From the Molecular Mechanisms of Tumor Promotion
Section, Laboratory of Cellular Carcinogenesis and Tumor Promotion and
§ Laboratory of Medicinal Chemistry, NCI, National
Institutes of Health, Bethesda, Maryland 20892 and the
¶ Georgetown Institute for Cognitive and Computational Science and
Departments of Oncology and Neuroscience, Georgetown University Medical
Center, Washington, D.C. 20007
Received for publication, November 6, 2000, and in revised form, February 5, 2001
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ABSTRACT |
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The C1 domains of conventional and novel
protein kinase C (PKC) isoforms bind diacylglycerol and phorbol esters
with high affinity. Highly conserved hydrophobic residues at or near
the rim of the binding cleft in the second cysteine-rich domain of PKC- The protein kinase C
(PKC)1 family of
serine/threonine kinases plays a central role in mediating the signals
that lead to divergent cellular functions (1, 2). The structure of the
PKCs is composed of an N-terminal regulatory region and a C-terminal
catalytic region. The regulatory region modulates enzymatic activities
by interacting with endogenous and exogenous activators and cofactors of PKCs through subdomains such as the pseudosubstrate region and the
C1 and C2 domains (3, 4). The twin C1 domains, a tandem repeat of a
cysteine-rich, zinc finger structure, are the binding sites for the
endogenous PKC ligand sn-diacylglycerol (DAG) and for the
phorbol ester tumor promoters (5, 6).
The C1 domain consists of a conserved 50 amino acid sequence possessing
the motif
HX12CX2CX13/14CX2CX4HX2CX7C
(C, cysteine; H, histidine; X, any other amino acid) and
coordinating two Zn2+ ions (7). The solution structure of
the C1b domain of PKC- The residues critical for maintaining the overall structure and ligand
binding by the C1 domain have been explored in several studies (9,
11-14). The 2 histidines and all but 1 of the 6 conserved cysteines
that coordinate the two Zn2+ ions, namely the residues at
positions 3, 8, 11, 21, 24, 27, and 38, are vital for structural
integrity and the interaction with ligands. Two loops at positions
7-12 and 20-27, which comprise most of the In addition to ligand binding, the C1 domain also contributes to
membrane interaction as does the C2 domain of the classical PKCs and
the pseudosubstrate region (15, 16). Less is known about the residues
responsible for membrane interaction.
Since its discovery as a conserved structural module, the C1 domain has
been found to be present in a range of novel proteins, distinct from
the PKCs, which constitute the superfamily of phorbol ester/DAG
receptor proteins. The chimaerins, Munc-13, PKD/PKC-µ, and RasGRP
exemplify subgroups within this emerging superfamily. The chimaerins,
RasGRP, and PKC isoforms differ in their ligand recognition, reflecting
both differences in lipid requirements as well as in intrinsic
specificity. Understanding the structural basis for ligand selectivity
remains largely unresolved. Phe-20 had been suggested to be involved in
differential affinity of Guided by computer modeling, NMR, and x-ray crystallography, we sought
in the present study to use site-directed mutagenesis to explore three
aspects of C1 domain functions in parallel: ligand recognition, lipid
interaction, and ligand selectivity. We mainly focused on four residues
at positions 13, 20, 22, and 24 along the rim of the ligand-binding
pocket that are exposed to the surface and positioned toward the
membrane (9). Residues of varying hydrophilicity such as Tyr, Asp, Lys,
and Arg were introduced at these positions in order to change the
overall surface hydrophobicity around the binding cleft, therefore
affecting their interaction with lipids and ligands. In order to
explore residues potentially important for determining the specific
binding activity of different C1 domains, we also mutated select
residues in PKC- Materials--
[20-3H]Phorbol 12, 13-dibutyrate
(PDBu) (20 Ci/mmol) was purchased from PerkinElmer Life
Sciences. PDBu was obtained from Alexis Biochemicals
(Pittsburgh, PA). Synthesis of
1-(4-methyl-3-(methylethyl)pentanoyl)-2-(3-methylbut-2-enoyl)-sn-glycerol (97F31, the branched DAG) will be reported
elsewhere.3
Phosphatidyl-L-serine was purchased from Sigma.
sn-1-Palmitoyl-2-oleoylphosphatidylserine (POPS) and
sn-1-palmitoyl-2-oleoylphosphatidylcholine (POPC) were purchased from Avanti Polar Lipids (Alabaster, AL). Reagents for expression and purification of glutathione S-transferase
(GST) fusion proteins were obtained from Amersham Pharmacia Biotech.
