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Originally published In Press as doi:10.1074/jbc.M701396200 on April 30, 2007

J. Biol. Chem., Vol. 282, Issue 28, 20467-20474, July 13, 2007
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Ceramide-1-phosphate Binds Group IVA Cytosolic Phospholipase a2 via a Novel Site in the C2 Domain*Formula

Robert V. Stahelin{ddagger}§1, Preeti Subramanian1, Mohsin Vora, Wonhwa Cho||, and Charles E. Chalfant**2

From the Department of Biochemistry, Medical College of Virginia Campus, Virginia Commonwealth University, Richmond, Virginia 23298-0614, the ||Department of Chemistry, University of Illinois at Chicago, Chicago, Illinois 60607-7061, the {ddagger}Department of Biochemistry and Molecular Biology, Indiana University School of Medicine-South Bend and §Department of Chemistry and Biochemistry and The Walther Center for Cancer Research, University of Notre Dame, South Bend, Indiana 46617, and **Research and Development, Hunter Holmes McGuire Veterans Affairs Medical Center, Richmond, Virginia 23249

Received for publication, February 16, 2007 , and in revised form, March 22, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Previously, ceramide-1-phosphate (C1P) was demonstrated to be a potent and specific activator of group IV cytosolic phospholipase A2{alpha} (cPLA2{alpha}) via interaction with the C2 domain. In this study, we hypothesized that the specific interaction site for C1P was localized to the cationic beta-groove (Arg57, Lys58, Arg59) of the C2 domain of cPLA2{alpha}. In this regard, mutants of this region of cPLA2{alpha} were generated (R57A/K58A/R59A, R57A/R59A, K58A/R59A, R57A/K58A, R57A, K58A, and R59A) and examined for C1P affinity by surface plasmon resonance. The triple mutants (R57A/K58A/R59A), the double mutants (R57A/R59A, K58A/R59A, and R57A/K58A), and the single mutant (R59A) demonstrated significantly reduced affinity for C1P-containing vesicles as compared with wild-type cPLA2{alpha}. Examining these mutants for enzymatic activity demonstrated that these five mutants of cPLA2{alpha} also showed a significant reduction in the ability of C1P to: 1) increase the Vmax of the reaction; and 2) significantly decrease the dissociation constant (K As) of the reaction as compared with the wild-type enzyme. The mutational effect was specific for C1P as all of the cationic mutants of cPLA2{alpha} demonstrated normal basal activity as well as normal affinities for phosphatidylcholine and phosphatidylinositol-4,5-bisphosphate as compared with wild-type cPLA2{alpha}. This study, for the first time, demonstrates a novel C1P interaction site mapped to the cationic beta-groove of the C2 domain of cPLA2{alpha}.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Group IV cytosolic phospholipase A2{alpha} (cPLA2{alpha})3 is the initial rate-limiting enzyme in eicosanoid biosynthesis in response to many inflammatory agonists (1, 2). The cellular activation of cPLA2{alpha} requires Ca2+-dependent membrane translocation of the enzyme, which is mediated by the N-terminal C2 domain (1-4). Cell-specific and agonist-dependent events coordinate translocation of cPLA2{alpha} to the nuclear envelope, endoplasmic reticulum, and Golgi apparatus via this domain (1-8). At these membranes, cPLA2{alpha} hydrolyzes membrane phospholipids to produce arachidonic acid, which initiates pathways leading to eicosanoid synthesis (1-8).

C2 domains were originally described in protein kinase C (9) and since have been identified in numerous proteins involved in lipid signaling. C2 domains are composed of about 120 amino acids forming a common fold of eight-stranded anti-parallel beta-sandwich. Most C2 domains bind to the membranes in a Ca+2-dependent manner via the three calcium binding regions (CBRs) that are located at one end of the beta-sandwich. These C2 domains are known to exhibit different Ca+2 binding affinities, which can be modulated by the presence or absence of phospholipids. Also, most of the C2 domains contain a cationic patch in the concave face of the beta-sandwich, known as the beta-groove (48). The C2 domain of cPLA2{alpha} binds two calcium ions via the hydrophobic calcium binding regions (CBR1 and CBR3) that are also critical to membrane binding and membrane penetration (10, 11). Recently, ceramide-1-phosphate (C1P) has been defined to be the membrane lipid that enhances the association of C2 domain of cPLA2{alpha} with membranes at lower calcium concentration (e.g. submicromolar) (12).

