©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Characterization of the Cysteine-rich Region of the Caenorhabditiselegans Protein Unc-13 as a High Affinity Phorbol Ester Receptor
ANALYSIS OF LIGAND-BINDING INTERACTIONS, LIPID COFACTOR REQUIREMENTS, AND INHIBITOR SENSITIVITY (*)

Marcelo G. Kazanietz , Nancy E. Lewin , Jay D. Bruns , Peter M. Blumberg (§)

From the (1) Molecular Mechanisms of Tumor Promotion Section, Laboratory of Cellular Carcinogenesis and Tumor Promotion, NCI, National Institutes of Health, Bethesda, Maryland 20892-4255

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

The Caenorhabditiselegans Unc-13 protein is a novel member of the phorbol ester receptor family having a single cysteine-rich region with high homology to those present in protein kinase C (PKC) isozymes and the chimaerins. We expressed the cysteine-rich region of Unc-13 in Escherichiacoli and quantitatively analyzed its interactions with phorbol esters and related analogs, its phospholipid requirements, and its inhibitor sensitivity. [H]Phorbol 12,13-dibutyrate [H]PDBu bound with high affinity to the cysteine-rich region of Unc-13 ( K= 1.3 ± 0.2 nM). This affinity is similar to that of other single cysteine-rich regions from PKC isozymes as well as n-chimaerin. As also described for PKC isozymes and n-chimaerin, Unc-13 bound diacylglycerol with an affinity about 2 orders of magnitude weaker than [H]PDBu. Structure-activity analysis revealed significant but modest differences between recombinant cysteine-rich regions of Unc-13 and PKC . In addition, Unc-13 required slightly higher concentrations of phospholipid for reconstitution of [H]PDBu binding. Calphostin C, a compound described as a selective inhibitor of PKC, was also able to inhibit [H]PDBu binding to Unc-13, suggesting that this inhibitor is not able to distinguish between different classes of phorbol ester receptors. In conclusion, although our results revealed some differences in ligand and lipid cofactor sensitivities, Unc-13 represents a high affinity cellular target for the phorbol esters as well as for the lipid second messenger diacylglycerol, at least in C. elegans. The use of phorbol esters or some ``specific'' antagonists of PKC does not distinguish between cellular pathways involving different PKC isozymes or novel phorbol ester receptors such as n-chimaerin or Unc-13.


INTRODUCTION

The phorbol esters, natural products derived from plants of the family Euphorbiaceae, have been extensively investigated in the last two decades on the basis of their potent activity as tumor promoters (1, 2) . The search for the receptors for this class of compounds led to the identification of PKC as their major target (3, 4, 5, 6, 7) . The complexity and heterogeneity in the biological responses to the phorbol esters suggested the existence of multiple receptors. Indeed, protein kinase C is now recognized to represent a complex family of isozymes. The ``classic'' isozymes , , and and the ``novel'' , , , and all bind phorbol esters with high affinity (8, 9) . The atypical isozymes, and , are unable to respond to the phorbol esters (10, 11) . Recently, two novel classes of receptors for the phorbol esters have been found. The members of the chimaerin family (-1 or n-chimaerin, -2, -1, and -2 chimaerin) possess a single cysteine-rich region at the N-terminal domain with high homology to those found in the PKC() isozymes. The C-terminal domain is unrelated to PKC but has high homology with the breakpoint cluster region protein. It functions as a GTPase-activating protein for the small GTP binding protein p21 (12, 13, 14, 15) . We have recently described that n-chimaerin binds phorbol esters with properties similar to PKC (16) . Unc-13, the only identified member of the third class of phorbol ester receptors, also contains a single cysteine-rich region as well as a domain with sequence homology to the C2 domain in PKC. The cDNA for Unc-13 was isolated from a Caenorhabditiselegans library and encodes a 1734-amino acid protein. Although the function of Unc-13 is not known, a mutation in the unc-13 gene is known to cause uncoordinated movement and abnormal accumulation of acetylcholine in the nervous system of the nematode (17) . Phorbol ester treatment of C. elegans likewise causes uncoordinated movement (18) .

