From the Department de Bioquímica y Biología Molecular (A), Facultad de Veterinaria, Universidad de Murcia, Apdo 4021, E-30100 Murcia, Spain
Received for publication, September 12, 2002, and in revised form, November 4, 2002
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ABSTRACT |
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In view of the interest shown in
phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2)
as a second messenger, we studied the activation of protein kinase C Phosphatidylinositol 4,5-bisphosphate
(PtdIns(4,5)P2)1
plays a key role in phosphoinositide signaling and regulates a wide range of processes at many subcellular sites. It is primarily detected
in the plasma membrane but is also found in secretory vesicles,
lysosomes, in the endoplasmic reticulum, the Golgi, and in the nucleus
(1-5). PtdIns(4,5)P2 can either bind to intracellular proteins and directly modulate their subcellular localization and
activity, or it can act as a precursor for the generation of different
second messengers. For example, several families of phospholipase C
enzymes are responsible for the hydrolysis of PtdIns(4,5)P2
in cells, leading to the production of diacylglycerol and inositol
1,4,5-trisphosphate (4, 6), which may, in turn, lead to the activation
of different proteins such as some PKC isotypes.
Protein kinase C (PKC) composes a large family of serine/threonine
kinases, which is activated by many extracellular signals and plays a
critical role in many signal-transducing pathways in the cell (7-9).
Based on their enzymatic properties, the mammalian PKC isotypes have
been grouped into smaller subfamilies. The first group, which includes
the classical isoforms In classical PKC isoenzymes, Ca2+-dependent
binding to membranes shows a high specificity for
1,2-sn-phosphatidyl-L-serine (11-14). Additionally, this group of isoenzymes is sensitive to other anionic phospholipids, including phosphatidic acid and polyphosphoinositides (15-16) and to a variety of amphipathic membrane compounds, such as
arachidonic acid and free fatty acids (17). Furthermore, in
vitro experiments have demonstrated that the presence of other anionic phospholipids in the vesicles decreases the requirement of
phosphatidylserine, suggesting that PKC activation, other than the
classical activation pathway (activation of phospholipase C and
production of diacylglycerol by hydrolysis of PtdIns(4,5)P2 and increase of intracytosolic Ca2+), could take place
in vivo (15).
In view of the interest in PtdIns(4,5)P2 as a second
messenger, several studies (11, 18-24) have addressed the activation of PKC isotypes by this class of lipid. However, the results show little consistency as to which of the different PKC isotypes are activated or as regards their specificity for the different lipids employed. These conflicting results are probably due to the different ways used by investigators to activate the enzyme.
It has been described that the C2 domains of several proteins, such as
synaptotagmin (25-29) and rabphilin 3A (30) among others, bind
PtdIns(4,5)P2 through co-linear sequences that consist of
highly basic amino acidic residues (3, 25, 26, 30, 31). These
lysine-rich sequences probably represent the inositol phosphate-binding
portions of larger phosphoinositide-binding domains. For example, in
synaptotagmin, this basic sequence is flanked by regions rich in
hydrophobic residues that could mediate acyl chain interactions
(3).
When we look at the amino acidic sequence of the PKC In this paper, we focus on the characterization of the
interaction mechanism between PKC Materials
1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine
(POPS), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine
(POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphate, and
phosphatidylinositol 4,5-bisphosphate (PIP2) were purchased from Avanti Polar Lipids Inc. (Birmingham, AL).
Phosphatidylinositol 3,4,5-trisphosphate and phosphatidylinositol
3-phosphate were purchased from Echelon Biosciences Inc. (Salt Lake
City, UT). Phosphatidylinositol and phosphatidylglycerol were purchased
from Lipid Products (Nutfield, Surrey, UK), and phorbol 12-myristate 13-acetate (PMA) was from Sigma.
Construction of Expression Plasmids
Rat PKC The cDNA fragment corresponding to residues 158-285 of the
PKC Expression and Purification of GST-PKC The pGEX-KG plasmid containing the PKC Cell Culture, Transfection, and Purification of PKC HEK293 cells were grown in Dulbecco's modified Eagle's medium
with 10% fetal calf serum. Transfection was performed with the Ca2+ phosphate method described by Wigler et al.
(36). Protein purification was performed as described by Conesa-Zamora
et al. (14).
Phospholipid Binding Measurements
Standard Assay--
The procedure described by Davletov
and Sudhof (37) was used with minor modifications. A total of 10 µg
of PKC PKC Kinase Activity Assay
The lipids used for the reaction were previously dried under a
stream of N2, and the last traces of organic solvent were
removed by keeping the samples under vacuum for 1 h. Lipids were
suspended in 20 mM Tris-HCl, pH 7.5, 0.05 mM
EGTA and vortexed vigorously to form multilamellar vesicles. A 20-µl
sample of the lipids was added to the reaction mixture (final volume,
150 µl), which contained 20 mM Tris-HCl, pH 7.5, 0.2 mg/ml histone III-S, 20 µM [ Structural Models
The PDB identifiers for the experimentally determined C2 and
ENTH domain structures used in the calculations were 1DSY (38) and 1HFA
(39). The Swiss-PDB Viewer 3.7 program by GlaxoSmithKline R&D Geneva
(57) was used to visualize the structures.
