From the Department of Chemistry, University of Illinois, Chicago,
Illinois 60607-7061
The C2 domains of conventional protein kinase C
(PKC) have been implicated in their
Ca2+-dependent membrane binding. The C2
domain of PKC-
contains several Ca2+ ligands that bind
multiple Ca2+ ions and other putative membrane binding
residues. To understand the roles of individual Ca2+
ligands and protein-bound Ca2+ ions in the membrane binding
and activation of PKC-
, we mutated five putative Ca2+
ligands (D187N, D193N, D246N, D248N, and D254N) and measured the
effects of mutations on vesicle binding, enzyme activity, and monolayer
penetration of PKC-
. Altered properties of these mutants indicate
that individual Ca2+ ions and their ligands have different
roles in the membrane binding and activation of PKC-
. The binding of
Ca2+ to Asp187, Asp193, and
Asp246 of PKC-
is important for the initial binding of
protein to membrane surfaces. On the other hand, the binding of another
Ca2+ to Asp187, Asp246,
Asp248, and Asp254 induces the conformational
change of PKC-
, which in turn triggers its membrane penetration and
activation. Among these Ca2+ ligands, Asp246
was shown to be most essential for both membrane binding and activation
of PKC-
, presumably due to its coordination to multiple Ca2+ ions. Furthermore, to identify the residues in the C2
domain that are involved in membrane binding of PKC-
, we mutated
four putative membrane binding residues (Trp245,
Trp247, Arg249, and Arg252).
Membrane binding and enzymatic properties of two double-site mutants
(W245A/W247A and R249A/R252A) indicate that Arg249 and
Arg252 are involved in electrostatic interactions of
PKC-
with anionic membranes, whereas Trp245 and
Trp247 participate in its penetration into membranes and
resulting hydrophobic interactions. Taken together, these studies
provide the first experimental evidence for the role of C2 domain of
conventional PKC as a membrane docking unit as well as a module that
triggers conformational changes to activate the protein.
 |
INTRODUCTION |
The protein kinase C
(PKC)1 family is a set of
serine/threonine kinases that transduce the myriad of signals
activating cellular functions and proliferation (1-3). More than 10 members of the PKC family have been identified by molecular cloning.
Based on common structural features, PKCs are generally classified into three groups; conventional PKC (
,
I,
II, and
subtypes),
novel PKC (
,
,
, and
subtypes), and atypical PKC (
and
subtypes). Conventional PKCs are activated by the
Ca2+-dependent translocation to the membrane
containing phosphatidyl serine (PS) and diacylglycerol (DG). It has
been proposed that the C2 domain of conventional PKCs is involved in
this Ca2+-dependent membrane binding activity
(4). On the other hand, novel PKCs and atypical PKCs that have either a
modified or no C2 domain can be activated in a
Ca2+-independent way (5, 6). In addition to PKC, the C2
domain has been found in a wide variety of proteins that are involved in diverse cellular functions (7). Sequence alignment of C2 domains of
these proteins suggests that all known C2 domains exhibit either type I
or type II topology, differing slightly in their
-strand
connectivity (7). The C2 domains of conventional PKCs and
synaptotagmins have type I topology and show significant sequence homology (see Fig. 1A). High resolution crystal structures
have been determined for the isolated C2 domains of synaptotagmin, phospholipase C-
1, and cytosolic phospholipase A2
(8-10). Despite noticeable variations in primary structures, all of
these proteins have highly homologous tertiary structural folds
consisting of eight antiparallel
-strands and connecting loops (Fig.
1B). These structures of C2 domains have defined much of
Ca2+ ligands in the Ca2+ binding sites, which
consist of three Ca2+ binding loops dubbed calcium binding
region 1 (CBR1), CBR2, and CBR3. Multiple Ca2+ ions have
been located within these sites, and the binding of these
Ca2+ ions shows positive cooperativity (11). A recent NMR
study assigned two Ca2+ ions (CA1 and CA2) bound to the C2
domain of a conventional PKC, PKC-
(12). A putative coordination
pattern of the two Ca2+ ions, based on this study and the
tertiary structure of homologous synaptotagmin C2A domain, is
illustrated in Fig. 1C. At present, the roles of individual
Ca2+ ligands and Ca2+ ions bound to these
ligands in the membrane binding and activation of conventional PKC are
not fully understood. Furthermore, other residues in the C2 domain that
are involved in membrane binding of conventional PKC have not been
identified. To address these questions, we mutated several residues in
the C2 domains of PKC-
, including five putative Ca2+
ligands and four putative membrane binding residues. Membrane binding
affinity, enzyme activity, and membrane penetrating power of these
mutants demonstrate that individual Ca2+ ions and their
ligands have distinct roles in the membrane binding and activation of
PKC-
. These studies also identify the C2 domain residues of PKC-
that are involved in its electrostatic and hydrophobic interactions
with membranes.
 |
EXPERIMENTAL PROCEDURES |
Materials--
1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine
(POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol
(POPG), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine (POPS), and 1,2-sn-dioleoylglycerol were purchased from
Avanti Polar Lipids, Inc. (Alabaster, AL) and used without further
purification. Hereinafter, DG refers to
1,2-sn-dioleoylglycerol. Tritiated POPC ([3H]POPC) was prepared from
1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine and
[9,10-3H]oleic acid using rat liver microsomes as
described (13, 14). Phospholipid concentrations were determined by
phosphate analysis (15). [
-32P]ATP (3 Ci/µmol) was
from Amersham Pharmacia Biotech, and cold ATP was from Sigma. Triton
X-100 was obtained from Pierce. Restriction endonucleases and other
enzymes for molecular biology were obtained from either Boehringer
Mannheim or New England Biolabs (Beverly, MA).
45CaCl2 (5.91 Ci/g) was purchased from American
Radiolabeled Chemicals (St. Louis, MO).
Mutagenesis--
Baculovirus transfer vectors encoding the
cDNA of PKC-
with appropriate C2 domain mutations were generated
by the overlap extension polymerase chain reaction (16) using
pVL1392-PKC-
plasmid (17) as a template. Briefly, appropriate
complementary synthetic oligonucleotides introducing the desired
mutation and two other primers at the beginning of the PKC-
gene and
around the NcoI site inside the PKC-
gene were used as
primers for polymerase chain reactions, which were performed in a DNA
thermal cycler (Perkin-Elmer) using Pfu DNA polymerase (Stratagene).
