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J. Biol. Chem., Vol. 280, Issue 43, 36452-36463, October 28, 2005
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*
1
From the
Department of Chemistry, University of Illinois, Chicago, Illinois 60607 and the Department of Microbiology and Immunology, Weill Medical College of Cornell University, New York, New York 10021
Received for publication, June 8, 2005 , and in revised form, July 13, 2005.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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(PKC) contains three membrane-targeting modules, two C1 domains (C1A and C1B) that bind diacylglycerol and phorbol ester, and the C2 domain that is responsible for the Ca2+-dependent membrane binding. Accumulating evidence suggests that C1A and C2 domains of PKC are tethered in the resting state and that the tethering is released upon binding to the membrane containing phosphatidylserine. The homology modeling and the docking analysis of C1A and C2 domains of PKC revealed a highly complementary interface that comprises Asp55-Arg252 and Arg42-Glu282 ion pairs and a Phe72-Phe255 aromatic pair. Mutations of these residues in the predicted C1A-C2 interface showed large effects on in vitro membrane binding, enzyme activity, phosphatidylserine selectivity, and cellular membrane translocation of PKC, supporting their involvement in interdomain interactions. In particular, D55A (or D55K) and R252A (or R252E) mutants showed much higher basal membrane affinity and enzyme activity and faster subcellular translocation than wild type, whereas a double charge-reversal mutant (D55K/R252E) behaved analogously to wild type, indicating that a direct electrostatic interaction between the two residues is essential for the C1A-C2 tethering. Collectively, these studies provide new structural insight into PKC C1A-C2 interdomain interactions and the mechanism of lipid-mediated PKC activation. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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, 
I, II, and 
subtypes), novel PKC (nPKC: 
, 
, 
, and 
subtypes), and atypical PKC (
and 
/
subtypes). cPKCs and nPKCs have two types of membrane-targeting domains, a tandem repeat of C1 domains (C1A and C1B) and a C2 domain, in the regulatory domain.
The C1 domain (
50 residues) is a highly conserved, cysteine-rich compact structure that was initially identified as the interaction site for 1,2-diacyl-sn-3-glycerol (DAG) and phorbol ester in PKCs (4-6). Recent studies have shown, however, that the C1 domains of PKCs have a wide range of affinities for DAG and phorbol ester (7-9). All C1 domains characterized so far have a common structure with exposed hydrophobic residues surrounding the ligand-binding pocket and a cluster of cationic residues in the middle of the molecule (see Fig. 1) (10-16). Owing to this unique structural feature, C1 domains can effectively penetrate the membrane containing anionic lipids. Although it was reported that PKCII C1B domain has stereospecificity for the phosphatidylserine (PS) headgroup (17), other PKC C1 domains do not distinguish among anionic phospholipids (8, 9).
The C2 domain (130 residues) is an eight-stranded sandwich protein that shows higher structural and functional diversity than the C1 domain (6, 18-20). The C2 domains of cPKCs bind multiple Ca2+ ions in their Ca2+-binding loops and thereby play a key role in Ca2+-dependent membrane binding of cPKCs (6, 18-20). The C2 domains of cPKCs have been shown to have definite PS specificity. In particular, recent structural (21) and mutational (22, 23) studies have demonstrated that the PS headgroup directly interacts with Ca2+ and including Asn189 several residues,, in the Ca2+ binding loops of PKC (see Fig. 1). All nPKCs contain a Ca2+-independent C2 domain in the amino terminus, but its role in membrane binding and activation of nPKCs is not fully understood.
Membrane binding and activation of cPKCs and nPKCs require DAG and PKC phosphorylation (and Ca2+ for cPKCs) and may also depend on binding to other lipids and proteins (1-3). Earlier in vitro membrane binding and activity assays indicated that PS is an essential activator of PKCs (24, 25). More recent studies have shown, however, that PS dependence (or selectivity) in membrane binding and activation varies among PKC isoforms: e.g. PKC and PKC show high PS selectivity, whereas PKC and PKC exhibit little PS selectivity (8, 9, 26, 27). Thus, it appears that PS selectivity of the intact PKC molecules is a complex phenomenon that does not simply reflect the PS specificity of isolated membrane targeting domains.
Extensive biochemical studies have been performed to elucidate the mechanisms by which cPKC and nPKC isoforms bind to the membrane and get activated. Our recent studies on PKC (28, 29), PKC (8), PKC (9), and PKC (27) have revealed that individual PKC isoforms follow distinct membrane-binding mechanisms and show different PS selectivity due in part to the differences in the accessibility and DAG affinity of their C1 domains. In the case of PKC, initial Ca2+- and PS-dependent membrane binding by the C2 domain is followed by the subsequent membrane penetration and DAG binding by the C1A domain, which eventually leads to enzyme activation (26, 28-30). In the resting state, the C1A domain is not available for DAG binding, because it is tethered to the C2 domain (or other parts of the molecule) via conserved Asp55 in the C1A domain. It has been proposed that PS specifically unleashes this interdomain tethering, because its carboxyl group effectively replaces that of Asp55; hence the PS selectivity (29) (see Fig. 6). The C1B domain is not directly involved in membrane binding and activation because of its extremely low DAG affinity (8). In contrast to PKC, PKC has high membrane binding and enzymatic activities without PS, because its C1A and C1B domains are readily accessible and have comparably high DAG affinities (8). Among nPKCs, PKC behaves like PKC, and PKC is similar to PKC with respect to PS selectivity and the DAG affinity and accessibility of their C1A and C1B domains (9, 27).
