Vitamin E isoforms have opposing regulatory effects on leucocyte recruitment during inflammation. Furthermore, in vitro, vitamin E isoforms have opposing effects on leucocyte migration across endothelial cells by regulating VCAM (vascular cell-adhesion molecule)-1 activation of endothelial cell PKCα (protein kinase Cα). However, it is not known whether tocopherols directly regulate cofactor-dependent or oxidative activation of PKCα. We report in the present paper that cofactor-dependent activation of recombinant PKCα was increased by γ-tocopherol and was inhibited by α-tocopherol. Oxidative activation of PKCα was inhibited by α-tocopherol at a 10-fold lower concentration than γ-tocopherol. In binding studies, NBD (7-nitrobenz-2-oxa-1,3-diazole)-tagged α-tocopherol directly bound to full-length PKCα or the PKCα-C1a domain, but not PKCζ. NBD-tagged α-tocopherol binding to PKCα or the PKCα-C1a domain was blocked by diacylglycerol, α-tocopherol, γ-tocopherol and retinol, but not by cholesterol or PS (phosphatidylserine). Tocopherols enhanced PKCα-C2 domain binding to PS-containing lipid vesicles. In contrast, the PKCα-C2 domain did not bind to lipid vesicles containing tocopherol without PS. The PKCα-C1b domain did not bind to vesicles containing tocopherol and PS. In summary, α-tocopherol and γ-tocopherol bind the diacylglycerol-binding site on PKCα-C1a and can enhance PKCα-C2 binding to PS-containing vesicles. Thus the tocopherols can function as agonists or antagonists for differential regulation of PKCα.
Tocopherols are antioxidant lipids that function by donating a hydrogen from the chromanol head hydroxy group to lipid radicals produced in lipid peroxidation chain reactions [1,2]. Tocopherols also have non-antioxidant functions and are reported to modulate disease, protein expression and cell signalling [3–5]. There are multiple natural isoforms of vitamin E, which differ in the number of methyl groups on the chromanol head, including the saturated α-, β-, γ- and δ-tocopherols, and unsaturated α-, β-, γ- and δ-tocotrienols. The most abundant forms of vitamin E in tissues and in the diet are α-tocopherol and γ-tocopherol. γ-Tocopherol has one less methyl group on the chromanol head than α-tocopherol.
We have reported previously that, in vivo, α-tocopherol decreases and γ-tocopherol increases leucocyte recruitment during allergic lung inflammation in mice [6–8]. Consistent with this, α-tocopherol decreases and γ-tocopherol increases endothelial cell signalling during leucocyte migration across endothelial cells in vitro [6–8]. During this leucocyte migration, leucocytes bind to the endothelial cell-adhesion molecules VCAM-1 (vascular cell-adhesion molecule 1) and ICAM-1 (intercellular adhesion molecule 1). VCAM-1 and ICAM-1 signal through activation of PKCα (protein kinase Cα) in the endothelial cells [9,10]. We have reported previously that the activation of PKCα by VCAM-1 is regulated by tocopherols [6–8]; α-tocopherol decreases and γ-tocopherol increases activation of endothelial cell PKCα [6–8]. It has also been reported that tocopherols regulate activation of PKC in other cell systems [6,11–16]; however, it is not known whether tocopherols can directly regulate PKCα.
PKCα is a serine/threonine kinase that utilizes the cofactors PS (phosphatidylserine), DAG (diacylglycerol) and calcium for activation [17–19]. PKCα comprises the domains C1a, C1b, C2, C3 and C4. C1a and C1b bind DAG and phorbol esters with differential affinities; the C1a domain preferentially binds DAG and the C1b domain preferentially binds phorbol esters. The C2 domain binds calcium and PS. The C3 catalytic domain binds ATP, and the C4 catalytic domain binds substrates. During cofactor-dependent (non-oxidative) activation, calcium recruits PKCα to the membrane where the C2 domain of PKCα directly interacts with PS in the membrane [18,20,21]. Upon C2 domain association with the membrane, the C1 domain and C2 domain fold of PKCα opens and the C1 domain then interacts with DAG in the membrane [22–24]. In addition to cofactor-dependent activation, we and others have shown that PKCα can be activated via direct oxidation of its regulatory domain [10,25]. In mild oxidizing conditions, H2O2 oxidizes thiols in the two zinc-finger regions within the C1a and C1b domains of PKCα, thus activating PKCα.
PKCα is transiently oxidized and activated during VCAM-1 signalling in endothelial cells . Briefly, VCAM-1 activates NOX2 (NADPH oxidase) that generates ROS (reactive oxygen species) for the oxidation and activation of PKCα . In addition, during VCAM-1 activation of PKCα, there is an increase in intracellular calcium, a PKCα cofactor , but there is no increase in the PKCα cofactor DAG; however, there is a reduction in endogenous cellular DAG . Thus the transient activation of PKCα by VCAM-1 is directly regulated by oxidation and the cofactors calcium and DAG. The total VCAM-1 activation of PKCα in cells is therefore the sum of the oxidative and cofactor-dependent activation.
