©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Low Affinity Binding of Phorbol Esters to Protein Kinase C and Its Recombinant Cysteine-rich Region in the Absence of Phospholipids (*)

Marcelo G. Kazanietz (1), Joseph J. BarchiJr. (2), James G. Omichinski (3), Peter M. Blumberg (1)(§)

From the (1)Molecular Mechanisms of Tumor Promotion Section, Laboratory of Cellular Carcinogenesis and Tumor Promotion, NCI, (2)Laboratory of Medicinal Chemistry, NCI, and (3)Laboratory of Chemical Physics, NIDDK, National Institutes of Health, Bethesda, Maryland 20892-4255

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
CONCLUSION
FOOTNOTES
REFERENCES

ABSTRACT

Binding of phorbol esters to protein kinase C (PKC) has been regarded as dependent on phospholipids, with phosphatidylserine being the most effective for reconstituting binding. By using a purified single cysteine-rich region from PKC expressed in Escherichia coli we were able to demonstrate that specific binding of [H]phorbol 12,13-dibutyrate to the receptor still takes place in the absence of the phospholipid cofactor. However, [H]phorbol 12,13-dibutyrate bound to the cysteine-rich region with 80-fold lower affinity in the absence than in the presence of 100 µg/ml phosphatidylserine. Similar results were observed with the intact recombinant PKC isolated from insect cells. When different phorbol derivatives were examined, distinct structure-activity relations for the cysteine-rich region were found in the presence and absence of phospholipid. Our results have potential implications for PKC translocation, for inhibitor design, and for PKC structural determination.


INTRODUCTION

Protein kinase C was the first receptor identified for the phorbol ester tumor promoters(1) . These compounds bind with high affinity (low nanomolar range) to the calcium-dependent PKC()isozymes , , , and , as well as to the calcium-independent PKC isozymes , , , and , but not to the atypical PKC isozymes and (2, 3) . Recently, the family of phorbol ester receptors was expanded with the discovery of n-chimaerin and Unc-13(4, 5) , two novel proteins that bind phorbol esters and related ligands similarly to the PKCs but which possess distinct effector regions(6, 7) . Deletion analysis of PKC has identified the two cysteine-rich regions in the regulatory domain as the sites of phorbol ester binding (8). Each of these cysteine-rich regions is a 50-amino-acid domain possessing the motif HXCXCXCXCXHXCXC (where H is histidine, C is cysteine, X is any other amino acid, and n = 13 or 14). An identical motif is present in n-chimaerin and Unc-13. Although the PKCs contain two cysteine-rich regions, a single recombinant cysteine-rich region is sufficient for binding [H]PDBu with high affinity(6, 9, 10, 11) .

The current models for the interaction of PKC with the phorbol esters consider phospholipid as an essential cofactor for binding to the enzyme(12, 13) . The anionic phospholipids fully reconstitute the phorbol ester binding, with phosphatidylserine being the most efficient (14, 15). In this study we examined the binding of [H]PDBu both to intact PKC and to the purified single cysteine-rich region of PKC in the absence of phospholipid cofactor. Our results clearly show that phorbol esters still bound to PKC in the absence of phospholipids, although with a reduced affinity.


EXPERIMENTAL PROCEDURES

Materials

[H]PDBu (20.7 Ci/mmol) was purchased from DuPont NEN (Boston, MA). Phorbol esters were obtained from LC Services Corp. (Woburn, MA). Phosphatidylserine, bovine -globulins, lysozyme, and DNase were purchased from Sigma.

Expression of PKC in Baculovirus

Recombinant PKC was expressed in Sf9 insect cells and partially purified as described in Ref. 3.

