EF-hand motifs of diacylglycerol kinase α interact intra-molecularly with its C1 domains.

Diacylglycerol kinase (DGK) α, which is activated by Ca(2+), contains a recoverin homology (RVH) domain, tandem repeats of two Ca(2+)-binding EF-hand motifs, two cysteine-rich C1 domains and the catalytic domain. We previously found that a DGKα mutant lacking the RVH domain and EF-hands was constitutively active and that the N-terminal region of DGKα, consisting of the RVH domain and EF-hand motifs, interacted intra-molecularly with the C-terminal region containing the C1 and catalytic domains. In this study, we narrowed down the interaction regions of DGKα. At the C-terminal region, the C1 domains are responsible for the intra-molecular interaction. At the N-terminal region, the EF-hand motifs mainly contribute to the interaction. Moreover, using highly purified EF-hand motifs and C1 domains, we demonstrate that they directly bind to each other. The co-precipitation of these two domains was clearly attenuated by the addition of Ca(2+). These results indicate that the Ca(2+)-induced dissociation of the intra-molecular interaction between the EF-hand motifs and the C1 domains of DGKα is the key event that regulates the activity of the enzyme.

Introduction

Diacylglycerol kinase (DGK) phosphorylates diacylglycerol (DG) to generate phosphatidic acid (PA) [1-5]. DG is an established activator of the conventional and novel protein kinase Cs, Ras guanyl nucleotide-releasing protein, Unc-13 and chimaerin [6,7]. PA also regulates various important signaling factors, such as phosphatidylinositol-4-phosphate 5-kinase, son of sevenless, Ras GTPase-activating protein, C-Raf and atypical protein kinase C [3,8-10]. Therefore, DGK is thought to function in a variety of physiological events by modulating the balance between two signaling lipids, DG and PA.

Ten mammalian DGK isozymes (α, β, γ, δ, ε, ζ, η, θ, ι and κ), which share two or three characteristic zinc finger-like C1 domains and the catalytic region of the enzyme, are divided into five groups according to their structural features [1-5]. Type I DGK isozymes (DGKs α, β and γ) commonly contain tandem repeats of two EF-hand motif domains and are classified as members of the EF-hand family of Ca2+-binding proteins. In addition to the Ca2+-binding EF hand motifs, all type I DGK isozymes contain an N-terminal recoverin homology (RVH) domain, two cysteine-rich C1 domains and the C-terminal catalytic region [1-5]. Interestingly, these isozymes exhibit different tissue- and cell-specific modes of expression. DGKα is most abundant in T-lymphocytes and the thymus [11,12], oligodendrocytes of the brain [13] and melanoma cells [14]. DGKα is involved in a wide variety of pathophysiological events [15], such as interleukin-2-dependent T-cell proliferation [16], T-cell anergy [17,18], hepatocyte growth factor-induced cell motility [19], melanoma apoptosis [14] and the progression of human hepatocellular carcinoma [20].

Calcium-mediated cellular signal transduction plays an important role in the control of the physiological functions in various types of cells [21,22]. Most of the EF-hand proteins, such as calmodulin (CaM), troponin C and calcineurin regulation subunit B, are relatively small molecules (10–20 kDa) [22] that play a specialized role as Ca2+-sensitive regulators of many target proteins, and their amino acid sequences are primarily composed of EF-hand motifs. In contrast, type I DGKs are relatively large for EF-hand proteins (80–90 kDa), and represent a fusion protein connected to EF-hand motifs (approximately 110 kDa) that are combined with other functional domains, including the RVH, C1 and catalytic domains [1-5]. Calcium-activated neutral protease, calpain [23,24] and inositol phospholipid-specific phospholipase C [25] are the only known examples of this type of fusion protein with a proven capacity for Ca2+.

