 |
INTRODUCTION |
Protein kinase C family
(PKC)1 is known to control
many cellular processes including metabolism regulation, receptor
signal transduction, cell growth and differentiation, and hormone and neurotransmitter secretion (1, 2). This family consists of 10 closely
related isoenzymes that can be divided into three groups according to
the type of activator they need. For example, conventional PKCs (
,
I,
II, and
) require the full complement of negatively charged
phospholipids, Ca2+, and diacylglycerol or phorbol esters
before they are activated. The novel PKCs (
,
,
, and
) on
the other hand do not require Ca2+, whereas the atypical
PKCs (
,
/
) do not require diacylglycerol or Ca2+
(3).
Conventional and novel PKCs share the property of using two
membrane-targeting modules for the sensitive, specific, and reversible regulation of their function. Thus, the C1 and the C2 domains are
involved in membrane translocation and subsequent enzyme activation (4). Many studies performed to date suggest a sequential mechanism in
the activation of PKC
by Ca2+ in which membrane
association is followed by increased catalytic activity (5-9).
However, it is still not clear whether these domains function in a
concerted way or as independent modules.
The first step of this process is probably the
Ca2+-dependent contact of the C2 domain with
negatively charged phospholipids at the plasma membrane (6, 8, 10-12).
Recent studies on different C2 domains reveal that, despite their
sequential and architectural similarity, they are functionally
specialized modules that exhibit distinct equilibrium and kinetic
behaviors that are probably optimized for different
Ca2+-signaling applications (13-14). Most of these studies
have been performed with isolated C2 domains and, in the case of
PKC
, the precise molecular mechanism driving the above interaction
and the consequences for the general functioning of the enzyme are still not well defined.
Our previous crystallographic studies of the isolated C2 domain of
PKC
bound to Ca2+ and the short chain lipid
1,2-dicaproyl-sn-phosphatidyl-L-serine show that
two or three Ca2+ ions bind to the domain, with one of them
(Ca1) bridging the protein directly to phosphatidylserine (PS).
Moreover, several residues in the domain seem to be involved in a
direct interaction with PS (15-16), and further studies show that both
Ca2+ and PS binding sites are important for in
vitro enzyme activation (9, 17, 18). Whether two or three calcium
ions are needed for plasma membrane translocation of PKC
and how PS
and diacylglycerol interact with the regulatory domain to orchestrate
membrane targeting and activation of PKC
in vivo are key
questions that remain to be answered.
To investigate the molecular determinants mediating the C2
domain-dependent plasma membrane localization of PKC
, we
generated a construct containing full-length PKC
tagged to green
fluorescent protein (PKC
-GFP). Additionally, different constructs
containing point mutations at the Ca2+ and PS binding sites
in the C2 domain of PKC
were produced and transfected into RBL-2H3
cells (a mast cell line that provides a useful model for studying
signal transduction mechanisms). The cells are activated by antigens
via the tyrosine kinase-dependent high affinity receptor
for IgE (Fc
RI) to induce the release of secretory granules and the
generation of cytokines. Activation of the receptor leads to
phospholipase C
activation and the consequent generation of inositol
1,4,5-triphosphate/Ca2+ and diacylglycerol, both of which
are regarded as PKC activators (19). Using confocal microscopy combined
with measurements of intracellular Ca2+ concentration in
time-lapse experiments, we have found that both Ca2+ and PS
binding sites in the C2 domain play important and differential roles in
the spatio-temporal localization of PKC
, suggesting a very accurate
and controlled mechanism driven primarily by the C2 domain.
 |
EXPERIMENTAL PROCEDURES |
cDNA Constructions--
N-terminal fusions of rat PKC
and
the different mutants to GFP were generated by inserting cDNAs into
the multiple cloning site of the pEGFP-N3 (Clontech
Laboratories, Inc., Palo Alto, CA) mammalian expression vector.
Briefly, cDNAs encoding PKC
and its mutants D187N, D246N/D248N,
D187N/D246N/D248N, N189A, R216A, R249A, and T251A were amplified by PCR
using the primers 5'-ATTCTCGAGCTATGGCTGACGTT and
3'-CCGGGTACCTACTGCACTTTGCAAGAT. XhoI/KpnI-digested PKC
and mutated
fragments were ligated with the
XhoI/KpnI-digested vector, thus generating the
different fusion constructs. Further details on site-directed
mutagenesis to generate the different mutants are reported by
Conesa-Zamora et al. (17). All constructs were confirmed by
DNA sequencing. The stability and viability of the mutated proteins
were studied by using specific activity measurements. It was
demonstrated that the mutants could be activated in a
PS-dependent manner, although not to the same extent as the
wild-type protein (9, 17). Moreover, earlier studies have shown that a
C-terminal GFP tag does not affect the catalytic activity or the
cofactor dependence of PKC
(20-22).