The method consisted of two steps. In the first step, two DNA fragments
overlapping at the mutation site were generated and purified on an
agarose gel. Then, these two fragments were combined to generate the
fusion product, which was further amplified by polymerase chain
reaction. The product was subsequently purified on an agarose gel,
digested with NcoI, and ligated to the pVL1392-PKC-
construct, which was digested with NcoI, dephosphorylated
with alkaline phosphatase to prevent self-ligation, and purified on an
agarose gel. The mutagenesis was verified by DNA sequencing of the
PKC-
gene using a Sequenase 2.0 kit (Amersham Pharmacia
Biotech).
Expression of PKC-
and Mutants in Baculovirus-infected
Sf9 Cells--
Wild type PKC-
and mutants were expressed in
baculovirus-infected Sf9 cells (Invitrogen, La Jolla, CA).
Transfection of Sf9 cells with mutant pVL1392-PKC-
constructs
was performed using the BaculoGoldTM Transfection Kit from
Pharmingen (San Diego, CA). Prior to transfection, endotoxins were
removed from plasmid DNA using LPS extraction kit (Qiagen, Valencia,
CA). Cells were incubated for 4 days at 27 °C, and the supernatant
was collected and used to infect more cells for the amplification of
virus. After three cycles of amplification, high-titer virus stock
solution was obtained. Sf9 cells were maintained as monolayer
cultures in TMN-FH medium (Invitrogen) containing 10% fetal bovine
serum (Life Technologies, Inc.). For protein expression, cells were
grown to 2 × 106 cells/ml in 500-ml suspension
cultures and infected with the multiplicity of infection of 10. The
cells were then incubated for 3 days at 27 °C. For harvesting, cells
were centrifuged at 1000 × g for 10 min, washed once
with Tris-HCl buffer, pH 7.5, and resuspended in 25 ml of extraction
buffer containing 20 mM Tris-HCl, pH 7.5, 10 mM
EGTA, 2 mM EDTA, 1 mM dithiothreitol, 50 µg/ml leupeptin, 1% Triton X-100, and 0.2 mM
phenylmethylsulfonyl fluoride. The suspension was homogenized in a
hand-held homogenizer chilled on ice. The extract was centrifuged at
50,000 g and 4 °C for 40 min. The supernatant was loaded
onto a 100-ml Q-Sepharose Fast Flow column (Amersham Pharmacia
Biotech). After washing with 100 ml of Buffer A (20 mM
Tris-HCl, pH 7.5, 1 mM EGTA, 1 mM EDTA, 1 mM dithiothreitol), the column was eluted with 200 ml of a
linear salt gradient to 0.5 M KCl in Buffer A. Active PKC
fractions were pooled, adjusted to 2 M KCl, and loaded onto
a 10 ml Poros PE column (Boehringer Mannheim) with a flow rate of 4 ml/min. A linear salt gradient from 2 to 0 M KCl in Buffer
A (total volume, 60 ml) was applied. Active PKC fractions were
concentrated and desalted in an Ultrafree-15 centrifugal filter device
(Millipore) and stored in Buffer A containing 50% glycerol at
20 °C. Protein concentration was determined by the bicinchoninic
acid method (Pierce).
Determination of PKC Activity--
Activity of PKC was assayed
by measuring the initial rate of [32P]phosphate
incorporation from [
-32P]ATP (50 µM, 0.6 µCi/tube) into the histone III-SS (400 µg/ml) (Sigma). The reaction
mixture contained large unilamellar vesicles (0.2 mM), 5 mM MgCl2, 12 nM PKC, and various
concentrations of CaCl2 (see under "Results") in 50 µl of 20 mM HEPES, pH 7.0. Protamine sulfate (200 µg/ml) was used as a substrate when the free enzyme concentration was
determined by PKC activity assay in vesicle binding measurements (see
below). Free calcium concentration was adjusted using a mixture of EGTA
and CaCl2 according to the method of Bers (18). Reactions
were started by adding MgCl2 to the mixture and quenched by
adding 50 µl of 1% aqueous phosphoric acid solution after a given
period of incubation (e.g. 5 min for histone) at room
temperature. Seventy-five-µl aliquots of quenched reaction mixtures
were spotted on P-81 ion-exchange papers (Whatman), washed four times
with 1% aqueous phosphoric acid solution, and washed once with 95%
aqueous ethanol. Papers were transferred into scintillation vials
containing 4 ml of scintillation fluid (Sigma), and radioactivity was
measured by liquid scintillation counting. The linearity of the time
dependence of the reaction was checked by monitoring the degree of
phosphorylation at regular intervals.
Protein Kinase C-Vesicle Binding--
The binding of PKC to
phospholipid vesicles was measured by a centrifugation assay using
sucrose-loaded large unilamellar vesicles (100 nm in diameter) (19).
Sucrose-loaded vesicles were prepared as described elsewhere (20).
Briefly, the lipid solution was added to a round-bottomed flask, and
organic solvent was removed by rotary evaporation. The lipid film was
suspended in 20 mM HEPES buffer, pH 7.0, containing 0.2 M sucrose and vortexed vigorously. Unilamellar vesicles
were prepared by multiple extrusion through a 0.1 µm polycarbonate
filter (Millipore) in a Liposofast microextruder (Avestin, Ottawa,
Ontario, Canada). The vesicle solution was diluted 5 times with 20 mM HEPES buffer, pH 7.0, containing 0.1 M KCl
and centrifuged at 100,000 × g for 30 min at 25 °C.
The supernatant was removed, and the lipid pellet was resuspended in
the same buffer solution. The final concentration of vesicle solution
was determined by measuring the radioactivity of a trace of
[3H]POPC (typically 0.1 mol %) included in all
phospholipid mixtures. For binding experiments, PKC (approximately 12 nM) was incubated for 15 min with sucrose-loaded vesicles
(0.1 mM), 1 µM bovine serum albumin, and
Ca2+ (or EGTA; see under "Results") in 150 µl of 20 mM HEPES (pH 7.0) containing 100 mM KCl. Bovine
serum albumin was added to minimize the loss of protein due to
nonspecific adsorption to tube walls. Vesicles were pelleted at
100,000 × g for 30 min using a Sorvall RC-M120EX
microultracentrifuge. Aliquots of supernatants were used for protein
determination by PKC activity assay using protamine sulfate as a
substrate. The fraction of bound enzyme was plotted against mol
percentage of anionic lipid in vesicles or against free
Ca2+ concentration.