Binding of phorbol esters to PKC occurs at the cysteine-rich regions (also called zinc fingers) in the regulatory domain (19) , where the motif H XC XC XC XC XH XC XC (C, cysteine; H, histidine; X, any other amino acid) is highly conserved between PKC isozymes, chimaerins, Unc-13, diacylglycerol kinase, and the oncogenes Vav and c-Raf (20, 21) . Each cysteine-rich region coordinates two Zn ions in its structure (22) . sn-1,2-diacylglycerol (DAG), a major second messenger produced through the hydrolysis of phosphatidylinositol 4,5-bisphosphate or PC, is the endogenous ligand that binds to the cysteine-rich region of PKC leading to the activation of the enzyme as well as its membrane translocation (23, 24) . Since phorbol esters bind to PKC in a fashion similar to DAG and similarly activate the enzyme, they have provided a powerful tool to assess the cellular roles of PKC in signal transduction. It is reasonable to assume that the non-kinase phorbol ester receptors may be also cellular targets for DAG. In this study, we analyzed the properties of the Unc-13 protein as a receptor for DAG/phorbol esters. A critical issue is that a previous report suggested that Unc-13 bound phorbol esters with an affinity much lower than that previously described for the PKC isozymes. A similar reduction in diacylglycerol affinity would render Unc-13 unresponsive to physiological concentrations of diacylglycerol. In these previous studies using a recombinant cysteine-rich region of the Unc-13 protein expressed in Escherichia coli, Ahmed and co-workers (20) had reported that [H]PDBu bound with an affinity of 67 nM. In contrast, we found that PKC isozymes and n-chimaerin expressed in baculovirus, as well as recombinant cysteine-rich regions of PKC isozymes expressed in bacteria, bind [H]PDBu with much greater affinity (0.1-1 nM). Although Unc-13 was first described as a phospholipid-independent phorbol ester receptor (17) , a second report suggested that the binding of phorbol esters to the cysteine-rich region of Unc-13 required the lipid cofactor (20) .

Given the lack of an extensive study of Unc-13 as a DAG/phorbol ester receptor, we decided to perform a detailed quantitative analysis of phorbol ester binding to the cysteine-rich region of Unc-13 and to compare it to a single cysteine-rich region of PKC (PKC isozyme). In contrast to previous reports, our results indicate that Unc-13 binds phorbol esters with high affinity as was also described for the PKC isozymes, although with modest differences in structure-activity relations and phospholipid requirements.


EXPERIMENTAL PROCEDURES

Materials

[H]PDBu (20.7 Ci/mmol) was purchased from DuPont NEN. [H]Bryostatin 1 (4.8 Ci/mmol) was prepared as we previously described for [H]bryostatin 4 (25) . Mezerein, thymeleatoxin, (-)-octyl-indolactam V, PDBu, and 12-deoxyphorbol ester were obtained from LC Services Corp. (Woburn, MA). OAG was purchased from Avanti Polar Lipids (Pelham, AL). Phospholipids were obtained from Sigma. Calphostin C was purchased from Calbiochem. Reagents for expression and purification of recombinant proteins in E. coli were purchased from Pharmacia Biotech Inc.