Expression and Purification of the C1b Region of PKC- [3H]PDBu Binding Assay--
[3H]PDBu
binding to the wild type and mutant GST-
For the determination of dissociation constants (Kd)
and number of binding sites (Bmax), typical
saturation curves with increasing concentrations of
[3H]PDBu (between 0.125 and 4 nM) were
performed in triplicate. Dissociation constants (Ki)
of the branched DAG 97F31 were determined by competition of
[3H]PDBu binding to GST-
For measuring [3H]PDBu binding in the absence of
phospholipids, the Kd was determined by competition
using nonradioactive PDBu as described previously with minor
modifications (18). 7-8 increasing concentrations of unlabeled PDBu
(10 nM to 10 µM) were used to compete 100 nM [3H]PDBu (specific activity 870.2 dpm/pmol) in a typical 250-µl reaction mix. 4-10 µg/tube
PKC- Large Unilamellar Vesicles (LUV)--
The preparation of the
sucrose-loaded vesicles was adopted, with minor modification, from the
procedure of Mosior and Epand (21). Aliquots of lipids, e.g.
POPS and POPC, in chloroform were mixed and dried under a stream of
N2. The lipids were subsequently resuspended in 170 mM sucrose in 20 mM Tris-Cl (pH 7.4) and
subjected to five freeze-thaw cycles by placing in a 42 °C water
bath and in dry ice alternately. LUV were obtained by 40 rounds of
extrusion through a 100-nm polycarbonate membrane in a LipoFast
liposome "factory" (Sigma). Lipid concentrations were monitored by
including a trace amount of [3H]DPPC before extrusion to
ensure an accurate final concentration after extrusion.
Vesicle Binding Assay--
An experimental approach similar to
that described above was used for the determination of
[3H]PDBu binding in the presence of LUV. Briefly, wild
type and mutant GST- [3H]PDBu Binding to PKC-
The wild type and mutant PKC-
We first determined the apparent [3H]PDBu binding
affinities of wild type and mutant PKC-
Analysis of [3H]PDBu binding in the absence of PS permits
the effects of the mutations on the interaction of the C1 domain with
ligand to be separated from those on the interaction of the C1 domain
with lipid or of the C1-ligand complex with lipid (Table II). In the absence of PS, the wild-type
PKC-
Since only properly folded protein is capable of binding to PDBu,
reflected by the changes of Bmax value, we were
only determining the binding properties of mutant protein that had
retained an intact structure. Meanwhile, the [3H]PDBu
binding curves for the wild-type and mutant PKC- Lipid Dependence of PKC-
The binding analysis ± lipid was carried out in the presence of
100% phosphatidylserine (100 µg/ml). Using POPS:POPC mixed vesicles,
we compared the lipid dependence of the wild type and the W22K mutant
of PKC- Binding of DAG to PKC- Implications for the Differential Structure Activities of Other C1
Domains--
Sequence alignment of C1 domains of different classes of
DAG/phorbol ester receptors and related proteins reveals specific changes of some generally conserved residues in the primary amino acid
sequences (Fig. 1). To explore the possible role of such residues in C1
domain function, we mutated the PKC-
The Arg-20 found in the C1 domain of PKC- Dissecting the mechanisms contributing to the selective activation
of members of the DAG/phorbol ester receptors is critical to the
understanding of the signaling through DAG and phorbol esters. Studies
on the PKC family showed that the C1 domain and pseudosubstrate region,
along with the C2 domain in Ca2+-dependent
classical PKCs, act as membrane targeting modules to trigger the
membrane association of PKC and a subsequent conformational change that
activates the kinases (24). Several regulatory factors have been
described that can contribute to isoform selectivity: 1) the
differential lipid requirements of members of this family (22, 24, 25);
2) the differential localization induced by the C1 domain ligands and
cofactors (26); 3) proteins that interact with PKCs, such as RACKs,
RICKs, and STICKs (3, 4); 4) regulation through intra- and
intermolecular phosphorylation (27, 28). Our current study is focused
on the interactions within the ternary complex formed between the C1
domain, the membrane, and the ligands for the C1 domain. Since detailed
structural information exists for the binary complex formed between the
C1b domain of PKC and its ligand as determined by NMR and x-ray
crystallography (8 -10, 14), we sought to use this domain as a model to
address the effect of lipid on the C1 domain and ligand interaction
with particular emphasis on the hydrophobic interactions within the
ternary complex.
The lipophilicity of C1 ligands is one element contributing to the
pattern of translocation of GFP-PKC- The ability of phorbol esters and related compounds to bind to C1
domains in the absence of phospholipids may have significant implications in the selective activation of the soluble, non-membrane associated PKCs. PKC is known to interact with both cytoskeletal and
nuclear proteins in the absence of lipids. However, the mode of their
binding and activation is less known. Prekeris et al. (33)
showed that PKC- We probed the determinants in the C1 domain for the selective
recognition of DAG versus phorbol esters. Although both DAG and phorbol ester are thought to bind to the same binding sites in the
C1 domain, differences in their mechanisms of interaction have been
described. The low and high affinity binding sites within the twin C1
domains of PKC- Members of the DAG/phorbol ester receptors exhibit different
patterns of ligand selectivity and lipid requirements (13, 23).
