From the
The Caenorhabditiselegans Unc-13 protein is a
novel member of the phorbol ester receptor family having a single
cysteine-rich region with high homology to those present in protein
kinase C (PKC) isozymes and the chimaerins. We expressed the
cysteine-rich region of Unc-13 in Escherichiacoli and quantitatively analyzed its interactions with phorbol esters
and related analogs, its phospholipid requirements, and its inhibitor
sensitivity. [
The phorbol esters, natural products derived from plants of the
family Euphorbiaceae, have been extensively investigated in the last
two decades on the basis of their potent activity as tumor promoters
(1, 2) . The search for the receptors for this class of
compounds led to the identification of PKC as their major target
(3, 4, 5, 6, 7) . The complexity
and heterogeneity in the biological responses to the phorbol esters
suggested the existence of multiple receptors. Indeed, protein kinase C
is now recognized to represent a complex family of isozymes. The
``classic'' isozymes
Binding of phorbol esters to PKC occurs at the cysteine-rich regions
(also called zinc fingers) in the regulatory domain
(19) , where
the motif
H X
Given the lack of an extensive study of Unc-13 as a
DAG/phorbol ester receptor, we decided to perform a detailed
quantitative analysis of phorbol ester binding to the cysteine-rich
region of Unc-13 and to compare it to a single cysteine-rich region of
PKC (PKC
[
Unc-13 is
also a high affinity receptor for the bryostatins, macrocyclic lactones
with an atypical pattern of biological activity as compared with the
typical phorbol esters. Scatchard analysis using
[
The cysteine-rich region in the regulatory domain was shown
to be the binding site for the phorbol esters in PKC. The high degree
of conservation in this region among several proteins other than PKC,
including the chimaerins, Unc-13, Vav, c-Raf, and diacylglycerol kinase
(20, 21) , suggested the possibility that several
signaling pathways may be regulated by diacylglycerol or their analogs,
the protein esters. We have previously reported that
n-chimaerin, a protein isolated from brain, closely resembles
the properties of PKC
The K
The phorbol esters
are commonly used as a tool to assess the role of PKC in biological
systems. The fact that Unc-13 as well as n-chimaerin
(16) so closely resemble PKC isozymes as phorbol ester receptors
suggests that many of the phorbol ester effects previously described
could potentially be non-PKC mediated. Furthermore, inhibitors targeted
to the cysteine-rich region, such as calphostin C, were unable to
distinguish between phorbol ester receptors. Alternative molecular
approaches to implicate a specific phorbol ester receptor class include
overexpression of single isoforms, dominant negative mutants, or
antisense. Pharmacologically, selective inhibitors targeted to the
kinase domain of PKC
(35, 36, 37) may likewise
become valuable tools for discriminating between PKC and non-PKC
phorbol ester receptors.
Similar potencies for diacylglycerol were
found for the cysteine-rich regions of Unc-13 and PKC
In summary, our findings suggest that Unc-13 represents
another member of the high affinity phorbol ester receptor family and
that, although modest differences were found with other cysteine-rich
regions, Unc-13 is a potential target for pathways regulated by
diacylglycerol/phorbol esters. Identification of mammalian homologs of
the Unc-13 protein as well as finding the biological role for Unc-13 at
the cellular level will represent important contributions to the
understanding of the biology of the phorbol esters.
Binding was performed in the
presence of PS and either 1 mM EGTA or 0.1 mM CaCl
Protein-ligand interactions were analyzed by competition of
[
Binding was performed using 300 µg/ml
phospholipid vesicles and 0.1 mM EGTA. Phospholipid vesicles
contained 80% PC and 20% of the phospholipid except when a single
phospholipid was used as indicated in the table. Values represent the
mean ± S.E. of three to five experiments (in parentheses) in
which determinations were done in triplicate. Results were normalized
to the binding in 80% PC, 20% PS vesicles as 100% binding. PI,
phosphatidylinositol; PIP, phosphatidylinositol 4-phosphate;
PIP
H]Phorbol 12,13-dibutyrate
[
H]PDBu bound with high affinity to the
cysteine-rich region of Unc-13 ( K
= 1.3 ± 0.2 nM). This affinity is similar
to that of other single cysteine-rich regions from PKC isozymes as well
as n-chimaerin. As also described for PKC isozymes and
n-chimaerin, Unc-13 bound diacylglycerol with an affinity
about 2 orders of magnitude weaker than [
H]PDBu.