Previous reports have indicated that activation of PKCα in cells can be altered by tocopherol treatment of cells or tissues, but it has not been reported whether tocopherols directly bind and regulate PKCα [6,11–16]. We report in the present paper that tocopherols directly bind and regulate PKCα. α-Tocopherol decreases and γ-tocopherol enhances PS-dependent activation of recombinant PKCα. Also, α-tocopherol ablates the γ-tocopherol-induced increase in PS-dependent activation of recombinant PKCα. Both α-tocopherol and γ-tocopherol significantly inhibit oxidative activation of PKCα; however, α-tocopherol inhibits oxidative-activation at 10-fold lower doses than γ-tocopherol. α-Tocopherol and γ-tocopherol enhance PKCα-C2 binding to PS-containing phospholipid layers. Moreover, these tocopherols directly bind to PKCα-C1a at the DAG-binding site. Thus α-tocopherol is an antagonist and γ-tocopherol is an agonist of PS-dependent PKCα activity. It is the sum of tocopherol isoforms' antioxidant and agonist/antagonist activities at the doses present in cells that yields the total tocopherol regulation of PKCα activity in a cell and tissue.
Cofactor-dependent PKC activity assay
The non-radioactive PKC assay kit (Calbiochem, catalogue number 539584) was used as described by the manufacturer, except for those reagents indicated below. Recombinant human His6-tagged rPKCα (Calbiochem, catalogue number 539650) or rPKCζ (Enzo Life Sciences, catalogue number BML-SE413) was used. PS supplied in chloroform/methanol (3:1,v/v) (Sigma–Aldrich, catalogue number P6641) and natural R,R,R-α-tocopherol (MP Biomedicals, catalogue number 02100562) or natural R,R,R-γ-tocopherol (Sigma–Aldrich, catalogue number 47785) in hexane was dried under nitrogen in an amber glass vial. For the kinase assay, PS was suspended in double-distilled water by three rounds of sonication in an iced Branson Model 1200 Ultrasonic water bath (40 kHz) for 1 min, followed by vortex-mixing for 30 s and being placed on ice. To suspend the tocopherols, a reaction mixture containing buffer, CaCl2 and PS was added according to the procedure described in the kit to generate final assay buffer concentrations of 6 mM MgCl2, 1 mM EDTA, 2 mM EGTA (pH 7.0), 2 mM CaCl2 and PS (at concentrations indicated in experiments). The negative control excluded the PKCα cofactors CaCl2 and PS. The tocopherols were suspended in buffer by three rounds of sonication in an iced Branson Model 1200 Ultrasonic water bath (40 kHz) for 30 s followed by vortex-mixing for 30 s. Then, 100 μM ATP (Calbiochem) was added and briefly vortex-mixed. PKCζ kinase activity assays were analysed in the presence of 30 μg/ml PS and 2 mM CaCl2. Reaction mixtures were brought to room temperature (21°C) for 10 min. Recombinant human rPKCα (Calbiochem, catalogue number 539650) or rPKCα (Enzo Life Sciences, catalogue number BML-SE413) was added to the tocopherol/reaction mixture, incubated for 5 min at room temperature, cooled on ice for 5 min, and then added to the substrate-coated plate from the Calbiochem PKC kit on ice. To initiate the kinase activity, the plate was placed in a room temperature water bath for 30 min. The reaction was stopped with 0.1 M H3PO4 and the plate was washed. The biotinylated anti-phospho-substrate antibody from the kit was added to all of the wells and incubated at room temperature for 1 h. Wells were washed and the secondary antibody from the kit (horseradish peroxidase conjugated to streptavidin) was added to all wells and incubated for 1 h at room temperature. The wells were washed and then o-phenylenediamine in substrate buffer [50 mM citric acid/sodium phosphate buffer (pH 5.0) plus H2O2] was added to the wells. When the colour change was sufficient (1–3 min), the reaction was stopped by adding 0.1 M H3PO4 to the wells. Absorbance was read on a luminescent plate reader at 492 nm. Data are presented as the relative fluorescence from the sample minus the fluorescence signal from the blank.
Oxidative activation of PKCα activity
Methods used were as described above in the cofactor-dependent protein kinase activity assay, except for the following: (i) glycerol from the commercial rPKCα kit was removed by dialysis since glycerol is an antioxidant, and (ii) no PS or CaCl2 was used in these assays since oxidative activation of PKCα is cofactor-independent. To remove glycerol, rPKCα was dialysed using 0.025 μm pore membrane (Millipore, catalogue number VSWP02500) against a reaction buffer containing iron [6 mM MgCl2, 50 mM Tris/HCl, 45 μM FeCl2, 1 mM EDTA and 2 mM EGTA (pH 7.0)] for 30 min on ice. Following the addition of tocopherol and reaction buffer with 45 μM FeCl2, oxidative activation of 15 ng of rPKCα was initiated by the addition of 1 or 10 mM H2O2 as described previously . After 2 min, the reaction was stopped with 9 mM DTT (dithiothreitol) as described previously . To examine PKC activity, the samples were then added to the substrate plate from the PKC kit and examined for the generation of fluorescence as described above in the cofactor-dependent protein kinase activity assay.