C1P is a new addition to a growing group of bioactive sphingolipids, which include ceramide and sphingosine-1-phosphate. Recent reports from our laboratory have shown ceramide kinase to be an upstream mediator of calcium ionophore- and interleukin-1beta-induced arachidonic acid release and eicosanoid synthesis. Further studies revealed that cPLA2{alpha} was required for C1P to induce arachidonic acid release (12). In a more recent study, we have shown that C1P allosterically activates cPLA2{alpha} and enhances the in vitro interaction of the enzyme with its membrane substrate phosphatidylcholine (PC) at the mechanistic level. Using surface dilution kinetics coupled with surface plasmon resonance (SPR) technology, C1P was demonstrated to regulate the association of cPLA2{alpha} with PC-rich micelles/vesicles via a novel undescribed site in the C2 domain. The current study identified this novel site to be on the beta-groove of cPLA2{alpha} and identified critical amino acids in this region required for the interaction of this bioactive sphingolipid with the enzyme. Importantly, this is the first study to map a site for interaction of C1P with a target protein.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—1-Palmitoyl-2-arachidonyl-sn-glycero-3-phosphocholine (PAPC) was purchased from Avanti%20Polar%20Lipids">Avanti Polar Lipids, Inc. (Alabaster, AL) and used without further purification. [14C]PAPC was purchased from American Radiolabeled Chemicals. A 1,2-dipalmitoyl derivative of phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2) was purchased from Cayman Chemical Co. (Ann Arbor, MI). Octyl glucoside and (3-(3-cholamidopropyl) dimethylammonio)-1-propane-sulfonate (CHAPS) were from Fisher Scientific. Pioneer L1 sensor chip was from Biacore AB (Piscataway, NJ). Triton X-100 was purchased from Pierce. Phospholipid concentrations were determined by a modified Bartlett analysis (13). Restriction endonucleases and enzymes for molecular biology were obtained from New England Biolabs (Beverly, MA). Ceramide-1-phosphate was prepared according to the published method by direct phosphorylation of D-erythro-C18:1-ceramide in 37% yield and >95% purity as determined by thin layer chromatography, 1H-NMR, 31P-NMR, and mass spectrometry analysis (14).

Construction of cPLA2{alpha} Mutants—The QuikChange site-directed mutagenesis kit (Stratagene) was used to introduce mutations in the pVL1393 vector with a His6 tag engineered to the C-terminal of cPLA2{alpha} gene. The three basic amino acids in the C2 domain of cPLA2{alpha} were mutated in combination to generate triple, double, and single mutants. Temperature cycling was performed according to manufacturer's instructions using Pfu DNA polymerase, which replicates both strands with high fidelity and without displacing the mutagenic primers. This generates a mutated plasmid containing staggered nicks. The product was treated with DpnI endonuclease, which specifically digests methylated and hemimethylated parent DNA template and selects for mutations containing synthesized DNA. The nicked vector DNAs containing the desired mutations were then transformed into Escherichia coli XL-10 Gold cells. The mutated vectors were sequenced to ensure the presence of only the desired mutation.

