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

2001; Elsevier BV; Volume: 276; Issue: 6 Linguagem: Inglês

10.1074/jbc.m008491200

1083-351X

Lenka Bittova, Robert V. Stahelin, Wonhwa Cho,

Erythrocyte Function and Pathophysiology

On the basis of extensive structure-function studies of protein kinase C-α (PKC-α), 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-α. 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-α, 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. On the basis of extensive structure-function studies of protein kinase C-α (PKC-α), 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-α. 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-α, 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. protein kinase C bovine serum albumin 1,2-sn-diacylglycerol 1,2-sn-dioleoylglycerol phosphatidylglycerol 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine phosphatidylserine surface plasmon resonance 3-[(3- cholamidopropyl)dimethylammonio]-1-propanesulfonate Protein kinases C (PKC)1are a family of serine/threonine kinases that play crucial roles in many different signal transduction pathways (1Nishizuka Y. FASEB J. 1995; 9: 484-496Crossref PubMed Scopus (2347) Google Scholar, 2Newton A.C. J. Biol. Chem. 1995; 270: 28495-28498Abstract Full Text Full Text PDF PubMed Scopus (1461) Google Scholar). 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 (α, βI, βII, and γ isoforms), novel PKC (δ, ε, η, and θ isoforms), and atypical PKC (ζ and λ/ι 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 (3Newton A.C. Curr. Opin. Cell Biol. 1997; 9: 161-167Crossref PubMed Scopus (843) Google Scholar). The C2 domain of conventional PKC is responsible for the Ca2+-dependent binding of protein to anionic membranes (4Edwards A.S. Newton A.C. Biochemistry. 1997; 36: 15615-15623Crossref PubMed Scopus (76) Google Scholar, 5Medkova M. Cho W. J. Biol. Chem. 1998; 273: 17544-17552Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 6Sutton R.B. Sprang S.R. Structure. 1998; 6: 1395-1405Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar, 7Verdaguer N. Corbalan-Garcia S. Ochoa W.F. Fita I. Gomez- Fernandez J.C. EMBO J. 1999; 18: 6329-6338Crossref PubMed Scopus (281) Google Scholar). 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 (8Bell R.M. Burns D.J. J. Biol. Chem. 1991; 266: 4661-4664Abstract Full Text PDF PubMed Google Scholar, 9Burns D.J. Bell R.M. J. Biol. Chem. 1991; 266: 18330-18338Abstract Full Text PDF PubMed Google Scholar, 10Kazanietz M.G. Bustelo X.R. Barbacid M. Kolch W. Mischak H. Wong G. Pettit G.R. Bruns J.D. Blumberg P.M. J. Biol. Chem. 1994; 269: 11590-11594Abstract Full Text PDF PubMed Google Scholar, 11Ono Y. Fujii T. Igarashi K. Kuno T. Tanaka C. Kikkawa U. Nishizuka Y. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 4868-4871Crossref PubMed Scopus (391) Google Scholar, 12Quest A.F. Bell R.M. J. Biol. Chem. 1994; 269: 20000-20012Abstract Full Text PDF PubMed Google Scholar, 13Zhang G. Kazanietz M.G. Blumberg P.M. Hurley J.H. Cell. 1995; 81: 917-924Abstract Full Text PDF PubMed Scopus (590) Google Scholar). Based on extensive structure-function studies on PKC-α, we have recently proposed a mechanism for thein vitro membrane binding and activation of PKC-α (14Medkova M. Cho W. J. Biol. Chem. 1999; 274: 19852-19861Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar). In this mechanism, PKC-α 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-α 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-α, 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. 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 (15Lands W.E. J. Biol. Chem. 1960; 235: 2233-2237Abstract Full Text PDF PubMed Google Scholar, 16Kim Y. Lichtenbergova L. Snitko Y. Cho W. Anal. Biochem. 1997; 250: 109-116Crossref PubMed Scopus (37) Google Scholar). Phospholipid concentrations were determined by phosphate analysis (17Kates M. Techniques of Lipidology. Elsevier, Amsterdam1986: 114-115Google Scholar). Fatty acid-free bovine serum albumin (BSA) was from Bayer Inc. (Kankakee, IL). [γ-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). Baculovirus transfer vectors encoding the cDNA of PKC-α with appropriate C1 domain mutations were generated by the overlap extension polymerase chain reaction using pVL1392-PKC-α plasmid as a template (18Medkova M. Cho W. Biochemistry. 1998; 37: 4892-4900Crossref PubMed Scopus (78) Google Scholar). 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-α 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-α gene containing a desired mutation, which was further amplified by polymerase chain reaction. The product was subsequently purified on an agarose gel, digested withNot I and Eco RI, 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). Wild type PKC-α and mutants were expressed in baculovirus-infected Sf9 cells (Invitrogen, La Jolla, CA) and purified as described previously (5Medkova M. Cho W. J. Biol. Chem. 1998; 273: 17544-17552Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 18Medkova M. Cho W. Biochemistry. 1998; 37: 4892-4900Crossref PubMed Scopus (78) Google Scholar). The transfection of Sf9 cells with mutant pVL1392-PKC-α 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. Activity of PKC was assayed by measuring the initial rate of [32P]phosphate incorporation from [γ-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 (19Bers D.M. Am. J. Physiol. 1982; 242: C404-C408Crossref PubMed Google Scholar). 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). The binding of PKC to phospholipid vesicles was measured by a centrifugation assay using large sucrose-loaded unilamellar vesicles (100 nm diameter) (20Rebecchi M. Peterson A. McLaughlin S. Biochemistry. 1992; 31: 12742-12747Crossref PubMed Scopus (175) Google Scholar). Sucrose-loaded vesicles were prepared as described previously (18Medkova M. Cho W. Biochemistry. 1998; 37: 4892-4900Crossref PubMed Scopus (78) Google Scholar). 