Cell Culture--
Rat basophilic leukemia (RBL-2H3) cells were
cultured at 37 °C in a humidified atmosphere of 5% CO2
in a growth medium of Dulbecco's modified Eagle's medium supplemented
with 15% (v/v) fetal calf serum, 50 units/ml penicillin, 50 µg/ml
streptomycin, and 4 mM glutamine. Cells were prepared for
confocal microscopy as described by Bolsover et al. (23).
Basically, harvested cells were resuspended in electroporation buffer
(120 mM NaCl, 5.5 mM KCl, 2.8 mM
MgCl2, 25 mM glucose, 20 mM Hepes,
pH 7.2) and 30 µg of cDNA. Cells were electroporated in a Bio-Rad
GenePulser with two 500-V pulses. The cells were immediately placed on
ice for 5 min before being plated on glass coverslips and incubated at
37 °C for 4-6 h, after which the growth medium was renewed. Cells
were used 24 h later after priming overnight with 500 ng/ml IgE-anti-dinitrophenyl (mouse monoclonal, Sigma). Coverslips were washed with 3 ml of extracellular buffer HBS (120 mM NaCl,
25 mM glucose, 5.5 mM KCl, 1.8 mM
CaCl2, 1 mM MgCl2, 20 mM HEPES, pH 7.2). All added substances were dissolved or
diluted in HBS. DiC8 and ionomycin were dissolved in Me2SO
and diluted to the final concentration with extracellular buffer
shortly before the experiment. During the experiment, the cells were
not exposed to Me2SO concentrations higher than 1%. All
the experiments were carried out at room temperature, and unless
otherwise stated, on at least four different occasions. In each
experiment, recordings were obtained from 2-6 cells.
Imaging of [Ca2+]i and
PKC-GFP--
[Ca2+]i was measured using Fura Red
(Molecular Probes, Inc., Eugene, OR). Stock solutions (2 mM) of the AM-ester form of the fluorescent
Ca2+ indicator were made using a solution of 2.5% (w/v)
Pluronic F-127 in absolute Me2SO. This stock was diluted
1000-fold in growth medium and applied to cells for 30 min at 25 °C
and 5% CO2. After incubation, the cells were washed with
HBS and mounted in a holder attached to the stage of a Leica inverted
confocal laser-scanning microscope. During imaging, cells were
stimulated with antigen (DNP-conjugated human serum albumin (HSA),
Sigma) or other agents as described.
Fluorescence confocal microscopy was used to monitor both the
translocation of EGFP fusion constructs and the intensity of the
Ca2+ indicator in response to different stimuli. Cells were
illuminated using a 488-nm laser line, and emitted light was
simultaneously sampled through a 515-540-nm band pass filter (EGFP
channel) and 590-nm long pass filter (Fura Red channel). The signals
from Fura Red and EGFP could not be completely separated (23). The same photomultiplier voltages were used throughout these experiments, meaning that the measurements made when only one dye was present were
also applicable to dual dye readings. Imaging of untransfected cells
loaded with Fura Red revealed minimal contamination of the EGFP channel
by the Fura Red signal; because the contamination represented less than
1% of the values recorded when EGFP was present, no correction was
performed. In contrast, imaging of transfected cells without Fura Red
revealed significant contamination of the Fura Red channel by the
signal from EGFP. The signals recorded in the Fura Red channel were
closely correlated with those recorded in the EGFP channel and had
values 24.7% as large. In the dual dye experiments we therefore
calculated the net Fura Red signal in any defined area of interest by
subtracting 24.7% of the EGFP signal from the overall signal in the
Fura Red channel. [Ca2+]i was then calculated
using
|
(Eq. 1)
|
where Kd is the apparent Ca2+
binding affinity of the Ca2+ indicator, determined by the
manufacturer (140 nM), I is the net Fura Red
fluorescence intensity of an individual cell,
Imin is the net Fura Red fluorescence intensity
of the cell at zero Ca2+, and Imax
is net Fura Red fluorescence intensity of the cell at saturating
Ca2+. Imin was replaced by
FI0, where F = (Imin/I0) = (Kd + Cr)/(CrS + Kd), I0 is the net Fura Red
fluorescence intensity of a resting unstimulated cell, calculated as
the average intensity of the first 10 images recorded in each experiment, Cr is the Ca2+ concentration
in resting unstimulated cells, measured as 136 ± 1 nM
in 256 untransfected cells loaded with the ratiometric Ca2+ indicator Fura-2 (see Bolsover et al.
