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J. Biol. Chem., Vol. 282, Issue 29, 21467-21476, July 20, 2007
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*
1121
3
From the
Department of Chemistry, University of Illinois, Chicago, Illinois 60607 and Division of Cell Biology, La Jolla Institute for Allergy and Immunology, La Jolla, California 92037
Received for publication, January 4, 2007 , and in revised form, May 31, 2007.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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is a novel PKC that plays a key role in T lymphocyte activation. PKC has been shown to be specifically recruited to the immunological synapse in response to T cell receptor activation. To understand the basis of its unique subcellular localization properties, we investigated the mechanism of in vitro and cellular sn-1,2-diacylglycerol (DAG)-mediated membrane binding of PKC. PKC showed phosphatidylserine selectivity in membrane binding and kinase action, which contributes to its translocation to the phosphatidylserine-rich plasma membrane in HEK293 cells. Unlike any other PKCs characterized so far, the isolated C1B domain of PKC had much higher affinity for DAG-containing membranes than the C1A domain. Also, the mutational analysis indicates that the C1B domain plays a predominant role in the DAG-induced membrane binding and activation of PKC. Furthermore, the Ca2+-independent C2 domain of PKC has significant affinity for anionic membranes, and the truncation of the C2 domain greatly enhanced the membrane affinity and enzyme activity of PKC. In addition, membrane binding properties of Y90E and Y90F mutants indicate that phosphorylation of Tyr90 of the C2 domain enhances the affinity of PKC for model and cell membranes. Collectively, these results show that PKC has a unique membrane binding and activation mechanism that may account for its subcellular targeting properties. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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, 
I, II, and 
subtypes), novel PKC (
, 
, 
, and subtypes), and atypical PKC (
and 
/
subtypes). Conventional and novel PKCs have two types of lipid binding domains, a tandem repeat of C1 domains (C1A and C1B) and a C2 domain, in the regulatory domains. The C1 domain (
50 residues) is a cysteine-rich compact module that was identified as the interaction site for sn-1,2-diacylglycerol (DAG) and phorbol ester (4-6). The C2 domain (130 residues) is an eight-stranded sandwich protein that is involved in Ca2+-dependent membrane binding for conventional isoforms (7-10). All novel PKCs contain a Ca2+-independent C2 domain in the amino terminus followed by the C1A and C1B domains in the regulatory domain (see Fig. 7A).
PKC is a novel PKC that is present predominantly in T lymphocytes and muscle cells. T cell activation requires sustained interaction of T cell receptor (TCR) with major histocompatibility complex-bound peptide antigen of the antigen-presenting cell at the cell-cell contact region, the so-called immunological synapse, where activated TCR molecules are clustered (11). Generation of DAG by phospholipase C1 isoform in the plasma membrane in response to TCR triggering is a key signal in the initiation of T cell activation which culminates in the cell proliferation and the execution of T cell effector functions (12). T cells express many PKC isoforms (, I, , , , , and ) (13); however, only PKC stably translocates to the immunological synapse (14), whereas other PKCs translocate to other regions of plasma membrane. Analysis of PKC knock-out mice showed the importance of PKC in regulating TCR-derived signals and demonstrated the requirement for PKC in activating the downstream elements AP-1, NF-
B, NFAT, and interleukin-2 in T cells (15, 16).
A great deal of effort has been made to understand the mechanism of selective targeting of PKC in T cells. It has been reported that lipid rafts play a significant role in the propagation of TCR signaling events by serving as a platform on which important signaling molecules, including phospholipase C1 (17, 18) and PKC (19), are recruited. Because lipid rafts are not restricted to the immunological synapse, lipid raft translocation of PKC may not fully account for the specific targeting of PKC; however, this suggests that PKC may specifically interact with lipid or protein components of lipid rafts. Vav1 protein, which is a guanine exchange factor for a small GTPase Rac and mediates actin polymerization, promotes translocation of PKC to the membrane (20). This suggests that PKC may also interact directly or indirectly with a cytoskeletal protein. Also, it was reported that TCR stimulation leads to rapid phosphorylation of PKC on Tyr90 in its C2 domain (see Fig. 7A)byLck kinase, which appears to be necessary for the recruitment of PKC to the immunological synapse (21). Although these studies have provided important clues, the exact mechanism underlying the specific targeting of PKC in T cells still remains unknown. The paucity of mechanistic information on PKC is in part attributed to the low stability of the protein, which makes it difficult to prepare recombinant wild type and mutant proteins in amounts sufficient for biophysical measurements.