Expression and Purification of Recombinant Cysteine-rich Regions in E. coli

Fragments encoding the cysteine-rich regions of Unc-13 and PKC were generated by PCR. For Unc-13, a mixed stage C. elegans library (Stratagene) was amplified, and the phage cDNA was isolated using standard techniques (26) and used as a template for the PCR reaction. The following oligonucleotides were used: 5`-ACTCTACAAGCATGGATCTACCCGATC-3` and 5`-CGATCTCTGATAACATTGACAAGTGA-3`, corresponding to nucleotides 1851-1877 and 2097-2122 in the sequence of Unc-13 as reported by Maruyama and Brenner (17) , respectively. The second cysteine-rich region of PKC was generated by PCR using the full-length mouse cDNA clone (27) as a template and the following oligonucleotides containing BamHI and EcoRI sites (underlined): 5`-TGAGGATCCCACCGATTCAAGGTTTATAAC-3` and 5`-AT-CGAATTCACACAGGTTGGCCACCTTCTC-3`. The corresponding PCR fragments were subcloned into the pCRII vector using the TA cloning system (Invitrogen). An EcoRI- EcoRI insert for Unc-13 and a BamHI- EcoRI insert for PKC were isolated and ligated in frame in pGEX vectors (Pharmacia) to get the pGEXUnc-13 and pGEX plasmids, respectively. The fragments were sequenced to confirm complete homology with the published sequences. To induce the expression of the GST-fused recombinant cysteine-rich regions, E. coli XL1-blue (Stratagene) was transformed with the pGEXUnc-13 and pGEX plasmids, the bacteria were grown at 30 °C to an A of 0.5-0.7 in 1 liter of LB media containing 50 µg/ml ampicillin, and the recombinant proteins were induced by the addition of 0.5 mM isopropyl--D-thiogalactopyranoside. After 5-6 h, the bacteria were pelleted at 4,000 g, resuspended in 20 ml of phosphate-buffered saline, and disrupted by sonication. Purification of GST-fusion proteins was done with glutathione-Sepharose 4B according to the manufacturer's instructions (Pharmacia). The purified recombinant proteins (in 50% glycerol) were kept at -70 °C. Although storage did not seem to affect the binding capacity of the PKC cysteine-rich region, some loss of activity was found in preparations of Unc-13 after several months at -70 °C, and therefore fresh preparations were used in all cases. Binding of [H]PDBu and [H]Bryostatin 1-[]PDBu binding was measured using the polyethylene glycol precipitation assay developed in our laboratory (28) . The assay was performed in a total volume of 250 µl, using 100 µg/ml phospholipid (100% phosphatidylserine, unless otherwise indicated), 1 mM EGTA (or 0.1 mM CaCl when indicated), different concentrations of [H]PDBu, and affinity-purified GST-fusion proteins (0.4 µg/ml). Detailed descriptions for Scatchard and competition assays can be found elsewhere (8, 28) . In binding assays performed with total cellular lysates as the source of receptor, pellets from 5-ml bacterial cultures were lysed by sonication in 1 ml of lysis buffer (50 mM Tris-Cl, pH 7.4, 1 mM EGTA, 50 µg/ml phenylmethylsulfonyl fluoride, and 250 µg/ml leupeptin), and 50 µl of lysate/tube were used. Incubation was carried out at 18 °C for 5 min. Inhibition of [H]PDBu binding by calphostin C was done using the conditions described in Ref. 16. Phospholipid preparations were obtained by sonication, as described in Ref. 29.

[H]Bryostatin 1 binding was measured by a filtration assay, using Triton X-100/phosphatidylserine-mixed micelles, in the presence of 0.1 mM EGTA, as we previously described (30) .


RESULTS

Expression of the Cysteine-rich Region of Unc-13 in E. coli

The GST expression system was used to express the cysteine-rich region of Unc-13 in E. coli. After isopropyl--D-thiogalactopyranoside induction, high levels of recombinant GST-Unc-13 were found in lysates of E. coli transformed with the pGEXUnc-13, as revealed by the appearance of a 33-kDa band in Coomassie Blue-stained polyacrylamide gels. By using glutathione-Sepharose 4B beads, the GST-Unc-13 was purified from bacterial lysates to about 90% purity. Similarly, high levels of expression were found in lysates of E. coli transformed with the pGEX plasmid after isopropyl--D-thiogalactopyranoside induction (Fig. 1 A). In contrast to the cysteine-rich region of PKC expressed as a GST-fusion protein, the majority of the recombinant GST-Unc-13 protein was insoluble when expressed in bacteria, and recovery after purification from the soluble fraction was comparatively lower. Overexpression of the recombinant cysteine-rich regions of Unc-13 and PKC resulted in high levels of [H]PDBu binding in bacterial lysates (Fig. 1 B). Recombinant cysteine-rich regions of Unc-13, PKC isozymes, and n-chimaerin bound Zn (Ref. 21 and data not shown), as was also shown by others (20) .


