(Received for publication, May 22, 1995; and in revised form, July 3, 1995)
From the
Phorbol esters bind with high affinity to protein kinase C (PKC)
isozymes as well as to two novel receptors, n-chimaerin and
Unc-13. The cysteine-rich regions present in these proteins were
identified as the binding sites for the phorbol ester tumor promoters
and the lipophilic second messenger sn-diacylglycerol. A
50-amino-acid peptide comprising the second cysteine-rich region of PKC
, expressed in Escherichia coli as a glutathione S-transferase (GST)-fusion protein, bound
phorbol 12,13-dibutyrate (PDBu) with high
affinity (K
=0.8 nM).
Using the cDNA of that cysteine-rich region as a template, a series of
37 point mutations was generated by site-directed mutagenesis, and the
mutated proteins were analyzed quantitatively for binding of
[
H]PDBu and, as appropriate, for binding of the
ultrapotent analog [
H]bryostatin 1. Mutants
displayed one of three patterns of behavior: phorbol ester binding was
completely abolished, binding affinity was reduced, or binding was not
significantly modified. As expected, five of the six cysteines as well
as the two histidines involved in Zn
coordination are
critical for the interaction of the protein with the phorbol esters. In
addition, mutations in several positions, including phenylalanine 3,
tyrosine 8, proline 11, leucines 20, 21, and 24, tryptophan 21,
glutamine 27, and valine 38 drastically reduced the interaction with
the ligands. The effect of these mutations can be rationalized from the
three-dimensional (NMR) structure of the cysteine-rich region. In
particular, the C-terminal portion of the protein does not appear to be
essential, and the loop comprising amino acids 20 to 28 is implicated
in the binding activity.
The protein kinase C (PKC) ()isozymes are a family of
related proteins that mediate one of the major cellular signal
transduction pathways which utilizes lipophilic second messengers. PKC
plays a central role in mediating the activation of growth factors,
neurotransmitters, and hormones and has been implicated in cellular
growth control and carcinogenesis (Weinstein, 1988). Diacylglycerol,
one of the products of the hydrolysis of inositol 1,4,5-diphosphate and
phosphatidylcholine, was described as the endogenous activator for most
of the PKC isozymes (Blumberg, 1991; Nishizuka, 1992). Both the
classical isoforms (PKC
, PKC
1, PKC
2, and PKC
)
and novel isoforms (PKC
, PKC
, PKC
, and PKC
)
are responsive to diacylglycerol. In contrast, the atypical isoforms
(PKC
and PKC
) are unresponsive to this lipophilic second
messenger (Ono et al., 1989b; Kazanietz et al., 1993;
Akimoto et al., 1994).
The phorbol esters, tumor-promoting
diterpene derivatives from plants of the family Euphorbiaceae, bind in
a phospholipid-dependent manner to the same site in PKC as do
diacylglycerols and activate the enzyme (Sharkey et al.,
1984). These compounds have become a valuable tool for studying the
mechanism of PKC activation and for elucidation of the cellular
pathways in which PKC is involved. Deletion analysis of PKC revealed
that the cysteine-rich regions present in the regulatory domain are the
sites of phorbol ester/diacylglycerol binding (Ono et al.,
1989a; Kaibuchi et al., 1989). Each of these cysteine-rich
regions is a 50- or 51-amino-acid domain possessing the motif
HXCX
CX
CX
CX
HX
CX
C,
where H is histidine, C is cysteine, X is any other amino
acid, and n is 13 or 14. These structures, also called zinc
fingers, are coordinated by two Zn
ions (Quest et
al., 1992). The motif is duplicated in tandem in both the
classical and novel PKC isozymes and is present only once in the
atypical PKC isozymes. Studies using recombinant cysteine-rich regions
revealed that a single copy of the motif is sufficient for the binding
of phorbol esters (Ono et al., 1989a; Burns and Bell, 1991;
Quest and Bell, 1994; Quest et al., 1994a; Kazanietz et
al. 1994a; Wender et al., 1995). This conclusion is
corroborated by the ability of the novel non-kinase proteins n-chimaerin and Unc-13, both of which contain a single copy of
the motif, to function as high affinity phorbol ester receptors (Hall et al., 1990; Ahmed et al., 1991, 1992; Maruyama and
Brenner, 1991; Areces et al., 1994). On the other hand, the
proto-oncogenes vav or c-raf, although they have a
single copy of the cysteine-rich domain that coordinates Zn
in their structure, do not bind phorbol esters (Ghosh et
al., 1994; Kazanietz et al., 1994a).