The notion of the intramolecular tethering (or masking) of C1 domains has been also supported by reports from other laboratories (31-35). For example, Conesa-Zamora (32) reported that the C2 domain of PKC is directly involved in the DAG-dependent binding of the C1 domain to membrane. Stubbs and coworkers (33) subsequently showed that the C2 domain of PKC was able to associate with the C1 domain. More recently, it has been reported that a cluster of Lys residues on the concave surface of the C2 domain interact with Asp55 (34, 35). Despite this mounting evidence, little is known about the nature of C1-C2 interdomain interactions and the residues involved in these interactions. In the present study, we performed computational modeling, in vitro membrane binding and activity measurements, and cellular membrane translocation measurements of PKC and mutants to identify the residues directly involved in the C1A-C2 interdomain interactions in PKC. The results not only provide new structural insight into PKC C1A-C2 interdomain interactions but also lend further support on our proposed mechanism of PKC activation.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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-32P]ATP (3 Ci/mmol) was from Amersham Biosciences. Restriction endonucleases and enzymes for molecular biology were obtained from New England Biolabs (Beverly, MA). Pioneer L1 sensor chip was from Biacore AB(Piscataway, NJ). Dulbecco's modified Eagle's medium (DMEM) and LipofectamineTM were from Invitrogen. Human embryonic kidney(HEK) 293 cell line, Zeocin, and ponasterone A were from Invitrogen (San Diego, CA).
Molecular Modeling of C1-C2 Domain Interactions—The homology model of the C1A domain of PKC was built (see Fig. 1) with the Nest (37) and Modeler (38) programs using the crystal structure of PKC C1B (11) (PDB id: 1PTR
[PDB]
) as the template and the alignment obtained with BLAST as a guide. The sequence identity between these two C1 domains is very high (42%). Zinc was docked into the homology model based on structure superposition with the C1B structure. The quality of the model is good based on the results from Verify3D (39) (data not shown), where Verify 3D scores are plotted as a function of residue number in the model; the scores are consistently positive and high (>0.15) indicative of a good structural fit for the C1A sequence. The protein docking programs Global Range Molecular Matching (GRAMM) (40) and DOCK4 (41) were used to dock the C1A homology model and the crystal structure of the PKC C2 domain (21) (PDB id: 1dsy
[PDB]
). GRAMM searches for geometric and hydrophobic complementarity between two proteins, whereas DOCK evaluates geometric and, more generally, chemical complementarity, although the user is required to define, as an input to the program, the docking surface on one protein, which is designated the "receptor" (here, the C2 domain). The parameters used for GRAMM are as follows: the matching mode was generic; the grid step was 6.8 Å; the repulsion was 6.5; the attraction double range was 0.0; the potential range type was grid-step; the projection was gray; the number of matches to output was 1000; and the angle of rotations was 20°. For DOCK4, we followed the recommended protocol, which defines how the system and information describing it is represented on the computational grid. Following docking, the results were corroborated by computational analysis of the predicted docked interface.
Expression Vector Construction and Mutagenesis—Baculovirus transfer vectors encoding the cDNA of PKC with appropriate C1 or C2 domain mutations were generated by the overlap extension PCR (42) using pVL1392-PKC- plasmid as a template (30). The PCR product was purified on an agarose gel, and the PKC gene was digested with NotI and EcoRI and subcloned into the pVL1392 vector digested with the same restriction enzymes. The mutagenesis was verified by DNA sequencing. Mammalian expression vectors for PKC and mutants with carboxyl-terminal enhanced green fluorescence protein (EGFP) tags were generated by subcloning the respective genes into the pIND (Invitrogen) with the spacer sequence, GGNSGG, as described previously (8).
Protein Expression and Purification—Full-length PKC and mutants were expressed in baculovirus-infected Sf9 cells. The transfection of Sf9 cells with pVL1392-PKC constructs was performed using a BaculoGoldTM transfection kit from BD Pharmingen. The plasmid DNA for transfection was prepared by using an EndoFree Plasmid Maxi kit (Qiagen) to avoid potential endotoxin contamination. Cells were incubated for 4 days at 27 °C, and the supernatant was collected and used to infect more cells for the amplification of virus. After three cycles of amplification, high-titer virus stock solution was obtained. Sf9 cells were maintained as monolayer cultures in TMN-FH medium (Invitrogen) containing 10% fetal bovine serum (Invitrogen). For protein expression, cells were grown to 2 x 106 cells/ml in 350-ml suspension cultures and infected with the multiplicity of infection of 10. The cells were then incubated for 60 h at 27 °C. PKC wild type and mutants were purified as described previously (30).
Determination of PKC Activity—Activity of PKC was assayed by measuring the initial rate of [32P]phosphate incorporation [-32 from P]ATP (50 µM, 0.6 µCi/tube) into the histone III-SS (400 µg/ml, Sigma). The reaction mixture contained large unilamellar vesicles (0.2 mM of total lipid concentration), 0.16 M KCl, 0.1 mM CaCl2, and 5 mM MgCl2 in 50 ml of 20 mM HEPES, pH 7.4. For experiments with different [Ca2+], the free calcium concentration was adjusted using the mixture of EGTA and CaCl2 according to the method of Bers (43). Reactions were started by adding 50 mM MgCl2 to the mixture, incubating the mixture for 10 min at room temperature, and quenching by the addition of 50 µl of 5% phosphoric acid. 75 µl of quenched reaction mixtures were spotted on P-81 ion-exchange paper, washed 4 times with a 5% solution of phosphoric acid, followed by 1 wash in 95% ethanol. Papers were transferred into scintillation vials containing 4 ml of scintillation fluid (Fisher Scientific), and radioactivity was measured by liquid scintillation counting.