Monolayer Measurements--
Surface pressure (
) of solution
in a circular Teflon trough was measured using a du Nouy ring attached
to a computer-controlled Cahn electrobalance (Model C-32) as described
previously (17, 21, 22). The trough (4 cm in diameter × 1 cm
deep) has a 0.5-cm-deep well for magnetic stir bar and a small hole
drilled at an angle through the wall to allow an addition of protein
solution. Five to 10 microliters of phospholipid solution in
ethanol/hexane (1:9 (v/v)) or chloroform was spread onto 10 ml of
subphase (20 mM HEPES, pH 7.0 containing either 0.1 or 0.5 mM of free Ca2+) to form a monolayer with a
given initial surface pressure (
o). The subphase was
continuously stirred at 60 rpm with a magnetic stir bar. Once the
surface pressure reading of monolayer had been stabilized (after
approximately 5 min), the protein solution (typically 50 µl) was
injected to the subphase, and the change in surface pressure (
)
was measured as a function of time at 23 °C. Typically, the 
value reached a maximum after 20 min. The maximal 
value depended
on the protein concentration at the low concentration range and reached
a saturation when the protein concentration was higher than 1 µg/ml.
Protein concentrations were therefore maintained above 1.5 µg/ml to
ensure that the observed 
represented a maximal value. The
critical surface pressure (
c) was determined by
extrapolating the 
versus
o plot to the
x axis.
Equilibrium Dialysis Measurements--
Equilibrium dialysis was
carried out at room temperature using a MEGATM System
microdialyzer (Pierce) with separated sample chambers. Dialysis
membranes with 3500 molecular weight cut-off were used. Prior to
calcium binding measurements, protein solutions were concentrated in
Ultrafree-4 centrifugal filter units (Millipore) and repeatedly washed
with 20 mM HEPES, pH 7.0 to remove EGTA and EDTA present in
the storage buffer. Twenty-five microliters of PKC-
and selected
mutant solutions (final concentration, 10-15 µM) in
individual sample chambers were dialyzed against 30 ml of 20 mM HEPES buffer, pH 7.0, containing 0.1 M KCl,
0.5 mM DTT, and 0.1 mM
45CaCl2 (specific activity of 7 µCi/ml).
Controls contained 25 µl of 20 mM HEPES, pH 7.0, instead
of a protein solution. After equilibration for 19 h, free and
total Ca2+ concentrations were determined by counting the
radioactivity of 5-µl aliquots from control and protein chambers,
respectively, from which the radioactivity of bound Ca2+
was calculated.
 |
RESULTS |
Design and Physical Properties of PKC-
Mutants--
According
to the model structure of the C2 domain of PKC-
shown in Fig.
1C, five
Ca2+-binding aspartyl residues can be classified into three
groups; CBR1 ligands, which primarily coordinate CA1
(Asp187 and Asp193), CBR2 ligands, which mainly
coordinate CA2 (Asp248 and Asp254), and
Asp246, which coordinates both Ca2+ ions. In
addition to Asp246, Asp187 and
Asp248 could also be involved in partial coordination to
the other Ca2+. Based on this assignment, the mutations of
Asp193 and Asp254 would only affect the binding
of CA1 and CA2, respectively, whereas the mutations of
Asp246 (and possibly Asp187 and
Asp248) would have effects on the binding of both
Ca2+ ions. Thus, it is possible to systematically analyze
the roles of the two Ca2+ ions in the membrane binding and
activation of PKC-
by selectively mutating their ligands and
separately measuring the effects of mutations on membrane binding and
activation. From crystal structures of C2 domains of phospholipase
C-
1 and cytosolic phospholipase A2, it has been proposed
that CBR1 and CBR3 are involved in binding to membranes (10, 23): more
specifically, CBR3 in membrane penetration and CBR1 in interfacial
contact with the lipid head group. There are two relatively conserved
tryptophans (Trp245 and Trp247) in the CBR3 of
PKC-
: they are absolutely conserved among conventional PKCs and
substituted for by either aromatic or hydrophobic residues in other
proteins (7). Given the importance of tryptophans in membrane-protein
interactions (24-26), we reasoned that the two tryptophans might be
involved in penetration into membranes. Also present in CBR3 are two
surface-exposed arginines that are relatively conserved among topology
I C2 domains (7) and might be involved in electrostatic interactions
between PKC-
and anionic phospholipids, such as PS. To assess the
roles of these residues, we generated two double-site mutants,
W245A/W247A and R249A/R252A. Unlike CBR3, CBR1 of PKC-
contains
neither conserved hydrophobic nor ionic residues. Because all nine
mutated residues are located in loop regions, the above mutations were
not expected to cause deleterious conformational changes. Indeed, all
seven mutants were expressed in baculovirus-infected insect cells as
well as wild type, indicating comparable thermodynamic stability and a lack of gross conformational changes. Furthermore, all mutants exhibited full membrane binding affinity and enzyme activity at saturating Ca2+, PS, and DG concentrations (see below),
again demonstrating that the mutations did not significantly disrupt
its tertiary structural fold.

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Fig. 1.
A, sequence alignment of C2 domains of
PKC- and synaptotagmin. 1 to 8 indicate
eight antiparallel -stands, and CBR1 to CBR3
indicate calcium binding loops. Five calcium-coordinating aspartates
are shown in open rectangles, and mutated tryptophans and
arginines are indicated by black rectangles above the
sequence. B, ribbon diagram of the C2 domain of synaptotagmin. Locations of
three calcium binding loops and CA1 are shown. C, a model
structure of Ca2+ binding loops of PKC- . This model is
based on sequence homology to synaptotagmin and a NMR calcium titration
study of PKC- (12).
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|
Properties of Ca2+ Ligand Mutants--
To
systematically analyze the effects of mutations on the
Ca2+-dependent membrane binding and activation
of PKC-
, we measured the following five properties: Ca2+
dependence of vesicle binding, PS content dependence of vesicle binding, Ca2+ dependence of enzyme activity, PS content
dependence of enzyme activity, and monolayer penetration. We previously
showed (17) that PKC-
displayed full vesicle binding affinity if the
POPS content was > 20 mol % in POPC/POPS vesicles containing 2.5 mol % DG under the conditions employed in these studies (see also Fig.