Expression and Purification of Recombinant Cysteine-rich Region

The second cysteine-rich region of PKC was generated by polymerase chain reaction using the full-length mouse cDNA clone (16) as a template and the following oligonucleotides containing BamHI and EcoRI sites (underlined) for unidirectional cloning: 5`-TGAGGATCCCACCGATTCAAGGTTTATAAC and 5`-ATCGAATTCACACAGGTTGGCCACCTTCTC. The 160-base pair fragment was subcloned in the pCR2000 vector (Invitrogen) and sequenced to confirm complete homology with the original sequence. The BamHI-EcoRI fragment was then isolated and ligated in frame in the pGEX-2TK vector (Pharmacia Biotech Inc.) to get the pGEXTK-CRR plasmid. In order to induce the expression of the recombinant cysteine-rich region (as a glutathione S-transferase-fusion protein) Escherichia coli JM101 cells were transformed with the pGEXTK-CRR plasmid, the bacteria grown up to an A of 0.5 in 1 liter of LB media containing 50 µg/ml ampicillin, and the recombinant protein induced by addition of 0.5 mM isopropyl-1-thio--D-galactopyranoside. Five hours later cells were pelleted at 4,000 g and resuspended in 20 ml of phosphate-buffered saline containing 3 mM dithiothreitol. Cells were lysed by homogenization, passed two times through a French press, and sonicated. Cellular debris was pelleted for 45 min at 15,000 g (4 °C), the remaining suspension was centrifuged subsequently at 30,000 g for 2 h (4 °C), and the supernatant was collected. For the purification of the glutathione S-transferase-fused cysteine-rich region of PKC , 10 ml of a 50% glutathione-Sepharose 4B suspension (Pharmacia) were added to the supernatant and incubated at 4 °C for 1 h. The glutathione-Sepharose 4B beads were collected by centrifugation (500 g, 5 min), and the glutathione S-transferase-fusion protein was then eluted by incubation of the beads with 1 ml of 10 mM reduced glutathione in 50 mM Tris-Cl, pH 8.0, as described by the manufacturer. The elution procedure was repeated an additional three times, and the collected fractions were pooled. The recombinant fusion protein showed >90% purity in Coomassie Blue-stained gels (data not shown). The solution was then treated with 250 units of thrombin for 4 h, time enough to cleave all of the recombinant cysteine-rich region from the glutathione S-transferase as checked by gel electrophoresis. In order to remove the thrombin, the eluate was made 0.5 M in NaCl and treated with 2 ml of benzamidine-Sepharose beads (Pharmacia) for 1 h at 25 °C. The beads were pelleted and the supernatant was partially desalted by ultrafiltration through an Amicon 3000 MW filter. The retentate was lyophilized and purified by HPLC on C4 silica gel using a gradient of 0.05% trifluoroacetic acid/HO: 0.05% trifluoroacetic acid in 90/10 CHCN/HO, from 75:25 to 25:75 over 30 min. The cysteine-rich region eluted at 14.8 min in this system, and the residual glutathione S-transferase eluted at 18 min (see Fig. 1, A and B). The protein obtained by this procedure was refolded as described previously(17) . Briefly, the protein was dissolved in 0.05% trifluoroacetic acid (pH 2.54), 2.5-3 molar eq (based on weight of protein) of Zn (as 50 mM ZnCl) were added, and the pH was slowly raised to 6.00 with dilute NaOH. The solution was concentrated on Centricon-3 filters. The cysteine-rich region purified by HPLC showed a single peak in the HPLC profile (Fig. 1B) and a single band in Coomassie Blue staining of SDS-polyacrylamide gels (Fig. 1C). [H]PDBu Binding-[H]PDBu binding was measured using the polyethylene glycol precipitation assay developed in our laboratory (18). For the determination of the K and B in the presence of phospholipid the assay was performed as follows. An assay mixture (250 µl) containing 50 mM Tris-Cl, pH 7.4, 100 µg/ml phosphatidylserine, 4 mg/ml bovine IgG, increasing concentrations of [H]PDBu, and a recombinant cysteine-rich region or intact PKC was incubated for 5 min at 37 °C. The samples were then chilled on ice for 5 min, and 200 µl of 35% polyethylene glycol in 50 mM Tris-Cl, pH 7.4, was added. The tubes were incubated for an additional 15 min to induce precipitation of the protein and were then centrifuged at 12,000 g for 15 min in a Beckman 12 microcentrifuge (at 4 °C). An aliquot (100 µl) of the supernatant was removed for determination of the free radioligand. The pellet was carefully dried with disposable wipes, and then the tip of the tube was cut off and the bound [H]PDBu was determined in a scintillation counter. Nonspecific binding was measured using 30 µM PDBu; the specific binding was calculated as the difference between the total and the nonspecific binding and was >80% even at the highest [H]PDBu concentration.