We have recently studied the mechanisms of membrane targeting and activation of conventional PKCs (PKC and PKC) (22-24) and a novel PKC (PKC and PKC) (25, 26). These studies have revealed that individual PKC isoforms follow distinct activation mechanisms due in part to the differences in the conformational flexibility and DAG affinity of their C1 domains. As a continuation of this line of investigation and as a first step toward the understanding of the mechanism of specific targeting of PKC in T cells, we investigated the mechanism by which DAG induces the in vitro and cellular membrane recruitment and activation of PKC with a particular emphasis on elucidating the roles of C1A, C1B, and C2 domains in these processes. To overcome the experimental difficulties associated with the low stability of PKC, we employed various sensitive in vitro and cellular techniques that allow quantification of the membrane binding and activity of PKC wild type and mutants. Results reveal that PKC has a unique membrane binding and activation mechanism, which derives mainly from unique properties of its two C1 domains and C2 domain.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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32P]ATP 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 cells, zeocin, ponasterone A, pIND vector, TMN-FH medium, and fetal bovine serum were from Invitrogen.
Expression Vector Construction and Mutagenesis—For bacterial expression of C2 (amino acids (aa) 1-123), C1A (aa 160-219), and C1B (aa 232-281) domains, the expression vectors were generated by subcloning the domain sequences from the human PKC cDNA into the pET21d vector (Novagen) between the restriction sites NcoI and XhoI. This vector is designed to introduce a carboxyl-terminal His6 tag for affinity purification of the expressed proteins. For the expression of the full-length PKC in insect cells, the baculovirus transfer vector was generated by subcloning the cDNA of PKC into the pVL1392 plasmid between NotI and BamHI sites. The 3'-end primer was designed to introduce a carboxyl-terminal His6 tag. The baculovirus transfer vectors for PKC mutants were prepared by the PCR mutagenesis using the pVL1392-PKC plasmid as a template. Mammalian expression vectors of PKC and mutants with carboxyl-terminal enhanced green fluorescence protein (EGFP) tags were generated by subcloning the corresponding genes with the spacer sequence, GGNSGG, into the pIND vector between restriction sites BamHI and NotI as previously described (25, 26).
Protein Expression and Purification—Bacterial expression of C1A, C1B, and C2 domains was performed in Escherichia coli strain BL21(DE3) (Novagen). The C2 and C1B domains were expressed as a soluble protein, whereas the C1A domain formed inclusion bodies. The isolated domains were expressed and purified as previously described (25, 26).
Full-length PKC and mutants were expressed in baculovirus-infected Sf9 cells. The pVL1392-PKC DNA was prepared for transfection by using an EndoFree Plasmid Midi kit (Qiagen) to avoid endotoxin contamination and transfected into Sf9 cells with BaculoGoldTM transfection kit (BD Pharmingen). Cells were incubated for 4 days at 27 °C, and the supernatant was collected and used for amplification of virus. After 3 cycles of amplification, high titer virus stock was obtained. Sf9 cells were maintained as monolayer cultures in TMN-FH medium containing 10% fetal bovine serum. For expression of the proteins, cells were grown to 2 x 106 cells/ml in 300-ml suspension cultures and infected with multiplicity of infection of 10. The cells were then incubated for 60 h at 27 °C.
For protein purification, cells were harvested at 1000 x g for 10 min, and the pellet was then washed once with sodium phosphate buffer, pH 8.0, and resuspended in 22 ml of extraction buffer containing 50 mM sodium phosphate, pH 8.0, 300 mM NaCl, 10 mM imidazole, 1% Triton X-100, 0.2 mM phenylmethylsulfonyl fluoride, and an EDTA-free protease inhibitor mixture tablet (Roche Applied Science). The suspension was homogenized in a hand-held homogenizer chilled on ice. The extract was centrifuged at 50,000 x g for 45 min at 4 °C. One ml of nickel-nitrilotriacetic acid-agarose (Qiagen) was added and incubated on ice while shaking at 80 rpm for 1 h. The mixture was poured into a 10-ml empty column, and the resin was washed first with 10 ml of 50 mM sodium phosphate, pH 8.0, containing 300 mM NaCl and 10 mM imidazole and subsequently with 10 ml of 50 mM sodium phosphate, pH 8.0, containing 300 mM NaCl and 15 mM imidazole. The column was then washed with 6 ml of the same buffer containing 20 mM imidazole and next with 3 ml of the same buffer containing 25 mM imidazole. The protein was then eluted from the column with 6 x 0.75-ml fractions of 50 mM sodium phosphate, pH 8.0, containing 300 mM NaCl and 300 mM imidazole. The eluted fractions were analyzed on an 8% sodium dodecyl sulfate-polyacrylamide gel (see supplemental Fig. S1). Fractions containing PKC were concentrated and desalted in an Ultrafree-15 centrifugal filter device (Millipore). Protein concentration was determined by the bicinchoninic acid method (Pierce).