Figure 1: Expression of recombinant cysteine-rich regions in E. coli. A, bacterial cultures overexpressing cysteine-rich regions of Unc-13 or PKC were lysed and subjected to SDS-polyacrylamide gel electrophoresis. The gels were then stained with Coomassie Blue. Positions of molecular weight markers are shown on the left. Total lysates are shown in lanesA and C. The corresponding GST-fusion proteins were affinity purified as described under ``Experimental Procedures'' and are shown in lanesB and D. B, binding of [H]PDBu to bacterial pellets (from 5-ml cultures) using 20 nM of the radioligand and 50 µl of lysate.



Binding of Phorbol Esters and Related Ligands to Unc-13

[H]PDBu binding to the purified cysteine-rich regions was measured. Scatchard analysis revealed that [H]PDBu bound to Unc-13 with high affinity in the presence of phosphatidylserine (Fig. 2). The dissociation constant ( K) for the ligand was 1.3 ± 0.2 nM ( n = 3). Calcium (0.1 mM) did not affect the binding of [H]PDBu to the receptor. Binding of [H]PDBu to the purified recombinant cysteine-rich region of PKC revealed similar affinity to that obtained for Unc-13, either in the presence or absence of calcium (). The Kof [H]PDBu for Unc-13 was substantially lower, i.e. higher binding affinity, than that first reported by Ahmed et al.(20) . The affinity of the ligand for the recombinant cysteine-rich regions was similar when the receptor was cleaved with thrombin (to separate it from the GST) and further purified by high pressure liquid chromatography (data not shown). Likewise, single cysteine-rich regions of PKC and n-chimaerin bound [H]PDBu with Kvalues of 2.4 ± 0.7 nM ( n = 3) and 2.0 ± 0.3 nM ( n = 3), respectively, in the presence of phosphatidylserine vesicles (Ref. 31 and data not shown).


Figure 2: [H]PDBu binding to the cysteine-rich regions of Unc-13 ( A) and PKC ( B). The recombinant GST-fusion proteins were incubated for 5 min (18 °C) with increasing concentrations of [H]PDBu (0.25-8 nM), 100 µg/ml phosphatidylserine, and 0.1 mM EGTA, and binding was measured using the polyethylene glycol precipitation assay. A representative experiment for each receptor is shown. Each experiment was performed three times. Each point represents the mean of three experimental values, generally with a standard error of <2%. Insets are the Scatchard plots derived from the corresponding binding curves.



To analyze receptor-ligand interactions, we quantitated competition of [H]PDBu binding with different classes of analogs, having different patterns of selectivity among PKC isozymes and different spectra of biological activities. The series of compounds tested, which included a 12-deoxyphorbol ester, an indole alkaloid (octylindolactam V), mezerein, a mezerein analog (thymeleatoxin), and a diacylglycerol (OAG), spanned a range of 3 orders of magnitude in binding affinity for PKC isozymes. Previous results from our laboratory revealed significant differences in structure-activity relations between PKC isozymes (8) . Phorbol esters and 12-deoxyphorbol esters, as well as mezerein and analogs, show preference for PKC , , and compared with PKC , , and (8) . n-chimaerin showed a pattern of ligand preference similar to that of PKC (16) . In this study, we found that the binding affinities for the different compounds tested were slightly weaker (2-5 times) for Unc-13 when compared with the PKC cysteine-rich region (). The Kfor the diacylglycerol OAG for binding to the cysteine-rich region of PKC was 142 nM, a value that is very similar to that obtained with PKC isozymes and n-chimaerin. Unc-13 bound OAG with slightly lower affinity ( K= 380 nM). The mezerein analog thymeleatoxin, which is the most dramatic example of selectivity between PKC isozymes ( i.e. 20-fold weaker affinity for PKC than PKC , see Ref. 8) bound with similar affinity to both the PKC and Unc-13 cysteine-rich regions.