Previously, we demonstrated that purified DGKα binds Ca2+ in 2:1 metal:enzyme stoichiometry with an apparent dissociation constant, Kd, of 300 nM [26,27]. The addition of Ca2+ in the presence of phosphatidylserine significantly activated the enzyme [26]. On the other hand, although DGK β and γ possess EF-hand motifs, the activities of these isozymes were not significantly affected by Ca2+ [27]. Intriguingly, a DGKα mutant lacking the RVH and the EF-hand motif domains translocated from the cytosol to the membranes [28,29] and became constitutively active [28-30]. DGKα was eluted at the monomer position (80 kDa) by gel filtration [31]. These results [28-31] imply the presence of an intra-molecular, not inter-molecular, interaction between the N-terminal region of DGKα, consisting of the RVH domain and EF hand motifs, and the C-terminal region, consisting of the C1 and catalytic domains, which masks its activity. Moreover, by expressing the N-terminal and C-terminal regions of DGKα separately, we recently showed that the two domains interact with each other and that this association is attenuated by the addition of Ca2+ [32]. Furthermore, we demonstrated that Ca2+-induced conformational changes in the DGKα-N-terminal region disrupted the intra-molecular association between the two regions of the enzyme [32]. However, the precise domains that mediate this binding are unknown.

In this study, we attempted to narrow down the interaction domains in both the N-terminal and C-terminal regions of DGKα, and we found an intra-molecular and direct interaction between the C1 domains of DGKα and its EF-hand motifs.

Results

The C1 domains of DGKα interact with the N-terminal region of the enzyme

We first attempted to narrow down the region of interaction in the C-terminal region of DGKα, which contains the C1 domains and catalytic region. To this end, the C1 domains and the catalytic region of DGKα that were separately fused with enhanced green fluorescence protein (EGFP) were constructed (Fig. 1). They were then expressed in mammalian COS-7 cells in addition to EGFP connected to the C-terminal region of DGKα (85 K), and proteins with the expected molecular weights, 46 K and 68 K, respectively, were obtained (Fig. S1). Glutathione S-transferase (GST, 26 K), and GST-fused with the N-terminal region of DGKα (48 K) (Fig. 1; Fig. S2) were produced by bacteria expression, followed by affinity purification using glutathione–Sepharose beads.

Fig. 1

Schematic representation of the DGKα mutants used in this study. RVHD, recoverin homology domain; EFHs, EF-hand motifs; C1Ds, C1 domains; CR, catalytic region; NTR, N-terminal region; CTR, C-terminal region.

We examined the physical interaction between the N-terminal region of DGKα and the C1 domain or catalytic region of the enzyme by conducting a co-precipitation analysis using the purified GST-fused DGKα-N-terminal region and lysates from COS-7 cells expressing the EGFP-DGKα-C1 domains or EGFP-DGKα-catalytic region. As previously reported [32], it was confirmed that the GST-DGKα-N-terminal region was co-precipitated with the EGFP-DGKα-C-terminal region (Fig. 2). As shown in Fig. 2, the C1 domains of DGKα were strongly co-precipitated with the DGKα-N-terminal region, while the catalytic region was not able to bind to the DGKα-N-terminal region. However, GST alone did not pull-down the EGFP-DGKα-C1 domains (Fig. 2). These results indicate that the C1 domains of DGKα are responsible for interacting with the N-terminal region of the enzyme.

Fig. 2

The DGKα-N-terminal region interacts with DGKα-C1 domains. (A) EGFP-DGKα-C-terminal region (CTR), -C1 domains (C1Ds) and -catalytic region (CR) were co-precipitated with GST alone or GST-DGKα-N-terminal region (NTR) in the absence of Ca2+ (with the addition of 5 mM EGTA). The precipitation of EGFP- and GST-tagged proteins was analyzed by Western blotting using anti-GST and anti-GFP antibodies. The data shown are representative of three independent experiments. Input: purified GST alone and GST-DGKα-NTR (upper panels) and COS-7 cell lysates expressing EGFP-DGKα-CTR, -C1Ds and -CR (lower panels). (B) The quantified relative intensities of the co-precipitated EGFP-DGKα-CTR, -C1Ds and -CR bands. The amount of EGFP-DGKα-CTR co-precipitated with GST-DGKα-NTR was set to 100%. The corresponding value of GST alone was subtracted. The error bars represent the standard deviation of three independent experiments. Statistical significance was determined using the student’s t-test (∗∗P < 0.01).