Cloning, protein expression and purification of PKCα domains C1a, C1b and C2
The GST (glutathione transferase)–PKCα-C1a fusion protein on a pGEX vector (a gift from Dr Alexandra Newton, University of California San Diego, San Diego, CA, U.S.A.) was expressed in BL-21 cells (GE Healthcare) and purified using glutathione–Sepharose beads 4B (GE Healthcare) according to standard methods with the following conditions: induction was 18 h at 25°C in the presence of 0.01 mM ZnSO4 and 1 mM IPTG (isopropyl β-D-thiogalactopyranoside); 50 μM ZnSO4 was added to all buffers following induction to allow proper folding of the GST–PKCα-C1a domain. Expression of GST–PKCα-C1a was determined by Coomassie Blue staining and Western blot analysis using an anti-GST antibody (Cell Signaling Technology, catalogue number 2622) and showed only two bands at 25 kDa (GST) and 31 kDa (GST–PKCα-C1a) (results not shown). GST–PKCα-C1a was stored in 25 mM Tris/HCl (pH 7), 75 mM NaCl and 50 μM ZnSO4 containing 50% glycerol.
PKCα-C1a, PKCα-C1b and PKCα-C2 domains were cloned into a pET21a vector with a His6 tag as described previously . To improve the expression and stability of PKCα-C1a and PKCα-C1b in Escherichia coli BL21 RIL codon plus (Stratagene) cells, EGFP [enhanced GFP (green fluorescent protein)] was inserted at the C-terminal of the C1 domain to produce EGFP-fused C1a, C1b and C2 domains. E. coli were grown in LB (Luria–Bertani) medium containing 100 μg/ml of ampicillin at 37°C until the D600 reached 0.8. Then, overexpression was induced by the addition of 0.5 mM IPTG for 6–10 h at 25°C. Cells were centrifuged at 6000 g for 15 min at 4°C, resuspended in 25 mM Tris/HCl (pH 7.4) containing 160 mM KCl, 1 mM PMSF and 5 mM DTT, and lysed by probe sonication at pulse level 3. The lysate was centrifuged at 2000 g for 20 min at 4°C. Ni-NTA (Ni2+-nitrilotriacetate; Qiagen) was added into the cell lysate and shaken for 30 min at 4°C. The mixture was applied to an anti-His6 column and the column was washed with 25 mM Tris/HCl (pH 7.4)/160 mM KCl/25mM imidazole. Proteins were eluted from the column by a gradient increase in imidazole in the buffer and then applied to an ion-exchange column for further purification. The purity and concentration of recombinant proteins were determined by SDS/PAGE and a bicinchoninic acid assay respectively.
SPR (surface plasmon resonance) for PKCα-C2 binding to LUVs (large unilamellar vesicles)
POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine), POPE (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine), POPS (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine) and the DAG derivative SAG (1-steroyl-2-arachidonyl-sn-glycerol) were from Avanti Polar Lipids. All SPR measurements were carried out at 24°C using a lipid-coated L1 chip (GE Healthcare) in the BIACORE X system as described previously . LUVs were extruded using a 100 nm-pore membrane as described previously . The SPR active surface and control surface were coated with POPC/POPE/POPS (70:20:10 mol%) and POPC vesicles respectively, as described previously . Alternatively, the control surface was coated with POPC/POPE/POPS/tocopherols in (70-x:20:10:x, x=0–10 mol%). After washing the sensor-chip surface with running buffer [25 mM Tris/HCl (pH 7.4) containing 0.16 M KCl], the active surface and control sensor-chip surface were coated with the indicated lipid composition to give the same RU (resonance unit) values. The level of lipid coating for both surfaces was kept at the minimum necessary for preventing non-specific adsorption to the sensor chips. This low surface coverage minimized the mass transport effect and kept the total protein concentration above the total concentration of protein binding sites on vesicles. For kinetic SPR measurements, the flow rate was 30 μl/min for association and dissociation phases. The protein association with the lipid layer is presented as the difference between the signals of the active chips and the background signals from the control chip.