Determination of PKC Activity—Activity of PKC was assayed by measuring the initial rate of [32P]phosphate incorporation from [-32P]ATP (50 µM, 0.6 µCi/tube) into myelin basic protein (200 µg/ml) (Sigma). The reaction mixture contained large unilamellar vesicles (0.2 mM total lipid concentration), 0.16 M KCl, and 5 mM MgCl2 in 20 mM HEPES, pH 7.4. The reaction was started by adding 50 mM MgCl2 to the mixture, and the mixture was incubated for 10 min at room temperature. The reaction was then quenched with 5% phosphoric acid. Seventy-five µl of the quenched reaction mixture was pipetted on a P-81 ion exchange paper and washed 3 times with 5% phosphoric acid followed by a wash with 95% ethanol. The paper was dried in an oven at 60 °C for 10 min. The paper was then transferred into a scintillation vial containing 4 ml of scintillation fluid (Fisher), and radioactivity was determined by liquid scintillation counting.
SPR Analysis—Equilibrium SPR measurements were performed at 23 °C in 10 mM HEPES, pH 7.4, containing 0.16 M KCl as described previously (24). The first flow cell was used as the control surface and coated with 5000 resonance units of POPC. The second flow cell was used as the active surface and coated with 5000 resonance units of varying lipid compositions (e.g. POPC/POPS/DiC18 = 69:30:1). After lipid coating, 30 µl of 50 mM NaOH was injected at a flow rate of 100 µl/min to wash away any unbound lipids. The base-line shift was maintained below 0.3 resonance units/min with continued washing before any binding measurements. The flow rate for equilibrium membrane binding measurements was set at 5 µl/min to allow sufficient time for the R values of the association phase to reach saturation response values (Req) (see Fig. 2A). After sensorgrams were obtained for 5 different concentrations of each protein within a 10-fold range of Kd(see Table 1), each of the sensorgrams was corrected for refractive index change by subtracting the control surface response from it. Req values were then plotted versus protein concentrations (C), and the Kd value was determined by a nonlinear least-squares analysis of the binding isotherm using an equation Req = Rmax/(1 + Kd/C). Each data set was repeated three times to calculate average and S.D. values.
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Immunoprecipitation and Immunoblotting—Confluent HEK293 cells cultured in a 10-cm2 plate were washed 3 times with ice-cold phosphate-buffered saline, pH 7.4, lysed in the cold lysis buffer (50 mM HEPES, pH 7.4, containing 100 mM KCl, 2 mM EGTA, 1% IGEPAL CA-630, and a protease inhibitor mixture (one tablet per 10 ml of buffer)), sonicated for 10 s, and centrifuged for 30 min. Sf9 cell extracts were obtained as described above. The protein content of cell lysates was determined by the bicinchoninic acid method (Pierce). The lysates of Sf9 cells and HEK293 cells were incubated with the mouse monoclonal anti-PKC antibody (Santa Cruz Biotechnology) for 4 h, and the immunocomplexes were allowed to precipitate overnight with the protein A-agarose beads. Proteins were separated on sodium dodecyl sulfate-polyacrylamide gel under reducing conditions and transferred on to a nitrocellulose membrane for immunoblotting analysis. Blots were treated with either 1 µg/ml mouse monoclonal anti-PKC antibody or 1 µg/ml anti-phosphotyrosine antibody light chain (BD Transduction Laboratories) followed by the incubation with the horseradish peroxidase-conjugated anti-mouse IgG antibody (0.01 ng/ml; Santa Cruz Biotechnology) and visualized with the ECL luminescence system (Amersham Biosciences).
Microscopy—Microscopy data were collected on a custombuilt combination laser scanning confocal and multiphoton microscope as described previously (25). All experiments were carried out at the same laser power and gains and offset setting on the photomultiplier tubes. Transfected cells were washed 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 the addition of DiC8. Then the translocation of protein was monitored at fixed intervals (every 7 s) after 150 µl of HEK buffer containing 0.1 mg/ml DiC8 was added. Control experiments were done with dimethyl sulfoxide. Images were analyzed using simFCS. Specifically, regions of interest in the cytosol were defined, and the average intensity in a square (1 µm x 1 µm) was 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. Intensity values corresponding to the place on the line indicating the edge of the cell were averaged. 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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—It has been long known that PS enhances the membrane affinity and activity of PKCs (27). Recent studies have revealed, however, that the PS dependence can vary significantly among PKC isoforms (24, 28). Among conventional PKCs, PKC (28) and PKCII (29) strongly prefer PS to other anionic phospholipids, such as phosphatidylglycerol (PG), whereas PKC shows little selectivity between PS and PG (24). Among novel PKCs, PKC shows a high degree of PS selectivity (25), whereas PKC does not display significant PS selectivity (26, 28). Because PKC is structurally very similar to PKC, it is expected to have high PS selectivity.