Monolayer Measurements—Surface pressure (
) of solution in a circular Teflon trough (4-cm diameter x 1-cm deep) was measured using a Wilhelmy plate attached to a computer-controlled Cahn electrobalance (Model C-32) as described previously (30). 5-10 µl of phospholipid solution in ethanol/hexane (1:9 (v/v)) was spread onto 10 ml of subphase (20 mM HEPES, pH 7.4, containing 0.16 M KCl and either 0.1 mM EGTA or 0.1 mM CaCl2) to form a monolayer with a given initial surface pressure (0). Once the surface pressure reading of monolayer had been stabilized (after 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 (
) was measured as a function of time. Typically, the value reached a maximum after 20 min. The maximal value at a given 0 depended on the protein concentration and reached a saturation value (i.e. [PKC] 
1.5 µg/ml). Protein concentrations in the subphase were therefore maintained above such values to ensure that the observed represented a maximal value. The critical surface pressure (c) was determined by extrapolating the versus 0 plot to the x-axis (44).
Surface Plasmon Resonance Analysis—Kinetics of vesicle-PKC binding was determined by the SPR analysis using a BIAcore X biosensor system (Biacore AB) and the L1 chip as described previously (29, 45). The first flow cell was used as a control cell and was coated with 5400 resonance units of POPC. The second flow cell contained the surface coated with vesicles with varying lipid compositions (e.g. POPC/POPS/DiC18 = 79:20:1) at 5400 resonance units. After lipid coating, 30 µ l of 50 mM NaOH was injected at 100 µl/min three times to wash out loosely bound lipids. Typically, no further decrease in SPR signal was observed after one wash cycle. After coating, the drift in signal was allowed to stabilize below 0.3 resonance unit/min before any binding measurements, which were performed at 25 °C and at a flow rate of 30 µl/min. 90 µl of protein sample was injected for an association time of 3 min, and the dissociation was then monitored for 10 min in running buffer. After each measurement, the lipid surface was typically regenerated with a 10-µl pulse of 50 mM NaOH. 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, five or more different concentrations (typically within a 10-fold range above or below the Kd) of each enzyme were used, and data sets were repeated three or more times. When needed, the entire lipid surface was removed with a 5-min injection of 40 mM CHAPS followed by a 5-min injection of 40 mM octyl glucoside at 5 µl/min, and the sensor chip was recoated for the next set of measurements. All data were analyzed using BIAevaluation 3.0 software (Biacore) to determine the rate constants of association (ka) and dissociation (kd) as described previously (29, 45), assuming 1:1 protein-membrane surface binding, because all kinetic sensorgrams agreed with this model. An equilibrium dissociation constant (Kd) was calculated from rate constants using an equation, Kd = kd/ka. Mass transport (46) was not a limiting factor in our experiments, because change in flow rate or ligand density did not affect kinetics of association and dissociation.
Cell Culture—A stable HEK 293 cell line expressing the ecdysone receptor (Invitrogen) was used for all experiments (23). Cells were cultured in DMEM supplemented with 10% fetal bovine serum at 37 °C in 5% CO2 and 98% humidity until 90% confluent. Cells were then passaged into 8 wells of a Lab-TechTM chambered cover glass for later transfection and visualization. Only cells between the 5th and 20th passages were used. For transfection, 80-90% confluent cells in Lab-TechTM chambered cover glass wells were exposed to 150 µl of unsupplemented DMEM containing 0.5 µg of endotoxin-free DNA and 1 µl of Lipofectamine reagent for 7 h at 37 °C. After exposure, the transfection medium was removed, and the cells were washed once with fetal bovine serum-supplemented DMEM, and overlaid with fetal bovine serum-supplemented DMEM containing Zeocin, and 5 µg/ml ponasterone A to induce protein production for 16-20 h.
Microscopy—Microscopy data were collected on a custom-built combination laser scanning confocal and multiphoton microscope as described previously (9). All experiments were carried out at the same laser power and gains and offset setting on the photomultiplier tubes. To clamp intracellular Ca2+ ([Ca2+]i) cells were loaded with 10 µM BAPTA-AM (Invitrogen) 30 min prior to imaging. Immediately before imaging, cells were washed once with 1 mM EGTA, following a wash twice with HEK buffer (1 mM HEPES, pH 7.4, containing 2.5 mM MgCl2, 140 mM NaCl, 5 mM KCl, and 6 mM sucrose). After washing, cells were overlaid with 150 µl of HEK buffer. Suitable cells were selected for imaging, and a single image was taken for each cell before addition of OPG or DiC8. Then, the translocation of protein and subcellular localization of lipid was simultaneously monitored at fixed intervals (every 15 s) after 150 µl of HEK buffer containing 0.1 mg/ml OPG (or DiC8) was added. Control experiments were done with dimethyl sulfoxide. Furthermore, control experiments done with HEK293 cells loaded with Fura Red, Calcium Crimson, or Fluo-4 (Invitrogen) indicated that addition of DiC8 to HEK293 cells does not increase [Ca2+]i, precluding the possibility that translocation response in these experiments is due to an increase in [Ca2+]i. Images were analyzed using simFCS. Specifically, regions of interest in the cytosol were defined, and the average intensity in a square (1 mm x 1 mm) obtained with respect to time. Membrane intensities were determined for each frame in individual cells by extending a line from cytosol to the outside of the cell and reading off the intensity with distance along the line. Lines were drawn in at least three places in each cell, and membrane intensity was determined. These values were averaged, and the resultant cytosolic intensity values were converted to a ratio for each frame: membrane/(membrane + cytosol). The values were then scaled for the entire time series from 0% to 100% of the obtained ratio for a given experiment. This allowed a comparison of ratiometric changes between experiments. Note that each experiment was repeated at least three times on a given day and was repeated at least two different days with different transfected cells.