Figure 1: Expression of cysteine-rich region of PKC in E. coli. A, the thrombin-cleaved glutathione S-transferase-fusion protein was purified on HPLC using a C4 silica gel column (conditions are given in the text). The cysteine-rich region eluted at 14.8 min as indicated by the arrow. B, HPLC pattern of the purified cysteine-rich region. C, 10 µg of the HPLC-purified protein was subjected to SDS-polyacrylamide gel electrophoresis (16% in Tricine) and stained with Coomassie Blue using standard techniques. Lane1, molecular weight markers; lane2, purified cysteine-rich region of PKC .



For measuring the parameters for [H]PDBu binding in the absence of phospholipids we determined the K from competition curves with nonradioactive PDBu. This experimental approach is described by Turner and Bylund (19) and Blumberg et al. (20). Typical saturation curves with increasing concentrations of [H]PDBu could not be performed because of the very high concentrations of radioligand needed. In a typical assay 6-8 increasing concentrations of the nonradioactive PDBu (in triplicate) were used to compete 100 nM [H]PDBu in a mixture containing 50 mM Tris-Cl, pH 7.4, 1 mM EGTA, and 4 mg/ml bovine IgG (or other carrier protein when indicated). After incubation (5 min, 37 °C), samples were chilled on ice for 5 min. Precipitation and separation of the free and bound radioligand were performed as described above. Nonspecific binding was determined in the presence of 100 µM PDBu and ranged between 25 and 45% of the total in the presence of [H]PDBu alone. The data were fitted to the theoretical inhibition curve, and the K was calculated from the equation K = ID - L, where ID is the concentration of nonradioactive ligand (PDBu) that displaced the binding of the radioligand by 50% and L is the concentration of free radioligand at the ID.

To measure competition of [H]PDBu binding by different phorbol esters, incubations were performed under similar conditions but using a fixed concentration of [H]PDBu (4 nM in the presence of phosphatidylserine and 100 nM in the absence of phosphatidylserine) and increasing concentrations of the nonradioactive ligand. In a typical competition assay, 6-8 different concentrations (in triplicate) of the competing ligand were used. The ID was determined for the competition curve in each case, and the K for the competing ligand was calculated by using the relationship K = ID/(1 + L/K), where K is the dissociation constant for [H]PDBu under the specific assay condition and L is the concentration of free radioligand at the ID. In general, triplicate determinations differed by <10%.


RESULTS

Using intact recombinant PKC expressed in Sf9 cells with the baculovirus system (3) we noted a low amount of [H]PDBu binding in the absence of added phosphatidylserine (Fig. 2A). Although small, this specific binding was significantly above background (p < 0.001). One explanation was that [H]PDBu could indeed bind to PKC in the absence of phospholipid but only did so with greatly reduced affinity. Although contrary to the generally accepted view in the field (12, 13), this explanation would fit both with the biochemical expectation that PKC per se must provide a partial binding site for its ligand as well as with experimental findings, outlined under ``Conclusion,'' of phorbol ester modulation of PKC properties in the absence of ligand.


Figure 2: [H]PDBu binding to PKC . A and B, binding of [H]PDBu (10 nM) to intact recombinant PKC expressed in baculovirus (A) or its second cysteine-rich region expressed in E. coli (B), either in the absence (-PS) or presence (+PS) of 100 µg/ml phosphatidylserine. C, specific binding of [H]PDBu (0.5-16 nM) to intact recombinant PKC in the presence of 100 µg/ml phosphatidylserine. The experimental values are expressed as mean ± S.E. of triplicate determinations in a single experiment. Two additional experiments gave similar results. D, binding of [H]PDBu to intact recombinant PKC in the absence of phospholipids was measured by competition of [H]PDBu (100 nM) with nonradioactive PDBu. K was determined from the formula, K = ID - L, as described under ``Experimental Procedures.'' Values represent mean ± S.E. of triplicate determinations in a single experiment. Two additional experiments gave similar results. Insets in C and D are the Scatchard plots derived from the corresponding binding curves. The K values presented in the top right-hand corner of each panel are expressed as mean ± S.E. from three experiments in each case. Note that the first point in the Scatchard plot in D corresponds to the binding of [H]PDBu in the absence of added cold ligand or 100% binding (171 ± 10 pmol/mg, n = 3). This value corresponds to approximately 1.5 10 dpm of [H]PDBu specifically bound per tube.



