©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Protein Kinase C Contains Two Activator Binding Sites That Bind Phorbol Esters and Diacylglycerols with Opposite Affinities (*)

(Received for publication, November 12, 1995)

Simon J. Slater Cojen Ho Mary Beth Kelly Jonathan D. Larkin Frank J. Taddeo Mark D. Yeager Christopher D. Stubbs (§)

From the Department of Pathology and Cell Biology, Thomas Jefferson University, Philadelphia, Pennsylvania 19107

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Based on marked differences in the enzymatic properties of diacylglycerols compared with phorbol ester-activated protein kinase C (PKC), we recently proposed that activation induced by these compounds may not be equivalent (Slater, S. J., Kelly, M. B., Taddeo, F. J., Rubin, E., and Stubbs, C. D.(1994) J. Biol. Chem. 269, 17160-17165). In the present study, direct evidence is provided showing that phorbol esters and diacylglycerols bind simultaneously to PKCalpha. Using a novel binding assay employing the fluorescent phorbol ester, sapintoxin-D (SAPD), evidence for two sites of high and low affinity was obtained. Thus, both binding and activation dose-response curves for SAPD were double sigmoidal, which was also observed for dose-dependent activation by the commonly used phorbol ester, 4beta-12-O-tetradecanoylphorbol-13-acetate (TPA). TPA removed high affinity SAPD binding and also competed for the low affinity site. By contrast with TPA, low affinity binding of SAPD was inhibited by sn-1,2-dioleoylglycerol (DAG), while binding to the high affinity site was markedly enhanced. Again contrasting with both TPA and DAG, the potent PKC activator, bryostatin-I (B-I), inhibited SAPD binding to its high affinity site, while low affinity binding was unaffected. Based on these findings, a model for PKC activation is proposed in which binding of one activator to the low affinity site allosterically promotes binding of a second activator to the high affinity site, resulting in an enhanced level of activity. Overall, the results provide direct evidence that PKCalpha contains two distinct binding sites, with affinities that differ for each activator in the order: DAG > phorbol ester > B-I and B-I > phorbol ester > DAG, respectively.


INTRODUCTION

Protein kinase C (PKC) (^1)comprises a family of isozymes that are pivotal in intracellular signal transduction(1, 2, 3, 4) . Activation of PKC requires association with the membrane and is induced by a number of activators and cofactors, the requirements for which differ for each isoform(2) . Thus, with the exception of the so called atypical PKC isoforms, the activity of membrane-associated PKC is potentiated by the second messenger, diacylglycerol, a product of hormone receptor-phospholipase C-catalyzed phospholipid hydrolysis.

In addition to the natural activators, including diacylglycerol, the enzyme is activated with high specificity by the tumor-promoting phorbol esters(5) . For this reason, phorbol esters are often used in the study of the mechanism of PKC activation, based on the assumption that they compete directly with diacylglycerols for a common binding site on the enzyme(6, 7) . Also it is commonplace in studies of the regulation of cellular processes to infer PKC involvement from a response elicited by phorbol esters(5) . However, a number of studies have provided evidence indicating that the activated conformational forms of PKC induced by diacylglycerol compared with phorbol esters may not be equivalent based on observations of marked differences in the biological (8, 9) and enzymatic properties(10, 11, 12, 13, 14, 15) . Thus, we recently showed that the level of activation induced by saturating concentrations of both diacylglycerol and phorbol ester together, in the same assay, was greater than that achievable by saturating concentrations of either activator in isolation(15) . This observation is not consistent with a model where activators simply compete for a single activator binding site or for two equivalent sites. We therefore proposed that discrete binding sites for diacylglycerol and phorbol esters exist on PKC, of low and high affinity, respectively(15) .

The diacylglycerol and phorbol ester binding sites on PKC have been shown to be confined to two cysteine-rich repeats (Cys-1 and Cys-2) within the C1 domain(16, 17, 18) . These subdomains are conserved in all PKC isoforms, apart from PKC, which only contains a single Cys subdomain that is incapable of binding phorbol esters(19, 20) . Based on this, a series of elegant deletion analysis experiments has revealed a minimal consensus sequence for phorbol ester binding to PKC consisting of a 43-amino acid peptide from the Cys-2 subdomain (21, 22, 23) . Recently, the crystal structure of the corresponding consensus motif from PKC in complex with a phorbol ester was derived (24) . This, along with determinations of the solution state structure (25, 26) and highly detailed site-directed mutagenesis experiments (27) , has allowed three-dimensional assignment of the amino acid residues involved in phorbol ester binding to the Cys-2 subdomain. Thus, while a single Cys subdomain has been shown to be sufficient for high affinity phorbol ester binding using peptide fragments(21) , both Cys-1 and Cys-2 subdomains may be capable of binding phorbol esters(17, 21, 25) . This is consistent with the existence of discrete diacylglycerol and phorbol ester binding sites as proposed in our recent study(15) .

