Roles of Ionic Residues of the C1 Domain in Protein Kinase C-alpha Activation and the Origin of Phosphatidylserine Specificity*

Lenka Bittova, Robert V. Stahelin, and Wonhwa ChoDagger

From the Department of Chemistry (M/C 111), University of Illinois at Chicago, Chicago, Illinois 60607-7061

Received for publication, September 15, 2000



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

On the basis of extensive structure-function studies of protein kinase C-alpha (PKC-alpha ), we have proposed an activation mechanism for conventional PKCs in which the C2 domain and the C1 domain interact sequentially with membranes (Medkova, M., and Cho, W. (1999) J. Biol. Chem. 274, 19852-19861). To further elucidate the interactions between the C1 and C2 domains during PKC activation and the origin of phosphatidylserine specificity, we mutated several charged residues in two C1 domains (C1a and C1b) of PKC-alpha . We then measured the membrane binding affinities, activities, and monolayer penetration of these mutants. Results indicate that cationic residues of the C1a domain, most notably Arg77, interact nonspecifically with anionic phospholipids prior to the membrane penetration of hydrophobic residues. The mutation of a single aspartate (Asp55) in the C1a domain to Ala or Lys resulted in dramatically reduced phosphatidylserine specificity in vesicle binding, activity, and monolayer penetration. In particular, D55A showed much higher vesicle affinity, activity, and monolayer penetration power than wild type under nonactivating conditions, i.e. with phosphatidylglycerol and in the absence of Ca2+, indicating that Asp55 is involved in the tethering of the C1a domain to another part of PKC-alpha , which keeps it in an inactive conformation at the resting state. Based on these results, we propose a refined model for the activation of conventional PKC, in which phosphatidylserine specifically disrupts the C1a domain tethering by competing with Asp55, which then leads to membrane penetration and diacylglycerol binding of the C1a domain and PKC activation.



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Protein kinases C (PKC)1 are a family of serine/threonine kinases that play crucial roles in many different signal transduction pathways (1, 2). At least 10 isoforms of mammalian PKCs have been identified to date and they all contain an amino-terminal regulatory domain linked to a COOH-terminal kinase domain. Based on structural differences in the regulatory domain, PKC isoforms have been generally subdivided into three classes; conventional PKC (alpha , beta I, beta II, and gamma  isoforms), novel PKC (delta , epsilon , eta , and theta  isoforms), and atypical PKC (zeta  and lambda /iota isoforms). Conventional PKCs are activated by the Ca2+-dependent translocation of proteins to the membrane containing anionic phospholipids, preferably phosphatidylserine (PS) and diacylglycerol (DAG). The membrane translocation is mediated by two types of membrane-targeting domains (C1 and C2 domains) in the regulatory region of conventional PKC (3). The C2 domain of conventional PKC is responsible for the Ca2+-dependent binding of protein to anionic membranes (4-7). The conventional PKC also contains a tandem repeat of cystein-rich, C1 domains (C1a and C1b) that provide a binding site for DAG and phorbol esters (8-13). Based on extensive structure-function studies on PKC-alpha , we have recently proposed a mechanism for the in vitro membrane binding and activation of PKC-alpha (14). In this mechanism, PKC-alpha initially binds to the membrane surface via the Ca2+-dependent membrane binding of the C2 domain. Once membrane-bound, PS specifically induces the insertion of the hydrophobic residues of the C1a domain into the membrane. The membrane penetration allows optimal DAG binding and drives the release of pseudo-substrate region from the active site, hence the PKC activation. Although this mechanism accounts for much of the temporal and spatial sequences of in vitro activation of conventional PKC, questions still remain as to how the C1 and C2 domains of PKC-alpha interact with each other during PKC activation and how PS specifically induces the membrane penetration of the C1a domain. To address these questions, we performed further structure-function studies on the C1a and C1b domains of PKC-alpha , with an emphasis on surface ionic residues. Results from these studies provide an important new clue to the understanding of the origin of PS specificity.


    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine (POPS), and 1,2-sn-dioleoylglycerol (DOG) were purchased from Avanti Polar Lipids, Inc. (Alabaster, AL) and used without further purification. Tritiated POPC ([3H]POPC) was prepared from 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine and [9,10-3H]oleic acid (American Radiochemical Co.) using rat liver microsomes as described (15, 16). Phospholipid concentrations were determined by phosphate analysis (17). Fatty acid-free bovine serum albumin (BSA) was from Bayer Inc. (Kankakee, IL). [gamma -32P]ATP (3 Ci/µmol) was from Amersham Pharmacia Biotech and cold ATP was from Sigma. Triton X-100 was obtained from Pierce Chemical Co. (Rockford, IL). Restriction endonucleases and enzymes for molecular biology were obtained from either Roche Molecular Biochemicals or New England Biolabs (Beverly, MA).

Mutagenesis-- Baculovirus transfer vectors encoding the cDNA of PKC-alpha with appropriate C1 domain mutations were generated by the overlap extension polymerase chain reaction using pVL1392-PKC-alpha plasmid as a template (18). Briefly, four primers, including two complementary oligonucleotides introducing a desired mutation and two additional oligonucleotides complementary to the 5'-end and 3'-end of the PKC-alpha gene, respectively, were used for polymerase chain reaction performed in a DNA thermal cycler (PerkinElmer Life Sciences) using Pfu DNA polymerase (Stratagene). Two DNA fragments overlapping at the mutation site were first generated and purified on an agarose gel. These two fragments were then annealed and extended to generate an entire PKC-alpha gene containing a desired mutation, which was further amplified by polymerase chain reaction. The product was subsequently purified on an agarose gel, digested with NotI and EcoRI, and subcloned into the pVL1392 plasmid digested with the same restriction enzymes. The mutagenesis was verified by DNA sequencing using a Sequenase 2.0 kit (Amersham Pharmacia Biotech).

Expression of PKC-alpha and Mutants in Baculovirus-infected Sf9 Cells-- Wild type PKC-alpha and mutants were expressed in baculovirus-infected Sf9 cells (Invitrogen, La Jolla, CA) and purified as described previously (5, 18). The transfection of Sf9 cells with mutant pVL1392-PKC-alpha constructs was performed using a BaculoGoldTM transfection kit from Pharmingen (San Diego, CA). The plasmid DNA for transfection was prepared by using an EndoFree Plasmid Maxi kit (Qiagen, Valencia, CA) to avoid potential endotoxin contamination.