The EF-hand motifs of DGKα interact with the C-terminal region of the enzyme

We next attempted to narrow down the interaction region at the N-terminal region of DGKα, which contains the RVH domain and EF-hand motifs. To this end, in addition to the GST-DGKα-N-terminal region, the RVH domain and the EF-hand motifs of DGKα (Fig. 1) were separately fused with GST, expressed in Escherichia coli cells and purified (Fig. S2). These proteins showed the expected molecular weights, 38 K and 36 K, respectively (Fig. S2). We confirmed that the GST-DGKα-N-terminal region was co-precipitated with EGFP-DGKα-C-terminal region (Fig. 3). The bands of the RVH domain and the EF-hand motifs that were pulled down were weaker than that of the N-terminal region. However, compared with the RVH domain, the EF-hand motifs exhibited markedly stronger interactions with the C-terminal region, and the observed band was an approximately three times more intense (Fig. 3). This result indicates that the EF-hand motifs of DGKα mainly contribute to the interaction with the C-terminal region of the enzyme.

Fig. 3

The DGKα-C-terminal region interacts with DGKα-EF-hand motifs. (A) EGFP-DGKα-C-terminal region (CTR) was co-precipitated with GST alone, GST-DGKα-N-terminal region (NTR), GST-DGKα-RVH domain (RVHD) or GST-DGKα-EF hand motifs (EFHs) in the absence of Ca2+ (with the addition of 5 mM EGTA). The precipitation of EGFP- and GST-tagged proteins was analyzed by Western blotting using anti-GFP and anti-GST antibodies. The data shown are representative of three independent experiments. Input: purified GST alone, GST-DGKα-NTR, -RVHD and -EFHs (upper panel) and COS-7 cell lysates expressing EGFP-DGKα-CT (lower panel). (B) The quantified relative intensities of the co-precipitated GST-DGKα-NTR, GST-DGKα-RVHD and GST-DGKα-EFHs bands. The amount of GST-DGKα-NTR co-precipitated with EGFP-DGKα-CTR was set to 100%. The value of GST alone was subtracted. The error bars represent the standard deviation of three independent experiments. Statistical significance was determined using the student’s t-test (∗∗P < 0.01).

EF-hand motifs of DGKα interact with the C1 domains of the enzyme

Because the N-terminal and C-terminal regions were used as partners of the C1 domains and EF-hand motifs in Figs. 2 and 3, respectively, we next confirmed that the EF-hand motifs of DGKα indeed associated with the C1 domains of the enzyme. We performed a co-precipitation analysis using the purified GST-fused DGKα-EF hand motifs and lysates of COS-7 cells expressing the EGFP-DGKα-C1 domains. As shown in Fig. 4, the DGKα-EF hand motifs co-precipitated with the EGFP-DGKα-C1 domains in the absence of Ca2+.

Fig. 4

DGKα-EF-hand motifs interact with DGKα-C1 domains. (A) GST alone or GST fused with DGKα-EF hand motifs (EFHs) was co-precipitated with EGFP-DGKα-C1 domains (C1Ds) in the absence (with the addition of 5 mM EGTA) or presence of Ca2+ (1 μM free-Ca2+, adjusted by adding EGTA/Ca2+ solution as calculated using Calcon software). The precipitation of EGFP- and GST-tagged proteins was analyzed by Western blotting using anti-GFP and anti-GST antibodies. The data shown are representative of three independent experiments. Input: purified GST alone and -EFHs (upper panels) and COS-7 cell lysates expressing EGFP-DGKα-C1Ds (lower panel). (B) The quantified relative intensities of the EGFP-DGKα-C1Ds co-precipitated with GST-DGKα-EFHs in the absence or the presence of Ca2+. The amount of EGFP-DGKα-C1Ds in the absence of Ca2+ was set to 100%. The corresponding value of GST alone was subtracted. The error bars represent the standard deviation of three independent experiments. Statistical significance was determined using the student’s t-test (∗∗∗P < 0.005).