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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C1 and C2 Domain Interactions—To gain structural insight into the putative C1A-C2 domain tethering in PKC, We first built a model structure of the C1A domain of PKC by homology modeling and performed docking analysis using this model structure and the crystal structure of the PKC C2 domain. We used the protein docking programs GRAMM and DOCK4, as these programs are well developed, have complementary strengths, and have been successful in a number of applications (47-49). As described under "Experimental Procedures," these programs evaluate geometric, hydrophobic, and electrostatic complementarity between the two domains. From the docking of a comparative model (albeit of high reliability) to a crystal structure, it is not expected to obtain more than qualitative results that may help guide and interpret experimental observations. Such predictions may identify interacting surfaces between two proteins or domains (50). Overall, 14 of 19 docked complexes obtained predict that the C1A domain binds to the surface of the C2 domain depicted in Fig. 1, and four of these complexes predict the Arg55-Glu252 ion pair. More specifically, three of the top ten complexes generated from GRAMM (40) and one of the nine total complexes generated from DOCK4 (41) showed a direct interaction between Asp55 (in C1A) and Arg252 electrostatic (in C2) (see panels D and E in Fig. 1). Another potential ion pair Arg42-Glu282 and an aromatic pair Phe72-Phe255 were also predicted in these four docked complexes. Importantly, all of these residues are highly conserved among cPKCs from a wide variety of organisms, indicating their functional and/or structural importance.
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are actually involved in the C1A-C2 tethering, we mutated these residues both individually and in combination. We also mutated additional residues that have been implicated in the C1-C2 domain interactions. We then measured the membrane-binding properties of these mutants by SPR analysis. The SPR analysis of membrane-protein interactions offers an advantage over other methods in that the effects of mutations of membrane-binding residues on membrane association (ka) and dissociation (kd) rate constants can be directly determined. We have recently shown that nonspecific electrostatic interactions driven by ionic residues mainly effect ka, whereas short-range specific interactions and hydrophobic interactions, which result from the membrane penetration of hydrophobic residues, largely influence kd (6, 20, 45). Our previous study showed that the D55A mutant of PKC has higher membrane affinity (i.e. smaller Kd) and longer membrane residence time (i.e. smaller kd) than wild type, particularly under sub-optimal conditions (e.g. at low Ca2+ concentration or without PS), due to greatly enhanced conformational freedom (or accessibility) of its C1A domain (29). We thus expected that mutation of other residues directly involved in the tethering of C1A domain would also decrease Kd and kd of PKC.
We first measured the binding of proteins to POPC/POPS/DiC18 (79:20:1) vesicles coated on the sensor chip at 5 µM Ca2+ (TABLE ONE). This Ca2+ concentration was selected to assess the basal membrane affinity of the mutants in comparison to the wild type. A lower Ca2+ concentration could not be employed, because many mutants showed no detectable membrane affinity by SPR analysis under such conditions. D55A exhibited a 19-fold increase in affinity (i.e. lower Kd), which was attributed to a 3-fold increase in ka and a 6-fold decrease in kd. A charge-reversal mutation D55K had essentially the same effect as the D55A mutation. Likewise, R252A (or R252E) had 10-fold lower Kd due mainly to smaller kd. As described above, the reduction in kd for all these mutants suggests that they have an enhanced capability of penetrating the membrane and binding DAG due to increased conformational freedom of the C1A domain. The modestly increased ka should derive from C1A domain cationic residues that are allowed to interact with anionic membranes more favorably due to the unleashing of C1A domain. As was the case with the Asp55-Arg252 ion-pair, mutation of other putative ion-pair residues (i.e. R42A and E282A) significantly (i.e. 8-fold) increased the vesicle affinity, which was primarily attributed to decreases in kd, indicating that these residues are also directly involved in the C1A-C2 tethering. Lastly, the two aromatic residues, Phe72 and Phe255, also seem to contribute to the tethering, as F72A and F255A mutations caused considerable increases (i.e. 4-fold) in vesicle affinity, again due mainly to decreases in kd.
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have been implicated in intramolecular interactions, membrane binding or PS selectivity. They include a cationic cluster (Lys197, Lys199, Lys205, Lys209, and Lys213) on the concave surface of the C2 domain (34, 35), and Asn189(21, 23) and Arg249 (21, 30) in the calcium-binding loop (see Fig. 1, B and C). To investigate the potential role of these residues in the C1A-C2 tethering, we prepared N189A, R249A, K197E/K199E/K209E/K211E (Q1), and K199E/K209E/K211E/K213E (Q2) mutants and measured their binding to POPC/POPS/DiC18 (79:20:1) vesicles. Two overlapping multiple-site mutants Q1 and Q2 were prepared because single-site mutations in this cationic cluster would not show a significant effect on the electrostatic properties of the protein. As summarized in TABLE ONE, all these mutations caused significant decreases in vesicle affinity, indicating that these residues are involved in membrane binding, either directly or indirectly, but not in C1A-C2 tethering. It is noteworthy that Arg249 located in the close proximity of Arg252 (see Fig. 1, B and C) does not contribute to the C1A-C2 domain tethering, which demonstrates the specific nature of the interdomain interactions. To corroborate the notion that Asp55-Arg252 and Arg42-Glu282 are important parts of the C1A-C2 tether, we prepared the double-site mutations, D55K/R252E and R42E/E282R, and measured their membrane-binding properties. If these residues are indeed involved in interdomain tethering through electrostatic interactions, then double charge reversal should abrogate the large positive effects of single-site mutations (e.g. an increase in vesicle affinity by D55K). We also characterized D55K/R249E as a control. As listed in TABLE ONE, the affinity of D55K/R252E (Kd = 22 nM) for POPC/POPS/DiC18 (79:20:1) vesicles was only slightly higher that of wild type (Kd = 36 nM) but much lower than those of corresponding single-site mutants (Kd = 1.8-2.7 nM). In contrast, D55K/R249E (Kd = 2.3 nM) was similar to D55K in that it had a 16-fold higher affinity than wild type. Likewise, the vesicle affinity of R42E/E282R was slightly higher that of wild type and significantly lower than those of R42A and E282A. These results thus lend further support on the notion that Asp55-Arg252 and Arg42-Glu282 pairs play a pivotal and specific role in the C1A-C2 interdomain tethering.