Binding of EGFP–PKCα-C1b to GUVs (giant unilamellar vesicles)
GUVs were prepared by electroformation as described previously . GUVs were grown in a sucrose solution (350 mM) while an electric field (3 V, 20 Hz frequency) was applied for 5 h at room temperature. GUVs comprised PC (phosphatidylcholine)/PE (phosphatidylethanolamine)/PS (65:20:10 mol%) with 5 mol% of SAG (positive control), α-tocopherol or γ-tocopherol. The 1–2 μl of sucrose-loaded GUV solution was added into an eight-well chamber containing 200 μl of 25 mM Tris/HCl buffer (pH 7.4), with 0.16 M KCl solution. GUVs, which were 5–30 μm in diameter, were mixed with 100 nM EGFP–PKCα-C1b and the fluorescence intensity was examined at room temperature using a custom-built, multi-photon, multi-channel microscope with SimFCS software as described previously . EGFP–PKCα-C1b was two-photon excited at 900 nm by a tunable Tsunami laser (Spectra Physics) and a 525±25 band-pass filter was used for emission. The images (256 pixels×256 pixels) were collected with the pixel dwell time of 32 ms using Peltier-cooled 1477P style Hamamatsu photomultiplier tubes. For determination of EGFP–PKCα-C1b binding, five GUVs were selected and, for each GUV, an averaged image of a total of ten frames was collected for further analysis by MATLAB. The total photon counts of the image were read into a 256×256 matrix to recreate the averaged image. Then a binary image mask was created using this image matrix by analysing the photon count histogram of the image. The image matrix and its binary mask were multiplied to extract the photon counts only from GUVs. The total photon counts of GUVs were divided by the total area of the pixels that constitute each GUV to yield the photon counts per pixel. Data are presented as the means±S.D. [average photon count per pixel of the GUV]/[average photon count per pixel outside the GUV].
Fluorescent tocopherol ELISA for binding to PKCα
A half-area 96-well plate (Costar, catalogue number 3690) was coated overnight with 2 μg/ml anti-His6 tag antibody (Abcam, catalogue number ab9108) in carbonate buffer [15 mM Na2CO3 and 35 mM NaHCO3 (pH 9.0)] and then washed with PBS/0.05% Tween 20 and blocked with PBS/3% BSA for 2 h. Saturation of His6-tagged-rPKCα binding to the anti-His6 tag antibody on the plate was determined by labelling with an anti-PKCα antibody (Abcam, catalogue number ab4124), which had been biotinylated using the EZ-Link Biotinylation Kit (Pierce, catalogue number 21343), and then addition of streptavidin–horseradish peroxidase/o-phenylenediamine. We found that 30 ng of rPKCα per well was the lowest concentration to saturate the plate, and thus 30 ng was used in the fluorescent lipid ELISA.
For the fluorescent lipid ELISA, NBD (7-nitrobenz-2-oxa-1,3-diazole)–α-tocopherol  was diluted in ethanol and briefly vortex-mixed. NBD–α-tocopherol is non-fluorescent in hydrophilic environments but fluoresces in hydrophobic environments, as described previously for NBD–α-tocopherol binding to αTTP (α-tocopherol transfer protein) . In this PKCα-binding assay, NBD fluorescence is increased when NBD–α-tocopherol binds to hydrophobic environments within the lipid-binding domains of PKCα. NBD–α-tocopherol in ethanol or an ethanol control was added to His6-tagged rPKCα (30 ng per well) (Calbiochem) in the reaction buffer from the PKC activity kit (Calbiochem, catalogue number 539584), generating a final concentration of 1% ethanol in reaction buffer [6 mM MgCl2, 1 mM EDTA, 2 mM EGTA (pH 7.0) and 2 mM CaCl2]. The NBD–α-tocopherol/rPKCα samples were protected from light for 5 min at room temperature and then applied to an anti-His6 antibody-coated ELISA plate. The plate was rotated at room temperature for 10 min and then washed ten times with PBS/0.05% Tween 20 to remove unbound NBD–α-tocopherol. Reaction buffer was added to the plate and relative fluorescence units were measured on a fluorescence reader at 469 nm excitation and 535 nm emission.
Suspension assay for tocopherol binding to rPKCα, rPKCζ and GST–rPKCα-C1a
The binding assay functions in a similar manner to previous studies on NBD–α-tocopherol binding to αTTP in which NBD becomes fluorescent when in hydrophobic environments within αTTP . NBD–α-tocopherol was not prepared in lipid vesicles since the hydrophobic environment of vesicles induces fluorescence. The specificity of binding is examined by competition with non-labelled tocopherols, known ligands of PKCα and control lipids such as cholesterol. Briefly, NBD–α-tocopherol and/or the competitors α-tocopherol (MP Biomedicals), γ-tocopherol (Sigma–Aldrich), α-tocotrienol (Cayman Chemicals, catalogue number 10008377), γ-tocotrienol (Cayman Chemicals, catalogue number 10008494), retinol (Sigma–Aldrich, catalogue number R732), DOG (1,2-dioctanoyl-sn-glycerol, Avanti) or cholesterol (Sigma–Aldrich, catalogue number C8667) were prepared in ethanol at 200× so that the final ethanol concentration in the assay was 1%. PS was prepared at 20× by adding reaction buffer to dried-down PS and alternating sonication and vortex-mixing for 30 s each, three times. To prepare the GST–rPKCα-C1a for the assay, it was dialysed (to remove storage glycerol) for 1 h at 4°C against the reaction buffer from the PKC activity kit (Calbiochem, catalogue number 539584). Full-length commercial rPKCα and rPKCζ were not dialysed. For the assay, rPKCα (0.1 μM), rPKCζ (0.1 μM) or GST–PKCα-C1a (0.2 μM) was added to the reaction buffer followed by addition of NBD–α-tocopherol. The samples were briefly vortex-mixed three times for 1 s. In assays with competitors, NBD–α-tocopherol was added to reaction buffer containing the indicated PKC enzyme, samples were vortex-mixed (three times for 1 s each), competitor was added, and samples were vortex-mixed again (three times for 1 s each). Samples were rotated for 30 min at room temperature while protected from light. Samples were plated on a half-area 96-well plate (Costar, catalogue number 3690) and relative fluorescence units were measured on a fluorescence reader at 469 nm excitation and 535 nm emission.