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to POPC/POPS/DiC18, POPC/POPG/DiC18, and POPC/POPI/DiC18 (all 69:30:1 in mole ratio) vesicles by the SPR analysis. As shown in Table 1, PKC exhibited 28- and 4-fold higher affinity for POPC/POPS/DiC18 vesicles than for POPC/POPG/DiC18 and POPC/POPI/DiC18 vesicles, respectively. The 28-fold PS selectivity over PG is even higher than the 10-fold PS selectivity observed for PKC under similar conditions (25). The PS selectivity of PKC was also evident in kinase activity measurements (see Fig. 1). PKC showed higher enzyme activity with POPC/POPS/DiC18 (99 - x:x:1) vesicles than with POPC/POPG/DiC18 or POPC/POPI/DiC18 (99 - x:x:1) vesicles at a given anionic lipid concentration. For example, PKC was 2-fold and 1.5-fold more active with POPC/POPS/DiC18 (69:30:1) vesicles than with POPC/POPI/DiC18 (69:30:1) and POPC/POPG/DiC18 (69:30:1) vesicles, respectively. This difference is smaller than expected from the Kd values of PKC for these vesicles, because it was necessary to perform the kinase assay under the condition in which a majority of enzyme molecules is membrane-bound (i.e. [lipids] >> Kd). Collectively, these results show that PKC has definite PS selectivity in membrane binding and kinase action.
DAG Affinity of C1A and C1B Domains and Their Roles in PKC Activation—We previously measured the DAG affinities of isolated C1 domains of several PKCs, which indicated that differential roles of C1A and C1B domains in the membrane targeting and activation of PKCs are ascribed to their different DAG affinities (24, 25). For PKC (22, 24) and PKC (25) whose C1A domain has much higher DAG affinity than C1B domain, the C1A domain plays a predominant role in these processes. For PKC (24) whose C1A and C1B domains have comparable DAG affinities, however, both C1 domains participate in the processes. PKC is similar to PKC in that both C1A and C1B domains are involved in the membrane binding and activation; however, the difference between the two PKC isoforms is that the C1A domain of PKC has about a 3-fold higher DAG affinity than its C1B domain (26). To see how the C1A and C1B domains of PKC are involved in its membrane binding and activation, we first expressed the isolated C1A and C1B domains of PKC. The C1B domain was expressed as soluble protein in E. coli, whereas the C1A domain was expressed as inclusion bodies, which were then solubilized in urea and refolded.
For C1 domains of other PKC isoforms, we determined Kd for binding to a soluble DAG (i.e. DiC8) monomer by isothermal titration calorimetry. In the case of PKC C1 domains, the isothermal titration calorimetry analysis was not practical due to difficulties encountered in expressing the proteins in amounts sufficient for the analysis. We, therefore, measured the binding of C1A and C1B domains to a longer chain DAG (i.e. DiC18) incorporated in the lipid bilayer by the SPR analysis that requires less protein samples than the isothermal titration calorimetry analysis. As shown in Table 1, the C1A and C1B domains had the Kd values of 1.9 µM and 26 nM, respectively, for the POPC/POPS/DiC18 (69:30:1) vesicles (notice that these Kd values are not for DiC18 monomers but for DiC18-containing vesicles; see Fig. 2 for SPR sensorgrams and binding isotherms). This is a unique finding in that no C1B domain has been shown to have much higher affinity for DAG or DAG-containing vesicles than its counterpart C1A domain in any other PKC isoforms characterized so far (22, 24-26). This also suggests that PKC might have a unique mechanism of DAG-mediated membrane binding and activation in which the C1B domain plays a predominant role.
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on its membrane binding and enzyme activity. Mutations were made on the hydrophobic residues whose counterparts in other PKCs have been shown to be important for their membrane binding (22, 24); i.e. W181G and L183G for the C1A domain and W253G and L255G for the C1B domain. We first measured the binding of these proteins to POPC/POPS/DiC18 (69:30:1) vesicles by the SPR analysis. As shown in Table 1, C1A domain mutants showed only modestly reduced affinities, whereas C1B domain mutants exhibited much lower affinities than the wild type. We then measured the kinase activities of these mutants in the presence of POPC/POPS/DiC18 (99 - x:x:1) vesicles. At all PS concentrations, C1B domain mutants (W253G and L255G) had much lower activity than the wild type and C1A domain mutants (W181G and L183G) (see Fig. 3A). Thus, these data underscore the importance of the C1B domain in the membrane binding and activation of PKC. Lastly, the PKC C1B domain did not distinguish between POPC/POPS/DiC18 (69:30:1) and POPC/POPG/DiC18 (69:30:1) vesicles (see Table 1), indicating that this domain by itself does not contribute to the PS selectivity of PKC.