However, the structural features that account for selective PKC isozyme
modulation are less known. To further our understanding of the
structure activity relations of other classes of DAG/phorbol ester
receptors, we probed the role of specific residues in other C1 domains
by mutating the corresponding residues in PKC- By mutating hydrophobic residues located at the rim of the ligand
binding cleft in PKC- (PKC-
C1b) were mutated to probe their roles in ligand
recognition and lipid interaction. [3H]Phorbol
12,13-dibutyrate (PDBu) binding was carried out both in the presence
and absence of phospholipids to determine the contribution of lipid
association to the ligand affinity. Lipid dependence was determined as
a function of lipid concentration and composition. The binding
properties of a high affinity branched diacylglycerol with
lipophilicity similar to PDBu were compared with those of PDBu to
identify residues important for ligand selectivity. As expected, Leu-20
and Leu-24 strongly influenced binding. Substitution of either by
aspartic acid abolished binding in either the presence or absence of
phosphatidylserine. Mutation of Leu-20 to Arg or of Leu-24 to
Lys caused a dramatic (340- and 250-fold, respectively) reduction in PDBu binding in the presence of lipid but only a modest
reduction in the weaker binding of PDBu observed in the absence of
lipid, suggesting that the main effect was on C1 domain -phospholipid
interactions. Mutation of Leu-20 to Lys or of Trp-22 to Lys had modest
(3-fold) effects and mutation of Phe-13 to Tyr or Lys was without
effect. Binding of the branched diacylglycerol was less dependent on
phospholipid and was more sensitive to mutation of Trp-22 to Tyr or
Lys, especially in the presence of phospholipid, than was PDBu. In
terms of specific PKC isoforms, our results suggest that the presence
of Arg-20 in PKC-
may contribute to its lack of phorbol ester
binding activity. More generally, the results emphasize the interplay
between the C1 domain, ligand, and phospholipid in the ternary
binding complex.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
was determined by NMR. The domain adopts a
globular fold allowing two non-consecutive sets of residues to form the
two separate zinc-binding sites (8). The x-ray crystallographic
structure of the C1b domain of PKC-
in complex with phorbol
13-acetate in the absence of phospholipid revealed that phorbol
13-acetate binds in a hydrophilic groove between two pulled-apart
strands at the tip of the domain. Phorbol ester binding caps the
hydrophilic groove and generates a contiguous hydrophobic surface
covering one-third of the domain, thereby facilitating the membrane
insertion of the domain (9). These conclusions are supported by
subsequent NMR analysis of the C1b domain of rat PKC-
in solution
titrated with lipid micelles in the presence and absence of phorbol
ester (10).
2 and
3 segments,
constitute the phorbol ester binding site. Mutations within this region
drastically affect the activity of the C1 domain (12).
2-chimaerin to constrained DAG analogs and
thymeleatoxin (17); the same residue was also suggested to be
responsible for weaker phorbol ester binding of the C1a domains of PKCs
(13). Arg-20 in PKC-
and -
may contribute, along with Gly at
position 11, to its lack of phorbol ester recognition. Trp-22 was
implicated in mediating differential lipid and ligand binding in
PKC-
and PKD/PKC-µ
(15).2 However, few studies
had thoroughly examined the roles of these residues in C1 domain function.
C1b to the unique residues appearing in the C1
domains of PKC-
, PKC-
/
, and n/
-chimaerin, as
well as in PKD/PKC-µ, which were candidates for their differential
binding activity and ligand selectivity, as suggested by sequence
comparisons and our structural modeling studies. The binding activities
of wild type and mutant PKC-
C1b domains for phorbol ester and DAG
were characterized in the presence and absence of phospholipid or in
the presence of lipid vesicles of different compositions. Our results
further our insight into the structural basis of C1 domain function.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Fused to
GST (GST-
C1b) in Escherichia coli--
The C1b domain of PKC-
was generated by polymerase chain reaction using the full-length mouse
PKC-
cDNA as template and was subcloned into a pGEX-2TK vector
(Amersham Pharmacia Biotech). The recombinant plasmid was expressed in
XL1-blue E. coli cells and purified to homogeneity as
described previously (18). Site-directed mutagenesis of the GST-
C1b
fusion proteins was performed with the Unique Site Elimination (U.S.E.)
system (Amersham Pharmacia Biotech) as described previously (12).