In the present study, the binding of phorbol ester, diacylglycerol, and also the potent PKC activator, bryostatin-I (B-I), to recombinant PKCalpha was investigated using a highly sensitive binding assay based on the fluorescent phorbol ester, sapintoxin-D (SAPD)(28) . Previous assays of phorbol ester binding have generally been based on the use of [^3H]PDBu. However, while this phorbol ester is relatively hydrophilic in nature, thus minimizing nonspecific binding to the membrane lipids, it also has a relatively low binding affinity for PKC (29) . Therefore, due to the limits imposed by nonspecific binding, low affinity phorbol ester interactions with PKC have not previously been detected. Although the possibility of a low affinity phorbol ester binding site was alluded to in a very recent study based on an inability to saturate binding of [^3H]PDBu to PKCalpha(29) . However, the affinity of SAPD for binding to PKCalpha is reported to be 10-fold greater than that of PDBu(29) , therefore allowing the possibility of detecting both high and low affinity phorbol ester binding. In the present study, it was found that PKCalpha contains two distinct specific binding sites of high and low affinity for SAPD, respectively. TPA competed for SAPD binding to both these sites. However, sn-1,2-dioleoylglycerol (DAG) inhibited SAPD binding to its low affinity site while enhancing binding at the high affinity site. Conversely, B-I displaced SAPD from the high affinity site while low affinity SAPD binding was unaffected. The data provide direct evidence that diacylglycerols, phorbol esters, and also B-I bind to two discrete sites on PKCalpha.


EXPERIMENTAL PROCEDURES

Materials

A peptide substrate corresponding to the PKC phosphorylation site of myelin basic protein (QKRPSQRSKYL, MBP) was custom synthesized by the Jefferson Cancer Institute Protein Chemistry Facility. Lipids were obtained from Avanti Polar Lipids, Inc. (Alabaster, AL). B-I was obtained from LC Services (Woburn, MA). SAPD, TPA, and 4alpha-12-O-tetradecanoylphorbol-13-acetate (4alpha-TPA) were from Sigma. ATP was from Boehringer Mannheim, and [-P]ATP was from DuPont NEN. All other chemicals were of analytical grade and obtained from Fisher.

Preparation, Purification, and Assay of Recombinant PKCalpha Activity

PKCalpha (bovine brain) was prepared using the baculovirus-insect cell expression system (30, 31) and purified as described previously(32) . Assays of activity followed a previously described method, which involved measurement of the initial rate of incorporation of P into MBP(15) . Large unilamellar vesicles (LUV), prepared as described elsewhere(33) , consisted of 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) and 1-palmitoyl-2-oleoylphosphatidylserine (POPS) (4:1, molar), with DAG incorporated as indicated. The LUV have been previously characterized and shown to be stable under similar assay conditions to those used here(34, 35) .

SAPD Binding Assay

Binding of SAPD to PKCalpha was quantified by measuring the resonance energy transfer (RET) from PKC tryptophans to the 2-(N-methylamino)benzoyl fluorophore of the phorbol ester. Thus, the fluorescence intensities at the emission maxima of PKC tryptophans and SAPD (333 and 425 nm, respectively; see Fig. 1), obtained upon excitation of the tryptophan fluorophore at 290 nm, were determined using a PTI Alphascan fluorimeter (Photon Technology Instruments, Princeton, NJ). The assay system (2 ml) consisted of 50 mM Tris/HCl, pH 7.4, 25 µM POPC:POPS LUV (4:1, molar), 0.1 mM CaCl(2), 5 ng/µl PKCalpha, and activators added as indicated. After incubation for 10 min at 30 °C in a stirred quartz cuvette (1-cm pathlength), SAPD was titrated into this assay system from a stock solution in dimethyl sulfoxide and the fluorescence intensities recorded as a function of phorbol ester concentration. The maximum level of dimethyl sulfoxide in the assay was 2% v/v, the presence of which had negligible effects on the observed fluorescence intensities, binding affinities, or on enzyme activity (results not shown). The absorbance at the excitation maximum of tryptophan (290 nm) of 25 µM SAPD (2.5 times greater than the maximum level used in the assay) was A = 0.01, therefore ensuring a linear relationship between fluorescence intensity and SAPD concentration. After correction for the contribution from direct excitation of the SAPD fluorophore, the resultant fluorescence intensities (F(i)) were normalized to that observed in the presence of all assay components apart from the phorbol ester SAPD (F(0)) according to: F = (F - F)/F. These data were then fitted by non-linear least squares analysis to a modified Hill equation, assuming a model involving two independent SAPD binding sites,