Recombinant Expression of cPLA2{alpha}—Recombinant human cPLA2{alpha} was expressed in Sf9 cells with a His6 tag using a baculovirus expression system and purified using a modified protocol as described previously (10, 15). Briefly, Sf9 cells were grown in suspension culture and infected with high titer recombinant baculovirus at a multiplicity of infection of 10 for 72 h after infection. The cells were then harvested and resuspended in 10 ml of extraction buffer (50 mM Tris, pH 8.0, 200 mM KCl, 5 mM imidazole, 10 µg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride) using a hand-held homogenizer. The cells were broken by 20 strokes with a Dounce homogenizer. The cell lysate was clarified by centrifugation at 100,000 x g for 45 min at 4 °C. The cleared lysate was batch-bound to 10 ml of nickel-nitrilotriacetic acid agarose for 30 min in a column. Once this solution passed through, the column was washed with 15 ml of Buffer 1 (50 mM Tris, pH 7.2, 0.2 M KCl, 10 mM imidazole, and 10% glycerol). Subsequently, the column was washed with 15 ml of Buffer 2 (50 mM Tris, pH 8.0, 0.1 M KCl, 15 mM imidazole, and 10% glycerol). Thirdly, the column was washed with 15 ml of Buffer 3 (50 mM Tris, pH 8.0, 0.1 M KCl, 20 mM imidazole, and 10% glycerol). The protein was eluted in 1-ml fractions using 10 ml of Buffer 4 (50 mM Tris, pH 8.0, 0.1 M KCl, 250 mM imidazole, and 10% glycerol). The enzyme fractions were monitored using SDS-PAGE, and fractions containing significant amounts of cPLA2{alpha} were pooled, concentrated, and desalted in an Ultracel YM-50 centrifugal filter device. Protein concentration was determined by the bicinchoninic acid method, and aliquots of 0.1 µg/µl were made using storage buffer (50 mM Tris, pH 7.4, 0.1 M KCl, and 30% glycerol). The recombinantly expressed enzyme was analyzed by SDS-PAGE and Coomassie Brilliant Blue staining, demonstrating a purity of ~85% for each cPLA2{alpha} (see supplemental Fig. 1).

Surface Plasmon Resonance Analysis—All SPR measurements were performed at 25 °C. A detailed protocol for coating the L1 sensor chip has been described elsewhere (16, 17). Briefly, after washing the sensor chip surface, 90 µl of vesicles containing various phospholipids (see Table 1) was injected at 5 µl/min to give a response of 6500 resonance units. An uncoated flow channel was used as a control surface. Under our experimental conditions, no binding was detected to this control surface beyond the refractive index change for either the C2 domain or cPLA2{alpha} (16, 18, 19). Each lipid layer was stabilized by injecting 10 µl of 50 mM NaOH three times at 100 µl/min. Typically, no decrease in lipid signal was seen after the first injection. Kinetic SPR measurements were done at the flow rate of 30 µl/min. 90 µl of protein in 10 mM HEPES, pH 7.4, containing 0.16 M KCl, 5% glycerol, and 10 mM Ca2+ was injected to give an association time of 90 s, whereas the dissociation was monitored for 500 s or more. The lipid surface was regenerated using 10 µl of 50 mM NaOH. After sensorgrams were obtained for five different concentrations of each protein within a 10-fold range of Kd, each of the sensorgrams was corrected for refractive index change by subtracting the control surface response from it. The association and dissociation phases of all sensorgrams were globally fit to a 1:1 Langmuir binding model: protein + (protein binding site on vesicle) {leftrightarrow} (complex) using BIAevalutation 3.0 software (Biacore) as described previously (16, 18, 19). The dissociation constant (Kd) was then calculated from the equation, Kd = kd/ka.A minimum of three data sets was collected for each protein. Equilibrium (steady-state) SPR measurements were performed with the flow rate of 5 µl/min to allow sufficient time for the R values of the association phase to reach saturating response values (Req). Req values were then plotted versus protein concentrations (C), and the Kd value was determined by a nonlinear least-squares analysis of the binding isotherm using an equation, Req = Rmax/(1 + Kd/C). Mass transport was not a limiting factor in our experiments since change in flow rate (from 2 to 80 µl/min) did not affect kinetics of association and dissociation. After curve fitting, residual plots and {chi}2 values were checked to verify the validity of the binding model. Each data set was repeated three times to calculate a standard deviation value.


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  		TABLE 1
cPLA2{alpha} and Mutant Membrane Binding Analysis All binding measurements were performed in 10 mM HEPES, pH 7.4, containing 0.16 M KCl, 10 µM Ca2+ and 5% glycerol.