Structure-activity analysis revealed significant but modest differences
between recombinant cysteine-rich regions of Unc-13 and PKC
. In
addition, Unc-13 required slightly higher concentrations of
phospholipid for reconstitution of [
H]PDBu
binding. Calphostin C, a compound described as a selective inhibitor of
PKC, was also able to inhibit [
H]PDBu binding to
Unc-13, suggesting that this inhibitor is not able to distinguish
between different classes of phorbol ester receptors. In conclusion,
although our results revealed some differences in ligand and lipid
cofactor sensitivities, Unc-13 represents a high affinity cellular
target for the phorbol esters as well as for the lipid second messenger
diacylglycerol, at least in C. elegans. The use of phorbol
esters or some ``specific'' antagonists of PKC does not
distinguish between cellular pathways involving different PKC isozymes
or novel phorbol ester receptors such as n-chimaerin or
Unc-13.
,
, and
and the
``novel''
,
,
, and
all bind phorbol
esters with high affinity
(8, 9) . The atypical
isozymes,
and
, are unable to respond to the phorbol esters
(10, 11) . Recently, two novel classes of receptors for
the phorbol esters have been found. The members of the chimaerin family
(
-1 or n-chimaerin,
-2,
-1, and
-2
chimaerin) possess a single cysteine-rich region at the N-terminal
domain with high homology to those found in the PKC
(
)
isozymes. The C-terminal domain is unrelated to PKC but has
high homology with the breakpoint cluster region protein. It functions
as a GTPase-activating protein for the small GTP binding protein p21
(12, 13, 14, 15) . We have recently
described that n-chimaerin binds phorbol esters with
properties similar to PKC
(16) . Unc-13, the only identified
member of the third class of phorbol ester receptors, also contains a
single cysteine-rich region as well as a domain with sequence homology
to the C2 domain in PKC. The cDNA for Unc-13 was isolated from a
Caenorhabditiselegans library and encodes a
1734-amino acid protein. Although the function of Unc-13 is not known,
a mutation in the unc-13 gene is known to cause uncoordinated
movement and abnormal accumulation of acetylcholine in the nervous
system of the nematode
(17) . Phorbol ester treatment of C.
elegans likewise causes uncoordinated movement
(18) .
C X
C X
C X
C X
H X
C X
C
(C, cysteine; H, histidine; X, any other amino acid) is highly
conserved between PKC isozymes, chimaerins, Unc-13, diacylglycerol
kinase, and the oncogenes Vav and c-Raf
(20, 21) . Each
cysteine-rich region coordinates two Zn
ions in its
structure
(22) . sn-1,2-diacylglycerol (DAG), a major
second messenger produced through the hydrolysis of
phosphatidylinositol 4,5-bisphosphate or PC, is the endogenous ligand
that binds to the cysteine-rich region of PKC leading to the activation
of the enzyme as well as its membrane translocation
(23, 24) . Since phorbol esters bind to PKC in a fashion
similar to DAG and similarly activate the enzyme, they have provided a
powerful tool to assess the cellular roles of PKC in signal
transduction. It is reasonable to assume that the non-kinase phorbol
ester receptors may be also cellular targets for DAG. In this study, we
analyzed the properties of the Unc-13 protein as a receptor for
DAG/phorbol esters. A critical issue is that a previous report
suggested that Unc-13 bound phorbol esters with an affinity much lower
than that previously described for the PKC isozymes. A similar
reduction in diacylglycerol affinity would render Unc-13 unresponsive
to physiological concentrations of diacylglycerol. In these previous
studies using a recombinant cysteine-rich region of the Unc-13 protein
expressed in Escherichia coli, Ahmed and co-workers
(20) had reported that [
H]PDBu bound with
an affinity of 67 nM. In contrast, we found that PKC isozymes
and n-chimaerin expressed in baculovirus, as well as
recombinant cysteine-rich regions of PKC isozymes expressed in
bacteria, bind [
H]PDBu with much greater affinity
(0.1-1 nM). Although Unc-13 was first described as a
phospholipid-independent phorbol ester receptor
(17) , a second
report suggested that the binding of phorbol esters to the
cysteine-rich region of Unc-13 required the lipid cofactor
(20) .