Intramolecular Tethering of C1A and C1B Domains—Our recent studies have indicated that the strong PS selectivity seen with PKC (22) and PKC (25) is ascribed to the specific PS-induced unleashing of their C1 domains that are tethered intramolecularly via highly conserved Asp or Glu (e.g. Glu178 of C1A and Glu246 of C1B for PKC; see Fig. 7A) in the resting state. On the other hand, lack of PS selectivity of PKC (24) and PKC (26, 28) is due to higher conformational flexibility of its C1 domains. Definite PS selectivity of PKC suggested that their C1 domains might be intramolecularly tethered. Thus, mutations of Glu178 and Glu246 would unleash C1A and C1B domains, respectively, thereby greatly enhancing the membrane binding and activation of PKC. Also, because the C1B domain plays a more direct role than the C1A domain in the membrane binding and activation of PKC, the mutation of Glu246 was expected to have a larger effect than that of Glu178, based on our previous studies on PKC (22) and PKC (25).
We, therefore, prepared the E178A and E246A mutants of PKC and compared their properties. Because of unusually low stability of E246A, this mutant was only partially purified (see supplementary Fig. S1), which precluded the SPR measurement due to high nonspecific binding to the sensor chip. We, therefore, performed the SPR analysis only for E178A. As listed in Table 1, this mutant had 2.2-fold higher affinity for POPC/POPS/DiC18 (69:30:1) vesicles than the wild type PKC, supporting the notion that Glu178 is involved in tethering of the C1A domain. To compare the effects of E178A and E246A mutations, we then measured the activity of these mutants by the kinase assay, which could be performed with smaller amounts of partially purified proteins. As shown in Fig. 3B, both E178A and E246A had significantly higher kinase activity than the wild type in the presence of POPC/POPS/DiC18 (99 - x:x:1) vesicles. At all PS concentrations, both mutants showed 2-fold higher activity than the wild type. This is again consistent with the notion that Glu178 and Glu246 of PKC restrict the flexibility of C1A and C1B domains, respectively, thereby keeping the enzyme inactive in the resting state. However, the comparable effects caused by E178A and E246A mutations were rather unexpected, given that the C1A domain has much lower DAG affinity and plays a less important role than the C1B domain in the membrane binding and activation of PKC. This result suggests that although the C1A domain is not directly involved in PKC activation, untethering of the C1A domain may be necessary for the function of the C1B domain because the C1A domain is located between the pseudosubstrate region and the C1B domain in the PKC molecule (see Fig. 7A). In other words, it may be necessary to unleash both C1A and C1B domains for full activation of PKC. This notion is supported by the finding that a double-site mutant, E178A/E246A, has much higher activity than the single-site mutants (see Fig. 3B). Thus, it appears that PKC has a unique membrane binding and activation mechanism in which the two C1 domains with distinctly different membrane affinities work in concert to achieve optimal membrane binding and enzymatic activation.
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—Although much is known about the Ca2+-dependent membrane binding properties the C2 domains of conventional PKCs and their roles in enzyme activation (7, 30, 31), less is known about the roles of Ca2+-independent C2 domains of novel PKCs. Our recent studies have shown that the isolated C2 domains of PKC (25) and PKC (26) do not have physiologically significant membrane affinity. Although these domains have been implicated in protein-protein interactions (32, 33), their roles in membrane targeting and activation of respective PKC isoforms have not been established.
The C2 domain of PKC shares high sequence homology with that of PKC. Unlike the C2 domain of PKC, however, the isolated C2 domain of PKC has significant affinity for PtdIns(4,5)P2-containing anionic membranes (34) (see also Table 1). The PKC C2 domain also bound POPC/POPE/POPS and POPC/POPE/POPG (both 60:20:20) vesicles with comparably lower affinity (see Table 1). Thus, the C2 domain selectively binds PtdIns(4,5)P2 and lacks PS selectivity.
To see if the C2 domain plays any role in the membrane binding and activation of PKC, we measured the effect of C2 domain deletion on the membrane binding and activation of full-length PKC. The SPR analysis showed that the C2 deletion mutant (
C2; devoid of residue 1-114) had 7-fold higher affinity for POPC/POPS/DiC18 (69:30:1) vesicles than wild type PKC (Table 1). Furthermore, C2 was much more active than the wild type in the presence of PS- or PG-containing vesicles (see Fig. 4A). Interestingly, C2 in the presence of a given concentration of PG was more active than the wild type in the presence of the same concentration of PS. These properties are in stark contrast to those of corresponding C2 domain deletion mutants of PKC (25) and PKC (26), both of which had wild type-like membrane affinity and enzyme activity. These data suggest that the C2 domain of PKC may be involved in keeping the enzyme in an inactive conformation, presumably by interacting with the C1A and C1B domains.