Briefly, the pGEX-
C1b plasmid was used as the template and a
PstI selection primer was used to convert a PstI
site to a SacII site within the pGEX plasmid. The mutated
residues are shown in Fig. 1. After two consecutive cycles of
restriction digestion and bacteria expansion, the full-length sequences
of the mutant plasmids were verified by sequencing, which was performed
by the DNA Minicore, Division of Basic Sciences, NCI, National
Institutes of Health (Bethesda, MD). The wild type and mutant
pGEX-
C1b plasmids were transformed into E. coli (XL-1 blue from Stratagene), and the positive clones were subsequently picked
and grown in LB medium supplemented with 100 µg/ml ampicillin. The
expression of GST fusion protein was induced by the addition of 0.5 mM
isopropyl-O-D-thiogalactopyranoside (IPTG). The
bacteria was harvested after 5 h of induction at 37 °C and the
GST-
C1b protein was purified using glutathione-Sepharose 4B beads
following the manufacturer's recommendation (Amersham Pharmacia). The
purity of the protein after eluting from the beads was verified by
SDS-PAGE and staining with Coomassie Blue.
C1b domains was measured
using the polyethylene glycol precipitation assay developed in our
laboratory (19) with minor modifications. The assay mixture (250 µl)
contained 50 mM Tris-Cl (pH 7.4), 100 µg/ml phosphatidylserine, 4 mg/ml bovine immunoglobulin G,
[3H]PDBu, and variable concentrations of competing
ligand. Incubation was carried out at 37 °C for 5 or 30 min. Samples
were chilled to 0 °C for 10 min, 200 ml of 35% polyethylene glycol
in 50 mM Tris-Cl (pH 7.4) was added, and the samples were
incubated at 0 °C for an additional 15 min. The tubes were
centrifuged in a Beckman 12 microcentrifuge at 4 °C (12,000 rpm, 15 min). A 100-µl aliquot of the supernatant was removed for the
determination of the free concentration of [3H]PDBu, and
the pellet was carefully dried. The tip of the centrifuge tube
containing the pellet was cut off and transferred to a scintillation vial for the determination of the total bound [3H]PDBu.
Aquasol was added both to an aliquot of the supernatant and to the
pellet, and radioactivity was determined by scintillation counting.
Nonspecific binding was measured using an excess of nonradioactive PDBu
(30 µM). Specific binding was calculated as the
difference between total and nonspecific binding (20).
C1b using 6-8 increasing
concentrations of the 97F31. ID50 values were determined
from the competition curve, and the Ki for the
competing ligand was calculated from its ID50 using the
relationship Ki = ID50/(1 + L/Kd), where L is the
concentration of free [3H]PDBu and Kd
is the dissociation constant for PDBu. Approximately 30 ng/tube
PKC-
C1b domain was used for each assay. In cases in which the
Kd of PDBu for the mutant PKC-
C1b domain was >20
nM, dissociation constants for PDBu were determined by
competition using non-radioactive PDBu competing with
[3H]PDBu (specific activity 870.2 dpm/pmol). Values
represent the mean of n experiments, as indicated, with
triplicate determinations for each point in each competition curve in
each experiment. Mutant protein (4 µg/tube) was used for each
competition assay. The amount of protein was adjusted for each assay so
that the specific binding was over 60-80% of total binding and the
total bound [3H]PDBu was below 30% of the total amount
of [3H]PDBu in the assay.
C1b domain was used in each assay, and the amount was adjusted
so that the specific binding was over 60-80% of total binding and the
total bound [3H]PDBu was below 30% of the total amount
of [3H]PDBu in the assay. Nonspecific binding, determined
in the presence of 100 µM PDBu, was 20-40% of total
bound in the absence of unlabeled PDBu. The data were fitted to the
theoretical inhibition curve, and the Kd was
calculated from the equation Kd = ID50
L, where ID50 is the concentration of the
nonradioactive PDBu that displaced the binding of the
[3H]PDBu by 50% and L is the concentration of
free radioligand at the ID50. Since some of the C1 domain
bound to the walls of the assay/centrifuge tube, both the pellet and
tube were counted.
C1b domains were incubated with
[3H]PDBu (2 nM), sucrose-loaded vesicles
comprising POPS and POPC in the presence of 50 mM Tris
buffer (pH 7.4), 100 mM KCl, and 5 mg/ml
-globulin. The
concentration of the total phospholipid in the assay was 200 mM unless otherwise indicated. Incubations were carried out
at 22 °C for 5 min, followed by polyethylene glycol precipitation as
described above for the [3H]PDBu binding assay. 15 ng to
4 µg/tube of protein was used in each assay, and the amount was
optimized so that the specific binding was over 60-80% of total
binding and the total bound [3H]PDBu was below 30% of
the total amount of [3H]PDBu in each binding assay.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
C1b Mutants in the
Presence or Absence of Lipid--
Mutations were introduced in
PKC-
C1b using site-directed mutagenesis as described under
"Experimental Procedures." The mutated residues and their positions
are indicated in Fig. 1A. The
three-dimensional structure of the four hydrophobic residues in
relation to the orientation of PDBu in complex with PKC-
C1b is
illustrated in Fig. 1B.