 


Figure 1
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  		FIGURE 1.
Structure of the cPLA2{alpha} C2 domain and full-length cPLA2{alpha} elucidating cationic residues involved in C1P binding. A, the cPLA2{alpha} C2 ribbon diagram is shown with two Ca2+ ions (spheres) bound to the domain. Aliphatic and aromatic residues (magenta) involved in PC binding and membrane penetration are shown on the top, whereas cationic residues (blue) involved in C1P binding are shown on the left side. B, cPLA2{alpha} is shown to demonstrate the positioning of the C1P binding site (blue) and phosphatidylinositol-4,5-[32P]phosphate binding site (red) in the full-length enzyme.

 
Mixed-micelle Assay for cPLA2{alpha}—cPLA2{alpha} activity was measured by a PC mixed micelle assay in a standard buffer composed of 80 mM Hepes (pH 7.5), 150 mM NaCl, 10 µM free Ca2+, 1 mM dithiothreitol. The assay also contained 0.3 mM PAPC with 250,000 dpm of [14C]PAPC, 2 mM Triton X-100, 26% glycerol, and 500 ng of purified cPLA2{alpha} protein in a total volume of 200 µl. To prepare the substrate, an appropriate volume of cold PAPC in chloroform, the indicated phospholipids, and [14C]PAPC in toluene/ethanol 1:1 solution were evaporated under nitrogen. Triton X-100 was added to the dried lipid to give 4-fold concentrated substrate solution (1.2 mM PAPC). The solution was probe-sonicated on ice (1 min on, 1 min off for 3 min). The reaction was initiated by adding 500 ng of the enzyme and was stopped by the addition of 2.5 ml of Dole reagent (2-propanol, heptane, 0.5 M H2SO4; 400:100:20, v/v/v). The amount of [14C]arachidonic acid produced was determined using the Dole procedure as described previously (20). All assays were conducted for 45 min at 37 °C. In this assay, our free calcium was calculated using the Maxchelator program utilizing the linear chelator, N-(2-hydroxyethyl)ethylene diamine-N,N',N'-triacetic acid, as described previously by our laboratory (12). Statistical and kinetic analysis was performed using SigmaPlot enzyme kinetics software, version 1.1 from SYSTAT software, Inc.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Structural analysis has shown that C2 domains have a common fold of conserved eight-stranded antiparallel beta-sandwich connected by surface loops (21-24). The surface loops are highly variable in terms of amino acid sequence and conformation and connect the beta-strands in two different topologies. Interestingly, a large number of C2 domains, including cPLA2{alpha}, contain a cationic patch (cationic beta-groove) (Fig. 1). Although the size and the electrostatic potential of the cationic beta-groove vary widely among C2 domains, its presence in most C2 domains implies an essential structural or functional role. The presence of these cationic residues in the beta-groove of cPLA2{alpha} was intriguing, as our previous data demonstrated that the C1P binding site resides in the C2 domain. To assess the importance of the beta-groove residues in cPLA2{alpha} membrane binding (Fig. 1), we prepared the following mutations: R57A, K58A, R59A, R57A/K58A, R57A/R59A, K58A/R59A, and R57A/K58A/R59A for membrane binding and activation studies.

Identification of the C1P Binding Site of cPLA2{alpha}—Herein, we employed SPR analysis for monitoring the affinity of wild-type and mutant cPLA2{alpha} for C1P-containing membranes. We have quantitatively measured the binding of cPLA2{alpha} and its C2 domain to a variety of lipid vesicles by SPR analysis (16, 17, 19, 25). To delineate the C1P binding site in cPLA2{alpha}, first, we compared the binding of wild-type cPLA2{alpha} with POPC vesicles and POPC vesicles containing 3 mol % C1P at 10 µM Ca2+. Lower Ca2+ concentrations were employed than in our previous study (19) to maximize the affinity disparity for C1P-containing vesicles between wild-type and mutants. Wild-type cPLA2{alpha} bound to PC vesicles with 49 nM affinity, similar to previous reports (15, 25), whereas interestingly, 3 mol % C1P in the vesicle increased the affinity of cPLA2{alpha} by nearly 10-fold (5.0 nM). This increased affinity was primarily due to a 4.4-fold slower dissociation rate (kd), whereas the association rate (ka) constant increased by 2-fold (Table 1). Based on our previous results, a slower dissociation rate caused by C1P suggests specific interactions with C1P or C1P-induced membrane penetration of the C2 domain (18, 49). To validate the Kd values determined from the kinetic SPR analysis, we also determined Kd by equilibrium SPR analysis (Fig. 2). The Kd value (44 ± 2.0 nM) calculated from the equilibrium binding isotherm agreed well with the Kd determined from the kinetic analysis (Kd = 49 ± 10 nM) for POPC vesicles, and that determined from equilibrium analysis with the addition of 3 mol % C1P (Kd = 4.1 ± 0.4 nM) was similar to the Kd (5.2 ± 0.4 nM) value determined from kinetic analysis.