isozyme). In contrast to previous reports, our results
indicate that Unc-13 binds phorbol esters with high affinity as was
also described for the PKC isozymes, although with modest differences
in structure-activity relations and phospholipid requirements.
Materials
[H]PDBu
(20.7 Ci/mmol) was purchased from DuPont NEN.
[
H]Bryostatin 1 (4.8 Ci/mmol) was prepared as we
previously described for [
H]bryostatin 4
(25) . Mezerein, thymeleatoxin, (-)-octyl-indolactam V,
PDBu, and 12-deoxyphorbol ester were obtained from LC Services Corp.
(Woburn, MA). OAG was purchased from Avanti Polar Lipids (Pelham, AL).
Phospholipids were obtained from Sigma. Calphostin C was purchased from
Calbiochem. Reagents for expression and purification of recombinant
proteins in E. coli were purchased from Pharmacia Biotech Inc.
Expression and Purification of Recombinant
Cysteine-rich Regions in E. coli
Fragments encoding the
cysteine-rich regions of Unc-13 and PKC were generated by PCR.
For Unc-13, a mixed stage C. elegans library (Stratagene) was
amplified, and the phage cDNA was isolated using standard techniques
(26) and used as a template for the PCR reaction. The following
oligonucleotides were used: 5`-ACTCTACAAGCATGGATCTACCCGATC-3` and
5`-CGATCTCTGATAACATTGACAAGTGA-3`, corresponding to nucleotides
1851-1877 and 2097-2122 in the sequence of Unc-13 as
reported by Maruyama and Brenner
(17) , respectively. The second
cysteine-rich region of PKC
was generated by PCR using the
full-length mouse cDNA clone
(27) as a template and the
following oligonucleotides containing BamHI and EcoRI
sites (underlined): 5`-TGAGGATCCCACCGATTCAAGGTTTATAAC-3` and
5`-AT-CGAATTCACACAGGTTGGCCACCTTCTC-3`. The corresponding PCR fragments
were subcloned into the pCRII vector using the TA cloning system
(Invitrogen). An EcoRI- EcoRI insert for Unc-13 and a
BamHI- EcoRI insert for PKC
were isolated and
ligated in frame in pGEX vectors (Pharmacia) to get the pGEXUnc-13 and
pGEX
plasmids, respectively. The fragments were sequenced to
confirm complete homology with the published sequences. To induce the
expression of the GST-fused recombinant cysteine-rich regions, E.
coli XL1-blue (Stratagene) was transformed with the pGEXUnc-13 and
pGEX
plasmids, the bacteria were grown at 30 °C to an
A
of 0.5-0.7 in 1 liter of LB media
containing 50 µg/ml ampicillin, and the recombinant proteins were
induced by the addition of 0.5 mM
isopropyl-
-D-thiogalactopyranoside. After 5-6 h,
the bacteria were pelleted at 4,000
g, resuspended in
20 ml of phosphate-buffered saline, and disrupted by sonication.