Unc-13 is also a high affinity receptor for the bryostatins, macrocyclic lactones with an atypical pattern of biological activity as compared with the typical phorbol esters. Scatchard analysis using [H]bryostatin 1 (Fig. 3) revealed a Kof 11.5 ± 2.0 nM for the cysteine-rich region of Unc-13 under our standard assay condition for bryostatin binding (30) . Because these assays are carried out in 0.1% Triton X-100, 20% phosphatidylserine, absolute potencies are not comparable with those determined in phosphatidylserine alone. The cysteine-rich region from PKC bound [H]bryostatin 1 with about 7-fold higher affinity than that of Unc-13 ( KPKC = 1.7 ± 0.2 nM). Phospholipid Dependence of [H]PDBu Binding-Reconstitution of phorbol ester binding by phospholipids depends both on the nature and concentration of the lipid cofactor (29) . Specific [H]PDBu binding to single cysteine-rich regions of PKC was shown to be phospholipid dependent (32) . PS is the most efficient of the phospholipids for reconstitution of binding, although other anionic phospholipids work as lipid cofactors as well (16, 29) . The concentration of PS required to reconstitute [H]PDBu binding by 50% (ED) for the Unc-13 cysteine-rich region was 58 ± 13 µg/ml; maximum binding was obtained at about 300 µg/ml PS. The cysteine-rich region of PKC showed a lower ED for PS (15 ± 2 µg/ml) and reached saturation at about 100 µg/ml (Fig. 4). Interestingly, under the same assay conditions, the full-length n-chimaerin as well as a PKC isoform (PKC ) showed ED values for reconstitution by PS of 14 and 12 µg/ml, respectively (16) .


Figure 3: Binding of [H]bryostatin 1 to recombinant cysteine-rich regions of Unc-13 ( A) and PKC ( B). Binding of [H]bryostatin 1 was measured with the GST fusion proteins using increasing concentrations of the radioligand (0.5-10 nM), phosphatidylserine-Triton X-100-mixed micelles, and 0.1 mM EGTA. Each experiment was performed four times, and for each recombinant protein a representative experiment is shown. The K values, expressed as mean ± S.E., are presented in the toprightcorner of the corresponding panel. Insets are the Scatchard plots derived from the corresponding binding curves.




Figure 4: Reconstitution of [H]PDBu binding to the cysteine-rich regions of Unc-13 and PKC by phosphatidylserine vesicles. Binding was performed using 0.1 mM EGTA and increasing concentrations of phosphatidylserine. Values represent the means of four experiments for either Unc-13 ( closedcircles) or PKC ( opencircles). Standard errors are represented by the errorbars.