C1A-C2 Tethering and PS Selectivity—Our mechanistic studies on various PKC isoforms have indicated that the PS selectivity of PKC derives, at least partially, from the disruption of the intramolecular tethering of PKC by PS (9, 29). Therefore, although wild-type PKC exhibits strong PS selectivity in membrane binding and enzymatic action, D55A of PKC and wild-type PKC that have unrestricted C1 domains show much reduced PS selectivity (29). To see if other PKC mutants also lose PS selectivity, we compared their relative binding affinities for POPC/POPS/DiC18 (79:20:1) and POPC/POPG/DiC18 (79:20:1) vesicles at 5 µM Ca2+ (see TABLE ONE; the last column). In accordance with previous studies (26), PKC exhibited 11-fold higher affinity for PS-containing vesicles than PG vesicles. Similarly, those mutants whose C1A-C2 tethering appears to be intact (see above) all showed significant PS selectivity (i.e. 7- to 14-fold). In contrast, all those mutants with disrupted C1A-C2 tethering (see above) displayed much reduced PS selectivity (i.e. 1.5- to 3.4-fold). These results further establish the correlation between the conformational freedom of the C1 domain and the PS selectivity. It should be noted that N189A also showed reduced PS selectivity, although this mutant appears to have the intact C1A-C2 tethering. This apparent paradox is due to the fact that Asn189 is directly involved in PS coordination to the C2 domain (21, 23), again illustrating the complex nature of PS selectivity of PKC (see "Discussion").
C1A-C2 Tethering and Plasma Membrane Selectivity—We have shown that many proteins with PS selectivity are recruited to the PS-rich inner plasma membrane in the cell (6, 23, 51). More generally, the cellular membrane-targeting specificity of a particular protein can be assessed by measuring its relative affinity for a set of vesicles whose lipid compositions recapitulate those of different cellular membranes (9, 23). For instance, PKC that is targeted to the plasma membrane strongly prefers the plasma membrane-mimicking vesicles to other membrane mimetics (e.g. nuclear membrane mimetic), whereas its E177A mutant (the homologue of PKC-D55A) that is recruited to all intracellular membranes binds all cell membrane mimetics with comparable affinities (9).
To see if PKC mutants with reduced PS selectivity would also lose their selectivity for the plasma membrane mimetic, we measured their affinities for the vesicles whose lipid headgroup compositions simulate those of inner plasma membrane and nuclear membrane, respectively. As listed in TABLE TWO, PKC bound to the plasma membrane mimetic 80-fold more strongly than to the nuclear membrane mimetic, which is consistent with its known plasma membrane localization behavior. It should be noted that this plasma membrane selectivity is more pronounced than the PS/PG selectivity determined above, because the plasma membrane is not only rich in PS but also the most anionic membrane in all cell membranes. Importantly, all PKC mutants with greatly reduced PS selectivity showed much lower preference (i.e. 
10-fold) for the plasma membrane mimetic than the wild type. This implies that these mutants may show different cellular membrane targeting behaviors from the wild type.
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and Mutations—The isolated C1 domain of PKC has an ability to effectively penetrate the lipid monolayer owing to the presence of exposed aromatic and aliphatic residues surrounding the DAG binding pocket (26, 28). However, the intact PKC molecule cannot display the same monolayer-penetrating activity until PS binding unleashes the C1A domain (28, 29). Thus, the wild type PKC shows high penetration activity only toward a PS-containing monolayer, whereas D55A is able to penetrate both PS- and phosphatidylglycerol (PG)-containing (or any anionic lipid-containing) monolayers equally well (28, 29). To corroborate the notion that PS specifically disrupts the C1A-C2 tethering mediated by the residues identified in this study, we measured the penetration of PKC and selected mutants into POPC/POPS (7:3) and POPC/POPG (7:3) mixed monolayers with the subphase containing 0.1 mM Ca2+ (or 0.1 mM EGTA). Because the monolayer measurements require larger amounts of proteins than the SPR measurements, only those mutants that can be expressed in relatively large quantities were employed in these measurements. Also, a higher Ca2+ concentration was used to carry out the monolayer measurements with smaller amounts of proteins. The phospholipid monolayer was spread at constant area, and the change in surface pressure () was monitored after the injection of protein into the subphase. DAG was not included in the lipid monolayers, because DAG itself was shown to have no effect on the monolayer insertion of PKCs (26, 52, 53). In general, is inversely proportional to 0 of the lipid monolayer, and an extrapolation of the versus 0 plot yields the critical surface pressure (c), which specifies the upper limit of 0 of a monolayer that a protein can penetrate into (44, 54). Because the surface pressure of cell membranes has been estimated to be in the range of 30-35 dynes/cm (55-57), the c value for a protein that penetrates cell membranes should be above 30 dynes/cm.
