experiments shown in Fig. 2. H. S. designed the experiments shown in Figs. 117 and and10,10, provided technical assistance, and contributed to the preparation of the figures. All authors reviewed the results and approved the final version of the manuscript.

*This work was supported by Ministry of Education, Culture, Sports, Science and Technology of Japan Grants 22370047, 23116505, 25116704, and 26291017; Japan Science and Technology Agency Grants AS221Z00794F, AS231Z00139G, and AS251Z01788Q; the Naito Foundation, the Hamaguchi Foundation for the Advancement of Biochemistry, the Daiichi-Sankyo Foundation of Life Science, the Terumo Life Science Foundation, the Futaba Electronic Memorial Foundation, the Daiwa Securities Health Foundation, the Ono Medical Research Foundation, the Japan Foundation for Applied Enzymology, the Food Science Institute Foundation, the Skylark Food Science Institute; the Food Science Institute Foundation, and the Asahi Group Foundation. The authors declare that they have no conflicts of interest with the contents of this article.

2The abbreviations used are:

DGK
diacylglycerol kinase
C1D
C1 domain
C1P
ceramide 1-phosphate
CL
cardiolipin
DG
diacylglycerol
PA
phosphatidic acid
PC
phosphatidylcholine
PE
phosphatidylethanolamine
PG
phosphatidylglycerol
PH
pleckstrin homology domain
PI
phosphatidylinositol
PI(3)P
PI 3-phosphate
PI(4)P
PI 4-phosphate
PI(5)P
PI 5-phosphate
PI(3,4)P2
PI 3,4-bisphosphate
PI(4,5)P2
PI 4,5-bisphosphate
PI(3,4,5)P3
PI 3,4,5-trisphosphate
PLC
phospholipase C
SM
sphingomyelin
Sph
sphingosine
RFP
red fluorescent protein.

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