Determination of PKC Activity-- Activity of PKC was assayed by measuring the initial rate of [32P]phosphate incorporation from [gamma -32P]ATP (50 µM, 0.6 µCi/tube) into the histone III-SS (400 µg/ml) (Sigma). The reaction mixture contained large unilamellar vesicles (0.1 mM), 5 mM MgCl2, 12 nM PKC, and 0.1 mM CaCl2 in 50 µl of 20 mM HEPES, pH 7.0. Protamine sulfate (200 µg/ml) was used to determine the free enzyme concentration in vesicle binding measurements (see below). Free calcium concentration was adjusted using a mixture of EGTA and CaCl2 according to the method of Bers (19). Reactions were initiated by adding MgCl2 to the mixture and quenched by adding 50 µl of 5% aqueous phosphoric acid solution after a given period of incubation (e.g. 10 min for histone) at room temperature. Seventy-five microliters of quenched reaction mixtures were spotted on P-81 ion-exchange papers (Whatman) and papers were washed 4 times with 5% aqueous phosphoric acid solution and washed once with 95% aqueous ethanol. Papers were then transferred into scintillation vials containing 4 ml of scintillation fluid (Sigma) and radioactivity was measured by liquid scintillation counting. The linearity of the time course of the reaction was checked by monitoring the degree of phosphorylation at regular intervals (e.g. 5 min).

Vesicle Binding Measurements-- The binding of PKC to phospholipid vesicles was measured by a centrifugation assay using large sucrose-loaded unilamellar vesicles (100 nm diameter) (20). Sucrose-loaded vesicles were prepared as described previously (18). The final concentration of vesicle solution was determined by measuring the radioactivity of a trace of [3H]POPC (typically 0.1 mol %) included in all phospholipid mixtures. For binding experiments, PKC (~12 nM) was incubated for 15 min with sucrose-loaded vesicles (0.1 mM), 1 µM BSA, and varying concentrations of Ca2+ in 150 µl of 20 mM Tris-HCl, pH 7.5, containing 0.1 M KCl. BSA was added to minimize the loss of protein due to nonspecific adsorption to tube walls. Vesicles were pelleted at 100,000 × g for 30 min using Sorvall RC-M120EX Microultracentrifuge. Aliquots of supernatants were used for protein determination by PKC activity assay using protamine sulfate as a substrate. The fraction of bound enzyme was plotted against the anionic lipid composition (mol %) of mixed vesicles. Mol % values of PS and PG giving rise to half-maximal vesicle binding and activity ([PS]1/2 and [PG]1/2) were estimated graphically from individual plots.

Monolayer Measurements-- Surface pressure (pi ) of solution in a circular Teflon trough (4 cm diameter × 1 cm deep) was measured using a Wilhelmy plate attached to a computer-controlled Cahn electrobalance (Model C-32) as described previously (18). Five to ten microliters of phospholipid solution in ethanol/hexane (1:9 (v/v)) or chloroform was spread onto 10 ml of subphase (20 mM Tris-HCl, pH 7.5, containing 0.1 M KCl and 0.1 mM free Ca2+) to form a monolayer with a given initial surface pressure (pi 0). The subphase was continuously stirred at 60 rpm with a magnetic stir bar. Once the surface pressure reading of monolayer had been stabilized (after about 5 min), the protein solution (typically 40 µl) was injected into the subphase through a small hole drilled at an angle through the wall of the trough and the change in surface pressure (Delta pi ) was measured as a function of time. Typically, the Delta pi value reached a maximum after 30 min. The maximal Delta pi value depended on the protein concentration and reached a saturation value (e.g. at [PKC-alpha >=  1 µg/ml). Protein concentrations in the subphase were therefore maintained above such values to ensure that the observed Delta pi represented a maximal value. The critical surface pressure (pi c) was determined by extrapolating the Delta pi versus pi 0 plot to the x axis (21).

Surface Plasmon Resonance (SPR) Measurements-- 400 µg/ml vesicle solutions were prepared in an appropriate flow buffer solution (typically 10 mM HEPES, pH 7.4, containing 0.15 M NaCl and varying concentrations of Ca2+). Before SPR measurements, the Biacore X (Biacore AB) instrument was allowed to equilibrate with the buffer until the drift in signal was less than 0.3 resonance units/min. The Pioneer L1 sensor chip was then coated with the vesicles at a flow rate of 5 µl/min. The immobilized lipid vesicles were washed with 10 µl of 10 mM NaOH at 100 µl/min flow rate to remove unattached vesicles. In control experiments, the fluorescence intensity of the flow buffer after rinsing the sensor chip coated with vesicles incorporating 10 mM 5-carboxyfluorescein (Molecular Probes) was monitored. Lack of detectable fluorescence signal indicated that the vesicles remained intact on the chip. Next, 25 µl of 0.1 mg/ml BSA was injected at 5 µl/min to block exposed sites on the chip surface, which was once again washed with 10 mM NaOH. All experiments were performed with a control cell in which a second sensor surface was coated with 0.1 mg/ml BSA at 5 µl/min and then washed with 10 mM NaOH. The drift in signal for both sample and control flow cells was allowed to stabilize below 0.3 resonance unit/min before any kinetic experiments were performed. All kinetic experiments were performed at 24 °C and a flow rate of 60 µl/min. A high flow rate was used to circumvent mass transport effects. The association was monitored for 90 s (90 µl) and dissociation for 4 min. The immobilized vesicle surface was then regenerated for subsequent measurements using 10 µl of 10-50 mM NaOH or 3 M NaCl. The regeneration solution was passed over the immobilized vesicle surface until the SPR signal reached the initial background value before protein injection. For data acquisition, 5 or more different concentrations (typically within a 10-fold range around the Kd) of each protein were used. After each set of measurements, the entire immobilized vesicles were removed by injection of 25 µl of 40 mM CHAPS, followed by 25 µl of octyl glucoside at 5 µl/min, and the sensor chip was re-coated with a fresh vesicle solution for the next set of measurements. All data were evaluated using BIAevaluation 3.0 software (Biacore). For each trial, the control surface response was subtracted out to eliminate any nonspecific binding and refractive index changes due to buffer change. Furthermore, the derivative plot was used to monitor potential mass transport effects. Once these factors were checked for each set of data, the association and dissociation phases of data were globally fit to a 1:1 Langmuir binding model: [protein·vesicle] left-right-arrow protein + vesicle. The dissociation phase was fit to the integrated rate equation, R = R0ekd(t-t0, where kd is the dissociation rate constant, R0 is the response at the start of fit data, and t0 is the time at start of fit data. The association phase is fit to the integrated rate equation: R = Req(1 - e-(kaC+kd)(t0)) RI, where Req = [kaC/(kaC + kd)] Rmax, RI = refractive index change, Rmax is the theoretical binding capacity, C is analyte concentration, and ka is the association rate constant. The curve fitting efficiency was checked by residual plots and chi 2. The dissociation constant (Kd) was then calculated from the equation, Kd = kd/ka.