As determined by
NMR (Hommel et al., 1994), in solution the second
cysteine-rich domain of rat PKC adopts a globular fold, where the
two Zn
ions are coordinated by two nonconsecutive
sets of Zn
-binding residues (three cysteines and one
histidine in each case) to form two separate metal-binding sites. The
topology can be described as two antiparallel
-sheets followed by
a C-terminal helix. The two metal-binding sites are located at either
end of one of the major
-sheets, with the histidines involved in
Zn
coordination buried in a hydrophobic cavity.
The postulated consensus sequence for binding of phorbol esters
includes 13 amino acids (Kazanietz et al., 1994a). In PKC
, one of the atypical isoforms lacking phorbol ester binding, a
single amino acid differs in the motif (glycine instead of proline in
position 11). However, restoration of the proline by site-directed
mutagenesis did not restore binding (Kazanietz et al., 1994a).
These results strongly suggested that other amino acids besides those
postulated for the consensus must be necessary for ligand binding.
Therefore, we decided to perform a more extensive mutagenesis study to
evaluate residues within the cysteine-rich region that may be relevant
for the interaction. Earlier studies indicated that double point
mutations of cysteines in PKC
cysteine-rich regions (those
corresponding to positions 31 and 34 in the consensus) abolished
phorbol ester binding (Ono et al., 1989a). Further, an elegant
deletional analysis (Quest et al., 1994b) clearly identified a
43-amino-acid fragment as the minimum necessary for the ligand
interaction. In our study we expressed a single recombinant
cysteine-rich region of PKC
(50 amino acids) in E. coli as a GST-fusion protein. A series of 37 point mutations in 25
different positions were made, and the corresponding proteins were
assayed for ligand binding using [
H]PDBu and
[
H]bryostatin 1. A unique aspect of this study is
that we were able to quantitate the interaction of the mutants with
[
H]PDBu by Scatchard analysis and thereby measure
quantitatively the changes in binding affinity as a consequence of the
point mutations. Our experiments led us to the identification of
several amino acid residues within the phorbol ester binding domain
that are critical for the interaction of the ligand with the receptor.
Elsewhere we have described the modeling of the structure of the second
cysteine-rich region domain of PKC
to the known structure of the
same domain in PKC
. We were able to take advantage of this
computer model together with our mutational analysis to develop
insights into the regions regulating phorbol ester and phospholipid
interactions.
Binding using
bacterial lysates was carried out using a fixed concentration of
radioligand (10 nM for either [H]PDBu or
[
H]bryostatin 1). Pellets from 1.5-ml cultures
were lysed by sonication in 500 µl of 50 mM Tris-HCI, pH
7.4, and 1 mM EGTA. Fifty µl per tube of the total lysate
were used both for the [
H]PDBu and
[
H]bryostatin 1 binding assays.
The phorbol ester receptors share a high degree of homology
in the cysteine-rich region. Alignment of these domains of the PKC
isozymes, of n-chimaerin and of Unc-13 shows a consensus of 13
residues (Kazanietz et al., 1994a). These conserved amino
acids include the cysteines and histidines involved in the coordination
of Zn atoms, as well as two glycines (positions 23
and 28 in the consensus) and a glutamine (position 27). In PKC
and PKC
, the atypical isoforms lacking phorbol ester binding, a
single proline at position 11 in the consensus is replaced by glycine
and arginine, respectively. The proto-oncogenes c-raf and vav share high homology with the phorbol ester receptors, and
the degree of conservation among the consensus residues is 77% and 92%,
respectively. Still, these two proteins are not able to bind phorbol
esters either with high or low affinity (Ghosh et al., 1994;
Kazanietz et al., 1994a). (
)Analysis of
conservation of amino acids within the 50-amino-acid domain in the
phorbol ester-binding proteins reveals regions with high and low
degrees of variability. In addition to those residues absolutely
conserved in the cysteine-rich region, several positions show very
restricted changes. Positions 8, 13, and 22 are occupied by aromatic
amino acids. Hydrophobic amino acids are present in positions 5, 20,
21,24, 29, 38, and 46. Regions of hypervariability are present at both
the N- and C-terminal ends (Fig. 1).
