From the
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
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
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
[
To measure competition of
[
Using intact recombinant PKC
A powerful approach to deal with this issue was to use
the recombinant cysteine-rich region of PKC
We confirmed that our measured binding of
[
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 [
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
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
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
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,
[
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.
Binding of the ligands to the second cysteine-rich
region of 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.
(
)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
HX
CX
CX
CX
CX
HX
CX
C
(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) .
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.
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
Recombinant
PKC in Baculovirus
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/H
O: 0.05%
trifluoroacetic acid in 90/10 CH
CN/H
O, 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
.
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%.
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 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 B
value 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).
(
)
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) .
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).
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.
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.
-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.
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.
Table: Structure-activity analysis of binding to the
second cysteine-rich region of PKC in the absence and presence of
phospholipids
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.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.