3). We therefore used POPC/POPS/DG (67.5:30:2.5) vesicles to measure
the Ca2+ dependence of vesicle binding for wild type and
mutants. The binding of Ca2+ ions to the isolated C2 domain
was shown to be consistent with the cooperative Hill model (11). The
concentration of Ca2+ giving rise to half-maximal binding
(or activity) ([Ca2+]1/2) was thus determined
from curve fitting of data to a Hill equation,
|
(Eq. 1)
|
where y, a, h, and [Ca2+] are relative
binding (or activity), arbitrary normalization constant, Hill
coefficient, and free Ca2+ concentration, respectively. As
shown in Fig. 2, the mutants exhibited a
wide range of Ca2+ dependences, and
[Ca2+]1/2 values varied from 2 µM
to >1 mM (Table I). A closer
examination of the Ca2+ dependences, however, revealed a
systematic pattern. Mutations of CBR1 ligands coordinating to CA1
(D187N and D193N) had more significant effects on the vesicle binding
than mutations of CBR3 ligands coordinating to CA2 (D248N and D254N).
This suggested that although both Ca2+ ions are involved in
the Ca2+-dependent vesicle binding of PKC-
CA1 might play a more important role than CA2. Among all
Ca2+ ligands, Asp246 appeared to be the most
important in the vesicle binding because the D246N mutation showed a
much more drastic effect on vesicle binding than any other mutation.
This might be due either to close proximity of Asp246 to
CA1 or to its coordination to both Ca2+ ions. On the other
hand, similar Ca2+ dependences of D248N and D254N suggested
that potential coordination of Asp248 to CA1 is not
significant. Finally, changes in Hill coefficient (1.2-1.5) for
mutants were not large enough to have any physical meaning (data not
shown). We then measured the dependence of vesicle binding of wild type
and mutants on the PS content of POPC/POPS vesicles containing 1 mol % of DG in the presence of 0.1 mM Ca2+. This
condition was selected to best illustrate the PS dependence because at
higher Ca2+ concentrations all PS dependences fell into too
narrow a range to compare (data not shown). As illustrated in Fig.
3, the mutants exhibited a wide range of
PS dependences, but the pattern was similar to Ca2+
dependences shown in Fig. 2. Because the origin of sigmoidal dependence
on PS is not fully understood, the plots were graphically analyzed to
determine the PS content resulting in half-maximal binding
([PS]1/2). As summarized in Table I, [PS]1/2 values
for mutants were in the order of D246N
D187N
D193N > D248N
D254N. Because the membrane binding of PKC-
takes
place through the formation of a protein-calcium-lipid complex (note
that this does not necessarily implicate the calcium bridge formation),
the lower the Ca2+ affinity of a mutant in the
presence of vesicles is, the higher PS would be required for vesicle
binding. Thus, the PS dependences of vesicle binding further support
the notion that Asp246 and CBR1 ligands (and CA1) are
essential for the membrane binding of PKC-
.

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Fig. 2.
Ca2+ dependence of vesicle
binding of PKC- and Ca2+ ligand mutants. Proteins
(12 nM) include wild type ( ), D187N ( ), D193N ( ),
D246N ( ), D248N ( ), and D254N ( ). Total lipid concentration of
POPC/POPS/DG (67.5:30:2.5) vesicles was 0.1 mM. Solid
lines represent theoretical curves constructed from parameters
determined from the nonlinear least squares fit using Equation 1.
Theoretical curves were not generated for those mutants the
Ca2+ dependence of which was not readily fitted into
Equation 1.
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Table I
Properties of C2 domain mutants of PKC-
See under "Experimental Procedures" for experimental conditions and
methods to calculate [Ca2+]1/2, [PS]1/2,
and c values. [Ca2+]1/2 values indicate
best-fit values ± S.D. determined from nonlinear least squares
analysis of data using Equation 1.
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Fig. 3.
Binding of PKC- and Ca2+
ligand mutants to POPC/POPS vesicles containing 1 mol % DG as a
function of the content of POPS. Proteins (12 nM)
include wild type ( ), D187N ( ), D193N ( ), D246N ( ), D248N
( ), and D254N ( ). Ca2+ concentration was 0.1 mM, and other experimental conditions were the same
as in Fig. 2.
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Next, we measured the PKC activity of wild type and mutants as a
function of free Ca2+ concentration and the PS content in
vesicles. If the role of Ca2+ ions is primarily to bring
PKC-
molecules to membranes, the relative enzyme activity of PKC-
mutants would directly reflect their relative vesicle binding affinity.
When PKC activity of mutants was measured using POPC/POPS/DG
(67.5:30:2.5) vesicles and histone in the presence of varying
concentrations of Ca2+, however, an unexpected pattern was
observed (Fig. 4). Wild type PKC-
showed comparable Ca2+ dependences for vesicle binding and
activity, indicating that Ca2+-dependent
vesicle binding and activation are coupled processes. In contrast, all
mutants but D193N required much higher Ca2+ concentrations
for half-maximal activity than for half-maximal vesicle binding,
suggesting that all Ca2+ ligands except Asp193
participate in coordination to a Ca2+ ion(s) that is
involved in not only the vesicle binding but also the activation of
PKC-
. Most notably, mutants of Asp187 and
Asp246, which are implicated in coordination to both
Ca2+ ions, resulted in drastic increases in
[Ca2+]1/2 (see Table I). This might be due to the
combination of reduced vesicle binding and activation. Less drastic but
significant decreases in activity were observed for mutants of
Asp248 and Asp254, which mainly coordinate to
CA2. Between the two, D248N required higher Ca2+ than D254N
for the same degree of activity, suggesting the relative closeness of
Asp248 to CA2. The dependences of PKC activity of mutants
on the PS content of vesicles (Fig. 5)
also exhibited a similar pattern. Taken together, these results
suggested that CA2 and its ligands (mostly CBR3 ligands) are essential
for the activation of PKC-
although CA2 and some of its ligands
(Asp248 and Asp254) are not critically involved
in membrane binding of protein. This in turn points to distinct roles
of the two Ca2+ ions and their ligands in the function and
regulation of PKC-
.