Figure 1: PKC tryptophan fluorescence and quenching by SAPD to produce resonance energy transfer. A, emission spectra. The fluorescence emission spectra (uncorrected) of PKCalpha (50 µg) with and without SAPD (2.5 µM) in the presence of POPC/POPS LUV (4:1, molar, 25 µM total lipid concentration) and Ca (0.1 mM) is shown. Under these conditions, SAPD bound to PKC, and the tryptophan emission at 340 nm, upon excitation at 290 nm, was quenched by RET to the SAPD (dotted line, before SAPD; heavy solid line, after SAPD). This led to enhanced fluorescence at 425 nm. For binding experiments, the signal at 425 nm was first corrected for the tryptophan emission tail obtained in the absence of SAPD with Ca (dotted line) and for the signal at 425 nm due to direct excitation of SAPD obtained in the absence of PKC (dotted line). The effect of LUV, Ca, and PKCalpha on the SAPD fluorescence emission obtained upon excitation at the absorption maximum of 362 nm is shown to be negligible, the three spectra centered at 425 nm (solid line) being almost identical. B, effects of Ca and EGTA. The fluorescence emission of the PKCalpha tryptophans at 333 nm (lower trace) was monitored simultaneously with the emission at 425 nm (upper trace) due to RET. The data reveal a quenching of the PKC tryptophans that is partially reversed upon addition of EGTA (1 mM), showing that membrane association is required for SAPD binding. Details were as otherwise described under ``Experimental Procedures.''



where F(h), F(h), F(l), and F(l) are the minimum and maximum fluorescence intensities, K(h) and K(l) are binding constants (defined as the SAPD concentration corresponding to a half-maximal fluorescence intensity increase), and nh and nl are the Hill coefficients for high and low affinity binding, respectively.


RESULTS

The aim of the present study was to test the hypothesis that PKCalpha contains two activator binding sites of low and high affinity. To accomplish this, SAPD binding to recombinant PKCalpha was determined in the presence and absence of either TPA, DAG, or B-I, present at levels that have been previously shown to correspond to those required to saturate both binding and activation(15, 36) . These data were compared with those obtained for the activation of PKCalpha induced by the same activator combinations used in the binding experiments.

In order to determine activator binding to PKCalpha, advantage was taken of the fluorescence properties of the 2-(N-methylamino)benzoyl fluorophore of SAPD, which has an excitation band centered at 362 nm that overlaps with the emission band of 333 nm measured for the PKCalpha tryptophans (Fig. 1A). This overlap facilitates RET between PKCalpha tryptophans and the SAPD fluorophore, allowing direct determination of SAPD binding without the need to separate bound from free ligand.

The addition of Ca to PKCalpha in the presence of LUV resulted in a slight blue shift in the tryptophan emission maximum obtained upon excitation at 290 nm (Fig. 1A). Addition of SAPD, at a concentration sufficient for occupation of both high and low affinity phorbol ester binding sites, in the presence of Ca resulted in RET, observed as a marked decrease in tryptophan fluorescence intensity and corresponding increase in the SAPD emission maxima (425 nm). This increase in SAPD fluorescence intensity was not due to an enhancement of the quantum yield of the SAPD fluorescence upon binding to PKC since the level of SAPD emission intensity, induced by excitation at the SAPD absorption maximum (362 nm), was unaffected by the addition of Ca, lipids (POPC/POPS LUV), or PKCalpha (Fig. 1A). Addition of EGTA reversed the level of RET (Fig. 1B) indicating that SAPD binding to PKCalpha is Ca-dependent. This parallels the previously reported effects of Ca chelation on PKC activity (e.g. see (37) ).