Figure 2
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  		FIGURE 2.
SPR binding analysis of cPLA2{alpha}. A, sensorgrams from kinetic measurements of cPLA2{alpha} to POPC/C1P (97:3) vesicles. cPLA2{alpha} was injected at 30 µl/min at varying concentrations (4, 8, 16, 32, and 64 nM). Solid lines represent the best-fit theoretical curves. RU, resonance unit. B, equilibrium SPR measurements of cPLA2{alpha} to POPC/C1P (97:3) vesicles. cPLA2{alpha} was injected at 2 µl/min at varying concentrations (1, 2, 4, 12, 25, 50, 100, and 200 nM), and Req values were measured. A binding isotherm (shown) was then generated form the Req versus the concentration of cPLA2{alpha}.A solid line represents a theoretical curve constructed from Rmax (215 ± 5) and Kd (5.2 ± 0.4 nM) values determined by nonlinear least squares analysis of the isotherm using equation Req = Rmax/(1 + Kd/C). 10 mM HEPES buffer, pH 7.4, with 0.16 M KCl and 10 µM Ca2+ was used for both sets of measurements.

 
Mutants of cPLA2{alpha} were first monitored for affinity to POPC vesicles to demonstrate that none of the mutants played a significant role in binding of cPLA2{alpha} to zwitterionic vesicles. Indeed, all mutants, including a triple cationic mutant (R57A/K58A/R59A), displayed little change in POPC vesicle affinity (Kd), with rate constant (ka and kd) values within respective error bar ranges (Table 1). To quantitatively assess the effects of the cationic mutants on C1P binding, we monitored their binding to POPC/C1P (97:3) vesicles in 10 µM Ca2+. Single mutants (R57A, K58A, and R59A) reduced binding 2.4-4.6-fold to POPC/C1P vesicles, whereas having little effect on POPC binding, suggesting their involvement in specific C1P binding. In support of this C1P-specific binding hypothesis, these mutations increased kd without significantly decreasing ka. Next, we monitored the binding of double and triple cationic mutants (R57A/R59A, K58A/R59A, and R57A/K58A/R59A) to POPC/C1P vesicles. All mutations reduced binding to POPC/C1P vesicles 4-6-fold, without effecting POPC vesicle binding. Furthermore, all three mutations primarily influenced kd (faster kd), supporting the specific nature of the interaction between these residues and C1P.


Figure 3
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  		FIGURE 3.
Activation of wild-type and mutant cPLA2{alpha} by C1P and PtdIns(4,5)P2. A, recombinant wild-type (•), R57A ({circ}), K58A ({blacktriangledown}), R59A ({triangledown}), R57A/K58A ({diamondsuit}), R57A/R59A ({blacksquare}), K58A/R59A ({square}), and R57A/K58A/R59A ({diamond}) mutants of cPLA2{alpha} (0.5 µg) were assayed in the presence of various mol % of dextro-erythro-C18:1 (D-E-C18:1) ceramide-1-phosphate ([C1P]/[Triton X-100 + PC + C1P]) for 45 min at 37 °C as described under "Experimental Procedures." The mol % of PC was fixed at 15 mol % of the [Triton X-100 + PC + C1P]. B, recombinant wild-type (WT) and mutants (R57A, R57A/K58A/R59A, R59A, and K58A/R59A) of cPLA2{alpha} (0.5 µg) were assayed in the absence (black bars) and presence (gray bars) of 2.5 mol % of PtdIns(4,5)P2 ([phosphatidylinositol(4,5)P2]/[Triton X-100 + PC + phosphatidylinositol(4,5)P2]) for 45 min at 37 °C. Data are presented as cPLA2{alpha} activity measured as nmoles of arachidonic acid produced/minute/milligram of recombinant cPLA2{alpha} ± standard error. Data are representative of six separate determinations on three separate occasions.