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Fig. 4.
Ca2+ dependence of enzyme
activities of PKC- and Ca2+ ligand mutants.
Proteins include wild type ( ), D187N ( ), D193N ( ), D246N
( ), D248N ( ), and D254N ( ). Total lipid concentration and PKC
concentration were 0.2 mM and 12 nM,
respectively, in 20 mM HEPES, pH 7.0, containing 0.1 M KCl, 5 mM MgCl2, histone III-SS
(400 µg/ml), and varying concentrations of Ca2+. Each
data point represents an average of two experiments. The
absolute value of maximal activity was 0.30 nmol/(µg · min).
Solid lines represent theoretical curves constructed from
parameters determined from the nonlinear least squares fit using
Equation 1.
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Fig. 5.
Dependence of activities of PKC- and
Ca2+ ligand mutants toward histone on the PS content in
POPC/POPS vesicles containing 1 mol % DG. Proteins include wild
type ( ), D187N ( ), D193N ( ), D246N ( ), D248N ( ), and
D254N ( ). Ca2+ concentration was 0.4 mM,
which was higher than that employed for vesicle binding measurements
(0.1 mM). Other experimental conditions were the same as in
Fig. 4.
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Our recent study showed that Ca2+ promotes the penetration
of PKC-
into membranes containing PS, which eventually results in PKC activation (17). If two Ca2+ ions indeed have distinct
roles, as indicated above, the mutants of their ligands might have
different effects on the membrane penetration of PKC-
. To test this
notion, we measured the interaction of PKC-
and selected mutants
with phospholipid monolayers. Lipid monolayers have proven to be a
sensitive tool for measuring lipid-protein interactions (27, 28). In
this system, the penetration of a protein into a phospholipid monolayer
at the air-water interface can be sensitively monitored at constant
area or at constant surface pressure. In these studies, a phospholipid
monolayer of a given initial surface pressure
o was
spread at constant area, and the change in surface pressure (
)
was monitored after the injection of the protein into the subphase.
Those proteins of which the actions involve the partial or full
penetration of membranes have an ability to penetrate into the
phospholipid monolayer with
o comparable to or higher
than that of biological membranes (approximately 31 dyn/cm) (29-32),
and vice versa. In general, 
is inversely proportional
to
o of the phospholipid monolayer, and an extrapolation of the 
versus
o plot yields the
critical surface pressure (
c) (28), which specifies an
upper limit of
o of a monolayer that a protein can
penetrate into. Therefore,
c should be above 31 dyn/cm
if the protein is able to penetrate into the membrane under a
physiological condition. Fig. 6 shows the

versus
o plot for wild type, D187N,
D193N, and D248N. As reported previously, PKC-
could penetrate
POPC/POPS (5:5) monolayers even when
o > 35 dyn/cm
(Table I), demonstrating its ability to penetrate biological membranes.
Note that these monolayer measurements were performed under the
condition in which wild type and D193N were fully active, whereas D187N
and D248N were only partially active (see Figs. 4 and 5). Under this
condition, a CBR1 ligand (CA1 ligand) mutant D193N, which exhibited
comparable decreases in vesicle binding and activity, showed a slight
reduction in monolayer penetrating power compared with wild type. In
contrast, two mutants, D187N and D248N, of which the enzyme activities
were reduced much more than their vesicle binding affinities were,
showed much lower penetrating power; as a result, they would not be
able to penetrate into biological membranes under the condition in
which wild type and D193N could (see Table I for their
c
values). Thus, these results strongly support the notion that the
binding of CA2 induces conformational changes of PKC-
, which in turn
trigger its membrane penetration and activation. On the other hand, the
binding of CA1 might simply anchor the protein to membrane
surfaces.

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Fig. 6.
Effect of the initial surface pressure of
POPC/POPS (5:5) mixed monolayers on the penetration of PKC- ( ),
D187N ( ), D193N ( ), and D248N ( ). The protein
concentration in the subphase was 20 nM. The subphase
contained 20 mM HEPES buffer, pH 7.0, containing 0.1 mM free Ca2+. Higher Ca2+
concentration (e.g. 0.5 mM) did not further
enhance the penetration of mutants. Each data point is from
a single measurement.
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Properties of R249A/R252A--
To evaluate the contributions of
Arg249 and Arg252 to the membrane binding and
activation of PKC-
, several properties of R249A/R252A were measured.
We first measured the Ca2+ dependence of its binding to
vesicles of different compositions. When the binding to POPC/POPS/DG
(67.5:30:2.5) vesicles was measured, wild type and R249A/R252A did not
show any significant difference in Ca2+ dependence (Fig.
7), implying that the two arginines are
not directly involved in the vesicle binding. It has been shown,
however, that DG greatly enhances the hydrophobic interactions between PKC and membranes, thereby rendering the relative contribution from
electrostatic interactions less important (17, 33). We therefore
measured the Ca2+ dependence of vesicle binding while
varying the DG content in the vesicles. As shown in Fig. 7, the smaller
the DG content was, the larger the difference in Ca2+
requirement for binding between wild type and R249A/R252A was. In the
absence of DG, apparent [Ca2+]1/2 values were 40 µM for wild type and > 1 mM for the
mutant. Similar results were obtained for the Ca2+
dependence of PKC activity determined in the presence of POPC/POPS/DG vesicles and histone (Fig. 8). Again, the
difference in Ca2+ requirement increased with the decrease
in DG content in vesicles. These results thus all point to the
importance of Arg249 and Arg252 in
electrostatic interactions between PKC-
and anionic membrane surfaces. This notion was further supported by two additional sets of
data. First, we measured the dependences of vesicle binding and enzyme
activity of wild type and R249A/R252A on the PS content of POPC/POPS/DG
vesicles at a fixed Ca2+ concentration. As shown in Fig.
9, R249A/R252A required a higher PS
content than wild type to achieve the same degree of vesicle binding
and activity. Also, the difference in PS dependence between wild type
and R249A/R252A was more pronounced with the decrease of DG content
(data not shown). Second, we measured the Ca2+ dependences
of binding of PKC-
and R249A/R252A to POPC/POPG/DG vesicles. It has
been shown that PKC-
binds, albeit less tightly, to phosphatidyl
glycerol-containing vesicles via mainly nonspecific electrostatic
interactions even in the presence of DG (17, 34). As shown in Fig.