The dose-dependent effects of increasing SAPD concentrations on RET between PKCalpha tryptophans and the phorbol ester revealed a double sigmoidal curve, indicating the existence of two separate SAPD binding sites (Fig. 2A). Binding of SAPD was found to be negligible in the absence of either Ca or phospholipid (Fig. 3A), suggesting that both high and low affinity SAPD binding sites are present on membrane-associated PKCalpha rather than being distributed between membrane-bound and soluble enzyme forms. The binding constants were K(h) = 150 ± 50 nM and K(l) = 2500 ± 300 nM for binding to the high and low affinity sites, respectively. In addition to varying markedly in affinity, these sites displayed differing degrees of SAPD binding cooperativity, the Hill coefficients for high and low affinity binding being nh = 1.1 ± 0.2 and nl = 2.9 ± 0.8, respectively. The SAPD-induced activity dose-response curve similarly consisted of a double sigmoidal curve, again indicating two activator sites (Fig. 2B). The binding constants determined from the activity data (K(h) = 100 ± 20 nM and K(l) = 2800 ± 200 nM, respectively) were similar to those for binding, as were the observed Hill coefficients (nh = 1.6 ± 0.5 and nl = 2.9 ± 0.7, respectively).


Figure 2: SAPD interaction with PKCalpha in terms of binding and enzyme activity: effect of TPA and 4alpha-TPA. A, binding. SAPD binding, in the presence of Ca and LUV, measured from RET between PKCalpha tryptophans and SAPD as a function of SAPD concentration, determined as described under ``Experimental Procedures'' and under Fig. 1A, is shown. Also shown is the effect of 2 µM TPA (open circles) and 2 µM 4alpha-TPA (open squares) on SAPD binding. B, activity. Dose-response curve for activation of PKCalpha by SAPD (closed circles) in the presence of 2 µM TPA (open circles) or 2 µM 4alpha-TPA (open squares) is shown. The TPA dose-response curve is shown for reference (closed triangles). Data are representative of triplicate determinations (error bars ± S.D.). Details were as otherwise described under ``Experimental Procedures.''




Figure 3: Effect of DAG on SAPD binding and activation. A, binding. The RET from PKCalpha tryptophans to SAPD, as a measure of SAPD binding to PKCalpha, at 425 nm determined as described under Fig. 1A is shown. Data are shown for 20 µg of PKCalpha, with (open circles) and without (filled circles) DAG (4 mol % of total lipid concentration). Also shown are RET in the absence of Ca (small triangle) or phospholipids (large triangles). B, PKC activation by SAPD. Dose-response curve for activation of PKCalpha by SAPD is shown. PKCalpha activity was assayed in the presence (open circles) or absence of (filled circles) DAG. Data are representative of triplicate determinations (error bars ± S.D.). Details were as otherwise described under ``Experimental Procedures.''



Effect of TPA and 4alpha-TPA on SAPD Binding and Activation

The dose-response curve for activation by the commonly used phorbol ester, TPA, was similar to that observed for SAPD and was also double sigmoidal, consistent with the existence of two distinct phorbol ester binding sites (Fig. 2B). In addition, TPA (2 µM) completely inhibited high affinity SAPD binding and markedly reduced low affinity binding (Fig. 2A), suggesting that TPA and SAPD both bind with low and high affinity to the same sites. Consistent with this, the level of activation induced by the same level of TPA was not further enhanced by increasing levels of SAPD, as would have been expected to result had the two activators bound to the two sites with unequal affinities (Fig. 2B).

The non-activating epimer of TPA, 4alpha-TPA, is often used in the study of phorbol ester interactions with PKC(38) . However, the binding properties of 4alpha-TPA to PKC have not previously been reported. The high affinity SAPD binding to PKCalpha while removed by TPA was only marginally affected by 4alpha-TPA (Fig. 2A). However, SAPD binding to the low affinity site was inhibited by both 4alpha-TPA and TPA. Consistent with this, in the presence of 4alpha-TPA, which alone does not activate PKC, the level of activity induced by SAPD levels corresponding to low affinity binding was reduced (Fig. 2B).

In a previous study(15) , we observed that the activity of a mixed PKCalpha,beta, mixed isoform preparation, induced by a maximum concentration of 1 µM TPA, was inhibited by 4alpha-TPA. The finding in the present study that TPA-stimulated PKCalpha activity was unaffected by 4alpha-TPA, within the same activator concentration range (Fig. 2B), may point to variations in the affinities of the two activator binding sites dependent on PKC isoform. This possibility is supported by the findings of a number of recent studies, which have indicated that phorbol ester (high) affinity binding varies markedly for different PKC isozymes(29, 39) .

Effect of DAG on SAPD Binding and Activation

The inclusion of DAG in the LUV preparation, at a concentration corresponding to that found to maximally activate PKC(15) , resulted in the complete removal of SAPD low affinity binding (Fig. 3A), while high affinity binding was enhanced. This was observed as a decrease in the binding constant for binding to the high affinity phorbol ester site (K(h) = 100 ± 7 nM) and an increase in the cooperativity (nh = 2.6 ± 0.2) of binding to this site. The data therefore indicate that DAG binds with higher affinity to the low affinity site relative to SAPD, suggesting that these two activators may bind with opposite affinities to the two sites. Further, the level of activation induced by SAPD in combination with a maximally activating concentration of DAG was greater than that obtainable with either DAG or SAPD separately, independent of SAPD concentration (Fig. 3B). This is inconsistent with equivalent competition between the two activators for either the low or high affinity phorbol ester binding sites.