 
Recent reports have demonstrated that PtdIns(4,5)P2 is able to increase cPLA2{alpha} affinity for the membrane as well as enhance cPLA2{alpha} activation (25-28). Unlike the C1P binding site that is located in the C2 domain, this binding site resides in the catalytic domain (25, 26). Therefore, it was expected that the above cationic site mutations would not affect the binding of cPLA2{alpha} to PtdIns(4,5)P2-containing vesicles. In fact, cationic mutants of full-length cPLA2{alpha} (Table 1) displayed analogous affinity to wild type for 3 mol % in POPC/PtdIns(4,5)P2 (97:3) vesicles. This again underscores the specific role of the C2 domain cationic beta-groove residues in C1P coordination. To demonstrate that a reduction in cationic charge by abolishing one to three cationic residues was not solely responsible for the reduction in C1P binding (i.e. a nonspecific electrostatic effect), we measured the binding of another triple cationic mutant (K541/543/544A) to the panel of lipids. This mutant displayed similar affinity to wild type for both POPC and POPC/C1P vesicles; however, the 3-fold increase with 3 mol % PtdIns(4,5)P2 was abolished for this mutation, which is in line with the proposed role of these amino acids in PtdIns(4,5)P2 binding (22).

Cationic Mutants of cPLA2{alpha} Fail to Respond to C1P without Effects on PtdIns(4,5)P2 Activation—Based on the SPR studies above, we predicted that the mutants of the cationic beta-groove of cPLA2{alpha} would demonstrate decreased response to C1P in vitro as compared with the wild-type cPLA2{alpha}. To determine whether our prediction was correct, we examined all of these cPLA2{alpha} mutants for activation with increasing mol % of C1P using a mixed-micelle assay. As shown in Fig. 3A (see also Table 2), C1P increased the Vmax value of wild-type cPLA2{alpha} by about 10-fold. For single mutants, R57A and K58A, however, CIP caused a smaller increase in the Vmax (Table 2). In accord with the SPR analysis, the triple mutant (R57A/K58A/R59A), the double mutants (R57A/K58A and R57A/R59A), and the single mutant (R59A) of cPLA2{alpha} had even smaller Vmax values in the presence of C1P (Fig. 3A and Table 2). Both the basal activity and the activation of cPLA2{alpha} by PtdIns(4,5)P2, were not significantly affected by cationic beta-groove mutations (Fig. 3B). These data again demonstrate that mutation of one or more basic amino acids (Arg57, Lys58, and Arg59) in this cationic beta-groove inhibits the response of cPLA2{alpha} to C1P without affecting basal activity or the response to PtdIns(4,5)P2. These data also support the specific nature of this interaction and a lack of structural defects due to mutagenesis of these amino acid residues.


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  		TABLE 2
KsA is the dissociation constant which is expressed in bulk concentration terms. Vmax is the true Vmax at an infinite bulk concentration of lipid substrate. App., appaernt