Because the EF-hand motifs of DGKα are known to bind to Ca2+ (Kd ≈ 300 nM) [26,27], we next attempted to determine whether the physical interaction between the EF-hand motifs and the C1 domains of DGKα was regulated by Ca2+. Adding 1 μM Ca2+ to the co-precipitation mixture markedly attenuated the co-precipitation of the EGFP-DGKα-C1 domain with the GST-DGKα-EF-hand motifs, with an approximate 100% decrease (Fig. 4). The result demonstrated that Ca2+ induced the dissociation of the physical interaction between the DGKα-EF hand motifs and the DGKα-C1 domains, and supports the previously suggested model that Ca2+-induced conformational changes of the EF-hand-containing N-terminal region of DGKα unmask the DGKα-C-terminal region containing the C1 domains and the catalytic region [28-30,32].

Because the C1 domains of DGKα were expressed in COS-7 cells and were not purified, we cannot exclude the possibility that contaminating factors may have affected the interaction between the C1 domains and the EF-hand motifs of the enzyme. Therefore, we next attempted to confirm that highly purified DGKα C1 domains were able to bind to the DGKα EF-hand motifs that we had previously purified (Fig. S2). The bacterially expressed, cysteine-rich C1 domain has not been successfully purified so far because this domain is insoluble and is recovered in inclusion bodies. To circumvent the problems relating to insolubility, we employed a cold shock-trigger factor (TF) expression system [33], and 6xHis-TF-fused C1 domains of DGKα were successfully expressed and purified (Fig. S3). It was confirmed that the purified 6xHis-TF-C1 domains had the expected molecular weight, 70 K (Fig. S3). As shown in Fig. 5, purified GST-DGKα-EF hand motifs clearly co-precipitated with purified 6xHis-TF-DGKα-C1 domains, indicating that these two domains directly bind to each other.

Fig. 5

Purified DGKα-C1 domains interact with purified DGKα-EF-hand motifs. (A) 6xHis-TF-fused DGKα-C1 domains (C1Ds) were expressed in bacteria and purified. GST alone or GST fused with DGKα-EF hand motifs (EFHs) was co-precipitated with 6xHis-TF-DGKα-C1Ds in the absence of Ca2+ (5 mM EGTA). The precipitation of 6xHis-TF- and GST-tagged proteins was analyzed by Western blotting using anti-6xHis and anti-GST antibodies. A representative result of three independent experiments is shown. Input: purified GST alone and -EFHs (upper panels) and purified 6xHis-TF-DGKα-C1Ds (lower panel).

Discussion

In this study, we demonstrated for the first time that the EF-hand motifs of DGKα interacted intra-molecularly with its C1 domains (Figs. 2–5). Moreover, the highly purified C1 domains were pulled down with the highly purified EF-hand motifs (Fig. 5), indicating that these domains directly interact with each other. We previously proposed the mechanisms by which DGKα is activated [32]. In that model, the N-terminal region of DGKα, consisting of the RVH domain and the EF-hand motifs, sterically masks the C-terminal region of the enzyme, consisting of the C1 and catalytic domains. In the presence of elevated levels of Ca2+, a conformational change that uncovers the C1 and catalytic domains is trigger. In the new model, it is clear that the EF-hand motifs interact with the C1 domains (Fig. 6) and that the interaction between them is Ca2+-sensitive (Fig. 5). Therefore, we have further refined the knowledge regarding the mechanism by which DGKα is activation.

Fig. 6

A schematic representation of the proposed intra-molecular interaction and activation mechanisms of DGKα. The EF-hand motifs interact with the C1 domains. The N-terminal region, consisting of the RVH domain and the EF-hand motifs, sterically masks the C-terminal region of the enzyme. Elevated levels of Ca2+ trigger a conformational change that uncovers the C1 and catalytic domains. RVHD, recoverin homology domain; EFHs, EF-hand motifs; C1Ds, C1 domains; CR, catalytic region.