Indeed, using 100 nM [H]PDBu and competition with increasing concentrations of nonradioactive PDBu, we were able to demonstrate specific PDBu binding to intact PKC in the absence of phospholipids with a K of 165 ± 15 nM (n = 3) (Fig. 2D). For comparison, the binding affinity in the presence of phosphatidylserine was 160-fold greater (K = 1.0 ± 0.2 nM, n = 3) (Fig. 2C). A concern in the interpretation of these findings was whether the low affinity binding could represent an artifact arising from lipid remaining in the preparation during purification.

A powerful approach to deal with this issue was to use the recombinant cysteine-rich region of PKC , a preparation that could be lipid extracted and then refolded again in aqueous solution. A single cysteine-rich region of PKC is the minimum element of the enzyme required for phorbol ester binding(9, 10, 11) . Initial experiments using 10 nM [H]PDBu and 0.5 nM HPLC-purified cysteine-rich region showed that phosphatidylserine was required for efficient reconstitution of high affinity ligand binding (Fig. 2B). The ED for phosphatidylserine was 15 ± 2 µg/ml (n = 4), and the maximum binding was achieved at 100 µg/ml phosphatidylserine (data not shown). The stoichiometry for [H]PDBu binding was 0.2, similar to that reported by Bell and co-workers in a recent paper (9). The dissociation constant (K) for binding of [H]PDBu to the cysteine-rich region in the presence of 100 µg/ml phosphatidylserine, using a 1 nM concentration of the receptor, was 1.9 ± 0.1 nM (Fig. 3A, ). A similar K (2.3 ± 0.1 nM, n = 4) was found by competing a fixed concentration of [H]PDBu (3 nM) with nonradioactive PDBu (data not shown).


Figure 3: [H]PDBu binding to the second cysteine-rich region of PKC . A, specific binding of [H]PDBu (0.5-16 nM) in the presence of 100 µg/ml phosphatidylserine. The concentration of cysteine-rich region in the assay is 1 nM. Values represent mean ± S.E. of triplicate determinations. B, binding to the second cysteine-rich region of PKC in the absence of phospholipids was measured by competition of [H]PDBu (100 nM) with nonradioactive PDBu, as described in Fig. 2. The concentration of receptor in the assay is 10 nM. Values represent mean ± S.E. of triplicate determinations. Insets in A and B are the Scatchard plots derived from the corresponding binding curves. The binding curves in A and B are representative of three experiments each and represent the fitted theoretical curves for noncooperative binding. The K values, expressed as mean ± S.E. (n = 3), are presented in the topright-hand corner of the corresponding panel. Note that the first point of the Scatchard plot in B corresponds to the specific binding of [H]PDBu in the absence of added cold ligand or 100% binding (7400 ± 400 pmol/mg, n = 3); this value corresponds to approximately 2 10 dpm [H]PDBu specifically bound per tube.



By using 100 nM [H]PDBu and 10 nM of the purified cysteine-rich region of PKC as a receptor we were able to determine that [H]PDBu bound in the absence of added phospholipids with a K of 157 ± 21 nM, about 80 times weaker affinity than in the presence of 100 µg/ml phosphatidylserine. The Bvalue in the absence of phospholipids was 83 ± 2% (n = 3) of that in the presence of phosphatidylserine. The slight decrease in recovered binding activity appears to reflect absorption to the walls of the incubation tube. Similar results were obtained with a glutathione S-transferase-fused cysteine-rich region. In this latter case, the K values for [H]PDBu in the presence and absence of phosphatidylserine were 0.8 ± 0.1 nM (n = 3) and 69 ± 20 nM (n = 2), respectively. Although the absolute K values seem to be slightly lower for the glutathione S-transferase-fusion protein than for the nonfusion protein, the shift in affinity remained the same (K ratio = 86). As described in earlier studies with recombinant PKC isozymes(3) , the kinetics of [H]PDBu binding was very fast at 37 °C, and maximum levels were attained at 2-5 min either in the presence or absence of phospholipids. [H]PDBu binding remained at a plateau for up to 30 min in the absence of phospholipids. In the presence of phosphatidylserine about 25% loss in [H]PDBu binding was found after 30 min of incubation. Experiments using glutathione S-transferase-fused cysteine-rich regions in which the cysteine residue at position 17 or 42 was replaced by glycine by site-directed mutagenesis showed no specific [H]PDBu binding either in the presence or absence of phospholipids (data not shown).()