(23)), and S = Imax/Imin, measured as
0.101 on this instrument. When excited at 488 nm, the fluorescence of
Fura Red falls on binding Ca2+. Imax
was replaced by SImin. Substitution of
Imin and Imax in Equation 1 therefore gives
|
(Eq. 2)
|
which was used to convert net Fura Red intensities into
Ca2+ values.
Image Analysis--
A series of confocal images were recorded
for each experiment at time intervals of 9.3 s. The time series
were analyzed using Lucida 4.0 software (Kinetic Imaging, Wirral, UK).
An individual analysis of protein translocation for each cell was
performed by tracing a line intensity profile across the cell (24). The relative increase in plasma membrane localization of the enzyme for
each time point was calculated by using the ratio r = (Imb
Icyt)/Icyt, where
Imb is the fluorescence intensity at the plasma membrane, and Icyt is the average cytosolic
fluorescence intensity. Mean values are given ±S.E.
Structural Models--
1DSY was used as the Protein Data Bank
identifier for the experimentally determined C2 domain structure (15).
The program used to visualize the structures was Swiss-PDB Viewer 3.7 program by GlaxoSmithKline (25).
 |
RESULTS |
Mutagenesis Rationale--
Fig.
1A illustrates a scheme based
on the structure of the C2 domain of PKC
bound to three
Ca2+ and to the short chain lipid
1,2-dicaproyl-sn-phosphatidyl-L-serine (15-16).
The side chains of the five aspartates (Asp-187, Asp-193, Asp-246,
Asp-248, and Asp-254) that directly coordinate Ca1 and Ca2 are shown.
Simple (D187N), double (D246N/D248N) and triple (D187N/D246N/D248N)
mutants were generated to study the role of these residues in the
plasma membrane targeting of the enzyme. D187N was chosen because the
crystallographic study showed that this aspartate residue forms a
bidentate interaction with Ca1, which is the ion involved in bridging
the protein residues with the phosphate group of the phospholipid
molecule (Fig. 1A). In the case of the double mutant
containing Asp-246 and Asp-248 substituted by Asn, key coordinations
with Ca1 and specially with Ca2 were abolished. Double (D246N/D248N)
and triple (D187N/D246N/D248N) substitutions of the aspartate residues
by Asn neutralized the highly negative charge exhibited by this region,
and it was thought that this might help understand whether this
neutralization of charges is the only mechanism required for membrane
targeting of the domain or a different mechanism operates this
interaction.

View larger version (18K):
[in this window]
[in a new window]
|
Fig. 1.
A, shown is a scheme based on the
model structure of Ca2+ binding loops of PKC C2 domain.
This model is based on the crystal structure of the C2 domain bound to
Ca2+ and the short chain lipid
1,2-dicaproyl-sn-phosphatidyl-L-serine (DCPS)
(15). Side chains of the residues involved in Ca2+
coordination are shown. The residues mutated in this work are
represented in dark gray (Asp-187, Asp-246, and Asp-248) and
additional unmutated residues are shown in light gray
(Asp-193 and Asp-254). B, scheme showing the side chain
residues (dark gray) involved in direct
1,2-dicaproyl-sn-phosphatidyl-L-serine
interactions (dotted lines). Note that Ca1 is located under
the phosphate group, whereas the phospholipid would form a cap over the
Ca2+ binding region, explaining the cooperative effect
found experimentally when Ca2+ and PS bind simultaneously
to PKC .
|
|
Fig. 1B shows the side chains of the residues involved in
the PS-binding site: Asn-189 (located in loop 1), Arg-216 (located in
loop 2), Arg-249 and Thr-251 (both located in loop 3). Thr-251 is a
special residue since it has been proposed to be involved in two types
of interaction. When the C2 domain only binds two Ca2+,
this residue interacts with the sn-1 acyl group oxygen of
the PS (15). However, when three Ca2+ are bound to the
domain, the side-chain oxygen of Thr-251 also coordinates the third
Ca2+, which seems to bind to the C2 domain only in the
presence of a very short phosphatidylserine analogue (16). Whether this third Ca2+ was necessary for PKC
plasma membrane
translocation was unclear as was the specific role of the threonine
residue. Thus, single substitutions by Ala of each amino acidic residue
located in the lipid-binding site were generated to study their role in
PKC
membrane localization. When these mutants were studied,
all of them exhibited enzyme activity at saturating Ca2+,
PS, and diacylglycerol concentrations, demonstrating that the mutations did not significantly disrupt the tertiary structural fold of
the mutant proteins (9, 17).