Effect of B-I on SAPD Binding and Activation

B-I inhibited SAPD binding to the high affinity phorbol ester site (Fig. 4A) when present at a concentration that has been determined in a separate experiment to correspond to that required to achieve a maximal level of activation (results not shown). However, low affinity SAPD binding was unaffected by B-I (Fig. 4A). This result was opposite to that obtained with DAG and suggests that B-I binds with greater potency to the high affinity site compared with SAPD, while it binds with a lower affinity than B-I at the low affinity phorbol ester binding site. Contrary to the enhanced activation induced by co-addition of DAG and SAPD compared with either activator in isolation, stimulation achieved with SAPD together with a maximally stimulating concentration of B-I was less than that induced by high SAPD concentrations and was not potentiated compared with that induced by B-I alone (Fig. 4B).


Figure 4: Effect of B-I on SAPD binding and activation. A, binding. The RET from PKCalpha tryptophans to SAPD, as a measure of SAPD binding to PKC, at 425 nm determined as described under Fig. 1A is shown. Data are shown for 20 µg of PKCalpha, with (open circles) and without (filled circles) 100 nM B-I. Details were as otherwise described under ``Experimental Procedures.'' B, PKC activation by SAPD. Dose-response curve for activation of PKCalpha by SAPD is shown. PKCalpha activity was assayed as described under Experimental Procedures`` with (open circles) and without (filled circles) 100 nM B-I. Data are representative of triplicate determinations (error bars ± S.D.).




DISCUSSION

In the present study, phorbol ester binding to PKCalpha was determined using a fluorescence binding assay employing the phorbol ester, SAPD. Both SAPD binding and activation dose-response curves indicated that PKCalpha contains two saturable phorbol ester binding sites of low and high affinity, respectively. TPA removed high affinity SAPD binding and also competed for the low affinity site. By contrast, DAG displaced the phorbol ester from the low affinity site while high affinity SAPD binding was enhanced. This coincided with an increased level of activity in the presence of both DAG and SAPD together, which was not observed with TPA in combination with SAPD. These observations suggest that DAG interacts with the two activator sites with opposite affinities to that of phorbol esters.

Indirect evidence for discrete activator sites for phorbol esters and diacylglycerol has been provided in studies from this (15) and other laboratories(14) . Thus, the level of PKC activity induced by DAG together with the phorbol ester, TPA, both at maximally stimulating concentrations, was greater than that achievable by either activator alone(15) . This observation is inconsistent with competition for a single activator binding site or equivalent competition for two sites with equal affinities for each activator. The observation that DAG inhibited SAPD binding to its low affinity site while enhancing high affinity SAPD binding indicates that the diacylglycerol has a higher affinity for the low affinity phorbol ester binding site than SAPD. Previous studies have indicated that a peptide corresponding to the Cys-2 subdomain binds phorbol ester with higher affinity than that corresponding to the Cys-1 subdomain(17, 25) . Thus, assuming that these differences in affinity are carried over into the intact enzyme, the results of the present study are consistent with the hypothesis that the Cys-1 and Cys-2 subdomains represent the low and high affinity binding sites for diacylglycerols and phorbol esters, respectively. However, this hypothesis remains to be proven.

The finding that binding of DAG to the low affinity site led to an increase in SAPD binding to the high affinity site indicates cooperativity between the two activator sites as shown by the observed increase in Hill coefficient. This would provide an explanation for the observed enhanced level of activation induced in the presence of both activators together. According to this, binding of an activator (e.g. diacylglycerol) to the low affinity site would lead to an enhancement of binding of a second activator (e.g. SAPD) to the high affinity site, thereby promoting enzyme activation. In the case of the addition of a single activator (e.g. diacylglycerol), binding of the activator to one of the two sites could promote binding of the same activator to the other site.

The level of activity induced by B-I, together with a concentration of SAPD sufficient to saturate binding to the low affinity site, was close to that induced by B-I alone and less than that achieved with the high concentration of SAPD in isolation. This observation suggests that SAPD binding to the low affinity site does not enhance activation induced by binding of B-I to the high affinity site. This therefore contrasts with the allosteric promotion of SAPD binding to the high affinity site induced by DAG and suggests that the effect of an activator on binding cooperativity may differ, possibly in a manner dependent on its affinity for the low affinity site. The importance of these findings should be viewed in the context of the dramatically different biological end points resulting from the cellular activation of PKC by B-I compared with phorbol esters(40) . Thus, for example, B-I is a potent anti-tumor agent while many phorbol esters are tumor promoters, despite the fact that both phorbol esters and B-I stimulate PKC activity(41) .