Figure 1:
Variability in the cysteine-rich
regions of phorbol ester binding proteins. The sequence of the second
cysteine-rich region of PKC is shown together with the variable
amino acids found in the cysteine-rich regions of PKC
(bovine),
PKC
(rat), PKC
(rat), PKC
(rat), PKC
(mouse),
PKC
(mouse), n-chimaerin (human), and Unc-13 (Caenorhabditis elegans). Sequences of the non-phorbol ester
binding proteins PKC
(mouse), PKC
(mouse), c-raf (human), and vav (mouse) are aligned for comparison. The
consensus represents all of the conserved amino acids in the phorbol
ester receptors. Positions mutated in this study in the second
cysteine-rich region of PKC
are shown in bold and underlined. *, residues necessary for phorbol ester
binding.
The second cysteine-rich
region of PKC was expressed in E. coli as a GST-fusion
protein. After isopropyl-O-D-thiogalactopyranoside
induction of bacterial cultures, high levels of recombinant proteins
were found in lysates as revealed by the appearance of a strong 33-kDa
band in Coomassie Blue-stained SDS-polyacrylamide gels (Fig. 2A). Overexpression of the cysteine-rich region
of PKC
resulted in high binding activity of
[
H]PDBu and [
H]bryostatin 1
in lysates (Fig. 2, B and C). Mutants of the
cysteine-rich region were likewise expressed as GST-fusion proteins in E. coli, and, in all cases, a 33-kDa band was observed in
bacterial lysates. In all cases, the GST-fusion proteins were recovered
predominantly from the soluble fraction of the bacterial lysates (data
not shown); they were purified from that fraction using
glutathione-Sepharose 4B beads. The purity of the preparations was
generally >80%. In some cases, a degradation product corresponding
to GST could also be observed in the polyacrylamide gels (Fig. 2A). The GST-fusion proteins could be detected
easily with an anti-GST antibody in Western blots, and they showed a
very high level of GST enzymatic activity (data not shown).
Figure 2:
Expression of the cysteine-rich region of
PKC mutants as GST-fusion proteins and screening with
[
H]PDBu and [
H]bryostatin
1. A, bacterial cultures overexpressing the wild-type
cysteine-rich region of PKC
or the mutants were lysed and
subjected to SDS-polyacrylamide gel electrophoresis (T). The
corresponding GST-fusion proteins were affinity-purified with
glutathione-Sepharose 4B beads (P). The gels were stained with
Coomassie Blue. Positions of the molecular weight markers are shown on
the left. Representative results for several mutants are
shown. Similar high expression was found with all of the 39 mutants. B, lysates from bacterial cultures overexpressing wild-type
and mutant cysteine-rich regions were subjected to
[
H]PDBu binding using a 10 nM
concentration of radioligand. One additional experiment gave similar
results. Representative experiments (performed in triplicate) are
shown. Results are expressed as mean ± S.E. C, binding
of [
H]bryostatin 1 (10 nM) to bacterial
lysates overexpressing wild-type and mutant cysteine-rich regions. Two
additional experiments gave similar results. Binding was performed in
duplicate, and the duplicates generally differed by less than 10%.
Representative experiments are shown. Assay conditions were chosen for
detection of weak binding (in B and C) and therefore
included high levels of cysteine-rich region in the lysates. As a
result, at least for the wild-type and some of the mutants showing high
binding, the total counts/min of [
H]PDBu or
[
H]bryostatin 1 bound reflect titration of the
ligand by excess receptor. In all of the mutants, the amino acid
indicated in the figure was mutated to
glycine.
Bacterial lysates for the different mutants were evaluated for
phorbol ester binding using [H]PDBu as a ligand.
In addition, and in order to detect low affinity interactions that
might not have been detected with this classical ligand, a more
sensitive assay using the ultrapotent analog
[
H]bryostatin 1 in the presence of
phosphatidylserine was also carried out (Fig. 2, B and C). As described previously (Kazanietz et al., 1994a,
1994b), by using nanomolar concentrations of
[
H]bryostatin 1, a ligand with an estimated
dissociation constant lower than 2.5 pM (DeVries et
al., 1988), we expect a gain in binding affinity of several orders
of magnitude. The specific mutants and the results of binding for both
radioligands using the lysates overexpressing high amounts of the
recombinant proteins are listed in Table 1. Mutations of the
cysteines in positions 14, 17, 31, 34, and 42 of the cysteine-rich
region completely abolished [
H]PDBu binding. No
binding of [
H]bryostatin 1 was detected in those
lysates. Mutation of the conserved histidine in position 1 completely
abolished [
H]PDBu binding. In contrast, low but
significant [
H]PDBu and
[
H]bryostatin 1 binding could still be detected
when histidine 39 was mutated, suggesting a very low affinity for the
ligand interaction in this latter case. After mutation of the cysteine
in position 50 to glycine, both [
H]PDBu and
[
H]bryostatin 1 binding were detected. Bell and
co-workers (Quest et al., 1994b) have previously shown that,
after deletion of this cysteine, phorbol ester binding still took
place, although a quantitative analysis was not performed in that case.