As reported previously (26), PKC showed weak penetration into the POPC/POPG monolayer (Fig. 2B) but had stronger penetration activity toward the POPC/POPS monolayer with c 30 dynes/cm in the presence of 0.1 mM Ca2+ in the subphase (Fig. 2A). In agreement with their membrane-binding properties, D55A, D55K (data not shown), R252A, and R252E (data not shown) all had high c values toward both POPC/POPS and POPC/POPG monolayers (c 35 dynes/cm) (Fig. 2, A and B). In contrast, D55K/R252E behaved like wild type PKC (Fig. 2, A and B), confirming that Asp55 and Arg252 are involved in tethering between the C1A and C2 domain. Again, R249A had no significant effect on the monolayer penetration of PKC (Fig. 2, A and B), confirming the specific nature of the Asp55-Arg252 ion pair. Interestingly, D55A, D55K, R252A, and R252E had high c values (c 35 dynes/cm) even in the presence of 0.1 mM EGTA in the subphase, whereas PKC wild type had a lower c value of 26 dynes/cm (Fig. 2C). It should be noted that, although all these proteins showed penetration into the monolayer with lower 0 without Ca2+, only those mutants with c > 30 dynes/cm would bind vesicles to some extent in the absence of Ca2+ (or at lower Ca2+ concentrations), because the surface pressure of large unilamellar vesicles has been estimated to be >30 dynes/cm (55-57). Taken together, these data verify the role of Asp55 and Arg252 in keeping the C1A domain from nonspecific membrane binding.
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and Mutants—To investigate how altered membrane-binding properties of PKC mutants affect their enzyme activities, we measured the kinase activity of PKC and mutants in the presence of POPC/POPS/DiC18 (99 - x:x:1) and POPC/POPG/DiC18 (99 - x:x:1) vesicles. Fig. 3 shows the relative enzyme activity of these proteins at two anionic lipid concentrations, 40 and 60 mol%. PKC showed high PS selectivity, i.e. much higher activity with PS than with PG at any given anionic lipid concentration. When compared with wild type, D55A and R252A exhibit a much lower degree of PS selectivity. At both 40 and 60 mol% of anionic lipids, D55A and R252A showed comparable activities with PS and PG. Furthermore, D55A and R252A were more active than wild type under all assay conditions (up to 170% of wild type activity). Similarly, R42A, E282A, F72A, and F255A all showed little PS selectivity, because they had much higher activity than wild type with PG vesicles. In contrast to these mutants, R249A retained the PS selectivity of wild type while being 20% less active than the wild type under all conditions. The activity of double-site mutants was also measured to investigate whether D55K/R252E or R42E/E282R could restore wild type activity and specificity. As shown in Fig. 3, D55K/R252E and R42E/E282R displayed wild type-like activity and PS selectivity, supporting the notion that these ionic pairs are part of a C1A-C2 tether. In contrast, D55K/R249E behaves similarly to D55K. Taken together, these kinase activity data verify that Asp55-Arg252 and Arg42-Glu282 ion pairs play a crucial role in the C1A-C2 tethering that keeps the membrane binding and enzymatic activities of PKC in check, which accounts for the PS selectivity of PKC in both membrane binding and kinase action.
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and Mutants—To see if altered membrane-binding properties of PKC mutants also dictate their cellular membrane targeting behaviors, we transfected HEK293 cells with PKC proteins with the carboxyl-terminal EGFP tags. Note that PKC and PKC-EGFP have similar affinity for the plasma membrane and nuclear membrane mimetics, showing that the EGFP tag does not influence the membrane affinity of PKC (see TABLE TWO). We have recently shown that PKC migrates very slowly to the plasma membrane in response to DiC8 addition at low [Ca2+]i in HEK293 cells, whereas D55A translocates to the plasma membrane much faster under the same conditions (29). To maintain low [Ca2+]i, cells were pretreated with BAPTA-AM in all measurements. Furthermore, we previously showed that DiC8 addition (0.1 mg/ml) did not alter [Ca2+]i (8) in HEK293 cells, meaning that PKC translocation under our conditions is due to DiC8 binding but not due to fluctuating Ca2+ levels.
As previously reported (8), PKC translocated slowly (Fig. 4A) to the plasma membrane upon DiC8 (or OPG) addition due to the inaccessibility of its C1A domain. In contrast, D55A (Fig. 4B), D55K (data not shown), R252A (Fig. 4C), and R252E (data not shown) all translocated much more rapidly under the same conditions. R42A (Fig. 4F), E282A (Fig. 4F), F72A (Fig. 4E), and F255A (Fig. 4E) also translocated to the plasma membrane rapidly, although not as fast as D55A and R252A. Furthermore, D55K/R252E (Fig. 4, D and F) and R42E/E282R (Fig. 4F) behaved similarly to wild type unlike the single-site mutants. Lastly, those PKC mutants with lower membrane affinities than wild type, i.e. R249A, Q1, Q2, and N189A, showed extremely slow cellular membrane translocation (Fig. 4E).
We previously showed that DiC8 and OPG are spontaneously distributed to all cellular membranes of HEK293 cells (9, 27). Therefore, any PKC isoform or mutant with no preference for the lipid composition of the plasma membrane could be recruited to other cellular membranes in response to DiC8 or OPG addition. Interestingly, both R252A and D55A with much reduced selectivity for the plasma membrane mimetic (see TABLE TWO) was localized not only to the plasma membrane but also to the perinuclear region in a large percentage of cells tested (85%, see Figs. 4 and 5). This suggests that the accessibility of the C1A domain and the resulting PS selectivity play an important role in the subcellular localization of PKC.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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-chimaerin (15), another C1 domain-containing protein, revealed how the C1 domain can be masked in the intact protein. Interestingly, our recent studies have indicated that the accessibility of C1 domains varies widely among PKC isoforms (8, 9, 27): i.e. C1 domains of PKC and PKC are readily accessible, whereas C1 domains of PKC and PKC are not. Full understanding of the origin of this difference would await the determination of the structures of intact PKC molecules. However, recent structural studies of isolated membrane targeting domains of PKCs as well as mutational and membrane-binding studies of isolated domains and intact proteins have provided important clues to the origin of divergent ligand affinities and accessibility of PKC C1 domains (8, 9, 21, 32-35). These studies have revealed that some PKC C1 domains have limited accessibilities, because they are intramolecularly tethered to other parts of PKC molecules. In the case of PKC, multiple lines of evidence have supported that the C1A domain is tethered to the C2 domain in the resting state (28, 29, 32-35). The present study provides the convincing evidence that the interdomain tethering takes place through specific electrostatic and hydrophobic interactions.