Intensive studies involving structure-activity relationships in combination with molecular modeling of a variety of PKC activators have revealed a three-dimensional structural unit (i.e. a pharmacophore) required for enzyme recognition(42) , which has been used to identify further novel activators and inhibitors(43) . Whether a compound has potential as a PKC agonist is generally determined by aligning groups, which are likely to be involved in binding with the conformationally fixed pharmacophore of the phorbol ester, PDBu. Using a similar approach, a recent study has proposed a model for the three-dimensional pharmacophore of diacylglycerol(44) . However, implicit in these studies is the assumption that diacylglycerol, phorbol esters, and other activators compete for the same single high affinity binding site. The existence of high and low affinity activator binding sites on PKCalpha requires that binding to these two sites cannot involve identical three-dimensional arrangements of participating groups. This is supported by the observed differences in binding affinities of TPA compared with 4alpha-TPA for the two sites relative to SAPD, which suggests that phorbol ester binding to the high affinity site is sensitive to the orientation of the 4-OH moiety, while the binding specificity of the low affinity site is independent of this requirement. Furthermore, the observation that diacylglycerol binds with opposite affinities compared with phorbol esters indicates that the pharmacophore for high affinity diacylglycerol binding does not correspond to that derived for PDBu. A similar conclusion was drawn by a previous molecular modeling study, based on comparisons of the molecular features required for phorbol ester and diacylglycerol-induced activation of PKC(45) . Similarly, the observation that B-I competes with SAPD for its high affinity site but not for low affinity binding indicates that the PDBu pharmacophore may be used to model the binding of B-I to the high affinity site. Indeed, a recent study has reported a close fit between groups involved in B-I binding and those of the phorbol ester pharmacophore(46) .

The cysteine-rich Cys-1 and Cys-2 subdomains, which are hypothesized to correspond to the low and high affinity activator binding sites, are common to each of the conventional (PKCalpha, -beta, -beta(I), and -) and novel PKC isoforms (PKC, -, -, -µ, and -). A recent study, showed that these PKC isoforms bind phorbol esters with markedly differing affinities(29) . Thus, as observed for PKCalpha, it is possible that these isoforms may also contain two binding sites, which may vary in their affinities for different activators. This possibility is currently under investigation in this laboratory.

In summary, it is shown that PKCalpha contains two distinct activator sites that bind activators with affinities in the order: DAG > phorbol ester > B-I and B-I > phorbol ester > DAG for the low and high affinity sites, respectively. This finding has important implications for the cellular activation of the enzyme. For example, the results indicate that natural regulators of PKC activity including diacylglycerol and also 1alpha,25-dihydroxyvitamin D(3)(47) can potentially bind simultaneously to the low and high affinity sites raising the possibility that PKC may function as an ``integrator'' of independent signaling pathways. Further, based on this model for PKC activation, it is shown that there may be a class of as yet undiscovered cellular compounds which alone do not stimulate PKC activity but on binding to one site may promote (or inhibit) the level of activation induced by the binding of another activator to the second site. Indeed, such a mechanism may apply to the modulation of PKC activity by hydrophobic drugs such as anesthetics and n-alkanols(48) . Finally, the differential binding of B-I and diacylglycerol to the two activator sites may, in part, explain the divergence in the enzymatic properties and in vivo effects of these agents.


FOOTNOTES

*
This work was supported by United States Public Health Service Grants AA08022, AA07215, AA07186, and AA07465. 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 should be addressed: Rm. 271 JAH, Dept. of Pathology and Cell Biology, Thomas Jefferson University, Philadelphia, PA 19107. Tel.: 215-955-5019; Fax: 215-923-2218; Stubbsc{at}jeflin.tju.edu.

(^1)
The abbreviations used are: PKCalpha, protein kinase Calpha; 4alpha-TPA, 4alpha-12-O-tetradecanoylphorbol-13-acetate; B-I, bryostatin-I; DAG, sn-1,2-dioleoylglycerol; LUV, large unilamellar vesicles; MBP, myelin basic protein peptide substrate; [^3H]PDBu, tritiated phorbol 12,13-dibutyrate; POPC, 1-palmitoyl-2-oleoylphosphatidylcholine; POPS, 1-palmitoyl-2-oleoylphosphatidylserine; RET, resonance energy transfer; SAPD, sapintoxin-D; TPA, 4beta-12-O-tetradecanoylphorbol-13- acetate.