Data were analysed using a one-way ANOVA followed by Tukey's multiple comparisons test (SigmaStat, Jandel Scientific). Results are presented as the means±S.E.M.
α-Tocopherol and γ-tocopherol differentially modulate cofactor-dependent rPKCα activity
We determined whether γ-tocopherol or α-tocopherol directly regulates PS cofactor-dependent activation of PKCα or oxidative activation of PKCα. In the presence of the cofactor calcium (2 mM CaCl2), γ-tocopherol consistently induced a significant, albeit small, increase in rPKCα activity in the presence of 15 μg/ml and 30 μg/ml PS (Figure 1A). This small increase is consistent with reports that γ-tocopherol induces a small significant increase in VCAM-1-activated PKCα in endothelial cells that then can result in large increases in leucocyte recruitment during inflammation in vivo [6,8]. At high PS (60 μg/ml) concentrations, rPKCα activity was elevated to the level of activity observed with 1 μM γ-tocopherol plus 15–30 μg/ml PS. In the absence of PS, γ-tocopherol did not increase the low rPKCα activity (results not shown). α-Tocopherol at 0.1–10 μM inhibited rPKCα activity in the presence of 15–60 μg/ml PS (Figure 1B). Furthermore, α-tocopherol at 1–50 μM ablated the γ-tocopherol (1 μM)-induced increase in rPKCα activity (Figure 1C). To determine whether the effects of γ-tocopherol and α-tocopherol on PKC activity were limited to DAG cofactor-dependent PKCs, we investigated whether tocopherols modulate PKCζ, which is active independent of the cofactor DAG . Neither γ-tocopherol nor α-tocopherol significantly modulated PKCζ activity (Figure 1D).
γ-Tocopherol enhances and α-tocopherol inhibits non-oxidative cofactor-dependent rPKCα activity
α-Tocopherol and γ-tocopherol inhibit oxidative activation of PKCα
VCAM-1-induced ROS oxidizes and directly activates PKCα . It has been reported that the oxidative activation of PKCα by H2O2 is accomplished by the Fenton reaction which requires catalysis by iron . Therefore iron was added to assay buffers to determine whether tocopherol regulates H2O2-induced activation of rPKCα. rPKCα was activated by H2O2 in the presence of FeCl2 (Figure 2A). Both γ-tocopherol and α-tocopherol inhibited H2O2-induced oxidative activation of rPKCα (Figure 2B). However, α-tocopherol (0.01 μM) was able to significantly inhibit rPKCα activity at lower doses that γ-tocopherol (0.1 μM) (Figure 2B).
α-Tocopherol and γ-tocopherol inhibit H2O2-induced oxidative activation of rPKCα
α-Tocopherol and γ-tocopherol enhance rPKCα-C2 binding to lipid layers containing PS
When PKCα is activated, it translocates to the plasma membrane and interacts with lipid cofactors, including PS. These membranes also contain tocopherols, but it is not known whether tocopherols in membranes regulate recruitment of PKCα. Therefore, using SPR, it was determined whether PKCα-C2 domain binding to lipid-coated surfaces was regulated by tocopherols. With SPR analysis, the relative change in binding differs among experiments; therefore comparisons among groups analysed by SPR were made within each experiment, and these are presented as separate panels in Figure 3. In Figure 3(A), there was a dose-dependent PKCα-C2 domain binding to PS-containing lipid surfaces [POPC/POPE/POPS (70:20:10 mol%)]. The PKCα-C2 domain did not bind in the absence of PS (results not shown). The PKCα-C2 domain did not bind to 90% POPC surfaces with 10 mol% α-tocopherol or 10 mol% γ-tocopherol (Figure 3B), indicating that α-tocopherol or γ-tocopherol alone was not sufficient for PKCα-C2 binding to a lipid surface without PS. Interestingly, addition of 5 mol% α-tocopherol or γ-tocopherol to a PS-containing lipid surface equally enhanced binding of PKCα-C2 as compared with the PS-containing lipid surface without tocopherols, as shown by the change in binding in Figure 3(C). However, just 1 mol% α-tocopherol enhanced the association of PKCα-C2 with the lipid layer (Figure 3D), whereas 5 mol% γ-tocopherol was required for enhanced association of PKCα-C2 (Figure 3E).