The ability of various phospholipids to reconstitute phorbol ester binding to Unc-13 was tested. Using a fixed concentration of phospholipids (300 µg/ml), we found that all the other acidic phospholipids (phosphatidylinositol, phosphatidylinositol 4-phosphate, phosphatidylinositol 4,5-bisphosphate, phosphatidylglycerol, phosphatidic acid) in a proportion of 20% in PC were able to reconstitute binding to the levels obtained with PS. Interestingly, partial reconstitution (38 and 50%, respectively) was observed with the neutral phospholipids PC and 20% PE, 80% PC. In contrast to the cysteine-rich region of Unc-13, at 300 µg/ml both acidic and neutral phospholipids were able to fully reconstitute binding to the cysteine-rich region of PKC (I). Concentration-response curves with the cysteine-rich region of PKC gave ED values for reconstitution of [H]PDBu binding of 86 ± 31 µg/ml and 111 ± 33 µg/ml for 20% PE, 80% PC and PC, respectively. Higher concentrations of neutral phospholipids were required for reconstitution of [H]PDBu binding to Unc-13. In this latter case, the ED for 20% PE, 80% PC was 182 ± 32 µg/ml, and the ED for PC was 444 ± 62 µg/ml. For comparison, [H]PDBu binding to full-length recombinant PKC (baculovirus expressed, see Ref. 8) was fully reconstituted by acidic phospholipids and only partially by neutral phospholipids, suggesting a different lipid requirement between the cysteine-rich region and the intact enzyme (I). It is possible that amino acid residues outside the cysteine-rich regions may contribute to the differences observed between the native protein and the phorbol ester binding domain, as suggested by Bell and co-workers (32) . Inhibition of [H]PDBu Binding to Unc-13 by Calphostin C-Calphostin C, a compound isolated from Cladosporium cladosporoides, was described as a PKC inhibitor acting at the regulatory domain of PKC (33) . We found here that calphostin C also inhibited phorbol ester binding to the cysteine-rich region of Unc-13 (Fig. 5). The ED for the inhibitor was 8.9 ± 1.1 µM. [H]PDBu binding to the cysteine-rich region of PKC was inhibited by calphostin C with an ED of 4.3 ± 0.6 µM. Similar inhibition patterns with calphostin C were also found for PKC and n-chimaerin in a previous report (16) . Our results indicate that the target site for calphostin C is the cysteine-rich region, and this compound, originally described as a selective PKC inhibitor, is not able to discriminate between the different classes of phorbol ester receptors.


Figure 5: Inhibition of [H]PDBu binding by calphostin C. Increasing concentrations of calphostin C were used to compete 3 nM [H]PDBu. Calphostin C was preincubated under ordinary fluorescent light (15 min) together with the reaction mix, as described in Ref. 16. Values represent the mean ± S.E. of a representative experiment in which determinations were performed in triplicate. Two other experiments gave similar results. Openbars, GST-Unc-13 cysteine-rich region; darkbars, GST-PKC cysteine-rich region; hatchedbars, full-length PKC .




DISCUSSION

The cysteine-rich region in the regulatory domain was shown to be the binding site for the phorbol esters in PKC. The high degree of conservation in this region among several proteins other than PKC, including the chimaerins, Unc-13, Vav, c-Raf, and diacylglycerol kinase (20, 21) , suggested the possibility that several signaling pathways may be regulated by diacylglycerol or their analogs, the protein esters. We have previously reported that n-chimaerin, a protein isolated from brain, closely resembles the properties of PKC as a phorbol ester receptor. Both receptors were virtually indistinguishable as determined by phorbol ester binding, structure-activity relations, phospholipid dependence, and inhibitor sensitivity (16) . In this paper, the quantitative analysis of the interaction between phorbol esters and Unc-13 likewise reveals that this protein from C. elegans behaves as a high affinity phorbol ester receptor. The cysteine-rich region of Unc-13 showed significant but modest differences in structure-activity relations and phospholipid dependence when compared with a similar domain in PKC .

The Kfor the ligand [H]PDBu for binding to the cysteine-rich region of Unc-13 expressed in E. coli was 1.3 nM, which is a much higher affinity than that originally described for this receptor expressed in bacteria (20) . The reduced binding affinity observed in that former study may reflect alterations in the conformation of the recombinant cysteine-rich region due to the conditions for the expression of the protein and/or the binding assay. Our results suggest that single cysteine-rich regions from PKC , Unc-13, and n-chimaerin (16) are high affinity binding sites in the corresponding proteins for both phorbol esters and diacylglycerol. In contrast, the cysteine-rich regions from the oncogenes vav and c- raf, as well as that from the PKC isozyme, were not able to bind phorbol ester or related ligands even with low affinity, although it is highly probable that a similar structure coordinating Zn also occurs in these proteins (21) .()