Purification of GST-fusion proteins was done with glutathione-Sepharose
4B according to the manufacturer's instructions (Pharmacia). The
purified recombinant proteins (in 50% glycerol) were kept at -70
°C. Although storage did not seem to affect the binding capacity of
the PKC
cysteine-rich region, some loss of activity was found in
preparations of Unc-13 after several months at -70 °C, and
therefore fresh preparations were used in all cases. Binding of [
H]PDBu and
[
H]Bryostatin
1-[
]PDBu binding was measured using
the polyethylene glycol precipitation assay developed in our laboratory
(28) . The assay was performed in a total volume of 250 µl,
using 100 µg/ml phospholipid (100% phosphatidylserine, unless
otherwise indicated), 1 mM EGTA (or 0.1 mM CaCl
when indicated), different concentrations of
[
H]PDBu, and affinity-purified GST-fusion
proteins (0.4 µg/ml). Detailed descriptions for Scatchard and
competition assays can be found elsewhere
(8, 28) . In
binding assays performed with total cellular lysates as the source of
receptor, pellets from 5-ml bacterial cultures were lysed by sonication
in 1 ml of lysis buffer (50 mM Tris-Cl, pH 7.4, 1 mM EGTA, 50 µg/ml phenylmethylsulfonyl fluoride, and 250 µg/ml
leupeptin), and 50 µl of lysate/tube were used. Incubation was
carried out at 18 °C for 5 min. Inhibition of
[
H]PDBu binding by calphostin C was done using
the conditions described in Ref. 16. Phospholipid preparations were
obtained by sonication, as described in Ref. 29.
H]Bryostatin 1 binding was measured by a
filtration assay, using Triton X-100/phosphatidylserine-mixed micelles,
in the presence of 0.1 mM EGTA, as we previously described
(30) .
Expression of the Cysteine-rich Region of Unc-13 in
E. coli
The GST expression system was used to express the
cysteine-rich region of Unc-13 in E. coli. After
isopropyl--D-thiogalactopyranoside induction, high levels
of recombinant GST-Unc-13 were found in lysates of E. coli transformed with the pGEXUnc-13, as revealed by the appearance of
a 33-kDa band in Coomassie Blue-stained polyacrylamide gels. By using
glutathione-Sepharose 4B beads, the GST-Unc-13 was purified from
bacterial lysates to about 90% purity. Similarly, high levels of
expression were found in lysates of E. coli transformed with
the pGEX
plasmid after
isopropyl-
-D-thiogalactopyranoside induction
(Fig. 1 A). In contrast to the cysteine-rich region of
PKC
expressed as a GST-fusion protein, the majority of the
recombinant GST-Unc-13 protein was insoluble when expressed in
bacteria, and recovery after purification from the soluble fraction was
comparatively lower. Overexpression of the recombinant cysteine-rich
regions of Unc-13 and PKC
resulted in high levels of
[
H]PDBu binding in bacterial lysates
(Fig. 1 B). Recombinant cysteine-rich regions of Unc-13,
PKC isozymes, and n-chimaerin bound
Zn (Ref. 21
and data not shown), as was also shown by others
(20) .
Figure 1:
Expression of recombinant cysteine-rich
regions in E. coli. A, bacterial cultures overexpressing
cysteine-rich regions of Unc-13 or PKC were lysed and subjected
to SDS-polyacrylamide gel electrophoresis. The gels were then stained
with Coomassie Blue. Positions of molecular weight markers are shown on
the left. Total lysates are shown in lanesA and C. The corresponding GST-fusion proteins were
affinity purified as described under ``Experimental
Procedures'' and are shown in lanesB and
D. B, binding of [
H]PDBu to
bacterial pellets (from 5-ml cultures) using 20 nM of the
radioligand and 50 µl of lysate.
Binding of Phorbol Esters and Related Ligands to
Unc-13
[H]PDBu binding to the
purified cysteine-rich regions was measured. Scatchard analysis
revealed that [
H]PDBu bound to Unc-13 with high
affinity in the presence of phosphatidylserine (Fig. 2). The
dissociation constant ( K
) for the ligand
was 1.3 ± 0.2 nM ( n = 3). Calcium (0.1
mM) did not affect the binding of
[
H]PDBu to the receptor. Binding of
[
H]PDBu to the purified recombinant cysteine-rich
region of PKC
revealed similar affinity to that obtained for
Unc-13, either in the presence or absence of calcium ().