By doing a Scatchard analysis for [
H]PDBu (Fig. 3), we found that this mutant showed no decrease in
affinity.
Figure 3:
Representative
[H]PDBu Scatchard plots for the second
cysteine-rich domain of PKC
(wild-type) and mutants. The
recombinant GST-fusion proteins were incubated for 5 min (18 °C)
with increasing concentrations of [
H]PDBu in the
presence of 100 µg/ml phosphatidylserine and 1 mM EGTA.
Binding was measured using the polyethylene glycol precipitation assay.
Representative experiments for the wild-type and 3 representative
mutants as indicated in the graph are shown. Each point represents the
mean of three experimental values, generally with a standard error of
<2%. Note different scales for [
H]PDBu for the
different mutants. For each of the GST-fusion proteins shown, similar
results were obtained in at least two additional
experiments.
The mutation results suggest that the lack of phorbol
ester binding by PKC may be rationalized by the combined effects
of the substitution of proline 11 to glycine and of leucine 20 to
arginine. The former change caused a 125-fold decrease in affinity to
the second cysteine-rich region of PKC
. The conversion of leucine
20 to glycine caused a 15-fold decrease in binding; the additional
effect when a positively charged residue is inserted in this site in
the hydrophobic loop still requires experimental determination.
In
addition to the cysteines and histidines, other amino acids in the
consensus are essential for phorbol ester binding. Mutation of
phenylalanine in position 3 to glycine completely abolished
[H]PDBu and [
H]bryostatin 1
binding. Substitution of the phenylalanine by another aromatic amino
acid (tryptophan) preserved phorbol ester binding. Interestingly, a
leucine was also tolerated in that position without any change in the
binding affinity, suggesting that any hydrophobic amino acid in
position 3, either aromatic or aliphatic, is sufficient to allow ligand
binding. Glutamine in position 27 is also essential for phorbol ester
binding. Mutation of this residue to glycine almost abolished
[
H]PDBu binding, although binding of the
ultrapotent analog [
H]bryostatin 1 was still
detectable, indicating a large reduction in binding affinity. An
introduction of a tryptophan in this position completely abolished
binding of both [
H]PDBu and
[
H]bryostatin 1. Mutation of glutamine 27 to
either valine or threonine reduced the affinity of
[
H]PDBu by factors of 28 or 131, respectively.
Replacement of the proline in position 11 by glycine produced a large
decrease in the affinity for [
H]PDBu, with a
decrease in K
of about 125-fold when compared to
the wild-type cysteine-rich region (Fig. 3).
Several
hydrophobic amino acids in the cysteine-rich regions seem to be
essential for phorbol ester binding. Replacement of tyrosine by glycine
in position 8 reduced the binding affinity of
[H]PDBu 60-fold. In contrast, the substitution of
phenylalanine in position 13 by glycine did not change the affinity for
the ligand. A highly lipophilic region located in the loop formed by
amino acids 20 to 27 was very sensitive to mutagenic changes. Mutation
of any of the three leucines in positions 20, 21, and 24 induced either
reduction or complete loss of [
H]PDBu binding.
Only the first of these leucine to glycine mutants gave measurable
[
H]PDBu binding (K
=
12 nM, 15-fold lower affinity than the wild type) (Fig. 3). Elimination of either leucine 21 or 24 induced a
complete loss of [
H]PDBu binding, and only low
levels of the ultrapotent ligand [
H]bryostatin 1
could be detected in lysates, suggesting a large reduction in ligand
affinity. Mutation of tryptophan in position 22 to glycine resulted in
a reduction in the affinity for [
H]PDBu of
31-fold. A phenylalanine in that same position could still be tolerated
with no significant changes in binding affinity. It is remarkable that
in the non-phorbol ester receptors PKC
, c-raf, and vav, several changes are found in this region (Fig. 1)
that could explain the lack of binding in these proteins. Mutation of
valine 38 completely abolished phorbol ester binding. In contrast,
replacement of valine in position 46 by glycine did not affect the
interaction with the ligand. In the deletion analysis of the second
cysteine-rich region of PKC
(Quest et al., 1994b),
[
H]PDBu binding was still found after deletion of
valine 46. Threonine in position 12 is highly conserved within the PKC
isozymes and Unc-13 and is a potential hydrogen bond acceptor for the
interaction with phorbol esters. A histidine in the same position is
found in the phorbol receptor n-chimaerin. Both PKC
and vav have an alanine in position 12. Mutagenesis analysis shows
that this residue is not essential for phorbol ester binding, since no
changes in [
H]PDBu binding were found after
replacing the threonine with glycine, serine, or valine. Another
potential hydrogen bond acceptor is found in position 15 where either
aspartic acid, glutamic acid, or serine is found in the phorbol ester
receptors. A glycine occupies that position in PKC
, and a lysine
is found in vav. Replacement of the aspartic acid by glycine
in the second cysteine-rich region of PKC
did not affect phorbol
ester binding. Substitution of the aspartic acid by asparagine, serine,
or tyrosine did not modify [
H]PDBu binding
either. Positions 30 and 39 are occupied by either a basic amino acid
or glutamine, suggesting a potential role as a hydrogen bond donor.