    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Roles of Cationic C1 Domain Residues in PKC Activation-- Figs. 1 and 2 illustrate amino acid sequences and model structures of C1a and C1b domains of PKC-alpha , respectively. In particular, Fig. 2 shows that the C1 domains of PKC-alpha have a polarized distribution of hydrophobic and ionic residues. The upper part of the molecule, where the DAG/phorbol ester binding pocket is located, contains a few aliphatic and aromatic residues whereas the middle part has a number of cationic residues. We have recently performed an extensive structure-function study on the hydrophobic residues of the C1a and C1b domains of PKC-alpha , which revealed their distinct roles in PKC activation (14). Hydrophobic residues in the C1a domain are essential for the membrane penetration and DAG-dependent activation of PKC-alpha , whereas those in the C1b domain are not directly involved in these processes. To assess the role of cationic residues of the C1 domains in the membrane binding and activation of PKC-alpha , we mutated several cationic residues in the C1a and C1b domains. Specifically, Lys62, Lys76, and Arg77 in the C1a domain and His127, Lys131, and Lys141 in the C1b domain were replaced by alanine (Fig. 1). Since all mutated residues are surface exposed (Fig. 2), these mutations were not expected to cause deleterious conformational changes. Indeed, all six mutants were expressed in baculovirus-infected insect cells as efficiently as wild type, suggesting comparable thermodynamic stability and lack of gross conformational changes.



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Fig. 1.   Sequence alignment of the C1a and C1b motifs of PKC-alpha . Mutated ionic residues are marked with asterisks.



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Fig. 2.   A proposed membrane binding mode of C1a and C1b motifs of PKC-alpha . The model structures of C1a and C1b domains shown in a ribbon diagram are built on the backbone of the C1b motif of PKC-delta with side chain replacements using a program Biopolymer (Molecular Simulation). The side chains of mutated cationic and anionic residues are highlighted in blue and red, respectively, and numbered. The upper part of C1a domain that partially penetrates into the membrane contains hydrophobic and aromatic side chains (shown in white).

We systematically analyzed the effects of the above mutations by measuring the anionic phospholipid dependence of vesicle binding and enzyme activity for wild type and mutants. First, we measured the PS dependence of binding to POPC/POPS mixed vesicles containing 1 mol % of DOG. As shown in Fig. 3, two C1a domain mutants, K62A and R77A, required significantly higher mol % of PS for vesicle binding ([PS]1/2 = 20 and 30 mol %, respectively) whereas another C1a mutant K76A and all C1b mutants behaved essentially the same as wild type ([PS]1/2 = 16 to 18 mol %). We then measured the kinase activity of wild type and mutants toward histone under the same conditions (i.e. in the presence of POPC/POPS/DOG mixed vesicles). In general, C1a domain mutants exhibited lower activity than did C1b domain mutants at a given PS concentration (Fig. 4). For instance, at 40 mol % PS wild type PKC-alpha and C1b domain mutants showed full activity. Under the same conditions, however, K62A and K76A showed only 44 and 55% of the wild type activity, respectively, although they are fully vesicle-bound (see Fig. 3). Most notably, R77A showed no detectable activity with up to 80 mol % PS although the protein should be fully vesicle-bound with 80 mol % PS (see Fig. 3). R77A exhibited full vesicle-binding affinity and enzymatic activity in the presence of 1 mol % of phorbol 12-myristate 13-acetate in the vesicles (data not shown), indicating that the extremely low activity of R77A is not due to deleterious conformational changes.



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Fig. 3.   Binding of PKC-alpha and C1 domain mutants to POPC/POPS/DOG vesicles as a function of POPS composition. Proteins include wild type (open circle ), K62A (black-triangle), K76A (black-square), R77A (black-diamond ), H127A (Delta ), K131A (), and K141A (diamond ). Total lipid concentration of POPC/POPS/DOG (99-x:x:1 in mol %) vesicles and PKC concentration were 0.1 mM and 12 nM, respectively, in 20 mM Tris-HCl buffer, pH 7.5, containing 0.1 M KCl, 0.1 mM Ca2+, and 1 µM BSA. Each data point represents an average of duplicate measurements.



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Fig. 4.   Dependence of enzymatic activity of PKC-alpha and C1 domain mutants on the POPS composition in POPC/POPS/DOG vesicles. Proteins include wild type (open circle ), K62A (black-triangle), K76A (black-square), R77A (black-diamond ), H127A (Delta ), K131A (), and K141A (diamond ). Total lipid concentration of POPC/POPS/DOG (99-x:x:1 in mol %) vesicles and PKC concentration were 0.1 mM and 12 nM, respectively, in 20 mM HEPES buffer, pH 7.0, containing 0.1 M KCl, 5 mM MgCl2, histone III-SS (400 µg/ml), and 0.1 mM Ca2+. Each data point represents an average of duplicate measurements. The absolute value of maximal activity is 0.20 ± 0.04 µmol/(mg/min).