The K
of [
H]PDBu
for Unc-13 was substantially lower, i.e. higher binding
affinity, than that first reported by Ahmed et al.(20) . The affinity of the ligand for the recombinant
cysteine-rich regions was similar when the receptor was cleaved with
thrombin (to separate it from the GST) and further purified by high
pressure liquid chromatography (data not shown). Likewise, single
cysteine-rich regions of PKC
and n-chimaerin bound
[
H]PDBu with K
values of 2.4 ± 0.7 nM ( n = 3)
and 2.0 ± 0.3 nM ( n = 3), respectively,
in the presence of phosphatidylserine vesicles (Ref. 31 and data not
shown).
Figure 2:
[H]PDBu binding to
the cysteine-rich regions of Unc-13 ( A) and PKC
( B). The recombinant GST-fusion proteins were incubated for 5
min (18 °C) with increasing concentrations of
[
H]PDBu (0.25-8 nM), 100 µg/ml
phosphatidylserine, and 0.1 mM EGTA, and binding was measured
using the polyethylene glycol precipitation assay. A representative
experiment for each receptor is shown. Each experiment was performed
three times. Each point represents the mean of three experimental
values, generally with a standard error of <2%. Insets are
the Scatchard plots derived from the corresponding binding
curves.
To analyze receptor-ligand interactions, we quantitated
competition of [H]PDBu binding with different
classes of analogs, having different patterns of selectivity among PKC
isozymes and different spectra of biological activities. The series of
compounds tested, which included a 12-deoxyphorbol ester, an indole
alkaloid (octylindolactam V), mezerein, a mezerein analog
(thymeleatoxin), and a diacylglycerol (OAG), spanned a range of 3
orders of magnitude in binding affinity for PKC isozymes. Previous
results from our laboratory revealed significant differences in
structure-activity relations between PKC isozymes
(8) . Phorbol
esters and 12-deoxyphorbol esters, as well as mezerein and analogs,
show preference for PKC
,
, and
compared with PKC
,
, and
(8) . n-chimaerin showed a
pattern of ligand preference similar to that of PKC
(16) .
In this study, we found that the binding affinities for the different
compounds tested were slightly weaker (2-5 times) for Unc-13 when
compared with the PKC
cysteine-rich region (). The
K
for the diacylglycerol OAG for binding
to the cysteine-rich region of PKC
was 142 nM, a value
that is very similar to that obtained with PKC isozymes and
n-chimaerin. Unc-13 bound OAG with slightly lower affinity
( K
= 380 nM). The
mezerein analog thymeleatoxin, which is the most dramatic example of
selectivity between PKC isozymes ( i.e. 20-fold weaker affinity
for PKC
than PKC
, see Ref. 8) bound with similar affinity
to both the PKC
and Unc-13 cysteine-rich regions.
H]bryostatin 1 (Fig. 3) revealed a
K
of 11.5 ± 2.0 nM for
the cysteine-rich region of Unc-13 under our standard assay condition
for bryostatin binding
(30) . Because these assays are carried
out in 0.1% Triton X-100, 20% phosphatidylserine, absolute potencies
are not comparable with those determined in phosphatidylserine alone.