10, R249A/R252A showed a higher
Ca2+ requirement than wild type in both the presence and
absence of DG. Note that wild type and R249A/R252A exhibited
indistinguishable Ca2+ dependence when binding to
POPC/POPS/DG (67.5:30:2.5) vesicles. Thus, these results again
indicated that Arg249 and Arg252 make
significant contributions to electrostatic interactions of PKC-
with
anionic membranes. Furthermore, the finding that R249A/R252A had
reduced affinity for both PS and phosphatidyl glycerol-containing
vesicles suggests that Arg249 and Arg252 are
not a part of the specific PS binding site. This notion was also
consistent with monolayer penetration behaviors of R249A/R252A. Our
previous study showed that PKC-
could selectively penetrate into
PS-containing monolayers. If Arg249 and Arg252
form a specific PS binding site, R249A/R252A would then show much
reduced monolayer penetration into PS-containing monolayers. As shown
in Fig. 11, R249A/R252A had essentially
the same monolayer penetration power as wild type. Taken all together,
these data show that Arg249 and Arg252 are
involved in nonspecific electrostatic interactions with anionic phospholipids.

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Fig. 7.
Ca2+ dependence of vesicle
binding of PKC- (open symbols) and R249A/R252A
(closed symbols). Vesicles (100 µM) were
composed of POPC/POPS/DG (67.5:30:2.5) ( and ), POPC/POPS/DG
(69.5:30:0.5) ( and ), and POPC/POPS (70:30) ( and ),
respectively. Experimental conditions for vesicle binding measurements
were the same as described in Fig. 2. Protein concentrations were 12 nM. Solid (wild type) and dotted
(R249A/R252A) lines represent theoretical curves constructed
from parameters determined from the nonlinear least squares fit using
Equation 1.
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Fig. 8.
Ca2+ dependence of enzyme
activities of PKC- (open symbols) and R249A/R252A
(closed symbols). Vesicles (100 µM) were
composed of POPC/POPS/DG (67.5:30:2.5) ( and ), POPC/POPS/DG
(69.5:30:0.5) ( and ), and POPC/POPS (69.9:30:0.1) ( and ),
respectively. Experimental conditions for activity measurements were
the same as described in Fig. 4. Each data point represents
an average of two experiments. The absolute value of maximal activity
was 0.34 nmol/(µg · min). Solid (wild type) and
dotted (R249A/R252A) lines represent theoretical
curves constructed from parameters determined from the nonlinear least
squares fit using Equation 1.
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Fig. 9.
Vesicle binding and enzyme activity of
PKC- ( and ), R249A/R252A ( and ), and W245A/W247A ( and ) as a function of the PS content of POPC/POPS vesicles
containing 1 mol % DG. Ca2+ concentration was 0.4 mM. Other experimental conditions for vesicle binding and
activity measurements were the same as in Figs. 2 and 4,
respectively.
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Fig. 10.
Ca2+ dependence of binding of
PKC- (open symbols) and R249A/R252A (closed
symbols) to POPC/POPG vesicles. Vesicles (100 µM) were composed of POPC/POPG/DG (67.5:30:2.5) ( and
) and POPC/POPG (70:30) ( and ), respectively. Experimental
conditions for vesicle binding measurements were the same as described
in Fig. 2. Solid (wild type) and dotted
(R249A/R252A) lines represent theoretical curves constructed
from parameters determined from the nonlinear least squares fit using
Equation 1.
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Fig. 11.
Effect of the initial surface pressure of
POPC/POPS (5:5) mixed monolayers on the penetration of PKC- ( ),
R249A/R252A ( ), and W245A/W247A ( ). The protein
concentration in the subphase was 20 nM. The subphase was
20 mM HEPES, pH 7.0, containing 0.5 mM free
Ca2+. Each data point is from a single
measurement.
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Properties of W245A/W247A--
Several properties of W245A/W247A
were measured to evaluate the contributions of Trp245 and
Trp247 to the membrane binding and activation of PKC-
.
First, the Ca2+ dependences of vesicle binding and activity
were measured and compared with that of wild type. As shown in Fig.
12, W245A/W247A had much higher
Ca2+ requirement for vesicle binding than wild type (see
also Table I), which indicated much reduced membrane binding affinity.
A comparable effect was seen with PKC activity measurements, indicating that the decreased activity mainly derived from the reduced membrane affinity. Because the two tryptophans are located within CBR3, we
measured the intrinsic Ca2+ binding affinities of wild type
and W245A/W247A in the absence of vesicles to preclude the possibility
that the high Ca2+ requirements for the membrane binding
and activation of W245A/W247A were due to the local disruption of
Ca2+ binding loops and the consequent decrease in intrinsic
Ca2+ affinity. Conventional PKCs and isolated C2 domains
can bind Ca2+ ions in the absence of phospholipids, albeit
with much reduced affinity (11, 35). Although accurate determination of
calcium dissociation constant was not performed due to the requirement for a large amount of protein and radiolabeled Ca2+, the
Ca2+ binding results illustrated in Fig.
13 demonstrate that wild type and
W245A/W247A have comparable intrinsic Ca2+ affinity,
whereas D187N has much reduced one. Thus, properties of W245A/W247A
shown in Fig. 12 should be mainly due to reduced membrane binding
affinity of this mutant. Furthermore, we measured the dependences of
vesicle binding and enzyme activity of wild type and W245A/W247A on the
PS content of POPC/POPS/DG vesicles at a fixed Ca2+
concentration (Fig. 9). Consistent with its other properties, W245A/W247A required much higher PS than wild type to achieve half-maximal vesicle binding and activity; again, the vesicle affinity
and the enzyme activity were reduced to comparable degrees. W245A/W247A
was, however, able to display full wild type activity at higher PS
contents (>60 mol %), indicating that the decrease in its activity
was not due to a deleterious gross conformation change. Finally, we
measured the monolayer penetrating ability of W245A/W247A to find out
if the tryptophans are involved in membrane penetration of PKC-
.