We confirmed that our measured binding of [H]PDBu to the cysteine-rich region of PKC in the absence of phospholipid reflected complex formation during the initial incubation at 37 °C. The K was independent of the volume of polyethylene glycol added after chilling of the complex (data not shown). Were either re-equilibration to have occurred at 0 °C or were the binding to have been a consequence of precipitate formation, then the K should have reflected the final [H]PDBu concentration after polyethylene glycol addition. We had previously shown that the slow rate of [H]PDBu release at 0 °C from the intact receptor in the presence of lipid is too slow to permit re-equilibration(21) .

In order to rule out the presence of contaminating lipids that might be responsible for the observed phorbol ester binding we performed a series of controls. First, the binding was not dependent on the specific carrier protein used for precipitation. Similar [H]PDBu binding was obtained in the absence of phospholipids when either DNase or lysozyme was used instead of bovine IgG (data not shown). Likewise, the cysteine-rich region bound [H]PDBu equally well in the absence of added phospholipid when the carrier was bovine IgG that had been subjected to lipid extraction (3 times) with chloroform. In order to test the efficiency of this extraction procedure we measured the recovery after the extraction of radiolabeled arachidonic acid and phosphatidylserine added to the IgG solution (about 1 10 cpm in each case). The solvent extraction removed 100% of the added [H]arachidonic acid and >90% of the added [C]phosphatidylserine. Finally, elemental analysis of the IgG revealed an upper limit for phosphorus of 0.000875% in the preparation (analysis performed by Galbraith Laboratories, Knoxville, TN). If we assumed that phosphorus was present at this limit of detection and was all attributable to phospholipids and that phosphatidylserine represented 20% of the total phospholipid, then phosphatidylserine would not be present after chloroform extraction at a concentration above 0.01 µg/ml. This concentration of phosphatidylserine is well below that required for supporting phorbol ester binding (data not shown).

Structure-activity analysis with a series of phorbol esters revealed that the different analogs tested also bound with lower potency to the cysteine-rich region of PKC in the absence of phospholipids (Table I). The ratios between the affinities in the absence and in the presence of phosphatidylserine differed for the different phorbol esters. Monoesters such as phorbol 12-decanoate and phorbol 13-decanoate, as well as the 12-deoxyphorbol derivative prostratin (12-deoxyphorbol 13-acetate), showed a smaller loss of affinity in the absence of phospholipids than did the phorbol 12,13-diesters. We conclude that structure-activity relations differ depending on the absence or presence of the phospholipid cofactor.


CONCLUSION

Since the discovery of PKC it has been considered that phospholipids are essential cofactors for the activation of the enzyme. The most effective of the naturally occurring phospholipids is phosphatidylserine, although all of the anionic phospholipids display cofactor activity(15) . Early studies from different laboratories including ours (2, 15) likewise showed that the binding of phorbol esters and related ligands to PKC was dependent on the presence of phospholipids. In the present study we could clearly characterize phorbol ester binding to their receptor in the absence of phospholipids, although with a lower affinity. By using PKC expressed in baculovirus we found that the intact enzyme bound [H]PDBu with an affinity of 165 nM in the absence of phospholipid. The dissociation constant was about 160-fold higher than that obtained in the presence of phosphatidylserine. Likewise, using the recombinant cysteine-rich region of PKC we showed [H]PDBu binding in the absence of phospholipid, with a 80-fold difference in affinity. Crucial technical features of the present experiments are that by using the recombinant cysteine-rich region of PKC we could achieve the high receptor levels necessary for the assays. Furthermore, using the recombinant cysteine-rich region of PKC we could highly purify this peptide by HPLC in the presence of organic solvents, avoiding any ambiguities concerning contamination with lipid in partially purified intact PKC purified under less harsh conditions.