In this study, we identified the EF-hand motifs of DGKα as a new target of the C1 domain. The C1 domain is included in a wide variety of important proteins, such as conventional and novel protein kinase Cs, Ras guanyl nucleotide-releasing protein, Unc-13 and chimaerin, and is known well to bind to DG and phorbol ester [6,7]. However, the protein target of C1 domain has not been clearly elucidated. With regard to its protein target, we previously reported that the N-terminal region of β2-chimaerin containing Src homology 2 and C1 domains, interacted with DGKγ [34]. However, the C1 domain alone did not bind to the catalytic region of DGKγ. The C1 domain in β2-chimaerin is located at the core of the structure, rather than being exposed, with the putative membrane-binding hydrophobic residues being occluded by intra-molecular contacts with other regions of the protein [35]. The present study showed that the C1 domain of DGKα bound intra-molecularly to the EF-hand motifs of the enzyme and that a Ca2+-sensitive interaction should regulate the activity of the enzyme (Figs. 2–5). Our results expand the knowledge regarding the target and function of C1 domain.

This study provides several new insights into the functions and regulatory mechanisms of EF-hand-containing proteins. First, although EF-hand-containing proteins, such as calmodulin [36], calpain [23,24], calcineurin regulation subunit B [37] and ALG-2 [38] bind the cofactors, Ca2+/calmodulin dependent protein kinase II, calpain small subunit 1, calcineurin and annexin XI, respectively, in an inter-molecular fashion, this study is, to our knowledge, the first demonstration that the EF-hand motifs, instead of the N-terminal region of DGKα containing the RVH domain and EF-hand motifs [32], can participate in an intra-molecular association. Second, we identified the C1 domains of DGKα as a new target of the EF-hand motif. Therefore, we expanded the binding protein list of the EF-hand motif.

Compared with the N-terminal region of DGKα, the EF-hand motifs alone and the RVH domain alone exhibited weaker interaction activities (Fig. 3). The result suggests that both the EF-hand motifs and the RVH domain are necessary to achieve the maximum binding activity with the C-terminal region. However, the EF-hand motifs exhibited markedly stronger interactions with the C-terminal region, corresponding to an approximately thrice strong intensity, than the RVH domain. This indicates that EF-hand motifs are mainly responsible for the binding to the C1 domains. Because Jiang et al. [30] previously reported that the EF-hand motifs and RVH domain act as a functional unit during the Ca2+-induced activation of DGKα, the RVH domain may sterically mask the catalytic site of the catalytic region.

In this study, we have shown that DGKα is quite unique among the EF-hand-containing proteins. Furthermore, our identification of a direct, intra-molecular, interaction between the EF-hand motifs and C1 domains of DGKα helps elucidate the activation mechanism of this pathophysiologically important enzyme [15]. However, further studies, including the determination of the tertiary structure of DGKα, are needed to explore the regulation of the activity of the enzyme in greater detail.

Experimental procedures

Plasmid constructs

The pGEX-6P-1-DGKα-N-terminal region (amino acids (aa) 1–200) construct (Fig. 1) was generated as previously described [32]. The pEGFP-DGKα-C-terminal region (aa 197–734) construct was prepared as previously described [39]. The cDNAs encoding the DGKα-RVH domain (aa 1–110) and DGKα-EF-hand motifs (aa 103–200) were generated from porcine DGKα-cDNA [11] and subcloned into pGEX-6P-1 (GE Healthcare Bio-Sciences, Tokyo, Japan) at the EcoRI/XhoI site. The cDNAs encoding the DGKα-C1 domains (aa 196–362) and DGKα-catalytic region (aa 363–734) were generated from porcine DGKα-cDNA and subcloned into pEGFP-C3 at the HindIII/PstI site. The cDNA encoding the DGKα-C1 domains (aa 196–362) was also subcloned into pCold TF DNA (Takara Bio, Otsu, Japan) at the HindIII/PstI site.

Expression and purification of GST fusion proteins

BL21 cells were transformed with the pGEX-6P-1 constructs. GST alone and GST-fusion proteins were expressed and purified according to the procedure recommended by the manufacturer (GE Healthcare Bio-Sciences). Specifically, 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, Kyoto, Japan), 1 mM EDTA (Dojindo, Kumamoto, Japan), 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 Bio-Sciences) at 4 °C. The purified proteins were dialyzed in phosphate-buffered saline containing 5 mM ethylene glycol tetraacetic acid (EGTA) (Dojindo).