Fig. 11 shows that Trp245 and Trp247 play an
important role in the membrane penetration of PKC-
. Thus, these
results corroborate the notion that the CBR3 of PKC-
containing
Trp245 and Trp247 is involved in membrane
penetration.

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Fig. 12.
Ca2+ dependence of vesicle
binding and enzyme activity of PKC- ( and ) and W245A/W247A
( and ). Experimental conditions for vesicle binding and
activity measurements were the same as described in Figs. 2 and 4,
respectively. Each data point represents an average of two
experiments. The absolute value of maximal activity was 0.30 nmol/(µg
· min). Solid (vesicle binding) and dotted
(activity) lines represent theoretical curves constructed
from parameters determined from the nonlinear least squares fit using
Equation 1.
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Fig. 13.
Ca2+ binding of PKC- ,
W245A/W247A, and D187N in the absence of phospholipid vesicles.
See under "Experimental Procedures" for experimental conditions and
the calculation of radioactivity of bound Ca2+ for each
protein. Each data point is an average of three
measurements. Note that the binding was determined at a fixed
Ca2+ concentration (100 µM) to qualitatively
determine relative Ca2+ binding affinities of wild type and
two mutants. Because this Ca2+ concentration is below a
saturating value (>1 mM) (12), the calculation of
stoichiometry based on the value of mol of Ca2+ per mol of
protein would only yield a lower estimate value. To circumvent
potential misinterpretation of data, the bound Ca2+ is thus
expressed in total radioactivity.
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DISCUSSION |
This report describes a systematic structure-function analysis on
the C2 domain of PKC-
. In particular, it represents the first
investigation to dissect the roles of different Ca2+ ions
and their ligands in the membrane binding and function of C2
domain-containing protein. The C2 domain has been found in a wide
variety of proteins and shown to be involved in Ca2+
signaling for some of these proteins (7). The ability of the C2 domain
to bind phospholipid vesicles in a Ca2+ dependent manner
has been demonstrated by functional expression and characterization of
recombinant C2 domain fragments of synaptotagmin (36, 37) and cytosolic
phospholipase A2 (7, 11, 38). The structural studies of C2
domains of synaptotagmin, phospholipase C-
1, and cytosolic
phospholipase A2 revealed the presence of three potential
Ca2+ binding sites but all three proteins bind two
Ca2+ ions in two of these sites. These studies were
analyzed using the model structure of PKC-
C2 domain with two bound
Ca2+ ions. This model is based on sequence homology to
synaptotagmin (see Fig. 1A) and a NMR titration study of
PKC-
-Ca2+ binding (12). It is thus possible that the
actual conformation of Ca2+ binding loops of PKC-
might
be slightly different from the model structure shown in Fig. 1,
B and C, and that a different number of
Ca2+ ions (e.g. three instead of two) might bind
to the Ca2+ binding loops. This uncertainty will be
resolved by determining the high resolution structure of PKC-
C2
domain complexed with Ca2+ ions. Nevertheless, the
conclusions from these structure-function studies as to differential
roles of Ca2+ binding ligands/Ca2+ ions and the
involvement of Arg249/Arg252 and
Trp245/Trp247 in electrostatic and hydrophobic
membrane binding, respectively, will not be significantly affected by
minor discrepancy between model and true structures.
Differential Roles of Ca2+ Binding Ligands and
Ca2+ Ions--
At least three roles have been proposed for
Ca2+ ions in the regulation of proteins containing the C2
domain. One possible role is to alter the surface electrostatic
potential of the C2 domain. This possibility has been ruled out by a
recent mutagenesis study of PKC-
II C2 domain (39). The
second postulated role is that Ca2+ ions provide a bridge
between PKC and membranes by coordinating to both protein and anionic
phospholipids. This is similar to the role of Ca2+ ions
proposed for annexins (40, 41). Finally, Ca2+ ions can
induce a conformational change of protein, which in turn triggers
interactions of PKC with membranes. The last mechanism has been
controversial due to conflicting observations from different structural
studies. For instance, a NMR study of PKC-
revealed no significant
conformational change induced by Ca2+ binding (12). On the
other hand, the coordination of multiple Ca2+ ions severely
disrupted the crystal of the C2 domain of synaptotagmin (8). A recent
study of the isolated C2 domain of cytosolic phospholipase
A2 showed that two Ca2+ ions bind to the C2
domain with positive cooperativity, which induces intradomain
conformational changes and drives the membrane interactions (11).
Similarly, our results indicate that Ca2+ ions not only
anchor the protein to membrane surfaces but also induce conformational
changes resulting in PKC activation. Most importantly, all evidence
indicates that individual Ca2+ ions are differentially
involved in these processes. Differential roles of two Ca2+
ions and their ligands are clearly illustrated in unparalleled Ca2+ (and PS) dependence of vesicle binding and that of
enzyme activity seen for most Ca+2 ligand mutants (D187N,
D246N, D248N, and D254N). For D193N, the decreased enzyme activity is
directly correlated with its reduced membrane affinity. Because
Asp193 would only coordinate CA1, this finding supports the
notion that CA1 is involved mainly in initial membrane anchoring of
protein. This notion is also consistent with monolayer data showing
that D193N can penetrate POPC/POPS monolayers almost as well as wild type. Other four mutants show much more pronounced decreases in activity than expected from their membrane affinity. It is interesting to find that all of these ligands are postulated to coordinate CA2.