 
Cationic Mutants of cPLA2{alpha} Fail to Decrease the Dissociation Constant (K As) in Response to C1P—In our previous studies, we have examined the kinetic interaction of C1P-cPLA2{alpha} using the surface dilution model (19). This model takes into account both two-dimensional surface interaction and the three-dimensional bulk interaction between an interfacial enzyme and lipid substrates (29-33). We previously demonstrated that C1P activates cPLA2{alpha} activity by lowering the apparent dissociation constant (K As) of the enzyme, thereby decreasing its dissociation from its membrane substrate (19). Thus, we examined whether mutation of these basic amino acids showed any effect on the ability of C1P to lower the dissociation constant (K As). As shown in Fig. 4, supplemental Fig. 2, and Table 2, C1P lowered the K As by 2.4-fold but had smaller effects on the triple mutants (R57A/K58A/R59A), the double mutants (R57A/K58A, R57A/R59A, and K58A/R59A), and the single mutant (R59A). These results corroborate the notion that the specific binding of C1P to the cationic beta-groove (Arg57/Lys58/Arg59) activates cPLA2{alpha} by lowering its membrane dissociation.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, for the first time, a novel interaction site for C1P has been identified for a target protein, specifically cPLA2{alpha}. C1P binds to a cationic patch (Arg57, Lys58, and Arg59) on the beta-groove of the C2 domain that is adjacent to but distinct from the membrane-penetrating CBRs. The interaction, with just 3 mol % C1P in the vesicles, increases cPLA2{alpha} affinity nearly 10-fold in 10 µM Ca2+. The affinity increase is due to a modest 2-fold increase in ka, and a more prominent 4.5-fold decrease in kd. Thus, C1P functions in increasing the membrane residence time of cPLA2{alpha}, reminiscent of other interactions of peripheral proteins with phosphatidylinositol and/or diacylglycerol (18, 34-36), which is generally attributed to the specific nature of the binding and/or membrane penetration induced via the interaction (16). In line with this specificity, mutations of cationic residues in the C2 domain (triple, double, or single), reduced binding to C1P-containing vesicles 2-6-fold without observable effects on PC or PC/PtdIns(4,5)P2 vesicles. Thus, mutagenesis of these cationic residues did not affect the structure of the enzyme. It is important to note that none of the C2 domain cationic mutants appreciably lowered PtdIns(4,5)P2 vesicle binding, demonstrating the unique nature of the C1P and PtdIns(4,5)P2 binding sites. In fact, it has been suggested that the PtdIns(4,5)P2 binding site resides in the catalytic domain (28, 31). Furthermore, furthering the validity of the C1P interaction, all mutations of the cationic groove residues increased kd and slightly decreased ka, similar to the effects of C1P on wild-type cPLA2{alpha} binding. Thus, these studies have established a role of C1P in the activation of cPLA2{alpha} via a novel binding site localized to the cationic beta-groove of the C2 domain.


Figure 4
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  		FIGURE 4.
The effect of C1P on the dissociation constant, K As, of wild-type, triple mutant (R57K/K58A/R59A), and single mutant (R59A) cPLA2{alpha} in the absence and presence of 4 mol % of C1P. Recombinant wild-type and mutant cPLA2{alpha} activity were measured as a function of PC molar concentration for 45 min at 37 °C. A, wild-type and R57A/K58A/R59A cPLA2{alpha} in the absence of D-e-C18:1 C1P (• and {circ}) and in the presence of 4 mol % D-e-C18:1 C1P ({blacktriangledown} and {triangledown}), respectively. B, wild-type and R59A cPLA2{alpha} in the absence of D-e-C18:1 C1P (• and {circ}) and in the presence of 4 mol % D-e-C18:1 C1P ({blacktriangledown} and {triangledown}), respectively. The PC mole fraction for all reactions was held constant at 0.137. Data are presented as cPLA2{alpha} activity measured as nmoles of arachidonic acid produced/minute/milligram of recombinant cPLA2{alpha} ± standard error. Data are representative of six separate determinations on three separate occasions.

 
Currently, the exact mechanism of stereospecific recognition of C1P by the cPLA2{alpha} C2 domain is unknown. Among three cationic residues investigated in this study, Arg59 seems to be most important because its mutation consistently has a bigger effect for C1P interaction than mutations of Arg57 and Lys58. This is intriguing in that Arg59 is more proximal than Arg57 and Lys58 to the calcium binding loops. Thus, when the Ca2+ binding loops interact with and partially penetrate the membrane, the cationic groove, Arg59 in particular, seems to be well positioned to bind an anionic lipid head group (37, 38). Under these conditions, C1P may serve as a bridge between cPLA2{alpha} and the membrane, similar to that proposed for the AP180 N-terminal homology domain (ANTH) domain of PtdIns(4,5)P2 (36, 39) interaction or the Ca2+-bridge suggested for the C2 domain of protein kinase C{alpha} (40). Alternatively, C1P could induce the more effective penetration of cPLA2{alpha} through the C2 and/or catalytic domains. Our earlier study demonstrated that the effects of C1P binding are more prominent on the isolated C2 domain than full-length cPLA2{alpha}, suggesting that C1P effects are more local to the C2 domain binding of cPLA2{alpha}. The current study opens an avenue to investigate the nature and orientation of cPLA2{alpha} as well as its isolated C2 domain at the C1P- and PtdIns(4,5)P2-containing membrane interface through lipid penetration analysis (10), EPR (37, 41, 42), x-ray reflectivity studies (38), or molecular dynamics simulations (41).