The Ca2+-binding Sites of the C2 Domain Are Critical
for PKC
Membrane Translocation Induced by the Activation of
Cell-surface Receptors--
Cells were transfected with PKC
-GFP
and loaded with Fura Red to monitor protein translocation and
[Ca2+]i simultaneously. To analyze the kinetics
of the two processes, a series of 63 images were taken at 9.3-s
intervals. To determine PKC
translocation, the relative increase in
plasma membrane over cytosolic fluorescence intensity (R)
was calculated at each time point.
Fig. 2A shows images of
antigen-stimulated RBL-2H3 cells expressing PKC
-GFP. In
unstimulated cells, the protein was evenly distributed throughout the
cytosol (Fig. 2A, 0 s), whereas stimulation with 40 ng/ml DNP-HSA resulted in oscillatory translocations of PKC
-GFP to
and from the plasma membrane (Fig. 2A, 149, 205, 223, and
279 s). The addition of ionomycin after 381 s also produced plasma membrane translocation (Fig. 2A, 437).

View larger version (28K):
[in this window]
[in a new window]
|
Fig. 2.
A, receptor-induced translocation of
PKC to the plasma membrane. The images shown were recorded at 0, 149, 205, 223, 279, 316, and 437 s. Antigen (40 ng/ml DNP-HSA) was
added after 83 s of recording. 1 µM ionomycin was
added after 381 s of recording. B, example of the time
course of Ca2+ signals after the addition of antigen
(Ag) and ionomycin. Apparent free Ca2+
concentration was calculated as stated under "Experimental
Procedures." C, example of the time course of plasma
membrane translocation of PKC . Note that there is a marked
correlation between Ca2+ spikes and membrane translocation
of the protein. R, relative plasma membrane
translocation.
|
|
When the time course of Ca2+ oscillations versus
the plasma membrane translocation of PKC
-GFP was compared (Figs.
2, B and C), intracellular Ca2+
spikes were seen to be highly correlated with the translocation of
PKC
-GFP. Maximum protein translocation occurred between 2 and
26 s after the first Ca2+ spike was generated. It is
interesting to note that the first Ca2+ spike was more
effective in producing the translocation of the protein to the plasma
membrane, whereas the second and third pulses, which showed the same
Ca2+ concentrations, were only capable of partially
anchoring the protein. The addition of ionomycin in the same experiment
produced a further increase in cytosolic Ca2+ and a
parallel translocation of PKC
-GFP that started to revert to the
cytosol 79 ± 23 s after ionomycin addition. It is
interesting to note that protein started to revert to the cytosol when
the intracellular Ca2+ concentration was still compatible
with protein translocation. Similar translocation profiles were
obtained in control experiments, in which ionomycin was added directly
to the cells without previous antigen stimulation (data not shown).
To investigate how the aspartate residues coordinate the different
Ca2+ ions and produce the membrane targeting of PKC
, we
generated several mutants that were also fused to GFP, as stated above. As shown in Fig. 3A, all the
mutants were localized homogeneously in the cytosol in unstimulated
cells. When the cells were stimulated by the addition of 40 ng/ml
DNP-HSA, no changes in plasma membrane translocation of the mutant
proteins were detected; similar results were obtained when ionomycin
was added. A comparison of Fig. 3, B and C,
clearly illustrates that although Ca2+ spikes were
generated in the cells transfected with the different mutant proteins,
these mutations strongly affected the
Ca2+-dependent translocation of the enzyme
(Fig. 3C). Additionally, none of the mutant proteins
translocated to the plasma membrane, although sustained cytosolic
Ca2+ concentrations ranging from 900 to 1400 nM
were produced in the cells analyzed by the addition of ionomycin. Taken
together these data suggest that the initial physiological
translocation of PKC
to the plasma membrane is driven
primarily in a Ca2+-dependent manner by the C2
domain and that the residues coordinating Ca1 and Ca2 are critical in
the in vivo protein-plasma membrane interaction.

View larger version (25K):
[in this window]
[in a new window]
|
Fig. 3.