We have previously shown that the isolated C1 domain (i.e. C1a + C1b) has essentially the same affinity for PS and phosphatidylglycerol (PG)-containing vesicles, indicating lack of a specific PS-binding site in the domain (14). This, in turn, suggests that the role of cationic residues in the C1 domains is to interact nonspecifically with anionic phospholipids. If this is the case, the mutations of the cationic residues of the C1 domains of PKC-alpha should not affect its PS specificity for vesicle binding affinity and enzyme activity. To test this notion, we measured the PG dependence of the binding of wild type and mutants to POPC/POPG/DOG mixed vesicles and compared it with the PS dependence shown in Fig. 3. As reported previously (18), PKC-alpha and all mutants required higher mol % of PG than PS for the same degree of vesicle binding (Fig. 5). As with the PS dependence of vesicle binding, only two C1a domain mutants, K62A and R77A, showed reduced binding affinity for PG-containing vesicles, while other mutants behaved like wild type. We then measured the PG dependence of kinase activity. As shown in Fig. 6, the PG dependence of activity compared well with the PS dependence (Fig. 4). In general, wild type and all mutants had much lower kinase activity in the presence of PG vesicles, and displayed significant activity only at high mol % of PG. Even at high mol % of PG, however, K62A and R77A exhibited much lower activity than wild type. For instance, R77A exhibited no detectable activity and K62A showed about 50% of wild type activity at 80 mol % PG. Thus, the PG dependence was qualitatively similar to corresponding PS dependence, indicating that the mutations of C1 domain residues affect the PS- and PG-dependent vesicle binding and activation of PKC-alpha to similar extents. Taken together, these results indicate that the cationic residues in the C1a domain, most notably Arg77, make important contributions to the membrane binding and activation of PKC-alpha by nonspecifically interacting with anionic membrane surfaces.



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Fig. 5.   Binding of PKC-alpha and C1 domain mutants to POPC/POPG/DOG vesicles as a function of POPG composition. Proteins include wild type (open circle ), K62A (black-triangle), K76A (black-square), R77A (black-diamond ), H127A (Delta ), K131A (), and K141A (diamond ). Experimental conditions are the same as described for Fig. 3.



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Fig. 6.   Dependence of enzymatic activity of PKC-alpha and C1 domain mutants toward histone on the POPG composition in POPC/POPG/DOG vesicles. Proteins include wild type (open circle ), K62A (black-triangle), K76A (black-square), R77A (black-diamond ), H127A (Delta ), K131A (), and K141A (diamond ). Experimental conditions are the same as described for Fig. 4. The absolute value of maximal activity is 0.20 ± 0.04 was µmol/(mg/min), as described for Fig. 4.

Roles of C1 Domain Aspartates-- Our model structures of C1a and C1b domains of PKC-alpha reveal the presence of single surface-exposed anionic residues, Asp55 (C1a) and Asp116 (C1b), located near the cationic patches (Fig. 2). Residues 55 and 116 are not perfectly matched in the sequence alignment but their relative location in the molecule should be similar based on our modeling (see Fig. 1). Conventional and novel PKCs invariably contain an anionic residue, predominantly Asp, in these positions. To determine the role of these unique aspartates, we replaced Asp55 in the C1a domain and Asp116 in the C1b domain with alanine and lysine, respectively. We then measured the vesicle binding and kinase activity of the mutants as a function of PS composition in POPC/POPS/DOG (99-x:x:1) mixed vesicles and also as a function of Ca2+. As shown in Fig. 7, D55A showed higher membrane affinity than wild type at a given PS composition in the range of 0 to 20 mol %. As a result, [PS]1/2 (approx 10 mol %) for D55A was significantly lower than that of wild type (approx 17 mol %). In contrast, D116A behaved similarly to wild type. A similar trend was seen with relative activity. In this case, the maximal activity of D55A was approx 35% higher than that of wild type even when both enzymes were fully activated. Fig. 8 shows the calcium dependence of PKC activity of the three proteins in the presence of POPC/POPS/DOG (69:30:1) vesicles. Again, D55A required less Ca2+ than wild type and D116A for activation and showed approx 40% higher maximal activity. Since our previous studies showed that Ca2+ and PS are required for triggering the membrane penetration and DAG binding of the C1a domain (14, 18), lower Ca2+ and PS requirements for D55A activation suggest that this mutant might have higher intrinsic activity to penetrate the membrane and bind DAG (see the monolayer penetration data below). This, in turn, implies that Asp55 might be involved in the specific tethering of C1a domain, which is relieved upon Ca2+-dependent binding to PS-containing membranes. The observed properties of D55A were not due to stronger nonspecific electrostatic interactions between the C1a domain and the anionic membrane caused by the removal of negative charge on the C1a domain, because D55K behaved essentially the same as D55A. On the basis of simple electrostatic effect, the former would have higher affinity and activity than the latter. To further test the notion that D55A exists in a more or less preactivated conformation, we measured the activity of wild type, D55A, and D116A in the presence of POPC/POPG/DAG (99-x:x:1) vesicles and 0.1 mM EGTA, which represents a highly nonproductive condition for PKC activation. As shown in Fig. 9, wild type and D116A exhibited extremely low PKC activity with POPG composition up to 80 mol % under these circumstances. In sharp contrast, D55A showed considerable residual activity: D55A was >10 times more active than wild type in a wide range of POPG concentration (i.e. 40-80 mol %). These data renders more credence to the notion that Asp55 is involved in the tethering of C1a domain, which keeps PKC-alpha in an inactive conformation.



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Fig. 7.   Relative binding affinity and enzyme activity of PKC-alpha and the C1 domain aspartate mutants as a function of PS composition. Wild type (open circle /), D55A (Delta /black-triangle), D55K (diamond /black-diamond ), and D116A (/black-square) were incubated with 0.1 mM POPC/POPS/DOG (99-x:x:1) vesicles. The relative affinity (open symbols and broken lines) and activity (filled symbols and solid lines) were measured as described in the legends to Figs. 3 and 4, respectively. The maximal activity of wild type toward histone is 0.20 ± 0.04 was µmol/(mg/min). Each data point represents an average of triplicate measurements.



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Fig. 8.   Dependence of enzyme activity of PKC-alpha and the C1 domain aspartate mutants on calcium concentration. Wild type (open circle ), D55A (Delta ), and D116A () (all 12 nM) were incubated with 0.1 mM POPC/POPS/DOG (69:30:1) vesicles in 20 mM HEPES buffer, pH 7.0, containing 0.1 M KCl, 5 mM MgCl2, histone III-SS (400 µg/ml), and varying concentrations of Ca2+. Each data point represents an average of duplicate measurements. The maximal activity of wild type toward histone is 0.20 ± 0.04 was µmol/(mg/min).



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Fig. 9.   Dependence of enzyme activity of PKC-alpha and the C1 domain aspartate mutants on PG composition. Wild type (open circle ), D55A (Delta ), and D116A () were incubated with 0.1 mM POPC/POPG/DOG (99-x:x:1) vesicles. The enzyme activity was measured as described in the legend to Fig. 4, except for 0.1 mM EGTA instead of 0.1 mM Ca2+. Each data point represents an average of triplicate measurements.