Our computational docking of the model structure of PKC C1A domain and the crystal structure of PKC C2 domain revealed good complementarity between the two domains in terms of surface geometry, hydrophobicity, and electrostatics. Most importantly, four best matching complexes among 19 good-fit combinations predict that the Asp55-Arg252 and Arg42-Glu282 pairs will form interdomain electrostatic interactions, whereas the Phe72-Phe255 pair will make favorable hydrophobic contact. It should be noted that computational docking of two proteins or domains could not produce direct and unambiguous structural information (58), especially when model structures are used. Despite this limitation, the docking analysis is still very useful and valuable, because it helps to formulate hypothetical models that can be experimentally tested. This is particularly so for those multidomain proteins like PKCs whose intact structures turn out to be very difficult to determine. In the present study, the ion pairs and the aromatic pair predicted by the docking analysis are shown to play a key role in the C1A-C2 interdomain interactions.
Mutations the four ionic residues (Arg42 and Asp55 in C1A and of Arg252 and Glu282 in C2), to either Ala or an opposite charge, dramatically enhance the accessibility of the C1A domain in the resting state of PKC, thereby converting the enzyme to a pre-activated form. Specifically, the mutants have higher membrane affinity, monolayer penetration activity, enzyme activity, and faster cellular membrane targeting than PKC wild type under suboptimal conditions, e.g. in the presence of low Ca2+ or nonspecific lipid PG. The notion that Asp55-Arg252 and Arg42-Glu282 ion pairs keep the C1A domain of PKC tethered in the resting state is corroborated by the finding that the double-site charge reversal mutations (R42E/E282R and D55K/R252E) abrogate the activating effects of single-site mutations and confer wild type-like properties.
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, showing that they are involved not in the C1A-C2 tethering but in membrane binding of the C2 domain.
The present study also sheds new light on the PS selectivity of PKC. As described in the introduction, the PS selectivity of PKC is a complex phenomenon that varies widely among PKC isoforms. It was long thought that the PS selectivity derives from the allosteric activation caused by binding of PS to a specific binding site (59). However, identification of the PS binding site has been elusive. A majority of C1 domains, including all PKC C1 domains, contain a cluster of basic residues in the middle of the molecule that drive the binding of the domains to anionic membranes by nonspecific electrostatic interactions (28). Although it was reported that the C1B domain of PKCII has stereospecificity for PS (17), this finding has not been substantiated in other PKC C1 domains. Our SPR measurements of many isolated PKC C1 domains have shown that they do not distinguish between PS and PG (8, 9, 27). The crystal structure of the PKC C2 domain complexed with a PS molecule (21) and subsequent mutational (22, 23) studies have established that the C2 domains of cPKCs do have a well defined PS binding site that consists of Ca2+ and several residues in the Ca2+ binding loops, including Asn189 that directly interacts with the serine headgroup of PS (see Fig. 1C). However, it should be emphasized that the divergent PS selectivity of PKC isoforms is not ascribed to the different PS binding affinities of individual C2 domains, because although PKC and PKC show dramatically different PS selectivity (8), their isolated C2 domains have comparable affinities for PS-containing vesicles (60). Instead, the PS selectivity of PKC isoforms is better correlated with the accessibility of their C1 domains. That is, only for those PKCs whose one or more C1 domains are tethered, the binding of PS (to the C2 domain for cPKCs) would lead to unleashing the C1 domain, resulting in allosteric activation by PS (8).
Consistent with this notion, all single-site mutations of Arg42, Asp55, Arg252, and Glu282 nearly abrogate PS selectivity of PKC in membrane binding and activation. Obviously, none of these residues are part of the PS binding site, because their mutations enhance the binding of PKC to PS-containing membranes. In this regard, it should be noted that the N189A mutation lowers the PS selectivity by selectively reducing the affinity for PS membranes, whereas mutations of the above four residues abolish the PS selectivity by enhancing the affinity of PKC for non-PS membranes preferentially. Again, the double charge-reversal mutants have the wild type-like PS selectivity and mutations of Arg249, and the Lys cluster has little effect on the PS selectivity of PKC. Lastly, loss of PS selectivity as a result of enhanced accessibility of C1A domain also strongly disputes the notion that the PS binding site is located in the C1 domain.
Although we did not directly quantify the energetics of the C1A-C2 interdomain interactions in PKC, the differences in Kd values between wild type and single-site mutants indicate that the Asp55-Arg252 ion pair makes the biggest contribution. Furthermore, dramatic effects of single-site mutations of any of four ionic residues on the membrane affinity of PKC indicate that the two ion pairs function synergistically rather than additively. Thus, the disruption of the Asp55-Arg252 ion pair (or the other pair) by PKC activators, such as PS, would readily expose C1A domain for DAG binding, resulting in enzyme activation.
We have shown for many membrane targeting domains and their host proteins that in vitro membrane binding properties of these proteins govern their cellular membrane targeting behaviors (6, 23, 61). In particular, their subcellular destination can be predicted by measuring the relative affinity of these proteins for vesicles the lipid compositions of which recapitulate those of different cellular membranes (23). Those PKC mutants with enhanced C1A accessibility translocate to the membrane much faster than PKC wild type in response to DAG addition at low [Ca2+]i. Because of loss of PS selectivity, these mutants also showed drastically reduced preference for the plasma membrane mimicking vesicles. As a result, some of these mutants show dual targeting to the plasma membrane and the nuclear membrane under the condition in which PKC is recruited exclusively to the plasma membrane. In conjunction with our previous results on other PKC isoforms (8, 9, 27), these results demonstrate that the subcellular targeting of PKC is greatly influenced by their C1 domain accessibility and consequent PS selectivity.