The involvement of the beta-groove in lipid binding was first suggested by Fukuda and coworkers who demonstrated the ability of the C2B domain of synaptotagmin II and IV to bind soluble inositol polyphosphates (43, 44). Subsequently, a number of other C2 domains have been shown to bind lipids through their beta-groove in both Ca2+-dependent and Ca2+-independent manners (45-47). Although most C2 domains reported to bind lipids through their beta-groove interact nonspecifically with phosphatidylinositides, such as PtdIns(4,5)P2, the cPLA2{alpha} C2 domain is one of the first C2 domains demonstrated to harbor such selectivity for anionic lipids, only displaying an affinity increase with C1P. Furthermore, this is the first known C2 domain to interact with a phosphorylated sphingolipid. Although this study opens an avenue to better understand the function of C1P in the recruitment of cPLA2{alpha} to the Golgi, it also serves as a framework to systematically study the unique nature of C2 domain lipid interactions with particular emphasis on the beta-groove.

In this study, for the first time, we have determined the amino acids (Arg57/Lys58/Arg59) critical for the C1P-cPLA2{alpha} interaction. The interaction site for C1P was localized to the cationic beta-groove of the C2 domain of the enzyme. Cationic mutants of cPLA2{alpha} demonstrated decreased response to C1P as shown by SPR and mixed-micelle activity assays. This effect was also shown to be specific to C1P as these mutants retained their response to PtdIns(4,5)P2. Thus, this study further defines a specific role for C1P in the activation of cPLA2{alpha}. The identification of the C1P binding site will now allow for "in-depth" studies on the requirement of the C1P-cPLA2{alpha} interaction for cPLA2{alpha} translocation to membranes.


   FOOTNOTES
 
* This work was supported by grants from the Veterans Affairs (Veterans Affairs Merit Review I) (to C. E. C.), from National Institutes of Health Grants HL072925 (to C. E. C.), CA117950 (to C. E. C.), GM52598 (to W. C.), GM53987 (to W. C.), and GM68849 (to W. C.) and American Heart Association AHA 5-30693 predoctoral fellowship (to P. S.). This work was also supported by an Indiana University Biomedical Research Grant (to R. V. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Formula The on-line version of this article (available at http://www.jbc.org) contains two supplemental figures and a supplemental table. Back

1 Both authors contributed equally to the manuscript. Back

2 To whom correspondence should be addressed: Dept. of Biochemistry, Rm. 2-016, Sanger Hall, Virginia Commonwealth University, 1101 East Marshall St., P. O. Box 980614, Richmond, VA 23298-0614. Tel.: 804-828-9526; Fax: 804-828-1473; E-mail: cechalfant{at}vcu.edu.

3 The abbreviations used are: cPLA2{alpha}, group IVA cytosolic phospholipase A2; C1P, ceramide-1-phosphate; CerK, ceramide kinase; PAPC, 1-palmitoyl-2-arachidonoyl-sn-phosphatidylcholine; POPC, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine; mol %, mole percentage of mixed-micelle; CHAPS, (3-(3-cholamidopropyl) dimethylammonio)-1-propane-sulfonate; PtdIns(4,5)P2, phosphatidylinositol-4,5-bisphosphate; SPR, surface plasmon resonance; PC, phosphatidylcholine; CBR, calcium binding region; D-E-C18:1, dextro-erythro-C18:1. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Darrell Peterson and Mario A. Saavedra for their help and support with the His6 tag purification. Virginia Commonwealth University was supported by National Institutes of Health NH1C06-RR17393for renovation.



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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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