The Origin of PS Specificity-- Since PS specifically allows the membrane penetration and DAG binding of C1a domain, we reasoned that PS with a carboxylic group in the head group might be able to release the hypothetical C1a domain tethering by competing with Asp55. If so, D55A (and D55K) should lose PS specificity in vesicle binding and activation due to lack of C1a domain tethering. To explore this possibility, we measured the vesicle binding and kinase activity of wild type and mutants as a function of anionic phospholipid composition in two different mixed vesicles, including POPC/POPS/DOG and POPC/POPG/DOG. As shown in Fig. 10, wild type PKC-alpha and D116A showed similar high PS specificity for vesicle binding; i.e. [PS]1/2 = 17 mol % and [PG]1/2 = 30-35 mol %. In contrast, D55A showed a lower degree of PS specificity; [PS]1/2 = 10 mol % and [PG]1/2 = 12 mol %. Thus, D55A binds PG-containing vesicles, for which conventional PKCs are known to have much lower affinity, as tightly as the wild type PKC-alpha binds PS-containing vesicles. This high affinity of D55A for PG vesicles leads to dramatically reduced PS specificity, when compared with wild type. The relative activities of the proteins determined in the presence of different mol % of POPS and POPG further support our model. All three proteins are essentially fully vesicle-bound when the anionic phospholipid composition of mixed vesicles is above 40 mol % (see Fig. 10). Thus, the relative activity of the proteins under these conditions should reflect mainly the effects of mutations on PKC activation. As shown in Fig. 11, the three proteins displayed distinctly different degrees of PS specificity. As reported previously, PKC-alpha showed high PS specificity, i.e. PS PG. When compared with wild type, D55A showed a much lower degree of PS specificity at both 40 and 60 mol % of anionic lipids. At 40 mol % of anionic lipids, PG was approx 75% as effective as PS in activating D55A. At 60 mol % of anionic lipids, activities on PS and PG were comparable. Also, D55A was more active than wild type under all assay conditions (up to 140% of wild type activity). On the other hand, D116A was about 15% less active than wild type in the presence of PS-containing vesicles but was more active than wild type in the presence of PG-containing vesicles. As a result, D116A showed considerable lower PS specificity than did wild type. Taken together, it is clear that Asp55 in the C1a domain plays an important role in the PS specificity for membrane binding and activation of PKC-alpha . On the other hand, Asp116 had little effect on the vesicle binding of PKC-alpha but modestly lowered the PS dependent activity while enhancing the PG dependent activity. Thus, Asp116 in the C1b domain might also be involved in PS specificity in PKC-alpha activation, albeit indirectly (see "Discussion").



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Fig. 10.   Binding of PKC-alpha and the C1 domain aspartate mutants to mixed vesicles as a function of PS and PG composition. Wild type (open circle /), D55A (Delta /black-triangle), and D116A (/black-square) were incubated with 0.1 mM POPC/POPS/DOG (99-x:x:1) vesicles (open symbols) or POPC/POPG/DOG (99-x:x:1) vesicles (closed symbols). Experimental conditions are the same as described for Fig. 3. Each data point represents an average of triplicate measurements.



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Fig. 11.   Enzymatic activity of PKC-alpha and C1 domain aspartate mutants toward histone at two different anionic lipid compositions. The kinase activity of wild type, D55A, and D116A was measured in the presence of POPC/POPS/DOG (open bars) and POPC/POPG/DOG (solid bars) mixed vesicles. Total lipid concentration and calcium concentration were 0.1 mM and DOG composition was 1 mol %.

Monolayer Measurements-- We have shown that PS specifically induces the penetration of C1a domain into the membrane (18). To corroborate the notion that PS specifically disrupts the tethering of C1a domain mediated by Asp55, we measured the penetration of PKC-alpha and two mutants, D55A and D116A, into POPC/POPS (6:4) and POPC/POPG (6:4) mixed monolayers (Fig. 12). If much reduced PS specificity of D55A in vesicle binding and activation derives from the loss of the specific C1a domain tethering, one would expect that D55A should have high monolayer penetration power regardless the nature of anionic phospholipids in the monolayer. In these measurements, a phospholipid monolayer of a given initial surface pressure pi 0 was spread at constant area and the change in surface pressure (Delta pi ) was monitored after the injection of the protein into the subphase. It has been shown (18, 22, 23) that those proteins whose actions involve the partial or full penetration of membranes, such as PKC-alpha , have an ability to penetrate into the phospholipid monolayer with pi 0 comparable to or higher than that of biological membranes (i.e. pi c >=  31 dyne/cm) (24). As reported previously (18), PS showed the unique ability to induce the Ca2+-dependent penetration of PKC-alpha into the monolayer: pi c approx  33 dyne/cm for the POPC/POPS (6:4) monolayer and 27 dyne/cm for the POPC/POPG (6:4) monolayer (Fig. 12). D116A again displayed a similar property with pi c approx  34 dyne/cm for the POPC/POPS monolayer and 29 dyne/cm for the POPC/POPG monolayer. In contrast, D55A showed no appreciable selectivity for PS and penetrated equally well into POPC/POPS and POPC/POPG monolayers (i.e. pi c approx  34 dyne/cm for both PS- and PG-containing monolayers). These results provide strong evidence for the notion that Asp55 is involved in the C1a domain tethering and that PS specifically disrupts the tethering and thereby induces the penetration of the hydrophobic residues of the C1a domain into the membrane.



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Fig. 12.   Effect of the initial surface pressure of monolayers on the penetration of PKC-alpha and mutants. Proteins used were wild type (open circle /), D55A (Delta /black-triangle), and D116A (/black-square) and their concentrations in the subphase were 1.5 µg/ml. Monolayers contained either POPC/POPS (6:4) (open symbols) or POPC/POPG (6:4) mixed monolayers (filled symbols). The subphase contained 20 mM Tris buffer, pH 7.5, containing 0.1 M KCl and 0.1 mM Ca2+. Each data point represents an average of duplicate measurements.