It was reported that TCR stimulation led to phosphorylation of PKC on Tyr90 in its C2 domain by Lck kinase, which may be important for targeting PKC to the immunological synapse in T cells (21). To see if the phosphorylation of Tyr90 controls the membrane binding of PKC, we prepared Y90E and Y90F mutants as mimetics of phosphorylated and dephosphorylated forms, respectively, and measured their membrane binding and kinase activity. As listed in Table 1, Y90E had 4.2-fold higher affinity for POPC/POPS/DiC18 (69:30:1) vesicles than Y90F, suggesting that Tyr90 phosphorylation enhances the membrane affinity of PKC. Also, Y90E was 2-3-fold more active than Y90F in the presence of POPC/POPS/DiC18 (99 - x:x:1) vesicles (Fig. 4B). Interestingly, Y90E showed slightly lower affinity for POPC/POPS/DiC18 (69:30:1) vesicles (Table 1) and lower kinase activity (Fig. 4B) than wild type. This implies that Tyr90 is largely phosphorylated in the recombinant PKC wild type expressed in insect cells and that the Y90E mutation cannot fully mimic the phosphotyrosine. To test this notion, we measured the total tyrosine phosphorylation of the extracts from SF9 cells overexpressing PKC wild type and Y90F, respectively. Fig. 5 shows that under the conditions where the wild type and Y90F were expressed in similar levels, the wild type showed strong Tyr phosphorylation, whereas the mutant exhibited no trace of Tyr phosphorylation. This strongly supports, albeit qualitatively, that the recombinant PKC expressed in insect cells has a significant extent of Tyr90 phosphorylation. Most importantly, the large difference in membrane affinity between Y90E and Y90F indicates that the phosphorylation of Tyr90 of PKC enhances its membrane affinity, which should in turn increase the overall enzyme activity of PKC.
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affect its cellular membrane targeting and to see if PKC follows the same mechanism in in vitro and cellular membrane binding, we monitored the DAG-dependent subcellular translocation of PKC and mutants, each tagged with EGFP at their carboxyl termini, in HEK293 cells. The cellular expression levels of different protein constructs were comparable in most cells (>80%) when assessed by visual inspection of EGFP fluorescence intensity and by Western blotting using PKC-specific antibodies (data not shown). Only those cells with similar PKC expression levels were used for further measurements.
We monitored by two-photon microscopy the spatiotemporal dynamics of EGFP-tagged PKC and mutants in response to a short-chain DAG, DiC8 (0.1 mg/ml). Fig. 6 shows the time-lapse images of EGFP-tagged proteins in representative cells. A minimum of quadruple measurements was performed for each protein with >5 cells monitored for each measurement. Typically, >80% of cell population showed similar behaviors with respect to DiC8-induced PKC translocation. In response to 0.1 mg/ml DiC8 stimulation, the wild type PKC-EGFP translocated mainly to the plasma membrane (Fig. 6A). The specific targeting of PKC to the PS-rich plasma membrane despite relatively random distribution of DiC8 in all cell membranes (25, 26) is consistent with its high PS selectivity, as seen with other PS-selective PKC isoforms (24, 25). The time-course analysis (Fig. 6B) shows that after an initial lag PKC essentially completed the translocation to the plasma membrane within 15 min.
When the cellular membrane translocation of PKC mutants was measured after DiC8 stimulation, a good correlation was seen between their in vitro membrane affinity (and/or enzyme activity) and cellular translocation efficiency. For instance, C2 (Fig. 6, A and C), E178A (Fig. 6, A and C), E246A (Fig. 6, A and C), and E178A/E246A (Fig. 6C), all of which showed higher in vitro membrane affinity and/or enzymatic activity than the wild type, were recruited to the plasma membrane much faster than the wild type. These mutants had no lag and immediately translocated to the plasma membrane upon DiC8 addition. In particular, C2 and E178A/E246A showed the fastest membrane translocation. Conversely, W253G and L255G (Fig. 6B) with greatly reduced membrane affinity showed much slower cellular membrane translocation than wild type. Also, W181G and L183G (see Fig. 6B) with wild type-like membrane affinities and activities migrated to the plasma membrane as fast as the wild type. Interestingly, Y90E (Fig. 6, A and C) translocated to the plasma membrane significantly faster than Y90F (Fig. 6, A and C) and the wild type, which further supports the notion that phosphorylation Tyr90 enhances the membrane binding affinity of PKC and thereby induced its membrane translocation and activation. Also, the finding that Y90F behaved similarly to the wild type implies that Tyr90 of PKC exists primarily as an unphosphorylated form in HEK293 cells, presumably due to lack of Lck in these cells. To test this notion, we also determined the extent of Tyr phosphorylation of the extracts from HEK293 cells overexpressing PKC wild type and Y90F, respectively. Fig. 5 shows that neither wild type nor Y90F was phosphorylated on Tyr. It should be noted that a comparable amount of wild type expressed in Sf9 cells showed strong Tyr phosphorylation. Thus, it is evident that Tyr90 is not phosphorylated in PKC expressed in HEK293 cells under our experimental conditions. Collectively, these cell translocation results indicate that cellular membrane targeting and in vitro membrane binding of PKC follow essentially the same mechanism.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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is an attractive model for this type of investigation because of its unique targeting to the immunological synapse in T cells and its importance in T cell activation. In the present study we employed various in vitro and cellular techniques that allow quantification of the membrane binding and activity of PKC wild type and mutants to gain new mechanistic insight into the membrane binding and activation of PKC.