A, confocal fluorescence images of
RBL-2H3 cells expressing the different Ca2+-binding site
mutants, D187N, D246N/D248N, and D187N/D246N/D248N. The images were
recorded at the different times stated. Antigen (40 ng/ml DNP-HSA) was
added after 83 s of recording, and 1 µM ionomycin
was added after 381 s of recording. B, examples of the
time courses of the Ca2+ signals after antigen
(Ag) and ionomycin additions. C, examples of the
time course of plasma membrane localization of the different mutants.
As shown, no translocation to the plasma membrane was observed for any
of them. R, relative plasma membrane translocation.
|
|
Ca2+ Binding Residues Are Involved in the C1
Domain-dependent Translocation of PKC
to the Plasma
Membrane--
To determine the role of the C1 domain in the
Ca2+-dependent membrane targeting of PKC
, we
examined the direct effect of increasing concentrations of the
membrane-permeant diacylglycerol (DiC8) on both cytosolic
Ca2+ and the kinetics of PKC
-GFP translocation. Neither
the Ca2+ signal nor PKC
-GFP targeting of the plasma
membrane was produced by increasing the concentrations of DiC8 alone
(Fig. 4, A and B).
Only when ionomycin was added to the medium did the protein translocate
to the membrane in a DiC8 concentration-dependent manner
(Fig. 4B). In fact, the higher the DiC8 concentration, the
longer the residence time of PKC
at the plasma membrane. It is
interesting to note that the protein translocation profile obtained
after the addition of ionomycin to the medium containing 10 µg/ml
DiC8 was very similar to that obtained when the cells were stimulated
previously with 40 ng/ml antigen (Fig. 2C), suggesting that
the concentration of diacylglycerol generated under physiological conditions might also be similar but probably only sufficient to anchor
the protein to the plasma membrane for a very short length of time
through the C1 domain, as has been suggested in previous reports (8,
26).

View larger version (18K):
[in this window]
[in a new window]
|
Fig. 4.
A, example of the time course of the
Ca2+ signal generated after the addition of the plasma
membrane-permeant diacylglycerol DiC8 (83 s) and ionomycin (381 s).
B, examples of the time course of the plasma membrane
translocation of PKC after the addition of 10, 25, 50, and 100 µg/ml DiC8. The results are representative experiments of 4-14 cells
obtained from at least four different experiments. R,
relative plasma membrane translocation.
|
|
Because the C1 and C2 domains have always been considered as
independent modules, it is striking that the effect of DiC8 is only
observed when the concentration of intracytosolic Ca2+
increases. The results obtained with the Ca2+ binding
mutants shed light on this issue. When the cells transfected with
PKC
D187N mutant were stimulated with increasing concentrations of
DiC8, no protein translocation was observed (n = 20 cells). However, when ionomycin was added (Fig.
5A) the mutant interacted with
the plasma membrane only in 50% of the cells analyzed with a rate
(R) of 0.6. Note, also, that after ionomycin stimulation the
translocation profiles reflected a slower and more transient process
compared with that of the wild-type protein that lasted at least
186 s with the same concentration of DiC8 (100 µg/ml). Moreover,
maximum protein translocation of PKC
D187N mutant was obtained
123 ± 11 s after ionomycin addition, whereas wild-type protein showed maximum translocation after 55 ± 10 s.

View larger version (17K):
[in this window]
[in a new window]
|
Fig. 5.
Comparison of the time course of DiC8-induced
plasma membrane translocation of the different Ca2+ binding
mutants, D187N (A), D246N/D248N (B),
and D187N/D246N/D248N (C). The relative plasma
membrane translocation (R) is represented as a function of
time. DiC8 was added after 83 s, and ionomycin was added after
381 s. The DiC8 concentrations used in each experiment were 25 ( ), 50 ( ), and 100 ( ) µg/ml.
|
|
Surprisingly, when the cells were transfected with the PKC
D246N/D248N mutant (Fig. 5B), the protein started to
translocate slowly to the plasma membrane after the addition of 50 or
100 µg/ml DiC8. This effect was clearly
concentration-dependent since 25 µg/ml DiC8 only produced
a transient translocation after ionomycin stimulation, whereas 50 and
100 µg/ml DiC8 promoted the irreversible plasma membrane localization
of the protein that was detectable after 115 ± 18 s
(n = 8 cells). Note also that measurable levels of
PKC
D246N/D248N mutant were recorded after 58 ± 4 s in
the presence of ionomycin when the cells were previously stimulated with 25 µg/ml DiC8 compared with the 86 ± 4 s needed in
the case of PKC
D187N mutant. Furthermore, the translocation of
PKC
D246N/D248N to the plasma membrane became almost independent of
ionomycin addition when 50 and 100 µg/ml DiC8 were used to stimulate
the cells.