Expression and purification of 6xHis-TF fusion protein

BL21 cells were transformed with the pCold-TF-DNA constructs. The TF-fusion proteins were expressed and purified according to the procedure recommended by the manufacturer (Takara Bio). Specifically, the expression of fusion proteins was induced with 0.1 mM isopropyl-1-thio-β-d-galactopyranoside at 15 °C for 24 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, 1 mM EDTA, 1 mM dithiothreitol, 20 μg/ml aprotinin, 20 μg/ml leupeptin, 20 μg/ml pepstatin, 20 μg/ml soybean trypsin inhibitor and 1 mM phenylmethylsulfonyl fluoride. The insoluble material was removed by centrifugation. The supernatants were purified by affinity chromatography on a Ni Sepharose 6 fast flow column (GE Healthcare) at 4 °C. The purified proteins were dialyzed in Tris–HCl buffer (pH 7.4) containing 5 mM EGTA.

Cell culture and transfection

COS-7 cells were maintained in Dulbecco’s modified Eagle’s medium (Wako Pure Chemical Industries, Osaka, Japan) containing 10% fetal bovine serum at 37 °C in an atmosphere containing 5% CO2. The cells were transfected with the cDNAs by electroporation (1 × 106 cells/2 mm gap cuvette, 110 V, 20.0 ms pulse length, one pulse) with the Gene Pulser Xcell™ Electroporation System (Bio-Rad Laboratories, Tokyo, Japan) according to the manufacturer’s instructions.

In vitro binding assay

COS-7 cells (∼1 × 107 cells/100-mm dish) expressing either enhanced green fluorescent protein (EGFP) alone or EGFP-tagged DGKα-CTR were lysed in 1 ml of 50 mM HEPES, pH 7.2, 1% (V/V) Nonidet P-40 (MP Biomedicals, Tokyo, Japan), 5 mM EGTA, 150 mM NaCl, 5 mM MgCl2, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride and Complete protease inhibitor mixture (1 tablet/50 ml, Roche Molecular Biochemicals, Tokyo, Japan). The mixture was centrifuged at 12,000g for 10 min at 4 °C. The resulting cell lysates (500 μl each) were incubated with 10 μg of GST and GST fusion proteins for 1 h at 4 °C. Then, 10 μl of glutathione–Sepharose beads were added to the lysates, and the mixture was incubated for 30 min at 4 °C with constant rocking. The beads were washed four times with 50 mM HEPES, pH 7.2, 0.1% (V/V) Triton X-100, 0.5 mM EGTA, 100 mM NaCl, 5 mM MgCl2 and 10% glycerol. The washed beads were boiled in 50 μl SDS sample buffer. The total lysates and precipitates were analyzed by Western blotting using anti-GST and anti-GFP monoclonal antibodies as described below.

One hundred fifty picomoles TF-fused DGKα-C1 domains was incubated with 150 pmol of GST and GST fusion proteins in 10 mM Tris–HCl, pH 7.4, 150 mM NaCl, and 5 mM EGTA for 1 h at 4 °C. Then, 10 μl of glutathione–Sepharose beads were added to the mixture, and the samples were incubated for 30 min at 4 °C with constant rocking. The beads were washed three times with 50 mM HEPES, pH 7.2, 0.1% (V/V) Triton X-100, 0.5 mM EGTA, 150 mM NaCl, 5 mM MgCl2 and 10% glycerol. The washed beads were boiled in 50 μl SDS sample buffer. The total lysates and precipitates were analyzed by Western blotting using anti-GST and anti-His tag monoclonal antibodies as described below.

Western blot analysis

The cell lysates and immunoprecipitates were separated using SDS–PAGE. The separated proteins were transferred to a PVDF membrane (Bio-Rad Laboratories, Tokyo, Japan) and blocked with 5% (w/w) skim milk. The membrane was incubated with anti-GST monoclonal antibody (B-14, Santa Cruz Biotechnology, Santa Cruz, CA), anti-GFP monoclonal antibody (B-2, Santa Cruz Biotechnology) or anti-6xHis monoclonal antibody (9C11, Wako Pure Chemical Industries) in 5% skim milk for 1 h. The immunoreactive bands were visualized using peroxidase-conjugated anti-mouse IgG antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA) and the ECL Western blotting detection system (GE Healthcare Bio-Sciences).