SPR Measurements-- The SPR analysis of membrane-protein interactions offers an advantage over other methods in that the effects of the mutations of membrane-binding residues on membrane association (ka) and dissociation (kd) rate constants can be directly determined (25, 26). In our recent study on the membrane binding of phospholipase A2, we showed that electrostatic interactions driven by ionic residues mainly affect ka whereas hydrophobic interactions resulting from the membrane penetration of hydrophobic residues largely influence kd.2 By means of the SPR analysis, we determined the values of ka and kd for PKC-alpha , D55A, and D116A at varying surface lipid compositions and calcium concentrations. First, we coated the sensor chip with POPC/POPS/DOG (69:30:1) vesicles and measured the binding in the presence of 0.1 mM Ca2+. As summarized in Table I, little variation of ka or kd was observed for the mutants when compared with wild type, hence comparable Kd values. This is consistent with our vesicle binding data (see Fig. 10), in which all three proteins exhibited the maximal binding under these conditions. Since there was a much larger difference in relative binding affinity with POPC/POPG/DOG (69:30:1) vesicles and 0.1 mM Ca2+ (see Fig. 10), we then measured the binding of the three proteins with the sensor chip coated with POPC/POPG/DOG (69:30:1) vesicles. In agreement with vesicle binding data, all three proteins showed lower affinity for the POPG-coated chip than for the POPS-coated chip. Again, D116A mutation did not significantly influence ka and kd under these conditions. However, D55A had 2.2-fold lower kd than wild type while having comparable ka, indicating that the mutation leads to the enhanced penetration into the POPG-containing vesicles. This is also consistent with the monolayer penetration data shown in Fig. 12. We then measured the binding to immobilized POPC/POPS/DOG (69:30:1) vesicles at the lowest possible Ca2+ concentration that gave rise to detectable SPR signal under our experimental conditions (i.e. 7 µM Ca2+). In accordance with Ca2+ dependence data in Fig. 8, D55A had 15-fold higher affinity (in terms of Kd) than wild type and D116A. Interestingly, enhanced affinity of D55A derived from both a 3.2-fold increase in ka and 4.6-fold decrease in kd. As was the case with binding to POPC/POPG/DOG (69:30:1) vesicles, the decrease in kd should be due to enhanced membrane penetration. On the other hand, the increased ka might originate from the contribution from C1a cationic residues that can readily interact with anionic membranes due to the lack of C1a domain tethering. This contribution would become more important when the C2 domain cannot effectively drive the membrane association at low calcium concentrations. Together, these data further supports the notion that Asp55 of PKC-alpha is involved in C1a domain tethering, the disruption of which allows the membrane penetration of the C1a domain, which in turn leads to more favorable interactions between C1a cationic residues and anionic membrane surfaces (see Fig. 2 and "Discussion").


                              
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Table I
Binding parameters for PKC-alpha and mutants determined from SPR analysis
Values represent the mean and standard deviation of five determinations.



    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Differential Roles of C1a and C1b Domains-- Both conventional and novel PKCs contain a tandem repeat of C1 domains, which serve as a binding site for DAG and phorbol esters (8-13). Irie et al. (27) recently reported that despite high sequence homology, isolated C1 domains of conventional and novel PKCs have different phorbol ester affinity, with dissociation constants ranging from 1 nM to >3 µM. Oancea et al. (28) also reported that isolated C1a and C1b domains of PKC-gamma showed different translocation patterns in the cell. A growing body of evidence indicates that C1a and C1b domains have distinct roles in the full-length PKC molecule. For instance, Slater et al. (29, 30) have reported that PKC-alpha contains two distinct binding sites with low and high affinity for phorbol esters and that DAG and phorbol esters bind to the two discrete sites with opposite affinity. Although these in vitro and cell studies have demonstrated distinct properties and roles of the C1a and C1b domains, no direct correlation between the intrinsic properties of individual C1 domains and their specific roles in different PKC isoforms has been established. Our recent study of PKC-alpha shed new light on the differential roles of C1a and C1b domain in the activation of conventional PKC (14). The study showed that differential roles of the two C1 domains in DAG-induced PKC activation are correlated with their different membrane penetration behaviors. That is, the C1a domain plays an essential role because its hydrophobic residues can penetrate into the membrane to bind DAG whereas the C1b domain does not because of lack of membrane penetration. Differential effects of cationic residue mutations described in this report corroborate the critical involvement of C1a domain in the membrane binding and activation of PKC-alpha . Among three cationic residues in the C1a domain, Arg77 is most essential for anionic vesicle binding and Lys62 also makes considerable contribution to vesicle binding, suggesting that these residues make immediate contact with anionic membrane surfaces. Interestingly, an orientation of the membrane-bound C1a domain that allows the penetration of its hydrophobic residues into the hydrophobic core of the membrane would also permit favorable contact of the two cationic residues with anionic membrane surfaces (see Fig. 2). These interactions are nonspecific Coulombic interactions, as the mutations reduce the binding to PS- and PG-containing vesicles to comparable extents. This notion is consistent with our previous finding that the vesicle binding and monolayer penetration of the isolated C1 domains (i.e. C1a + C1b) showed no PS specificity (14). Unlike C1a domain, C1b domain appears to bind the membrane in an orientation that allows neither the penetration of its hydrophobic residues nor electrostatic interactions between its cationic residues with anionic membrane surfaces. Note that C1a domain mutants display much larger decreases in activity than expected from their reduced membrane affinity. In particular, R77A shows no activity even under the conditions where all enzyme molecules are vesicle-bound. Furthermore, R77A showed markedly reduced penetration into the POPC/POPS (6:4) monolayer (pi c = 26 dyne/cm) when compared with wild type (data not shown). This indicates that the electrostatic interactions of the C1a cationic residues with anionic phospholipids are important not only for membrane binding but also for membrane penetration, DAG binding, and activation of PKC-alpha . It has been shown that multiple anionic phospholipids, including some PS molecules, are required for conventional PKC activation (31, 32). It is not likely that a conventional PKC molecule contains multiple specific binding sites for these anionic phospholipids. Our results indicate that the cationic residues in the C1a domain can provide nonspecific electrostatic interaction sites for the anionic phospholipids.