α-Tocopherol and γ-tocopherol enhance the PKCα-C2 association with PS-containing vesicles
α-Tocopherol and γ-tocopherol enhance rPKCα-C1a, but not rPKCα-C1b, binding to lipid GUVs
Since membrane binding of the PKCα-C1a and PKCα-C1b domain is difficult to monitor by SPR analysis, we measured its binding to membranes containing tocopherols by fluorescence microscopy using EGFP–PKCα-C1a, EGFP–PKCα-C1b and GUVs comprising POPC/POPE/POPS (65:20:10 mol%) or POPC/POPE/POPS (65:20:10 mol%) with 5 mol% α-tocopherol, γ-tocopherol or the positive control SAG. Fluorescence microscopy of the vesicles and fluorescence intensity analysis of the GUV surfaces showed that EGFP– PKCα-C1a and EGFP–PKCα-C1b bound the GUVs containing the positive control SAG (Figure 4). The EGFP–PKCα-C1a bound the GUVs containing α-tocopherol or γ-tocopherol (Figure 4B). In contrast, EGFP–PKCα-C1b did not bind to the GUVs containing α-tocopherol or γ-tocopherol (Figure 4A). This indicates that α-tocopherol or γ-tocopherol induce the membrane binding of the PKCα-C1a domain, but not the PKCα-C1b domain.
α-Tocopherol and γ-tocopherol bind PKCα-C1a but do not bind PKCα-C1b
NBD–α-tocopherol directly binds full-length rPKCα
Although in Figures 1 and 2 it was shown that α-tocopherol or γ-tocopherol regulated activation of PKCα, and in Figure 3 it was shown that these tocopherols regulated recruitment of PKCα to PS-containing membranes, it is not known whether tocopherols directly interact with PKCα. Therefore it was invesitgated whether tocopherols bind to full-length His6-tagged rPKCα using anti-His6 tag-coated/BSA-blocked ELISA plates and NBD-tagged α-tocopherol. In this assay, NBD–α-tocopherol becomes fluorescent when inserted into a hydrophobic pocket in PKCα, as has been described for NBD–α-tocopherol binding to αTTP . We did not use NBD–γ-tocopherol because it is difficult to synthesize and is not available. We determined that, for this assay, the maximum His6-tagged rPKCα binding to the anti-His6-tag-coated ELISA plates was 30 ng/well as measured by labelling with an anti-PKC antibody (Figure 5A). To examine NBD–α-tocopherol binding, NBD–α-tocopherol was added to rPKCα; this was then applied to an anti-His6 antibody-coated ELISA plate; the plate was washed and then fluorescence was determined in a fluorescence plate reader. NBD–α-tocopherol directly bound to rPKCα with a significant increase in the fluorescence signal at 5 μM NBD–α-tocopherol (Figure 5B). Although this lipid ELISA assay demonstrates that NBD–α-tocopherol directly binds to rPKCα, there was high background from lipid binding to BSA used for blocking the plate (results not shown).
NBD–α-tocopherol binds full-length rPKCα
For greater assay sensitivity, NBD–α-tocopherol was added to rPKCα in solution or control buffer, and the change in fluorescence was determined as NBD–α-tocopherol fluoresces when it binds in a hydrophobic environment. NBD–α-tocopherol bound to rPKCα at 1 μM NBD–α-tocopherol (Figure 6A). To examine the specificity of tocopherol binding to rPKCα, 5 μM NBD–α-tocopherol was added to rPKCα in the presence of increasing doses of the unlabelled amphipathic lipids α-tocopherol, γ-tocopherol, α-tocotrienol, γ-tocotrienol or cholesterol. α-Tocopherol, γ-tocopherol, α-tocotrienol and γ-tocotrienol competed with NBD–α-tocopherol binding to rPKCα (Figures 6B–6D). In contrast, the negative control cholesterol did not compete with NBD–α-tocopherol binding to rPKCα (Figure 6B).
Tocopherol binds to rPKCα but not rPKCζ
Next it was investigated whether DAG competes with NBD–α-tocopherol binding, since DAG contains structural similarities to tocopherols in that they each have a hydroxy group and lipid tail. At just 5 μM DOG, there was complete ablation of 5 μM NBD–α-tocopherol binding to rPKCα as compared with NBD–α-tocopherol background fluorescence in the presence of DOG without rPKCα (Figure 6E). Furthermore, retinol, which is reported to bind to the DAG site of the C1a domain [33,34], competed for NBD–α-tocopherol binding to rPKCα (Figure 6G). In contrast with competitors of the C1a domain, PS, which binds the PKCα-C2 domain, did not compete for NBD–α-tocopherol binding to rPKCα (Figure 6F). The dose-dependent increase in fluorescence in the presence of PS without rPKCα (Figure 6F) probably occurred as a result of an NBD–α-tocopherol association with hydrophobic environments within PS complexes. It was also determined whether NBD–α-tocopherol binds to PKCζ because PKCζ is activated independently of DAG . NBD–α-tocopherol did not bind PKCζ at the 5 μM dose used in rPKCα-binding studies and still did not bind at 50 μM NBD–α-tocopherol (Figure 6H). Thus tocopherols directly bind to rPKCα, but not PKCζ. Moreover, tocopherols, tocotrienols and PKCαC1a-binding lipid cofactors compete with the binding of NBD–α-tocopherol to rPKCα.