The novel PKC family can be subdivided into two groups, / group and / group, based on sequence similarity. The present study demonstrates that PKC has a distinctly different membrane binding and activation mechanism from PKC despite strong overall structural similarities. PKC is similar to PKC in one aspect; i.e. both have PS selectivity in membrane binding and kinase action. It should be noted that our result is at odds with an earlier qualitative study indicating that PKC phosphorylates moesin more effectively in the presence of PG than in the presence of PS (35). Our quantitative SPR analysis clearly shows that PKC has much higher affinity for PS-containing vesicles than for PG-containing vesicles, which is corroborated by the kinase activity assay. Perhaps, the PG selectivity seen in the previous study is due to the use of a different PKC substrate (note that myelin basic protein was used in our study) and different kinase assay conditions. Our previous studies on PKC (22, 24-26, 28) and other proteins (36-38) have shown that PS selectivity of a protein contributes to specific targeting of the protein to the PS-rich plasma membrane. Thus, PS selectivity of PKC as well as the selectivity of its C2 domain for PtdIns(4,5)P2 should drive specific plasma membrane targeting of this PKC isoform. Indeed, our cell study shows that PKC is exclusively localized to the plasma membrane in HEK293 cells under the condition in which DAG molecules are randomly distributed in all cell membranes. Our recent studies have also shown a good correlation between the PS selectivity of PKC isoforms and the conformational restriction of their C1 domains via conserved anionic residues (Glu178 of C1A and Glu246 of C1B for PKC) (22, 24-26, 28). Likewise, the present study indicates that the C1 domains of PKC are also conformationally restricted, as E178A and E246A mutations significantly increase the in vitro membrane affinity (measured only for E178A) and kinase activity of PKC and also enhance the efficiency of plasma membrane translocation of PKC in HEK293 cells. Also, a lack of PS selectivity seen with isolated C1B and C2 domains further supports the notion that PS selectivity of PKC derives not from specific binding of PS to either C1 or C2 domains but from PS-mediated disruption of interdomain interactions tethering C1 domains.
PKC is unique among PKCs in two key aspects. First, its C1B domain has much higher DAG affinity than the C1A domain and, thus, consequently plays a more important role in membrane binding and activation of the enzyme. Second, it has a Ca2+-independent C2 domain with relatively high membrane affinity. The C2 domain suppresses the membrane binding of the rest of the molecule and thereby attenuates the enzyme activity in the resting state. Our SPR analysis of isolated C1 domains and SPR and activity measurements of hydrophobic site mutants of PKC clearly show that the C1B domain is the main player in DAG-dependent membrane binding and activation of PKC. In all other PKC isoforms whose DAG-dependent membrane binding and activation have been systematically investigated, the C1A domain has higher DAG affinity than the C1B domain; only for PKC, C1A and C1B domains have similar DAG affinities.5 This unique C1B dominance in PKC raises an interesting question about the activation mechanism of PKC. In all conventional and novel PKCs, the pseudosubstrate region that blocks the active site of the enzyme in the resting state is located in the immediate amino-terminal side of the C1A domain. Thus, the conformational change of the protein that leads to the movement of the C1A domain (typically as a result of membrane penetration and binding to membrane-incorporated DAG) would remove the adjacent pseudosubstrate region from the active site (22, 39). In the case of PKC, the movement of the C1B domain accompanying its DAG binding and membrane penetration would be transmitted to the pseudosubstrate region through the intervening C1A domain (see Fig. 7B). This notion is supported by the finding that the E178A mutation in the C1A domain significantly increases the in vitro membrane affinity and kinase activity of PKC as well as its cellular translocation efficiency despite the fact that the C1A domain is not directly involved in DAG-dependent membrane binding. Furthermore, E178A/E246A is more active than E246A and translocates to the plasma membrane faster than E246A in HEK293 cells. This type of contribution from a nonessential C1 domain has not been seen with other PKCs, including PKC (25), and would not be expected unless a concerted movement of C1A and C1B domains is required for PKC activation.
