(Received for publication, November 12, 1995)
From the
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 PKC. 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, 4
-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 PKC
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.
Protein kinase C (PKC) ()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 PKC 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
[
H]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
[
H]PDBu to PKC
(29) . However, the
affinity of SAPD for binding to PKC
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 PKC
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 PKC
.
Figure 1:
PKC tryptophan fluorescence
and quenching by SAPD to produce resonance energy transfer. A, emission spectra. The fluorescence emission spectra (uncorrected)
of PKC (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
PKC
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 PKC
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, F
, F
, and F
are the minimum and maximum
fluorescence intensities, K
and K
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.
The aim of the present study was to test the hypothesis that
PKC contains two activator binding sites of low and high affinity.
To accomplish this, SAPD binding to recombinant PKC
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
PKC
induced by the same activator combinations used in the binding
experiments.
In order to determine activator binding to PKC,
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 PKC
tryptophans (Fig. 1A). This overlap facilitates RET between
PKC
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 PKC
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 PKC
(Fig. 1A). Addition of EGTA reversed the level of RET (Fig. 1B) indicating that SAPD binding to PKC
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 PKC 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 PKC
rather than being distributed between
membrane-bound and soluble enzyme forms. The binding constants were K
= 150 ± 50 nM and K
= 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
= 100 ± 20 nM and K
= 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 PKC in terms of
binding and enzyme activity: effect of TPA and 4
-TPA. A, binding. SAPD binding, in the presence of Ca
and
LUV, measured from RET between PKC
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 4
-TPA (open squares) on SAPD binding. B, activity. Dose-response curve for activation of PKC
by SAPD (closed circles) in the presence of 2 µM TPA (open circles) or 2 µM 4
-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 PKC 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 PKC
, 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 PKC
by SAPD is shown. PKC
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.''
The non-activating epimer of
TPA, 4-TPA, is often used in the study of phorbol ester
interactions with PKC(38) . However, the binding properties of
4
-TPA to PKC have not previously been reported. The high affinity
SAPD binding to PKC
while removed by TPA was only marginally
affected by 4
-TPA (Fig. 2A). However, SAPD binding
to the low affinity site was inhibited by both 4
-TPA and TPA.
Consistent with this, in the presence of 4
-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 PKC,
,
mixed
isoform preparation, induced by a maximum concentration of 1 µM TPA, was inhibited by 4
-TPA. The finding in the present study
that TPA-stimulated PKC
activity was unaffected by 4
-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) .
Figure 4:
Effect of B-I on SAPD binding and
activation. A, binding. The RET from PKC 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 PKC
, 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 PKC
by SAPD is shown. PKC
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.).
In the present study, phorbol ester binding to PKC was
determined using a fluorescence binding assay employing the phorbol
ester, SAPD. Both SAPD binding and activation dose-response curves
indicated that PKC
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
PKC 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 4
-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 (PKC, -
,
-
, 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 PKC
, 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 PKC 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 1
,25-dihydroxyvitamin D
(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.