Mutation of the lysine 30 to asparagine did not affect phorbol ester
binding. Moreover, introduction of a glutamic acid in that position
produced only a minor reduction (about 4-fold) in the affinity for
[
H]PDBu binding. Similarly, substitution of
lysine 41 by glycine did not affect phorbol ester binding. Mutation to
valine of methionine 36, a residue conserved in the second copy of
cysteine-rich regions of PKC, did not change the affinity for
[
H]PDBu. No changes in binding were also observed
after mutation of lysine 4 to valine.
Cysteine-rich regions in PKC, n-chimaerin, and
Unc-13 are the binding sites for the second messenger diacylglycerol as
well as for the phorbol ester tumor promoters. The second cysteine-rich
region of PKC expressed as a GST-fusion protein binds
[
H]PDBu with an affinity of around 1 nM,
similar to that described for the intact enzyme expressed in
baculovirus (Kazanietz et al., 1993). Recombinant
cysteine-rich regions of n-chimaerin or Unc-13 behave
similarly as phorbol ester receptors (Areces et al., 1994;
Kazanietz et al., 1995). The thorough mutagenesis analysis
that we performed revealed many essential amino acids within the
protein relevant for the binding activity.
Determination by NMR of
the three-dimensional structure of a single PKC cysteine-rich region
from PKC revealed that it adopts a globular fold coordinating two
Zn
ions by a noncontiguous set of residues (Hommel et al., 1994). Cysteines 14, 17, and 40 and histidine 39
coordinate the first Zn
. Cysteines 31, 34, and 50 and
histidine 1 coordinate the second metal ion. Since the two
Zn
ions are an integral part of the structure,
mutations in the cysteines or histidines may disrupt the conformation
necessary for phorbol ester binding. Still, some tolerance can be found
for coordination of the Zn
atoms. Our results clearly
show that cysteine 50 (which coordinates the second Zn
in the protein) is not necessary for phorbol ester binding. Quest et al. (1994b) have found that a C-terminal deletion in the
cysteine-rich domain in PKC
did not abolish
[
H]PDBu binding and therefore defined a minimal
43-amino-acid peptide necessary for binding activity. That deletion
included valine 46, a highly conserved residue in all the phorbol ester
receptors, which according to our results is not necessary for binding.
NMR analysis revealed that this amino acid is very important for the
folding of the C-terminal
-helix. Taking all these data together,
we conclude that the C-terminal portion of the cysteine-rich domain is
not involved in the binding of the phorbol esters. Our result also
indicate a clear dissociation between phorbol ester binding and
Zn
coordination, as suggested by Bell's group
(Quest et al., 1994b). A small but detectable
[
H]bryostatin 1 binding is detected after
mutation of the histidine 39 to glycine, suggesting that even after
partially losing the coordination of the first Zn
binding can still take place, although probably with a very low
affinity.
Several amino acids seem to be critical for the structure
of the cysteine-rich domain, and, therefore, large conformational
changes should be expected after mutation. Modeling the second
cysteine-rich region of PKC to that published for PKC
(
)facilitated interpretation of the changes observed after
mutation. For example, the side chain of phenylalanine 3 is involved in
a hydrophobic interaction with the side chains of histidine 1 and
leucine 29 (or, for other cysteine-rich domains, other hydrophobic
amino acids in position 29), and, therefore, it is extremely important
for the overall fold of the structure. This is supported by the finding
that binding is completely abolished after mutation to glycine but
still retained after mutation to tryptophan or leucine. Tyrosine 8 and
proline 11 are part of a big loop (residues 7 to 12). The side chain in
tyrosine 8 points toward the outside of the protein, and only
hydrophobic residues are tolerated in this position, therefore
suggesting a possible involvement in phospholipid interaction. The side
chain of proline 11 has some hydrophobic interactions with leucine 20.