When PKC
D187N/D246N/D248N mutant (Fig. 5C) was tested,
it was translocated to the plasma membrane at only 25 µg/ml DiC8, and
it showed a faster kinetics (68 ± 5 s; n = 13 cells) of translocations than PKC
D246N/D248N mutant. These
results demonstrate a strong correlation between the degree of
neutralization of the aspartate residues that coordinate the
Ca2+ ions and the accessibility of DiC8 to the C1 domain.
Direct Interaction of the Ca2+ Binding Region with
Phospholipids Is Needed for the Stable Localization of PKC
--
We
also generated point mutations of the residues involved in the
PS-binding site; Asn-189, Arg-216, Arg-249, and Thr-251 were mutated to
Ala and fused to GFP. It is important to note that all these constructs
exhibited a very similar degree of
Ca2+-dependent activation to the wild-type
protein except Thr-251, which showed a slightly decreased
Ca2+/phospholipid-dependent activation (17).
When those constructs were transfected into RBL-2H3 cells, all of them
were expressed and uniformly distributed in the cytosol in resting
conditions, except the PKC
R249A mutant, which was also localized in
the nucleus (Fig. 6A). Fig. 6,
B and C, illustrate how PKC
N189A and PKC
R216A mutants partially translocated to the plasma membrane upon
antigen stimulation. However, there were two significant differences
compared with the wild-type protein. First, the delay before maximum
translocation ranged between 31 and 53 s for PKC
N189A and
between 30 and 74 s for PKC
R216A, whereas the wild-type protein only needed 2-26 s; second, the mutated proteins only translocated once after the first Ca2+ spike, none of the
other Ca2+ spikes producing plasma membrane translocation
of these two mutants. Furthermore, when ionomycin was added,
intracellular Ca2+ concentrations increased to 1000 nM or above in all cells (Fig. 6B), and the
localization profiles of PKC
N189A and PKC
R216A showed a very
transient process compared with that seen in the case of the wild-type
protein. In particular, the half-maximal plasma membrane dissociation
time of PKC
N189A mutant was 73 ± 9 s and of PKC
R216A
mutant was 76 ± 16 s compared with the wild-type PKC
of
108 ± 12 s, suggesting that these mutations produce a slight
alteration in the Ca2+-dependent membrane
targeting of the enzyme.

View larger version (23K):
[in this window]
[in a new window]
|
Fig. 6.
A, confocal fluorescence images of RBL
cells expressing the different lipid-binding site mutants N189A, R216A,
R249A, and T251A. The images were recorded at the different times
stated. Antigen (40 ng/ml DNP-HSA) was added after 83 s, and 1 µM ionomycin was added after 381 s of recording.
B, examples of the time course of the Ca2+
signals after antigen (Ag) and ionomycin addition.
C, examples of the time course of plasma membrane
translocation of the different mutants. R, relative plasma
membrane translocation.
|
|
PKC
R249A and PKC
T251A mutant did not translocate to the plasma
membrane even after ionomycin addition (Fig. 6C). Because both these proteins interact with one of the carbonyl group of the
fatty acyl chains of the phospholipid (15), these results suggest that
the protein might use these residues to anchor to the membrane
interface, resulting in greater stabilization of the protein-membrane complex.
When RBL-2H3 cells transfected with these phospholipid binding mutants
were stimulated with increasing concentrations of DiC8, no plasma
membrane translocation was observed until ionomycin was added to the
stimulation medium (Fig. 7). The fact
that both PKC
N189A and PKC
R216A mutants translocated to a
similar extent to the wild-type protein suggests that the C1 domain can
overcome the slight lack of function of these C2 domain mutants (Fig.
7, A and B). In contrast, the PKC
R249A mutant
did not translocate to the plasma membrane either in the presence of
DiC8 or ionomycin (Fig. 7C). The PKC
T251A mutant
targeted the plasma membrane in a transient manner (Fig.
7D), and the half-maximal plasma membrane dissociation time
of 114 ± 14 s (n = 10 cells) was much
shorter than that exhibited by wild-type PKC
, PKC
N189A, and
PKC
R216A. These results suggest that the role played by Arg-249 and
Thr-251 in the C2 domain membrane anchorage cannot be overcome by DiC8 acting through the C1 domain and that the interactions existing between
these residues and the plasma membrane might be essential for the
membrane targeting of PKC
.

View larger version (14K):
[in this window]
[in a new window]
|
Fig. 7.