The cysteine-rich region from PKC
bound
[
H]bryostatin 1 with about 7-fold higher affinity
than that of Unc-13 ( K
PKC
=
1.7 ± 0.2 nM). Phospholipid Dependence of [
H]PDBu
Binding-Reconstitution of phorbol ester binding by
phospholipids depends both on the nature and concentration of the lipid
cofactor
(29) . Specific [
H]PDBu binding
to single cysteine-rich regions of PKC was shown to be phospholipid
dependent
(32) . PS is the most efficient of the phospholipids
for reconstitution of binding, although other anionic phospholipids
work as lipid cofactors as well
(16, 29) . The
concentration of PS required to reconstitute
[
H]PDBu binding by 50% (ED
) for the
Unc-13 cysteine-rich region was 58 ± 13 µg/ml; maximum
binding was obtained at about 300 µg/ml PS. The cysteine-rich
region of PKC
showed a lower ED
for PS (15 ±
2 µg/ml) and reached saturation at about 100 µg/ml
(Fig. 4). Interestingly, under the same assay conditions, the
full-length n-chimaerin as well as a PKC isoform (PKC
)
showed ED
values for reconstitution by PS of 14 and 12
µg/ml, respectively
(16) .
Figure 3:
Binding of
[H]bryostatin 1 to recombinant cysteine-rich
regions of Unc-13 ( A) and PKC
( B). Binding of
[
H]bryostatin 1 was measured with the GST fusion
proteins using increasing concentrations of the radioligand
(0.5-10 nM), phosphatidylserine-Triton X-100-mixed
micelles, and 0.1 mM EGTA. Each experiment was performed four
times, and for each recombinant protein a representative experiment is
shown. The K values, expressed as mean ± S.E., are
presented in the toprightcorner of the
corresponding panel. Insets are the Scatchard plots
derived from the corresponding binding
curves.
Figure 4:
Reconstitution of
[H]PDBu binding to the cysteine-rich regions of
Unc-13 and PKC
by phosphatidylserine vesicles. Binding was
performed using 0.1 mM EGTA and increasing concentrations of
phosphatidylserine. Values represent the means of four experiments for
either Unc-13 ( closedcircles) or PKC
( opencircles). Standard errors are represented by the
errorbars.
The ability of various
phospholipids to reconstitute phorbol ester binding to Unc-13 was
tested. Using a fixed concentration of phospholipids (300 µg/ml),
we found that all the other acidic phospholipids (phosphatidylinositol,
phosphatidylinositol 4-phosphate, phosphatidylinositol
4,5-bisphosphate, phosphatidylglycerol, phosphatidic acid) in a
proportion of 20% in PC were able to reconstitute binding to the levels
obtained with PS. Interestingly, partial reconstitution (38 and 50%,
respectively) was observed with the neutral phospholipids PC and 20%
PE, 80% PC. In contrast to the cysteine-rich region of Unc-13, at 300
µg/ml both acidic and neutral phospholipids were able to fully
reconstitute binding to the cysteine-rich region of PKC
(I). Concentration-response curves with the cysteine-rich
region of PKC
gave ED
values for reconstitution of
[
H]PDBu binding of 86 ± 31 µg/ml and
111 ± 33 µg/ml for 20% PE, 80% PC and PC, respectively.
Higher concentrations of neutral phospholipids were required for
reconstitution of [
H]PDBu binding to Unc-13. In
this latter case, the ED
for 20% PE, 80% PC was 182
± 32 µg/ml, and the ED
for PC was 444 ±
62 µg/ml. For comparison, [
H]PDBu binding to
full-length recombinant PKC
(baculovirus expressed, see Ref. 8)
was fully reconstituted by acidic phospholipids and only partially by
neutral phospholipids, suggesting a different lipid requirement between
the cysteine-rich region and the intact enzyme (I). It is
possible that amino acid residues outside the cysteine-rich regions may
contribute to the differences observed between the native protein and
the phorbol ester binding domain, as suggested by Bell and co-workers
(32) . Inhibition of [
H]PDBu Binding to
Unc-13 by Calphostin C-Calphostin C, a compound isolated
from Cladosporium cladosporoides, was described as a PKC
inhibitor acting at the regulatory domain of PKC
(33) . We found
here that calphostin C also inhibited phorbol ester binding to the
cysteine-rich region of Unc-13 (Fig. 5). The ED
for
the inhibitor was 8.9 ± 1.1 µM.