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 mmTris-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]12 and [PG]12) were estimated graphically from individual plots. Surface pressure (π) 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 (18Medkova M. Cho W. Biochemistry. 1998; 37: 4892-4900Crossref PubMed Scopus (78) Google Scholar). Five to ten microliters of phospholipid solution in ethanol/hexane (1:9 (v/v)) or chloroform was spread onto 10 ml of subphase (20 mmTris-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 (π0). 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 (Δπ) was measured as a function of time. Typically, the Δπ value reached a maximum after 30 min. The maximal Δπ value depended on the protein concentration and reached a saturation value (e.g. at [PKC-α] ≥ 1 μg/ml). Protein concentrations in the subphase were therefore maintained above such values to ensure that the observed Δπ represented a maximal value. The critical surface pressure (πc) was determined by extrapolating the Δπversus π0 plot to the x axis (21Verger R. Pattus F. Chem. Phys. Lipids. 1982; 30: 189-227Crossref Scopus (130) Google Scholar). 400 μg/ml vesicle solutions were prepared in an appropriate flow buffer solution (typically 10 mm HEPES, pH 7.4, containing 0.15m 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 K d) 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] ↔ protein + vesicle. The dissociation phase was fit to the integrated rate equation, R =R 0 e k d(t−t 0, where k d is the dissociation rate constant,R 0 is the response at the start of fit data, andt 0 is the time at start of fit data. The association phase is fit to the integrated rate equation:R = R eq(1 −e −(k aC+k d)(t0)) + RI, where R eq = [k aC/(k aC +k d)] Rmax, RI = refractive index change, R max is the theoretical binding capacity, C is analyte concentration, andk a is the association rate constant. The curve fitting efficiency was checked by residual plots and χ2. The dissociation constant (K d) was then calculated from the equation, K d =k d/k a. Figs. 1 and 2illustrate amino acid sequences and model structures of C1a and C1b domains of PKC-α, respectively. In particular, Fig. 2 shows that the C1 domains of PKC-α 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-α, which revealed their distinct roles in PKC activation (14Medkova M. Cho W. J. Biol. Chem. 1999; 274: 19852-19861Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar). Hydrophobic residues in the C1a domain are essential for the membrane penetration and DAG-dependent activation of PKC-α, 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-α, 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.Figure 2A proposed membrane binding mode of C1a and C1b motifs of PKC -α. The model structures of C1a and C1b domains shown in a ribbon diagram are built on the backbone of the C1b motif of PKC-δ with side chain replacements using a program Biopolymer (Molecular Simulation). The side chains of mutated cationic and anionic residues are highlighted inblue and red, respectively, andnumbered. The upper part of C1a domain that partially penetrates into the membrane contains hydrophobic and aromatic side chains (shown in white).View Large Image Figure ViewerDownload Hi-res image Download (PPT) 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]12 = 20 and 30 mol %, respectively) whereas another C1a mutant K76A and all C1b mutants behaved essentially the same as wild type ([PS]12 = 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-α 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.Figure 4Dependence of enzymatic activity of PKC -α and C1 domain mutants on the POPS composition in POPC/POPS/DOG vesicles. Proteins include wild type (○), K62A (▴), K76A (▪), R77A (♦), H127A (Δ), K131A (■), and K141A (⋄). 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 0.1m 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).View Large Image Figure ViewerDownload Hi-res image Download (PPT) 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 (14Medkova M. Cho W. J. Biol. Chem. 1999; 274: 19852-19861Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar). This, in turn, suggests that the role of cationic residues in the C1 domains is to interactnonspecifically with anionic phospholipids. If this is the case, the mutations of the cationic residues of the C1 domains of PKC-α 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 (18Medkova M. Cho W. Biochemistry. 1998; 37: 4892-4900Crossref PubMed Scopus (78) Google Scholar), PKC-α 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-α 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-α by nonspecifically interacting with anionic membrane surfaces.Figure 6Dependence of enzymatic activity of PKC -α and C1 domain mutants toward histone on the POPG composition in POPC/POPG/DOG vesicles. Proteins include wild type (○), K62A (▴), K76A (▪), R77A (♦), H127A (Δ), K131A (■), and K141A (⋄). 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.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Our model structures of C1a and C1b domains of PKC-α 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]12 (≈10 mol %) for D55A was significantly lower than that of wild type (≈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 ≈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 ≈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 (14Medkova M. Cho W. J. Biol. Chem. 1999; 274: 19852-19861Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar, 18Medkova M. Cho W. Biochemistry. 1998; 37: 4892-4900Crossref PubMed Scopus (78) Google Scholar), 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-α in an inactive conformation.Figure 8Dependence of enzyme activity of PKC -α and the C1 domain aspartate mutants on calcium concentration. Wild type (○), D55A (Δ), and D116A (■) (all 12 nm) were incubated with 0.1 mmPOPC/POPS/DOG (69:30:1) vesicles in 20 mm HEPES buffer, pH 7.0, containing 0.1 m KCl, 5 mmMgCl2, 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).View Large Image Figure ViewerDownload Hi-res image