Although this is the first study that demonstrated phospholipid-independent phorbol ester binding to PKC, some previous reports in the literature had suggested that phorbol esters could induce phospholipid-independent effects. Nakadate and Blumberg (22) observed a low level of PKC activation by PDBu in the absence of any phospholipid cofactor. In addition, DaSilva et al.(23) presented evidence for phospholipid-free activation of rat brain PKC by phorbol esters measured as increases in both phosphotransferase activity and autophosphorylation. Finally, a recent report by Weinstein and co-workers (24) found that phorbol 12-myristate 13-acetate enhanced calcium binding to the regulatory domain of PKC -1 in the absence of phosphatidylserine. Although most of these studies did not rigorously rule out the presence of lipids that could support phorbol ester binding, they further corroborate the concept of a phospholipid-independent interaction between PKC and phorbol esters.

Our findings have three conceptual implications. First, they relate to the mechanism of PKC translocation by phorbol esters. The observation that insoluble phorbol esters, e.g. phorbol 12,13-dioleate, still bound to PKC had argued that PKC recognized membrane-associated ligand(18) . A similar conclusion of course follows from the binding activity of natural diacylglycerols, which are also insoluble in aqueous solution. Those findings had suggested that translocation did not represent the induced association of PKC with the membrane but rather represented a reduced rate of release of PKC from the membrane, causing net accumulation. Our findings that phorbol esters can bind to PKC in the absence of phospholipid now suggest that induction of transfer of PKC to the membrane is also possible. Although these distinct mechanisms do not make different predictions regarding the equilibrium binding constant, they make different predictions about rate. It is perhaps relevant that different ligands differ markedly in the rates of PKC translocation that they induce(26, 27) .

In intact cells, [H]PDBu binds with a K of 10-50 nM(18) . The K of 165 nM, which we derived from [H]PDBu binding to PKC in the absence of phospholipid, is the same order of magnitude as the K reported for PDBu in intact HL-60 cells, which is 50 nM. Although direct experimental evaluation is required, it is clear that the concept that cytosolic PKC binds phorbol esters is plausible.

The typical phorbol esters are not optimized for binding in the absence of phospholipid, moreover. Our limited structure-activity analysis indicates that derivatives differ in their relative dependence on phospholipids for binding. Phorbol 12-decanoate, for example, bound with only 6-fold weaker affinity in the absence of phospholipid, compared with an 80-fold difference for PDBu. Novel analogs that preferentially bind to cytosolic PKC and stabilize the enzyme in the cytosol represent potential inhibitors of PKC. PKM, the catalytic domain of PKC liberated by proteolysis, provides a partial analogy. Although enzymatically active, PKM is cytosolic rather than membrane-bound and functionally induces only a few biological responses compared with the intact PKC(25) .

Finally, our present results demonstrate a direct interaction between phorbol esters and a cysteine-rich region in PKC in the absence of lipid cofactors. Identification of the amino acid residues in this domain responsible for the phorbol ester binding is crucial to understanding the interaction at the molecular level. The technical obstacles to NMR and x-ray analysis of the receptor-ligand interactions should be greatly simplified by the lack of a requirement for phospholipids to support binding.

  
Table: Structure-activity analysis of binding to the second cysteine-rich region of PKC in the absence and presence of phospholipids

Binding of the ligands to the second cysteine-rich region of PKC was analyzed by competition of [H]PDBu either in the absence (-PS) or the presence of 100 µg/ml phosphatidylserine (+PS), as described under ``Experimental Procedures.'' Values represent the mean ± S.E. of the number of experiments in parentheses.



FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence and reprint requests should be addressed: Bldg. 37, Rm. 3A01, NIH, 37 Convent Dr. MSC 4255, Bethesda, MD 20892-4255. Tel.: 301-496-3189; Fax: 301-496-8709.

The abbreviations used are: PKC, protein kinase C; PDBu, phorbol 12,13-dibutyrate; HPLC, high pressure liquid chromatography; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine.

M. G. Kazanietz, S. Wang, G. W. A. Milne, N. E. Lewin, H. L. Lin, and P. M. Blumberg, manuscript in preparation.


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