[
H]PDBu binding to the cysteine-rich region of
PKC
was inhibited by calphostin C with an ED
of 4.3
± 0.6 µM. Similar inhibition patterns with
calphostin C were also found for PKC
and n-chimaerin in
a previous report
(16) . Our results indicate that the target
site for calphostin C is the cysteine-rich region, and this compound,
originally described as a selective PKC inhibitor, is not able to
discriminate between the different classes of phorbol ester receptors.
Figure 5:
Inhibition of
[H]PDBu binding by calphostin C. Increasing
concentrations of calphostin C were used to compete 3 nM
[
H]PDBu. Calphostin C was preincubated under
ordinary fluorescent light (15 min) together with the reaction mix, as
described in Ref. 16. Values represent the mean ± S.E. of a
representative experiment in which determinations were performed in
triplicate. Two other experiments gave similar results. Openbars, GST-Unc-13 cysteine-rich region; darkbars, GST-PKC
cysteine-rich region; hatchedbars, full-length PKC
.
as a phorbol ester receptor. Both receptors
were virtually indistinguishable as determined by phorbol ester
binding, structure-activity relations, phospholipid dependence, and
inhibitor sensitivity
(16) . In this paper, the quantitative
analysis of the interaction between phorbol esters and Unc-13 likewise
reveals that this protein from C. elegans behaves as a high
affinity phorbol ester receptor. The cysteine-rich region of Unc-13
showed significant but modest differences in structure-activity
relations and phospholipid dependence when compared with a similar
domain in PKC
.
for the
ligand [
H]PDBu for binding to the cysteine-rich
region of Unc-13 expressed in E. coli was 1.3 nM,
which is a much higher affinity than that originally described for this
receptor expressed in bacteria
(20) . The reduced binding
affinity observed in that former study may reflect alterations in the
conformation of the recombinant cysteine-rich region due to the
conditions for the expression of the protein and/or the binding assay.
Our results suggest that single cysteine-rich regions from PKC
,
Unc-13, and n-chimaerin
(16) are high affinity binding
sites in the corresponding proteins for both phorbol esters and
diacylglycerol. In contrast, the cysteine-rich regions from the
oncogenes vav and c- raf, as well as that from the PKC
isozyme, were not able to bind phorbol ester or related ligands
even with low affinity, although it is highly probable that a similar
structure coordinating Zn
also occurs in these
proteins
(21) .
(
)
. In
addition, diacylglycerols also bound with similar potency to PKC
isozymes and n-chimaerin
(8) . Given that
diacylglycerol and the phorbol esters interact at the same binding
sites, we could assume that the non-PKC targets also mediate cellular
signaling pathways mediated by the second messenger diacylglycerol. The
fact that different lipid requirements exist for different
cysteine-rich regions complement our previous results that
phospholipids differentially regulate phorbol ester binding to
different PKC isozymes
(8) . At the intracellular level, such a
difference in lipid cofactor requirement could constitute a major
regulatory factor for ligand-protein interaction for the different
receptors.
Table: Dissociation constants
(K ) for
[
H]PDBu for the cysteine-rich
regions of Unc-13 and PKC
. Each K
value
represents the mean ± S.E. The number of experiments is shown in
parentheses.
Table: Structure-activity analysis of binding to
the cysteine-rich regions of Unc-13 and PKC
H]PDBu (3 nM) binding with the
individual compounds using 100 µg/ml PS and 1 mM EGTA. Six
to eight increasing concentrations (in triplicate) of the competing
ligand were used. The ID
values were determined from the
competition curves, and the corresponding K
values for the ligands were obtained (8). Values represent the
mean ± S.E. of the number of experiments in parentheses.
Table: Reconstitution of
[H]PDBu binding by different
phospholipids
, phosphatidylinositol 4,5-bisphosphate; PA,
phosphatidic acid; PG, phosphatidylglycerol.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.