The Role of C1 Domain Aspartates and the Origin of PS Specificity-- A consensus model of conventional PKC activation holds that the Ca2+-dependent binding of PKC to PS and DAG (or phorbol esters) triggers conformational changes of PKC, resulting in the removal of the pseudosubstrate from the active site and PKC activation (2). Our previous studies provided specific mechanistic details for this general model: Ca2+ and PS induce the membrane binding of protein and the specific membrane penetration of the C1a domain of PKC-alpha to allow its interactions with DAG, which also drives the removal of the pseudosubstrate from the active site. Based on cellular translocation studies, Oancea and Meyer (33) proposed that the DAG-binding site of PKC-gamma (i.e. two C1 domains) is inaccessible to DAG in the resting state because it is clamped to the catalytic domain by the pseudosubstrate. The present study indicates that a single aspartate residue Asp55 is involved in tethering of the C1a domain of PKC-alpha to the protein molecule in the resting state, thereby rendering the DAG-binding site inaccessible to DAG in the membrane. The mutation of Asp55 to alanine (or lysine) dramatically changed the membrane binding, activation, and monolayer penetration of PKC-alpha . In particular, D55A shows much higher vesicle affinity, activity, and monolayer penetration power than wild type under nonactivating conditions, i.e. with PG and in the absence of (or at low) Ca2+, indicating that D55A has enhanced conformational flexibility that allows it to be activated much more easily than wild type. As a result, D55A shows much reduced PS specificity, which is reminiscent of the isolated C1 domains (i.e. C1a + C1b) (14), suggesting that its behaviors are dictated mainly by the C1 domains due to the disruption of C1a domain tethering. A recent study by Johnson et al. (34) suggested that a PS-specific binding site is located in the C1 domain of PKC-beta II, based on the finding that the isolated C1b domain has higher affinity for PS than for other anionic phospholipids. It should be noted that the reduced PS specificity of D55A cannot be accounted for by this model for at least two reasons. First, D55A has higher affinity for PS vesicles and higher activity in the presence of PS vesicles than wild type (see Fig. 7), which precludes the possibility that Asp55 is directly involved in PS binding. Second, no mutation of C1a domain cationic residues has a significant effect on PS specificity of binding and activity, showing that these residues do not serve as a PS-binding site. Thus, it is also unlikely that Asp55 indirectly influences PS specificity by interacting with the PS-binding site in the C1 domain. Furthermore, the model cannot fully explain the enhanced penetration of D55A into PG-containing monolayers and its elongated membrane residence time at PG-containing vesicles and at low Ca2+. These data are more consistent with the notion that the disruption of Asp55-mediated tethering leads to the nonspecific membrane penetration of the C1a domain, which results in dramatically enhanced binding affinity for non-PS anionic phospholipid aggregates and much higher activity in the presence of nonspecific lipids. This in turn suggests that the specific Ca2+- and PS-dependent membrane penetration and activation of PKC-alpha involves the disruption of Asp55-mediated C1a domain tethering. The role of Asp116 of the C1b domain in PKC-alpha activation is less clearly defined. The wild type-like vesicle affinity and monolayer penetration of D116A indicate that Asp116 is not directly involved in PKC activation and PS specificity. D116A, however, shows consistently lower activity than wild type in the presence of POPC/POPS/DOG vesicles and has significantly higher activity under nonspecific conditions, e.g. in the presence of POPC/POPG/DOG vesicles (see Fig. 11). In particular, D116A has >5-fold higher basal activity than wild type in the absence of calcium and lipid cofactors (data not shown). Thus, Asp116 might be indirectly involved in PS specificity of PKC activation by suppressing the level of nonspecific activation. This would supplement the direct role of Asp55 of the C1a domain in the PS-specific PKC activation.

The understanding of the exact chemical nature of the C1a domain tethering and the identification of residues that interact with Asp55 would require high-resolution structural information of the full-length PKC molecule. Based on several lines of evidence supporting the importance of C1-C2 interdomain interactions in PKC regulation, we speculate that Asp55 interacts with a C2 domain residue in the calcium-binding loop. Although either the C1 or C2 domain alone is capable of recruiting conventional PKC to the membrane, a concerted action of both domains is absolutely required for full activation of the enzyme and, in particular, for its PS specificity. For instance, our previous study with the isolated C1 and C2 domains of PKC-alpha indicated that a primary determinant of PS specificity resides in the C2 domain (14), yet the present study shows that the mutation in the C1a domain has a dramatic effect on the PS specificity. Furthermore, the full-length PKC-alpha , which shows much more pronounced PS specificity than does the isolated C2 domain, exhibits its full PS specificity only in the presence of C1 domain ligand, DAG, or phorbol esters. The close interaction of C1 and C2 domains has also been implicated in the regulation of a novel PKC from Aplysia (35). A recently determined crystal structure of the C2 domain of PKC-alpha ·calcium·PS complex provides an important clue to understanding the nature of putative C1-C2 inter-domain interaction and the origin of PS specificity (7). In this structure, the phosphate oxygen of a PS molecule specifically coordinates with a calcium ion, while its carboxylate interacts with the backbone and side chain nitrogens of Asn189 in the calcium binding pocket (see Fig. 13). The structure raises an intriguing possibility that Asp55 in the C1a domain specifically interacts with Asn189 in the Ca2+ binding pocket of the C2 domain in the resting state to keep the protein in an inactive conformation, as schematically illustrated in Fig. 13. We propose that upon membrane binding of PKC, which is driven by electrostatic interactions involving the C2 domain-bound calcium ions and cationic residues in the C1a domain, the carboxylate group of PS (one or more molecules) might unleash the putative tethering by replacing Asp55. This might allow the C1a domain to penetrate into the membrane and bind DAG. The molecular motion accompanying the membrane penetration would then remove the pseudosubstrate from the active site, hence the activation. Undoubtedly, the corroboration of this hypothetical model entails further studies, including the mutation of Asn189 and other C2 domain residues that might interact with Asp55. Also, it remains to be seen whether or not the model can account for the activation of other PKCs. As such, the model provides a basis for further investigation of the molecular mechanisms underlying the subcellular targeting and activation of PKC isoforms.



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Fig. 13.   A proposed mechanism of the in vitro membrane binding and activation of conventional PKC. In this model, the C1a domain and the C2 domain are tethered via hydrogen bond between Asp55 and a C2 domain residue (e.g. Asn189). When the protein binds to PG-containing membranes (case A), the C1a-C2 tethering remains intact, and consequently PKC remains largely inactive. When the protein binds to PS-containing membranes (case B, see the inset), however, the carboxylate of PS releases Asp55 of the C1a domain from the tethering, resulting in the membrane penetration and DAG binding of the C1a domain and PKC activation.



    ACKNOWLEDGEMENT

We thank John Rafter for helpful suggestions and discussions.


    FOOTNOTES

* This work was supported in part by National Institutes of Health Grant GM53987.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Established Investigator of the American Heart Association. To whom correspondence should be addressed. Tel.: 312-996-4883; Fax: 312-996-2183; E-mail: wcho@uic.edu.