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Fig. 1.
A, the mutagenic scheme on PKC- C1b
and the sequence alignment of the cysteine-rich regions from different
classes of DAG/phorbol ester receptors. The sequence of the
second cysteine-rich domain of PKC-
, PKC-
C1b, is shown together
with the mutated amino acids and the sites of mutation as marked with
*. The elements of secondary structure are shown above the sequence. In
the consensus, the PKC-
and -
sequences are from bovine
(b) and the PKC-µ and chimaerin sequences are from human
(h). Residues which are the same as the mutations made in
PKC-
C1b are shown in bold. B, the modeled complex
structure of PDBu in complex with the C1b domain of PKC-
(see
Footnote 4). It is of note that based upon our predicted model for PDBu
and the x-ray structure for phorbol 13-acetate, PDBu and phorbol
13-acetate form the same hydrogen bonding network with
PKC-
C1b.
C1b domains were expressed in E. coli as GST fusion proteins. Protein expression was induced by the
addition of IPTG. In initial experiments, the expression level after
induction with IPTG was monitored every hour. An incubation time of
4-5 h after the addition of IPTG was found to be optimal and was then
used subsequently for all GST PKC-
C1b fusion proteins. The fusion
proteins were predominantly expressed in the cytoplasm and were
isolated from the soluble fraction by binding to glutathione-Sepharose 4B beads. The purified proteins were detected on SDS-PAGE with an
apparent molecular mass of 33 kDa. All mutant proteins appeared the
same size on SDS-PAGE as the wild-type protein. No shift in molecular
weight was observed (data not shown).
C1b domains in the presence
of phosphatidylserine (Table I).
[3H]PDBu bound to the wild type PKC-
C1b domain with a
Kd of 0.49 nM, in good agreement with
our previous value (12, 18). The mutants varied markedly in their
affinities for PDBu. Replacement of Leu-20 or Leu-24 with Asp led to
complete loss of measurable binding. Several hundred-fold loss of
activity was observed for L20R and for L24K. In contrast, only a 3-fold
effect was observed for L20K, comparable to that for W22K. The other
mutations caused less than a 2-fold change. The
Bmax values of all the mutants were reduced
compared with the wild type, with the L20K, L20F, and L24K mutants
being 3-6-fold lower. This reduction presumably reflects stability or
ease of folding and did not correlate with the changes in binding
affinity. Due to the variable Bmax, the amount
of protein used was optimized to give above 60-80% specific binding
for [3H]PDBu and below 30% of total
[3H]PDBu bound in each binding assay.
[3H]PDBu binding to mutants of C1b domain in PKC-
C1b domain in the
presence of 100 µg/ml PS. Seven increasing concentrations (in
triplicate) of [3H]PDBu were used. Values represent the
mean ± S.E. of the number of experiments in parentheses. ND, not
detected; mut., mutant.
C1b domain showed a 70-fold decrease in [3H]PDBu
binding affinity. This value agrees well with that reported previously
(18). As in the presence of lipid, neither the L20D nor L24D mutants
displayed detectable [3H]PDBu binding. Dramatically, the
L20R and L24K mutants, which showed 340- and 250-fold weaker binding
than the wild type in the presence of lipid, showed decreases of only
6.7- and 2.9-fold, respectively, compared with wild type in the absence
of phospholipid; indeed, their absolute binding affinities were similar
in the presence or absence of phospholipid. It thus appears that these mutations have limited effect on the ligand binding per se
but interfere with formation of the complex with phospholipid. The L20K
mutant showed a modest (3.2-fold) decrease in binding affinity in the
absence of lipid, similar to its decrease in the presence of
phospholipid (3.2-fold), suggesting that the effect was on the ligand
binding. Conversely, the W22K mutant showed virtually no decrease in
the absence of phospholipid (1.1-fold) compared with a 2.9-fold
decrease in the presence, suggesting that its influence was on lipid
interaction.
[3H]PDBu binding to mutants of the C1b domain in PKC- in
the absence of PS
C1b domain are
consistent with homogeneous binding kinetics. Computer modeling studies
showed that mutations introduced at the hydrophobic residues at the rim
of the binding cleft are unlikely to introduce structural changes to
the overall folding of the zinc finger
protein.4
C1b Mutants--
To evaluate
interaction of the C1 domain-ligand complex with phospholipids
directly, we determined the dose dependence of [3H]PDBu
binding to the wild type and mutant PKC-
C1b domains as a function of
phospholipid concentration, using LUV (Fig.