Comparison of the time course of DiC8-induced
plasma membrane translocation of the different lipid-binding mutants,
N189A (A), R216A (B), R249A
(C), and T251A (D). The relative
plasma membrane translocation (R) is represented as a
function of time. DiC8 was added after 83 s, and ionomycin was
added after 381 s. The DiC8 concentrations used in each experiment
were 25 ( ), 50 ( ), and 100 ( ) µg/ml. Ag,
antigen.
|
|
 |
DISCUSSION |
Role of Ca2+ Ions in the C2
Domain-dependent Translocation of PKC
--
The results
obtained in this work suggest that a very sensitive mechanism exists
whereby the Ca2+ binding region of the C2 domain drives
PKC
membrane targeting in vivo. Substitutions of Asp-187
and Asp-246/248 by Asn had a dramatic effect on the
Ca2+-dependent membrane targeting of the
enzyme, confirming previous biochemical studies in which similar
substitutions produced a severe impairment of
Ca2+-dependent phospholipid binding (8, 9, 28).
It is important to note that Asp-187 mainly coordinates Ca1, and the
single substitution of an aspartate residue by asparagine was not
expected to produce such a damaging effect, since the other four
aspartate residues involved remain intact and could coordinate at least
one Ca2+ ion. Thus, these results confirm the hypothesis
proposed in the crystallographic model whereby Ca1 serves as a bridge
between some of the aspartate residues located in the protein and the phosphate group of the PS (15, 16).
Similar function has been attributed to Ca1 in the case of
synaptotagmin I (C2A domain) (29, 30). In contrast, Ca1 seems not to be
essential for the membrane binding of cytosolic phospholipase A2 (31). Thus, the varied results obtained with the
different proteins suggest that the subtle structural differences
existing among C2 domains are probably the basis of the different
functional mechanisms exhibited by the proteins that hold these domains.
Additionally, it was observed that the Ca2+ binding mutant
proteins associated with the plasma membrane in a
DiC8-dependent manner was strongly correlated with the
number of aspartate residues neutralized in each mutant (Fig. 5). This
effect could well correspond to the neutralization of charges that
occurs after Ca2 binds to the C2 domain (13, 32, 33) and which probably
produces a conformational change that in turn allows diacylglycerol
access to the C1 domain. This hypothesis is supported by several
studies that have shown through different techniques that the
full-length enzyme undergoes a general conformational change upon
Ca2+ binding (6, 12, 34-36). Whether or not this
conformational change also produces the liberation of the
pseudosubstrate region from the catalytic domain before C1 domain
binding to diacylglycerol cannot be easily answered, but previous works
in our lab have shown that a significant activation of the enzyme can
occur in the only presence of PS and Ca2+, further
supporting this hypothesis (9, 17).
The need of a third Ca2+ ion (Ca3) to anchor PKC
to the
plasma membrane is still under debate. In this work, no
Ca2+-dependent translocation of PKC
T251A
was observed, suggesting that if there is a need for a third
Ca2+ to anchor the enzyme to the plasma membrane, this ion
has to bind to the domain before PKC
interacts with the phospholipid bilayer. In addition, the reduced plasma membrane association exhibited by PKC
T251A mutant in the presence of DiC8
implies that this residue might be involved in stabilizing C2 domain
membrane docking before diacylglycerol binds to the C1
domain. Taking into account the low affinity exhibited by
this C2 domain for a third Ca2+ in solution (14, 16), it is
more likely that this stabilization might be due to a direct
interaction between Thr-251 and the phospholipid bilayer, but the
mediation of a third Ca2+ in this process cannot be
completely ruled out.
Role of the Phosphatidylserine Binding Region in the C2
Domain-dependent Translocation of PKC
--
Another
important issue addressed in this work was the role of the amino acidic
residues at the Ca2+ binding region of the C2 domain of
PKC
involved in direct binding with the phosphatidylserine at the
membrane surface. In this case, we observed that mutation to Ale of the
side chains of Asn-189 and Arg-216 had a different effect on the
Ca2+-dependent translocation of PKC
than did
the same substitutions in Arg-249 and Thr-251. In the first two cases,
the mutations resulted in a weaker membrane association of the
proteins. However, after the addition of diacylglycerol and
ionomycin, these mutants bound to the membrane in a similar way to the
wild-type protein, suggesting that the interactions exerted by these
two residues located in loops 1 and 2 are important for stabilizing the
protein-membrane complex, although they are not crucial for the protein
to anchor to the membrane.