The present study, in conjunction with the previous studies from several laboratories, provides a unifying view of the mechanism of cPKC activation. On the basis of elegant cell studies using various truncation mutants of PKC, Oancea and Meyer (31) reported that the DAG binding site in the C1 domains is not accessible in an "inactive" conformation and becomes accessible by the Ca2+-dependent membrane binding of the C2 domain. This model is similar to our proposed model for PKC activation with respect to C1 domain tethering (29); however, a main difference between the two models is that the model by Oancea and Meyer suggests that the C1 domains are clamped in an inactive conformation by strong interactions between the pseudosubstrate and the active site, not by the direct interactions of the C1 domains with the C2 domain (or other parts of the molecule). Also, our subsequent study showed that the C1 domains of PKC are much more accessible to DAG binding in the resting state than those of PKC (8). Strong support for our model for PKC activation was recently provided by Slater et al. (33) who showed that direct C1-C2 interdomain interactions retain the PKC molecule in an inactive conformation. The study presented here provides more structural insights into the C1-C2 interdomain interactions, thereby lending further support for our proposed mechanism of PKC activation. In the refined model illustrated in Fig. 6, membrane binding of PKC is initiated by Ca2+-dependent adsorption of the C2 domain to the anionic membrane surface. Binding of a PS molecule in the membrane to the Ca2+-binding loops of the C2 domain would cause a local conformational change that moves the side chain of Arg252 and consequently disrupts its interactions with Asp55. This would result in unleashing of the C1A domain, which then penetrates the membrane and binds DAG. This movement of the C1A domain would not only enhance the membrane binding energy but also remove the pseudosubstrate from the active site, leading to enzyme activation. Our model proposes that the PKC-DAG interaction comes after the disruption of the C1A-C2 interaction caused by specific Ca2+-dependent PS binding. It may seem to be at odds with the previous report showing that PKC can also bind DAG-containing membranes at low to no calcium (62). It should be noted, however, that this type of binding is much weaker than Ca2+-dependent binding and is not conducive to enzyme activation (62). Our model is also consistent with the model proposed for PKCII activation by Newton and coworkers in which Ca2+ binding to the C2 domain induces the low affinity membrane adsorption of PKCII, which then diffuses in the plane of the membrane to find DAG through the C1 domain and gets activated (63, 64). A main difference is that our model proposes the presence of interdomain tethering that is specifically unleashed by the PS binding.
Due to divergent structural and functional properties of C1A, C1B, and C2 domains of different PKC isoforms, a significant degree of variation is expected in their activation mechanisms. For example, PKC contains all those ionic residues involved in the C1A-C2 tethering in PKC yet has fully accessible C1 domains and shows no PS selectivity (8). Thus, it appears that the C1A-C2 interdomain interactions require precise surface complementarity that is somehow deficient in PKC. Also, PKC behaves similar to PKC in terms of high PS selectivity and low accessibility of its C1A domain (9), despite the fact that its Ca2+-independent C2 domain has extremely low affinity for lipids (9), including PS, and that the deletion of the C2 domain has little effect on the membrane binding and activation of PKC (9). Thus, the C1A domain of PKC should interact with other parts of the molecule, presumably the C1B domain or the catalytic domain (65). Furthermore, the inter-molecular tethering of the C1B domain may be crucial for the regulation of the activity of some PKC isoforms. Obviously, further studies are needed to fully answer these questions. Nevertheless, our model for PKC activation should serve as a framework with which to systematically investigate the mechanisms of activation for different PKC isoforms.
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FOOTNOTES |
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1 To whom correspondence should be addressed: Dept. of Chemistry (M/C 111), University of Illinois at Chicago, 845 West Taylor St., Chicago, IL 60607-7061. Tel.: 312-996-4883; Fax: 312-996-2183; E-mail: wcho{at}uic.edu.
2 The abbreviations used are: PKC, protein kinase C; BAPTA-AM, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester; CHAPS, (3-[3-cholamidopropyl)dimethylammonio]-1-propanesulfonate; cPKC, conventional protein kinase C; DAG, sn-1,2-diacylglycerol; OPG, 1-octanoyl-2-(8-pyrenyloctanoyl)-sn-3-glycerol; DiC8, sn-1,2-dioctanoylglycerol; DiC18, sn-1,2-dioleoylglycerol; DMEM, Dulbecco's modified Eagle's medium; EGFP, enhanced green fluorescent protein; HEK, human embryonic kidney; nPKC, novel protein kinase C; POPC, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine; POPE, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine; POPG, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol; POPI, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoinositol; POPS, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine; PG, phosphatidylglycerol; PS, phosphatidylserine; SPR, surface plasmon resonance.
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REFERENCES |
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TOPABSTRACT |
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), D55A (
), R249A (
), R252A (
), and D55K/R252E (
), and their concentrations in the subphase were 1.5 µg/ml. The subphase contained 20 mM HEPES, pH 7.4, containing 0.16 M KCl and 0.1 mM Ca2+ (or 0.1 mM EGTA). A, POPC/POPS (7:3) monolayer with 0.1 mM Ca2+; B, POPC/POPG (7:3) monolayer with 0.1 mM Ca2+; C, POPC/POPS (7:3) monolayer with 0.1 mM EGTA. Each data point represents an average of duplicate measurements.