Furthermore, since proline changes the angle of the peptide backbone,
the conformational change produced as a consequence of its mutation to
glycine may be responsible for the large loss in phorbol ester binding
affinity. Although the PKC
has a glycine in this position, its
mutation to proline did not restore binding (Kazanietz et al.,
1994a), suggesting that further differences in other amino acids must
contribute to the lack of binding in this PKC isozyme. Glutamine 27 is
conserved in all the PKC isozymes and buried deep in the protein.
Although its backbone does not seem to have strong interactions with
other residues, its side chain folds inside the protein, interacts with
the side chains of asparagine 7, proline 11, and leucine 21, and also
forms a weak hydrogen bond with the backbone of tyrosine 8. Changes in
position 27 were not tolerated, and binding was totally lost or
substantially reduced after mutation, suggesting that the size and
nature of the glutamine 27 are extremely important to keep the
structure in the right conformation for the interaction with the
phorbol esters. Valine 38, a conserved amino acid (only in the first
cysteine-rich region of PKC
and PKC
is an isoleucine
found), is critical for phorbol ester binding. Its hydrophobic side
chain is buried deep inside the protein and interacts with the side
chains of phenylalanine 3, leucine 29, cysteine 42, and valine 46 to
form a hydrophobic core inside the protein.
The loop of amino acids
20 to 27 is an essential part of the structure necessary for phorbol
ester binding. Mutations of residues within that loop strongly affect
the activity. Position 20 is occupied by leucine or other hydrophobic
amino acids in the phorbol ester receptors (an arginine is found in PKC
). Its hydrophobic chain points outside toward the protein surface
and does not seem to be very important for the overall structure of the
protein. Its mutation to glycine produce a 15-fold loss in binding
activity, probably a consequence of a reduced interaction with the
lipid. The side chain of leucine 21 folds inside the protein and
interacts with the side chains of threonine 12, glutamine 27, and
asparagine 37. Its backbone forms a hydrogen bond with the backbone of
threonine 12. The loss in binding after mutation to glycine clearly
supports its importance in the protein folding. In the case of
tryptophan 22 and leucine 24, no strong backbone or side chain
interactions are found. Both side chains point toward the outside of
the protein and, therefore, may be critical for the interaction with
either the phospholipid or the phorbol ester lipophilic chains. The
mutant tryptophan 22 to glycine, in contrast to the one to
phenylalanine, is not able to interact physically with phospholipid
vesicles in an ultracentrifugation assay similar to the one described
by Quest et al. (1994a), (
)suggesting that a
hydrophobic residue in position 22 may be involved in the interaction
of PKC with membrane phospholipids. In PKC
, the positive charge
of the arginine in position 20 may be disruptive to the interaction of
this region with the phorbol esters and phospholipids.
Fig. 4shows a three-dimensional view of the second
cysteine-rich region of PKC delta and a summary of the amino acids
within the domain relevant for phorbol ester binding. The residues
analyzed by mutagenesis displayed one of three patterns of behavior:
phorbol ester binding was completely abolished, binding affinity was
reduced, or binding affinity not significantly modified. Our
experimental results support the importance of the loop of amino acids
in positions 20 to 27 in phorbol ester binding. Others (Ichikawa et
al., 1995) have postulated a role for this loop in phorbol ester
binding based on the exposed hydrophobic surface of the hairpin loop
which presumably interacts with the membrane phospholipids. As
described in detail elsewhere, computer modeling provides
strong support for this region being the site of phorbol ester binding.
Finally, crystallographic analysis of the second cysteine-rich region
of PKC
has demonstrated directly that phorbol ester binds in the
absence of phospholipid (Zhang et al., 1995) in a narrow
groove at the tip of the domain, near the loop created by amino acids
20-27. Bound phorbol esters replace bound water molecules which
form bridging hydrogen bonds in the
-sheets and create a
hydrophobic cap that stabilizes the membrane-bound form of the enzyme.
Figure 4:
Three-dimensional structure of the second
cysteine-rich domain of PKC . The specific residues analyzed by
mutagenesis are indicated in the figure. Phorbol ester binding was
completely abolished, red; binding affinity was reduced, blue; or binding affinity was not significantly modified, green. The two Zn
ions are shown in yellow.