In contrast, interactions established by the side chain of Arg-249 were
very critical since its mutation inhibited the ability of the protein
to interact with the plasma membrane even in the presence of saturating
Ca2+ and diacylglycerol. This effect might be because of
the loss of certain interactions with the phospholipid moieties since
this mutant exhibited very similar
Ca2+-dependent activation to wild-type PKC
,
as demonstrated in previous work (17). Moreover, PKC
is not the only
case that has been reported since it has been shown recently (37) that
the Ca2+ binding affinity of a homologue residue mutated in
the C2A domain of synaptotagmin I does not differ from observations
made in wild-type protein. In the crystallographic model of the C2
domain of PKC
(15), the guanidinium group of Arg-249 has been
proposed to interact through hydrogen bonding to the sn-2
acyl group oxygen and additionally to the ester oxygen of the
sn-1 acyl chain. Because the Arg-216 side chain establishes
hydrogen bonding only with the carbonyl group of the sn-2
acyl chain, it seems that the interactions occurring between Arg-249
and the phospholipid in the C2 domain membrane anchorage are more
critical than the corresponding interaction of Arg-216. It should also
be mentioned that in the crystallographic model, extra hydrophobic
interactions were established between the aliphatic carbons of Arg-249
and the acyl chains of
1,2-dicaproyl-sn-phosphatidyl-L-serine, and this
is supported by the drastic effect observed in the membrane targeting
of the enzyme when Arg-249 was substituted by Ala. Although it is not
clear that the location and orientation of these acyl chains of the
phospholipid shown in the crystallographic study are the same as their
corresponding location in a bilayer, it is possible that hydrophobic
interactions further increase the importance of Arg-249 for the C2
domain membrane docking and orientation. It is important to take into
account that there are hydrophobic residues (Trp-245 and Trp-247)
surrounding Arg-249 in loop 3 that have been implicated in the partial
membrane penetration of PKC
(6). Similar evidence has been proffered
in the case of the C2A domain of synaptotagmin I, where direct
interactions of residues located in loop 3 and loop 2 with a soluble
phosphatidylserine were reported (38-39).
Conclusions--
Together, these results provide a deeper
molecular understanding for the sequential mechanism of PKC
activation driven by the C2 domain (Fig.
8). In our model, increase in
intracellular Ca2+ produces the binding of Ca1 and Ca2 when
the protein is still in the cytosol, leading to the membrane targeting
of the enzyme through the C2 domain (Fig. 8b). Ca1 is
responsible for bridging the protein to the specific phospholipid
molecules, which are also recognized with the help of Asn-189 and
Arg-216, whereas Ca2 is responsible for keeping Ca1 in its proper
location and for inducing a conformational change in PKC
, which
partially penetrates and orientates in the phospholipid bilayer through loop 3 (Arg-249 and Thr-251). Only after this has occurred can the C1
domain find the diacylglycerol generated in the membrane surface,
enabling the protein to become fully activated (Fig. 8c).
Whether or not the C2 domain-dependent translocation of
PKC
itself is able to transduce signals downstream, representing a distinct, diacylglycerol-independent signaling mode is still not known
and will have to be further studied.

View larger version (19K):
[in this window]
[in a new window]
|
Fig. 8.
Proposed mechanism for C2
domain-dependent translocation of PKC
to the plasma membrane. In resting conditions, where
cytosolic Ca2+ is ~160 nM, the protein is
found in the cytosol (a). When the Ca2+ level
increases upon cross-linking of IgE receptors, at least two
Ca2+ ions bind to the C2 domain to form a
2-Ca2+-PKC complex with high membrane binding affinity
and which undergoes a conformational change (b). After that
the C2 domain can penetrate the interface of the bilayer through loop 3 when it orientates itself, leading to a more stable interaction that,
in turn, allows the C1 domain to access the diacylglycerol
(DAG) generated in the membrane, fully activating the enzyme
(c). Note that C1a and C1b have been placed based on the
model described by Cho and Medkova (27). R, relative plasma
membrane translocation; Cat, catalytic
domain.
|
|
The present study provides new insights into the molecular mechanism
underlying the dynamic nature of PKC
redistribution in
vivo. Our results have dissected a sequential mechanism that controls the function of the C2 domain of PKC
. Because the different PKC isoenzymes have been implicated in the malignant transformation and
proliferation of cells, they have become a very attractive target for
anticancer drug design (40). One of the main problems in using this
strategy is the ubiquity and the lack of specific inhibitors/activators
for each isoenzyme. For this reason, an accurate definition of the
residues involved in the Ca2+/lipid-dependent
translocation is a very useful tool for the design of new drugs that
can act specifically in this critical activation step.