2). The mutants examined were those whose
binding affinities were measurable and dependent on the presence of
lipid. The F13K and F13Y and L20K mutants showed either no or little
shift, whereas the W22K and W22Y mutants showed intermediate shifts in
the dose-response curves. The results of the lipid reconstitution thus
show good qualitative agreement with the conclusions from the binding
analysis in the presence and absence of phospholipid.
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Fig. 2.
[3H]PDBu binding to
PKC- C1b and its mutants as a function of
concentration of phospholipid. Binding was carried out as
described under "Experimental Procedures." The final concentration
of phospholipid in the form of LUVs in the reaction was 0.2 mM and the vesicles were composed of 70 mol% POPS and 30 mol% POPC. Values represent the mean ± S.E. of at least three
experiments per mutant.
C1b as a function of the mol% POPS at a constant total
concentration of lipid of 0.2 mM (Fig.
3A). The mol% POPS for 50%
reconstitution under these conditions shifted from 13.5% for the wild
type to 46.5% for the W22K mutant. Lipid conditions that are marginal
for reconstitution of ligand binding should be reflected in a decreased
apparent affinity for ligand binding. Thus, at POPS:POPC (50:50), which
is saturating for binding of PDBu to the wild type but not for the W22K
mutant of PKC-
C1b, the Kd for the mutant was
6.38 ± 1.11 nM compared with 0.35 ± 0.15 nM for the wild type, a difference of 20-fold, whereas no
or less difference in relative Kd value was seen in the absence of lipid or under lipid conditions that gave rise to
maximal reconstitution (Fig. 3B). We have described
elsewhere similar differential effects of lipid composition on ligand
binding to different phorbol ester receptors with different lipid
requirements, namely PKC-
and RasGRP (22).
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Fig. 3.
The differential phospholipid dependence of
mutant W22K compared with the wild type
PKC- C1b. A, the reconstitution
of [3H]PDBu binding to PKC-
C1b and its mutant W22K by
mol% POPS. Binding was measured in the presence of 2 nM
[3H]PDBu with increasing mol% of POPS with POPC as the
remainder. The final concentration of LUV was 0.2 mM.
B, comparison of the [3H]PDBu binding
affinities of W22K and wild type PKC-
C1b under different lipid
conditions. The Kd for PKC-
C1b and W22K in the
absence of PS, at 50 mol% POPS and POPC, and at 100% PS were
determined by Scatchard analysis. Values represent the mean ± S.E. of four separate experiments.
C1b Mutants in the Presence or Absence of
Lipid--
97F31 is a synthetic unconstrained branched DAG with
hydrophobicity similar to that of PDBu (log P = 3.88 compared with 3.43 for PDBu). It was selected for the current study
from a series of branched DAGs3 based on its low log
P value with retention of PKC binding affinity. We
determined the binding to wild type and mutant PKC-
C1b domains of
this DAG in the presence and absence of lipid and compared these
results with those for PDBu (Table III).
Whereas PDBu binding affinity to wild type PKC-
C1b decreased 68-fold
in the absence of phosphatidylserine, the binding affinity of the DAG
only decreased by 20-fold. Thus, in the absence of phospholipids, the
DAG bound with only 15-fold weaker affinity than did PDBu. These
results suggest a weaker contribution of the phospholipid to the DAG
binding. Second, the W22K or W22Y mutants showed reduced affinity in
the absence of phospholipid as well as a reduction in the presence of
phospholipid, suggesting an effect of this residue on the C1 domain-DAG
interaction, which was not seen for PDBu. Finally, the L24K mutant
appeared to have a substantial effect on DAG binding even in the
absence of phospholipid, although the high concentration of ligand used
in the measurements might raise some concerns. Reflecting the
differences both in the structural requirements for binding and the
influence of lipid on the binding for PDBu compared with the DAG, the
relative affinities for PDBu and the DAG in the presence of
phospholipid ranged from as little as 4-fold for the L20R mutant to
250-fold for the W22K mutant.
Inhibition of [3H]PDBu binding to mutants in the C1b domain
of PKC- by 97F31
C1b domain to introduce these
residues, which appear in the other C1 domains.
clearly contributes to its
lack of phorbol ester binding activity. As described above, the L20R in
PKC-
C1b resulted in a >300 fold decrease in [3H]PDBu
binding affinity. Together with the 600-fold decrease in binding
affinity attributable to P11G (11), also found in PKC-
, these
mutations could account for a 2 × 105 decrease in
binding affinity. Phe-20 appears in
2-chimaerin and in the C1a
domain of most PKC isoforms and was a candidate for contributing to
ligand selectivity of
2-chimaerin. We find that mutation of Leu-20
in PKC-
C1b to Phe resulted in no changes in its affinity toward
PDBu, the constrained DAG analog B8-DL-B8, or thymeleatoxin, three
compounds that were differentially recognized by
2-chimaerin (17,
23) (Table IV). Lys-22 appears in the C1b
domain of the PKD family. Although we found only a modest difference in
the lipid dependence for the PKC-
C1b mutant W22K, the residue at
this position seemed to be differentially important for DAG binding.