NBD–α-tocopherol directly and specifically binds to the PKCα-C1a domain
Next we investigated whether NBD–α-tocopherol directly binds to the PKCα-C1a domain, using GST–PKCα-C1a. GST was not removed from the GST–PKCα-C1a because C1a is relatively hydrophobic and the GST tag stabilizes the PKCα-C1a domain in solution. NBD–α-tocopherol bound to GST–PKCα-C1a as compared with GST alone at a 5 μM tocopherol dose, which is the same dose that optimally bound full-length rPKCα (Figures 6A and 7A). This NBD–α-tocopherol binding to GST–PKCα-C1a was competed with unlabelled α-tocopherol (Figure 7B), γ-tocopherol (Figure 7C) and DOG, the PKCα-C1a cofactor (Figure 7D), but not the control amphipathic lipid cholesterol (Figure 7E). DOG (5–10 μM) enhancement of NBD–α-tocopherol binding to PKCα-C1a (Figure 7D) suggests that perhaps DOG binding to the PKCα-C1a domain exchanges with NBD–α-tocopherol, facilitating tocopherol binding. In summary, NBD–α-tocopherol directly binds full-length rPKCα and the PKCα-C1a domain, resulting in regulation of PKCα activity.
α-Tocopherol, γ-tocopherol and DOG compete with NBD–α-tocopherol for binding to PKCα-C1a
In these studies, we demonstrate that α-tocopherol and γ-tocopherol directly modulate cofactor-dependent activation of rPKCα and oxidative activation of rPKCα. This regulation occurs through direct binding of tocopherol to the C1a domain of PKCα and through enhancement of PKCα binding to PS in tocopherol-containing lipid layers. These innovative studies are the first to demonstrate that tocopherols directly bind and modulate PKCα activity.
The PKCα-C1a regulatory domain contains a high-affinity binding site for DAG and retinol [33,34]. Cofactor binding to PKCα-C1a is influenced by cofactor fatty acid chain length and saturation, and by the hydroxy group donation of a hydrogen to a recipient atom in the PKCα-C1a domain [23,35–37]. We found that tocopherols, which have a reactive hydroxy group on the chromanol head and an unsaturated lipid tail, compete with DAG binding to the PKCα-C1a domain. We also report that the binding of tocopherols regulate PKCα activity. γ-Tocopherol elevates cofactor-dependent PKCα activity and α-tocopherol inhibits cofactor-dependent PKCα activity. Furthermore, 1 μM α-tocopherol blocks this enhancing effect of 1 μM γ-tocopherol. Therefore γ-tocopherol serves as an agonist and α-tocopherol serves as an antagonist of cofactor-dependent PKCα activity. Without PS, γ-tocopherol does not increase the low PKCα activity (results not shown), suggesting that PS is necessary for γ-tocopherol enhancement of cofactor-dependent PKCα activity. At high PS concentrations (60 μg/ml) without tocopherol, PKCα activity is elevated to the level of PKCα activity with tocopherol and 30 μg/ml PS, suggesting that high PS induces maximal activation of PKCα activity without further agonist regulation through the PKCα-C1a domain.
Tocopherols also function as antioxidants. In the present paper we report that both α-tocopherol (0.01 μM) and γ-tocopherol (0.1 μM) inhibited the oxidative activation of rPKCα, suggesting an antioxidant function for tocopherols. However, α-tocopherol significantly decreased H2O2 activation of PKCα at a 10-fold lower dose compared with γ-tocopherol, even though α-tocopherol and γ-tocopherol have approximately equal antioxidant ability towards lipids in solution [38–40] and an equal ability to bind PKCα (Figure 6B). Therefore tocopherol isoforms differ in their antioxidant capacity towards PKCα.