Published, JBC Papers in Press, October 11, 2000, DOI 10.1074/jbc.M008491200

2 R. V. Stahelin and W. Cho, submitted for publication.


    ABBREVIATIONS

The abbreviations used are: PKC, protein kinase C; BSA, bovine serum albumin; DAG, 1,2-sn-diacylglycerol; DOG, 1,2-sn-dioleoylglycerol; PG, phosphatidylglycerol; POPC, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine; POPG, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol; POPS, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine; PS, phosphatidylserine; SPR, surface plasmon resonance; CHAPS, 3-[(3- cholamidopropyl)dimethylammonio]-1-propanesulfonate.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


1. Nishizuka, Y. (1995) FASEB J. 9, 484-496[Abstract/Free Full Text]
2. Newton, A. C. (1995) J. Biol. Chem. 270, 28495-28498[Free Full Text]
3. Newton, A. C. (1997) Curr. Opin. Cell Biol. 9, 161-167[CrossRef][Medline] [Order article via Infotrieve]
4. Edwards, A. S., and Newton, A. C. (1997) Biochemistry 36, 15615-15623[CrossRef][Medline] [Order article via Infotrieve]
5. Medkova, M., and Cho, W. (1998) J. Biol. Chem. 273, 17544-17552[Abstract/Free Full Text]
6. Sutton, R. B., and Sprang, S. R. (1998) Structure 6, 1395-1405[Medline] [Order article via Infotrieve]
7. Verdaguer, N., Corbalan-Garcia, S., Ochoa, W. F., Fita, I., and Gomez- Fernandez, J. C. (1999) EMBO J. 18, 6329-6338[Abstract/Free Full Text]
8. Bell, R. M., and Burns, D. J. (1991) J. Biol. Chem. 266, 4661-4664[Free Full Text]
9. Burns, D. J., and Bell, R. M. (1991) J. Biol. Chem. 266, 18330-18338[Abstract/Free Full Text]
10. Kazanietz, M. G., Bustelo, X. R., Barbacid, M., Kolch, W., Mischak, H., Wong, G., Pettit, G. R., Bruns, J. D., and Blumberg, P. M. (1994) J. Biol. Chem. 269, 11590-11594[Abstract/Free Full Text]
11. Ono, Y., Fujii, T., Igarashi, K., Kuno, T., Tanaka, C., Kikkawa, U., and Nishizuka, Y. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 4868-4871[Abstract]
12. Quest, A. F., and Bell, R. M. (1994) J. Biol. Chem. 269, 20000-20012[Abstract/Free Full Text]
13. Zhang, G., Kazanietz, M. G., Blumberg, P. M., and Hurley, J. H. (1995) Cell 81, 917-924[Medline] [Order article via Infotrieve]
14. Medkova, M., and Cho, W. (1999) J. Biol. Chem. 274, 19852-19861[Abstract/Free Full Text]
15. Lands, W. E. (1960) J. Biol. Chem. 235, 2233-2237[Medline] [Order article via Infotrieve]
16. Kim, Y., Lichtenbergova, L., Snitko, Y., and Cho, W. (1997) Anal. Biochem. 250, 109-116[CrossRef][Medline] [Order article via Infotrieve]
17. Kates, M. (1986) Techniques of Lipidology , 2nd Ed. , pp. 114-115, Elsevier, Amsterdam
18. Medkova, M., and Cho, W. (1998) Biochemistry 37, 4892-4900[CrossRef][Medline] [Order article via Infotrieve]
19. Bers, D. M. (1982) Am. J. Physiol. 242, C404-408[Abstract]
20. Rebecchi, M., Peterson, A., and McLaughlin, S. (1992) Biochemistry 31, 12742-12747[Medline] [Order article via Infotrieve]
21. Verger, R., and Pattus, F. (1982) Chem. Phys. Lipids 30, 189-227[CrossRef]
22. Bazzi, M. D., and Nelsestuen, G. L. (1988) Biochemistry 27, 6776-6783[Medline] [Order article via Infotrieve]
23. Souvignet, C., Pelosin, J. M., Daniel, S., Chambaz, E. M., Ransac, S., and Verger, R. (1991) J. Biol. Chem. 266, 40-44[Abstract/Free Full Text]
24. Demel, R. A., Geurts van Kessel, W. S. M., Zwaal, R. F. A., Roelofsen, B., and van Deenen, L. L. M. (1975) Biochim. Biophys. Acta 406, 97-107[Medline] [Order article via Infotrieve]
25. Schuck, P. (1997) Annu. Rev. Biophys. Biomol. Struct. 26, 541-566[CrossRef][Medline] [Order article via Infotrieve]
26. Myszka, D. G. (1997) Curr. Opin. Biotechnol. 8, 50-57[CrossRef][Medline] [Order article via Infotrieve]
27. Irie, K., Oie, K., Nakahara, A., Yanai, Y., Ohigashi, H., Wender, P. A., Fukuda, H., Konishi, H., and Kikkawa, U. (1998) J. Am. Chem. Soc. 120, 9159-9167[CrossRef]
28. Oancea, E., Teruel, M. N., Quest, A. F., and Meyer, T. (1998) J. Cell Biol. 140, 485-498[Abstract/Free Full Text]
29. Slater, S. J., Kelly, M. B., Taddeo, F. J., Ho, C., Rubin, E., and Stubbs, C. D. (1994) J. Biol. Chem. 269, 4866-4871[Abstract/Free Full Text]
30. Slater, S. J., Ho, C., Kelly, M. B., Larkin, J. D., Taddeo, F. J., Yeager, M. D., and Stubbs, C. D. (1996) J. Biol. Chem. 271, 4627-4631[Abstract/Free Full Text]
31. Hannun, Y. A., Loomis, C. R., and Bell, R. M. (1985) J. Biol. Chem. 260, 10039-10043[Abstract/Free Full Text]
32. Orr, J. W., and Newton, A. C. (1992) Biochemistry 31, 4667-4673[Medline] [Order article via Infotrieve]
33. Oancea, E., and Meyer, T. (1998) Cell 95, 307-318[Medline] [Order article via Infotrieve]
34. Johnson, J. E., Giorgione, J., and Newton, A. C. (2000) Biochemistry 39, 11360-11369[CrossRef][Medline] [Order article via Infotrieve]
35. Pepio, A. M., and Sossin, W. S. (1998) Biochemistry 37, 1256-1263[CrossRef][Medline] [Order article via Infotrieve]


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