The phorbol esters are commonly used as a tool to assess the role of PKC in biological systems. The fact that Unc-13 as well as n-chimaerin (16) so closely resemble PKC isozymes as phorbol ester receptors suggests that many of the phorbol ester effects previously described could potentially be non-PKC mediated. Furthermore, inhibitors targeted to the cysteine-rich region, such as calphostin C, were unable to distinguish between phorbol ester receptors. Alternative molecular approaches to implicate a specific phorbol ester receptor class include overexpression of single isoforms, dominant negative mutants, or antisense. Pharmacologically, selective inhibitors targeted to the kinase domain of PKC (35, 36, 37) may likewise become valuable tools for discriminating between PKC and non-PKC phorbol ester receptors.

Similar potencies for diacylglycerol were found for the cysteine-rich regions of Unc-13 and PKC . In addition, diacylglycerols also bound with similar potency to PKC isozymes and n-chimaerin (8) . Given that diacylglycerol and the phorbol esters interact at the same binding sites, we could assume that the non-PKC targets also mediate cellular signaling pathways mediated by the second messenger diacylglycerol. The fact that different lipid requirements exist for different cysteine-rich regions complement our previous results that phospholipids differentially regulate phorbol ester binding to different PKC isozymes (8) . At the intracellular level, such a difference in lipid cofactor requirement could constitute a major regulatory factor for ligand-protein interaction for the different receptors.

In summary, our findings suggest that Unc-13 represents another member of the high affinity phorbol ester receptor family and that, although modest differences were found with other cysteine-rich regions, Unc-13 is a potential target for pathways regulated by diacylglycerol/phorbol esters. Identification of mammalian homologs of the Unc-13 protein as well as finding the biological role for Unc-13 at the cellular level will represent important contributions to the understanding of the biology of the phorbol esters.

  
Table: Dissociation constants (K ) for [H]PDBu for the cysteine-rich regions of Unc-13 and PKC

Binding was performed in the presence of PS and either 1 mM EGTA or 0.1 mM CaCl. Each Kvalue represents the mean ± S.E. The number of experiments is shown in parentheses.


  
Table: Structure-activity analysis of binding to the cysteine-rich regions of Unc-13 and PKC

Protein-ligand interactions were analyzed by competition of [H]PDBu (3 nM) binding with the individual compounds using 100 µg/ml PS and 1 mM EGTA. Six to eight increasing concentrations (in triplicate) of the competing ligand were used. The ID values were determined from the competition curves, and the corresponding Kvalues for the ligands were obtained (8). Values represent the mean ± S.E. of the number of experiments in parentheses.


  
Table: Reconstitution of [H]PDBu binding by different phospholipids

Binding was performed using 300 µg/ml phospholipid vesicles and 0.1 mM EGTA. Phospholipid vesicles contained 80% PC and 20% of the phospholipid except when a single phospholipid was used as indicated in the table. Values represent the mean ± S.E. of three to five experiments (in parentheses) in which determinations were done in triplicate. Results were normalized to the binding in 80% PC, 20% PS vesicles as 100% binding. PI, phosphatidylinositol; PIP, phosphatidylinositol 4-phosphate; PIP, phosphatidylinositol 4,5-bisphosphate; PA, phosphatidic acid; PG, phosphatidylglycerol.



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: Molecular Mechanisms of Tumor Promotion Section, Laboratory of Cellular Carcinogenesis and Tumor Promotion, NCI, National Institutes of Health, 37 Convent Dr. MSC 4255, Bldg. 37, Rm. 3A01, 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; GST, glutathione S-transferase; PC, phosphatidylcholine; PS, phosphatidylserine; PE, phosphatidylethanolamine; OAG, 1-oleoyl 2-acetyl glycerol; DAG, sn-1,2-diacylglycerol; PCR, polymerase chain reaction.

Recent reports suggested that the vav protoncogene binds phorbol esters (34). Although our results strongly argue against the presence of either high or low affinity binding sites in vav, the basis for the discrepancy between the studies is currently not known.


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