Clearly, Asp246 is the most essential ligand for both
Ca2+-dependent vesicle binding and activation
of PKC-
. Drastically reduced membrane affinity and enzyme activity
of this mutant is not due to deleterious conformational changes as it
shows the activity toward histone comparable to that of wild type under a certain condition; e.g. in the presence of POPS/DG
(97.5:2.5) vesicles and 0.6 mM Ca2+ (data not
shown). In the crystal structure of synaptotagmin, carboxylates of
Asp172 (corresponding to Asp187 of PKC-
) and
Asp178 (Asp193 of PKC-
) are slightly closer
to CA1 (2.63 and 2.79 Å, respectively) than that of Asp230
(Asp246 of PKC-
) (2.99 Å) (8); yet D246N has lower
vesicle affinity than D187N and D193N. Thus, it is likely that the
critical role of Asp246 in vesicle binding derives from its
ability to coordinate both Ca2+ ions tightly. Similarly,
its essential role in activation might originate from its ability to
coordinate both Ca2+ ions and, presumably, its proximity to
CA2. Overall, properties of mutants of the CA2 ligands can be best
accounted for by assuming that CA2 is involved in PKC activation. It
has been generally proposed that the activation of conventional PKC
involves conformational changes of protein, including the removal of
the pseudosubstrate region from the active site of PKC (42). Our recent
study showed that the penetration of PKC-
into PS-containing
membranes is a part of these conformational changes and is also
essential for its interactions with DG (17). Thus, much reduced
monolayer penetration ability of D187N and D248N indicates that the
binding of CA2 to PKC-
leads to its activation by triggering the
membrane penetration of protein. Taken all together, our results
indicate that CA1 is primarily involved in initial membrane anchoring, whereas CA2 is more directly involved in conformational changes. It
should be noted that the deactivation of CA2 ligands also significantly impaired the membrane binding of PKC-
. Thus, the distinction between
the two Ca2+ ions is not that obvious as far as the
membrane anchoring role is concerned. As for inducing conformational
changes, however, all evidence indicates that CA1 plays no direct role.
It is likely that the anchoring role of CA1 (and CA2) is achieved by
the formation of Ca2+ bridge between PKC and phospholipid
head group(s). It is less clear, however, how CA2 would induce
conformational changes of PKC-
. Further studies are necessary to
address these questions.
Arg249/Arg252 and
Trp245/Trp247--
Because the C2 domain is
responsible for the Ca2+-dependent membrane
binding of many proteins, it is reasonable to postulate that the domain
contains essential membrane binding residues. For most peripheral
membrane-binding proteins, both electrostatic and hydrophobic
interactions play roles in their membrane binding, although their
relative contributions vary with the type of proteins (see, for
example, Refs. 24, 43, and 44). The C2 domain of conventional PKC
contains several putative membrane binding residues. Recent mutagenesis
studies of PKC-
II showed that cationic residues in
3
and
4 strands and a loop connecting
5 and
6 strands (see Fig.
1A) are not involved in membrane binding (39, 45). Our
studies positively identify those residues that are involved in
electrostatic interactions (Arg249/Arg252) and
hydrophobic interactions (Trp245/Trp247). These
studies thus provide the first experimental evidence for the notion
that the C2 domain of conventional PKC is directly involved in membrane
binding. Both W245A/W247A and R249A/R252A show the similar
Ca2+ (and PS) dependences for membrane binding and for
enzyme activity, indicating that these residues are involved primarily
in membrane binding but not in subsequent activation steps. These
properties of mutants are due neither to the deleterious gross
conformational change of protein, which would abolish enzyme activity,
nor to local conformational perturbation of calcium binding loops,
which would lower intrinsic calcium affinity. It has been shown that the membrane binding and activation of conventional PKC requires the
binding of a large number of PS molecules (and other anionic phospholipids) to PKC (34, 46, 47). Despite relatively high PS
specificity of PKC, the presence of PS-specific binding site(s) has
been disputed based on the ability of certain synthetic phospholipids to simulate the effects of PS (48). In any event, the binding of
multiple PS molecules (and other anionic phospholipids) would require
the presence of PS binding site(s) on the surface of PKC molecule
because such binding could not be mediated by PKC-bound Ca2+ ions alone. Our data show that Arg249 and
Arg252 form a part of binding sites for anionic
phospholipids. It is evident from the similar phosphatidyl glycerol and
PS dependences of R249A/R252A that this site is not specific for PS.
The crystal structure of synaptotagmin shows that Arg233
and Lys236, which correspond to Arg249 and
Arg252 of PKC-
, respectively, form a cationic patch on
the surface of CBR3 that has been proposed to make direct contact with
membranes (10, 23). Thus, Arg249 and Arg252 are
properly located for their putative role as a nonspecific binding site
for anionic phospholipids. A question still remains as to whether or
not a separate PS-specific site(s) exists in another part of PKC
molecule, the answer of which awaits further structure-function
studies.
Drastically reduced vesicle affinity and enzyme activity of W245A/W247A
underscore the importance of these residues in the membrane binding of
PKC-
. In particular, its significantly lower monolayer-penetrating
power compared with wild type indicates that the tryptophans are
involved in membrane penetration of PKC-
and resulting interactions
with the hydrophobic interior of membranes. This penetration is,
however, different from the CA2-induced penetration, which results in
PKC activation, in that the W245A/W247A mutation shows no further
reducing effect on PKC activity than expected from the decrease in
vesicle affinity. Unlike Arg249 and Arg252,
Tyr239 and Phe231 of synaptotagmin, which
correspond to Trp245 and Trp247 of PKC-
, are
not fully surface-exposed. Thus, the penetration by Trp245
and Trp247 appears to be a part of conformational changes
of PKC-
induced by CA2 binding. This particular conformational
change does not directly lead to PKC activation but enhances the
membrane binding of PKC-
. Judging from the extent of reduction in
vesicle affinity, Trp245 and Trp247 make
significant contribution to total membrane binding energy of PKC-
.
Evaluation of contributions of individual membrane binding modules of
PKC-
to electrostatic and hydrophobic membrane-protein interactions
will require further systematic structure-function analyses.
In summary, these studies clearly indicate distinct roles of calcium
ions bound to the C2 domain of PKC-
in its membrane binding and
activation and identify residues that are directly involved in
electrostatic and hydrophobic membrane binding, thereby defining the
role of C2 domain as a membrane docking unit of PKC-
and as a module
that triggers conformational changes to activate the protein. Two
calcium ions and cationic Arg249/Arg252 of the
C2 domain all contribute to the initial membrane binding of PKC-
,
which is mainly electrostatic in nature. Although the sequence of
events involved in membrane binding and subsequent activation of
PKC-
is not fully understood, the formation of the complex of
protein-calcium ions-phospholipids induces conformational changes and
membrane penetration of PKC-
, which not only strengthen its membrane
binding (largely hydrophobic in nature) but also lead to enzyme
activation. Because minor but definite structural and functional
differences of isolated C2 domain fragments have been found, it is
premature to speculate whether or not the regulation of PKC-
by its
C2 domain might represent a general mechanism of Ca2+
signaling by the C2 domain. These studies pave the way toward a better
understanding of mechanism of C2 domain-mediated Ca2+
signaling for other membrane-binding proteins.