PKC activity is also reported to positively correlate with membrane bilayer curvature, non-bilayer phases and dehydration of the membrane by DAGs in the presence of calcium [41,42]. Using cell-free systems, it has been reported that the DAG polar head group spacing and the degree of acylated chain saturation contributes to PKC activation [37,43]. In the present study, α-tocopherol, at 5-fold lower concentrations than γ-tocopherol, enhanced the PKCα-C2 domain interaction with PS-containing lipid surfaces without direct tocopherol interaction with the PKCα-C2 domain. This may be consistent with the significantly greater partitioning of α-tocopherol than γ-tocopherol into polyunsaturated lipid-rich domains for differential regulation of membrane structure . Since it is reported that the PKCα-C2 domain binds to membranes and then, subsequently, the PKCα-C1a domain associates with DAG , it suggests that tocopherols influence the association of the PKCα-C2 domain with PS in the membrane, and then tocopherols in the membrane compete with membrane DAG for binding to the PKCα-C1a domain. Thus α-tocopherol and γ-tocopherol directly bind the PKCα-C1a domain, regulate cofactor-dependent activation of PKCα, regulate oxidative activation of PKCα and regulate recruitment of PKCα to lipid membranes. This suggests that antioxidant and non-antioxidant effects of these tocopherols contribute to the overall regulation of PKCα activity in cells and tissues.
Tocopherols have been reported to modulate PKCα activation in cells . In cells, PKCα is recruited to the cell membrane where it interacts with PS and DAG [18,22,45]. PKCα is also transiently activated by oxidation . We have reported previously that α-tocopherol pre-treatment of endothelial cells inhibits VCAM-1-induced oxidative activation of PKCα . However, during VCAM-1 activation of PKCα, in addition to oxidative activation of PKCα, there is also generation of calcium  and consumption of the PKCα cofactor DAG , suggesting a contribution of both oxidative activation of PKCα and cofactor-dependent activation of PKCα during VCAM-1 signalling in endothelial cells. Therefore α-tocopherol may inhibit VCAM-1 signalling by functioning both as an antioxidant and as an antagonist of PKCα. In contrast, γ-tocopherol, which is at one-tenth the tissue concentration of α-tocopherol, elevates VCAM-1 activation of PKCα and ablates the inhibitory effects of α-tocopherol on VCAM-1 activation of PKCα in endothelial cells . Since in tissues γ-tocopherol is at one-tenth the concentration of α-tocopherol , but we report in the present paper that 10-fold more γ-tocopherol than α-tocopherol was required to have equal antioxidant capacity towards PKCα, it suggests that, in cells, γ-tocopherol has a much lower total antioxidant capacity towards PKCα than α-tocopherol. Therefore non-antioxidant functions for γ-tocopherol are consistent with the potent γ-tocopherol enhancement of VCAM-1 activation of PKCα in cells. This enhancing effect of γ-tocopherol in cells may occur through the direct cofactor-dependent agonist activation of PKCα by γ-tocopherol and/or γ-tocopherol's enhancement of PKCα recruitment to PS-containing membranes as observed in the present study. An enhancing effect of γ-tocopherol on cofactor-dependent PKCα activity is consistent with a contribution of cofactor-dependent (calcium and DAG) activation of PKCα during VCAM-1 signalling [10,26]. In vivo, the anti-inflammatory effect of α-tocopherol and pro-inflammatory effect of γ-tocopherol on leucocyte recruitment [6,8,46] is the sum of tocopherol isoform antioxidant and agonist/antagonist functions.
In summary, α-tocopherol inhibits and γ-tocopherol elevates PKCα activity in the presence of PS. In contrast, both α-tocopherol and γ-tocopherol inhibit PS-independent oxidative activation of PKCα, although α-tocopherol significantly inhibits this oxidative PKCα activation at one-tenth the concentration required for γ-tocopherol inhibition. α-Tocopherol and γ-tocopherol modulate PKCα activity by enhancing association of the PKCα-C2 domain to PS-containing lipid layers and the tocopherols directly bind to the PKCα-C1a domain. Thus tocopherols can function as antioxidants and function as PKCα agonists or antagonists for the regulation of PKCα activity in cells.
enhanced GFP (green fluorescent protein)
giant unilamellar vesicle
intercellular adhesion molecule 1
large unilamellar vesicle
protein kinase C
reactive oxygen species
surface plasmon resonance
α-tocopherol transfer protein
vascular cell-adhesion molecule 1
Christine McCary in the research laboratory of Joan Cook-Mills performed the experiments in Figures 1 and 2 and 5–7, and participated in preparation of the paper. Youngdae Yoon in the research laboratory of Wonhwa Cho performed the experiments in Figures 3 and 4, and participated in preparation of the paper. Candace Panagabko in the research laboratory of Jeffrey Atkinson synthesized the NBD–α-tocopherol that was used in Figures 5–7. Wonhwa Cho and Jeffrey Atkinson participated in experimental design and preparation of the paper. Joan Cook-Mills guided the design of the research and experimental approaches, and participated in the preparation of the paper.
We thank Dr Sean Davidson, Dr Cara Gottardi, Dr Phil Howles and Dr Alexandra Newton for their helpful advice and suggestions regarding protein–lipid interactions. We thank Dr David Escobar and Dr Rigen Mo for helpful advice on the expression of the GST–PKCα-C1a domain.
This work was supported by the National Institutes of Health [grant numbers R01 AT004837 (to J.M.C-M), GM68849 (to W.C.)].