Two other mutants in PKC-
C1b, V38Y and K41Q, showed no substantial
differences compared with wild-type protein (Table IV). Tyr-38 is
present in the C1a domain of PKC-µ and Gln-41 in both
- and
-chimaerins. Expression of the V38Y mutant was problematic,
presumably reflecting the important role of Val-38 in stabilizing
folding of the C1 domain.
Comparison of the binding activities of PKC-C1b mutants and wild
type protein with relevance to differential C1 domain functions
C1b
domains was determined by Scatchard analysis in the presence of 100 µg/ml PS. Binding by thymelcatoxin and
(Z)-(1-(hydroxymethyl)-4-[4-methyl-3-(methylethyl)-pentylidene]-3-oxo-2-oxolanyl)methyl
4-methyl-3-(methylethyl)pentanoate (B8-DL-B8) (36) was evaluated by
competition of [3H]PDBu binding. Values were also determined
for native PKC-
and for other C1 domain-containing proteins with
endogenous sequence differences corresponding to the mutations
introduced into the isolated C1b domain of PKC-
. Values represent
the mean ± S.E. of the number of experiments in parentheses. ND,
not detected.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
in vivo, reflecting the importance and specificity of hydrophobic interactions between the
C1 ligand and the membrane (29). The C1 domain itself makes important
contributions to hydrophobic interactions both with the ligand and the
membrane. Data from NMR and x-ray crystallographic studies indicated a
number of conserved hydrophobic residues: Tyr-8, Met-9, Phe-13, Leu-20,
Tyr-22 (in PKC-
) or Trp-22 (in PKC-
, -
, -
, -
), Leu-24,
and Ile-25, which are exposed at the surface of the protein and form a
hydrophobic cap to facilitate the membrane insertion of the protein
(14). Our previous molecular modeling studies showed that Leu-20,
Leu-24, and Trp-22 contributed to the hydrophobic interactions between
PDBu and PKC-
C1b, but Phe-13 did not have any significant
hydrophobic interaction with PDBu (30). To further evaluate the role of
these hydrophobic residues in the membrane binding and the ligand
selectivity, we mutated these four highly conserved hydrophobic
residues at the rim of the binding cleft. Our results indicated that
the [3H]PDBu binding to mutants in which Leu-20 and
Leu-24 were changed to hydrophilic residues (with the exception of
L20K) were greatly reduced in the presence of lipids but showed little
changes from wild type in the absence of lipids, reflecting a greater
contribution to the interaction with the membrane. In contrast, the
binding affinities of the mutants at residues Phe-13 and Trp-22 were
less affected. The order of their overall impact on lipid interaction was Leu-24 > Leu-20 > Trp-22 > Phe-13. The nature of
the substitution, as expected, greatly influenced the outcome. The
order of the impact of the residues introduced at these positions was
Asp > Arg > Lys > Trp > Phe. Introducing the
negatively charged aspartic acid fully abolished ligand binding
either in the presence or absence of phospholipids. In contrast,
mutating to a hydrophobic residue, phenylalanine, did not change the
affinity of PKC-
C1b. In an initial study probing the structural
determinants in PKC-
C1b for ligand recognition by site-directed
mutagenesis, a L24G mutation completely abrogated the
[3H]PDBu binding and W22G and L20G mutants displayed
partial activity, whereas W22F and F13G mutants showed affinity similar
to that of wild type PKC-
C1b (12). Molecular dynamics simulations
showed that mutation of Leu-24 to Gly significantly altered the
backbone conformation of Gly-23 and residue 24 and mutation of Leu-20
to Gly significantly affected the backbone conformations of Val-25 and
Leu-26.4 These results suggest a role for Leu-20 and Leu-24
in maintaining the binding site conformation in addition to their role
in ligand-PKC and lipid-PKC interactions. Molecular dynamics simulation
showed that mutation of Phe-13 to Gly did not significantly alter the conformation of the binding site. Taken together with the present study, our results showed that Phe-13 played a minimal role in ligand-PKC and lipid-PKC interactions. In comparison, a recent report
by Medkova and Cho (15) on the interplay of the C1 and C2 domains of
PKC-
in its membrane binding and activation indicated a significant
role for the hydrophobic residues Trp-58 and Phe-60 (equivalent to the
Trp-22 and Leu-24 in PKC-
C1b) in the C1a domain of PKC-
for the
membrane penetration and activation of PKC-
, whereas those in the
C1b domain of PKC-
were found to be not directly involved in these
processes. Both we and others have described the non-equivalent roles
of the C1a and C1b domains as well as marked differences in the C1a and
C1b domains of different isoforms (15, 31, 32).