For conventional PKCs, membrane binding and activation of a protein is triggered by binding of Ca2+ to the C2 domain that subsequently targets the protein to the membrane, which is followed by membrane penetration and DAG binding of one or both C1 domains (22, 39). On the other hand, membrane binding and activation of novel PKCs is mediated primarily by DAG-C1 interactions, although protein-protein interactions can also play an important role in cellular membrane targeting. For PKC (25) and PKC (26), isolated C2 domains did not have physiologically significant membrane affinity, and their truncation did not have appreciable effect on in vitro and cellular membrane binding and activity of respective PKC isoforms. In contrast, the C2 domain of PKC binds PtdIns(4,5)P2-containing vesicles with relatively high affinity, and its deletion greatly enhances the membrane affinity and enzyme activity both in vitro and in HEK293 cells. These results indicate that the C2 domain is directly involved in membrane binding and that the C2 domain in the resting state suppresses the membrane interaction of the C1B domain, perhaps through interdomain interactions with C1 domains. The involvement of the C2 domain in tethering C1 domains of PKC is supported by the finding that C2 and E178A/E246A have similarly fast cell membrane translocating properties (see Fig. 6C). As proposed for PKC (39), initial membrane binding of the C2 domain may allow PS molecules in the membrane to liberate the C1B (and C1A) domain from the intramolecular, interdomain tethering.
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C2 domain? Our SPR and cell translocation data suggest that phosphorylation of Tyr90, which was reported to be catalyzed by Lck kinase in T cells and might be important for PKC targeting to the immunological synapse (21), contributes at least partially to triggering the membrane recruitment of PKC. It does not appear that phosphorylation of Tyr90 increases the membrane affinity of the C2 domain per se because neither Y90E nor Y90F mutation affects the membrane affinity of the isolated C2 domain (see Table 1). Therefore, it is likely that Tyr90 phosphorylation induces conformational changes that position C2 and C1B domains in the intact PKC molecule for better membrane interactions. The finding that Y90F has significantly lower membrane affinity and kinase activity than Y90E suggests that before Tyr90 phosphorylation PKC may not productively interact with the membrane probably because the initial C2 domain-mediated membrane adsorption does not lead to the membrane penetration of the C1B domain (see Fig. 7B). Obviously, further studies are needed to determine the exact role of Tyr90 phosphorylation in PKC activation, which is beyond the scope of this investigation.
On the basis of our results, we propose a hypothetic model of PKC membrane recruitment and activation illustrated in Fig. 7B. In the resting state of PKC, neither C2 nor C1B domain is fully exposed for optimal membrane interactions; hence, low membrane affinity and enzyme activity. Phosphorylation of Tyr90 induces conformational changes that position C2 and C1B domains for improved membrane interactions and thereby trigger the membrane recruitment of PKC. Initial binding of the C2 domain to the plasma membrane containing PtdIns(4,5)P2 and PS would then unleash the C1A and C1B domains by the action of PS that disrupts the interdomain tethering between the C2 domain and C1 domains via Glu178 and Glu246, respectively. Membrane penetration and DAG binding of the C1B domain and the concomitant movement of the C1A domain pull the pseudosubstrate region from the active site, resulting in enzyme activation. Although further studies are needed to test this hypothetical model, the model provides a framework within which to systematically investigate the mechanism by which PKC is specifically targeted to the immunological synapse in T cells.
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FOOTNOTES |
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The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. S1.
1 These authors equally contributed to this work.
2 Current address: Dept. of Biochemistry and Molecular Biology, Indiana University School of Medicine-South Bend and Department of Chemistry and Biochemistry and The Walther Center for Cancer Research, University of Notre Dame, South Bend, IN 46617.
3 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.
4 The abbreviations used are: PKC, protein kinase C; CHAPS, (3-[3-cholamidopropyl)dimethylammonio]-1-propane sulfonate; DAG, sn-1,2-diacylglycerol; 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; POPC, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine; 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; POPE, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine; PG, phosphatidylglycerol; PS, phosphatidylserine; PtdIns(4,5)P2, phosphatidylinositol-4,5-bisphosphate; SPR, surface plasmon resonance; TCR, T cell receptor.
5 It should be noted that different results have been reported for the phorbol ester-mediated membrane binding and activation of PKC isoforms; e.g. PKC-C1B has higher phorbol ester affinity than PKC-C1A (25, 40). This is because many C1 domains, either as isolated domains or in the context of the full-length proteins, have totally different affinities for DAG and phorbol esters (25). This finding, therefore, cautions against using phorbol esters alone to elucidate the mechanisms of DAG-mediated membrane binding and activation of PKCs.
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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, mutants, and isolated domains determined by SPR analysis
), POPC/POPI/DiC18 (99 - x:x:1) (PI, 
), or POPC/POPG/DiC18 (99 - x:x:1) (
) vesicles in 20 mM HEPES buffer, pH 7.4, containing 0.16 M KCl, 5 mM MgCl2, and myelin basic protein (200 mg/ml). Each data point represents an average of triplicate measurements. The relative activity was calculated in comparison with the maximal activity (0.016 µmol/mg/min) of wild type achieved with POPC/POPS/DiC18 (19:80:1) vesicles.
), W181G (
), W253G (
), and L255G (