ACKNOWLEDGEMENTS

We are grateful to Dr. R. M. Bell for providing a PKCalpha baculovirus preparation.


REFERENCES

  1. Nishizuka, Y. (1995) FASEB J. 9, 484-496 [Abstract/Free Full Text]
  2. Stabel, S., and Parker, P. J. (1991) Pharmacol. & Ther. 51, 71-95
  3. Hug, H., and Sarre, T. F. (1993) Biochem. J. 291, 329-343 [Medline] [Order article via Infotrieve]
  4. Kikkawa, U., Kishimoto, A., and Nishizuka, Y. (1989) Annu. Rev. Biochem. 58, 31-44 [CrossRef][Medline] [Order article via Infotrieve]
  5. Blumberg, P. M. (1991) Mol. Carcinogen. 4, 339-344 [Medline] [Order article via Infotrieve]
  6. Sharkey, N. A., Leach, K. L., and Blumberg, P. M. (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 607-610 [Abstract]
  7. Sharkey, N. A., and Blumberg, P. M. (1985) Biochem. Biophys. Res. Commun. 133, 1051-1056 [Medline] [Order article via Infotrieve]
  8. Kreutter, D., Caldwell, A. B., and Morin, M. J. (1985) J. Biol. Chem. 260, 5979-5984 [Abstract/Free Full Text]
  9. Morin, M. J., Kreutter, D., Rasmussen, H., and Sartorelli, A. C. (1995) J. Biol. Chem. 262, 11758-11763 [Abstract/Free Full Text]
  10. Bazzi, M. D., and Nelsestuen, G. L. (1989) Biochemistry 28, 9317-9323 [Medline] [Order article via Infotrieve]
  11. Bazzi, M. D., and Nelsestuen, G. L. (1989) Biochemistry 28, 3577-3585 [Medline] [Order article via Infotrieve]
  12. Bazzi, M. D., and Nelsestuen, G. L. (1988) Biochemistry 27, 7589-7593 [Medline] [Order article via Infotrieve]
  13. Bazzi, M. D., and Nelsestuen, G. L. (1988) Biochem. Biophys. Res. Commun. 152, 336-343 [Medline] [Order article via Infotrieve]
  14. Kazanietz, M. G., Krausz, K. W., and Blumberg, P. M. (1992) J. Biol. Chem. 267, 20878-20888 [Abstract/Free Full Text]
  15. Slater, S. J., Kelly, M. B., Taddeo, F. J., Rubin, E., and Stubbs, C. D. (1994) J. Biol. Chem. 269, 17160-17165 [Abstract/Free Full Text]
  16. Ono, Y., Fujii, T., Igarashi, K., Kuno, T., Tanaka, C., Kikkawa, U., and Nishizuka, Y. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 4868-4871 [Abstract]
  17. Burns, D. J., and Bell, R. M. (1991) J. Biol. Chem. 266, 18330-18338 [Abstract/Free Full Text]
  18. Bell, R. M., and Burns, D. J. (1991) J. Biol. Chem. 266, 4661-4664 [Free Full Text]
  19. Ono, Y., Fujii, T., Ogita, K., Kikkawa, U., Igarashi, K., and Nishizuka, Y. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 3099-3103 [Abstract]
  20. Kazanietz, M. G., Bustelo, X. R., Barbacid, M., Kolch, W., Mischak, H., Wong, G., Pettit, G. R., Bruns, J. D., and Blumberg, P. M. (1994) J. Biol. Chem. 269, 11590-11594 [Abstract/Free Full Text]
  21. Quest, A. F. G., and Bell, R. M. (1994) J. Biol. Chem. 269, 20000-20012 [Abstract/Free Full Text]
  22. Quest, A. F. G., Bardes, E. S. G., and Bell, R. M. (1994) J. Biol. Chem. 269, 2953-2960 [Abstract/Free Full Text]
  23. Quest, A. F. G., Bardes, E. S. G., and Bell, R. M. (1994) J. Biol. Chem. 269, 2961-2970 [Abstract/Free Full Text]
  24. Zhang, G., Kazanietz, M. G., Blumberg, P. M., and Hurley, J. H. (1995) Cell 81, 917-924 [Medline] [Order article via Infotrieve]
  25. Wender, P. A., Irie, K., and Miller, B. L. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 239-243 [Abstract]
  26. Ichikawa, S., Hatanaka, H., Takeuchi, Y., Ohno, S., and Inagaki, F. (1995) J. Biochem. (Tokyo) 117, 566-574