bound to actin in a phorbol
ester-dependent manner. Similarly, F-actin also interacts
with other members of PKC family, and the isoform specificity is
associated with their differential dependence on phorbol esters and
Ca2+ (34). STICKs, RACKS, and RICKS were also described to
recruit the inactive or activated non-membrane-bound PKCs to the
different intracellular locations (3, 4). Here, a 70- and 20-fold weaker binding affinity in the absence of lipids was detected for the
wild-type C1 domain by PDBu and the DAG. Design of ligands that are
selective for either protein-associated or free PKC could cause
antagonism of typical responses modulated by membrane-associated PKC.
, described by Slater et al. (35), were
reported to show the opposite affinities for DAG and phorbol ester. Two
modes of interaction between DAG and the C1 domain, namely
sn-1 and sn-2, were proposed based on
computer-guided, molecular docking analysis (36, 37). Strikingly,
constrained DAG analogs developed from this modeling with branched side
chains to maximize the hydrophobic interaction between DAG and the
hydrophobic residues near the binding cleft exhibited binding potencies
approaching that of PDBu (36). Our results demonstrated significant
differences in the affinities of the mutants for the DAG
versus PDBu, reflecting the existence of specific
determinants for the recognition of DAG. Specifically, mutant L24K
showed a unique pattern of recognition of 97F31. In addition, the
mutants at Trp-22 exhibited a large reduction in their binding affinity
to DAG but not to PDBu. Our molecular dynamics simulation revealed that
the side chain of Trp-22 was very flexible,4 which would
allow the side chain to easily adopt different conformations when
interacting with different ligands. Thus, introducing different side
chains at this position could affect the ligand selectivity. The fact
that the same mutated residue (Lys-22) occurs in all members of
PKD/PKC-µ family implies a potential different selectivity for DAG
and consequently a possible functional difference compared with the
other members of the PKC family. This hypothesis is currently under investigation.
C1b. We found that
L20R resulted in >300-fold loss of apparent affinity toward PDBu and
>30-fold loss of affinity to the branched DAG in the presence of PS,
as well as a 10-fold shift in lipid dependence. The atypical PKCs,
PKC-
and PKC-
/
, are unable to bind C1 domain ligands despite
the high homology to other C1 domains. An attempt at restoring the DAG
binding activity of PKC-
by mutating Gly at position 11 to Pro was
unsuccessful (11). Here, we identified another possible site that may
account for the inactivity of PKC-
. Efforts to introduce a second
mutation, R20L, in addition to G11P in order to restore the activity of
PKC-
, are under way.
C1b to hydrophilic residues, we demonstrated the critical role played by the hydrophobic residues in the C1 domain
for C1 domain/lipid interaction and for ligand recognition. Our
findings provide further insight into the structural basis for
regulation of DAG/phorbol ester-generated signaling pathways, enhancing
our ability both to predict behavior from sequence as well as exploit
sequence differences for design of selective ligands.
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FOOTNOTES |
---|
* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Bldg. 37, Rm. 3A01, National Cancer Institute, 37 Convent Dr., MSC 4255, Bethesda, MD 20892-4255. Tel.: 301-496-3189; Fax: 301-496-8709;
E-mail: blumberp@dc37a.nci.nih.gov.
Published, JBC Papers in Press, March 14, 2001, DOI 10.1074/jbc.M010089200
2 Q. J. Wang, T.-W. Fang, V. E. Marquez, and P. M. Blumberg, manuscript in preparation.
3 K. Nacro, D. M. Sigano, S. Yan, M. C. Nicklaus, L. L. Pearce, N. E. Lewin, S. H. Garfield, P. M. Blumberg, and V. E. Marquez, manuscript in preparation.
4 Pak, Y., Enyedy, I. J., Varady, J., Kung, J. W., Lorenzo, P. S., Blumberg, P. M., and Wang, S. (2001) J. Med. Chem., in press.
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ABBREVIATIONS |
---|
The abbreviations used are: PKC, protein kinase C; PKD, protein kinase D; DAG, diacylglycerol; PAGE, polyacrylamide gel electrophoresis; PDBu, phorbol 12,13-dibutyrate; LUV, large unilamellar vesicle; POPS, sn-1-palmitoyl-2-oleoylphosphatidylserine; POPC, sn-1-palmitoyl-2-oleoylphosphatidylcholine; GST, glutathione S-transferase; IPTG, isopropyl-O-D-thiogalactopyranoside; PS, phosphatidylserine.
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