  27. Kazanietz, M. G., Wang, S., Milne, G. W. A., Lewin, N. E., Liu, H. L., and Blumberg, P. M. (1995) J. Biol. Chem. 270, 21852-21859 [Abstract/Free Full Text]
  28. Taylor, S. E., Gafur, M. A., Choudhury, A. K., and Evans, F. J. (1981) Experientia 37, 681-682 [Medline] [Order article via Infotrieve]
  29. Dimitrijevic, S. M., Ryves, W. J., Parker, P. J., and Evans, F. J. (1995) Mol. Pharmacol. 48, 259-267 [Abstract]
  30. Burns, D. J., Bloomenthal, J., Lee, M. H., and Bell, R. M. (1990) J. Biol. Chem. 265, 12044-12051 [Abstract/Free Full Text]
  31. Stabel, S., Schaap, D., and Parker, P. J. (1991) Methods Enzymol. 200, 670-673 [Medline] [Order article via Infotrieve]
  32. Slater, S. J., Kelly, M. B., Taddeo, F. J., Ho, C., Rubin, E., and Stubbs, C. D. (1994) J. Biol. Chem. 269, 4866-4871 [Abstract/Free Full Text]
  33. MacDonald, R. C., MacDonald, R. I., Menco, B. P., Takeshita, K., Subbarao, N. K., and Hu, L. R. (1991) Biochim. Biophys. Acta 1061, 297-303 [Medline] [Order article via Infotrieve]
  34. Boni, L. T., and Rando, R. R. (1985) J. Biol. Chem. 260, 10819-10825 [Abstract/Free Full Text]
  35. Epand, R. M. (1994) Anal. Chem. 218, 241-247
  36. de Vries, D. J., Herald, C. L., Pettit, G. R., and Blumberg, P. M. (1988) Biochem. Pharmacol. 37, 4069-4073 [Medline] [Order article via Infotrieve]
  37. Mosior, M., and Newton, A. C. (1995) J. Biol. Chem. 270, 25526-25533 [Abstract/Free Full Text]
  38. Ashendel, C. L. (1985) Biochim. Biophys. Acta 822, 219-242 [Medline] [Order article via Infotrieve]
  39. Marquez, C., Martinez, A. C., Kroemer, G., and Bosca, L. (1992) J. Immunol. 149, 2560-2568 [Abstract/Free Full Text]
  40. Blumberg, P. M., and Pettit, G. R. (1992) in New Leads and Targets in Drug Research, Alfred Benzon Symposium 33 (Krogsgaard-Larsen, P., Brogger Christensen, S., and Kofod, H., eds) pp. 273-285, Munksgaard, Copenhagen, Denmark
  41. Hennings, H., Blumberg, P. M., Pettit, G. R., Herald, C. L., Shores, R., and Yuspa, S. H. (1987) Carcinogenesis 8, 1343-1346 [Abstract]
  42. Wender, P. A., Koehler, K. F., Sharkey, N. A., Dell'Aquila, M. L., and Blumberg, P. M. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 4214-4218 [Abstract]
  43. Wang, S., Zaharevitz, D. W., Sharma, R., Marquez, V. E., Lewin, N. E., Du, L., Blumberg, P. M., and Milne, G. W. A. (1994) J. Med. Chem. 37, 4479-4489 [Medline] [Order article via Infotrieve]
  44. Rando, R. R., and Kishi, Y. (1992) Biochemistry 31, 2211-2218 [Medline] [Order article via Infotrieve]
  45. Leli, U., Hauser, G., and Froimowitz, M. (1989) Mol. Pharmacol. 37, 286-295 [Abstract]
  46. Wender, P. A., Cribbs, C. M., Koehler, K. F., Sharkey, N. A., Herald, C. L., Kamano, Y., Pettit, G. R., and Blumberg, P. M. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 7197-7201 [Abstract]
  47. Slater, S. J., Kelly, M. B., Taddeo, F. J., Larkin, J. D., Yeager, M. D., McLane, J. A., Ho, C., and Stubbs, C. D. (1995) J. Biol. Chem. 270, 6639-6643 [Abstract/Free Full Text]
  48. Slater, S. J., Cox, K. J. A., Lombardi, J. V., Ho, C., Kelly, M. B., Rubin, E., and Stubbs, C. D. (1993) Nature 364, 82-84 [CrossRef][Medline] [Order article via Infotrieve]

©1996 by The American Society for Biochemistry and Molecular Biology, Inc.