Characterization of the PtdIns(4,5)P2 and PKC
In order to study whether Ca2+ can affect the PKC Binding Mechanism of PtdIns(4,5)P2 to PKC
Fig. 2B shows the results obtained when
PKC
When the second mutant, PKC Characterization of the PtdIns(4,5)P2-PKC
Strikingly, very different results were obtained when these experiments
were performed in the absence of Ca2+ (Fig. 3B).
No PKC PtdIns(4,5)P2-dependent Activation of
PKC
In the absence of Ca2+, vesicles containing
PtdIns(4,5)P2 increased the enzyme activity more than 6 times that obtained with POPS-containing vesicles, which also suggested
an important role for the PtdIns(4,5)P2-binding site in
enzyme activation under these conditions (Fig. 4B). These
data correlated well with those obtained in the binding assay, because
full translocation of the enzyme to the phospholipid vesicles occurred
in the presence of histone and PMA.
Note also that the PtdIns(4,5)P2-dependent
specific activity of PKC Demonstrating the Existence of the New PtdIns(4,5)P2
Site in Full-length PKC
In contrast, when POPS was included in the lipid vesicles instead of
PtdIns(4,5)P2 (Fig. 5B), PKC
It is also interesting to note that the maximal specific activity
obtained when the PKC
Another way in which we attempted to distinguish between the
contributions of the PMA and Ca2+ to the full activation of
the enzyme was by measuring the specific activity of the wild-type
protein and of the two mutants in the presence of Ca2+ and
in the absence of PMA. Under such conditions, the activation of PKC
Parallel binding experiments were performed under the same conditions
used to measure the specific activity of the enzyme. Model membranes
containing POPC and 10 mol % PtdIns(4,5)P2 were generated, and vesicle membrane binding was measured in the presence and in the absence of 0.3 mol % PMA. In the case of POPC membranes, only 10% of wild-type and mutants were bound to the vesicles (Fig. 8). In a similar way to what happened in
the activation assays, when PtdIns(4,5)P2 was included in
the membranes, 78% of wild-type PKC
Taken together, these results suggest that the lysine-rich cluster of
the C2 domain is the most important site in the
PtdIns(4,5)P2-dependent activation of PKC Specificity of Phosphoinositides for PKC
Under these conditions, PtdIns(3,4,5)P3 activated
only 57% of the total activity displayed by PtdIns(4,5)P2
suggesting that the latter is more efficient in activating the protein,
probably because it docks better to the binding site, whereas some
degree of steric impedance makes PtdIns(3,4,5)P3 less
capable of activating the enzyme. In the case of PtdIns(3)P, the
maximum activation reached was only 25% that obtained with
PtdIns(4,5)P2, demonstrating that PKC Classically, PtdIns(4,5)P2 has been defined as the
precursor of the mediators, diacylglycerol and inositol
(1,4,5)P3, after hydrolysis by hormone-sensitive
phospholipase C enzymes (41). However, new experiments are now
revealing another signaling mode, which is controlled by intact
PtdIns(4,5)P2 rather than its hydrolysis products. For
example, this phosphoinositide has been implicated in the control of
membrane dynamics, cell shape, and cytoskeleton rearrangement (42-43).
Thus, PtdIns(4,5)P2 has emerged as a highly versatile
signaling molecule in its own right, although it remains to be
clarified how the functions of the proteins that interact with these
lipids are coordinated.
In this work, we have defined a new PtdIns(4,5)P2-binding
site located in a crevice of the C2 domain of PKC Experiments performed with the isolated domain revealed that in the
absence of Ca2+ this site is more specific for
PtdIns(4,5)P2 than other negatively charged phospholipids.
Thus, two major and different phospholipid-binding sites can be defined
in the isolated domain as follows: first, the Ca2+ binding
region located on top of the domain, which binds Ca2+ and
phosphatidylserine and probably PtdIns(4,5)P2 but with
relatively low affinity; and second located in the crevice formed by
However, the scenario changed when binding and activity assays were
performed with full-length protein, in which case Ca2+ and
PMA were also necessary to obtain full activation of the enzyme when
PtdIns(4,5)P2 was used as activator. The binding
experiments carried out in the absence of Ca2+ showed that
the protein needs to be in a particular conformation/state to be
accessible to negatively charged phospholipids and PMA. This agrees
with previous reports that have described how, in the absence of
Ca2+, the pseudosubstrate is clamped to the catalytic
domain, and the binding of PS/Ca2+ and diacylglycerol is
necessary to promote the liberation of the pseudosubstrate, leading to
an "open conformation" and full activation of the enzyme (10,
44).2
The use of PKC Further support for a double and distinct phospholipid-binding site in
the C2 domain was obtained when the mutants were activated with POPS-
or PtdIns(4,5)P2-containing vesicles and compared with the
wild-type protein (Fig. 5). The D246N/D248N substitution was more
critical in the PS-dependent activation, whereas the
K209A/K211A substitution was more critical in the
PtdIns(4,5)P2-dependent activation.
Concerning the role of the C1 domain in the
PtdIns(4,5)P2-dependent activation of the
enzyme, the above experiments showed that this domain makes a small
contribution to full binding and activation of the enzyme, which would
take place in a third step in the sequential activation mechanism.
Recent structure-function studies have demonstrated that several
positively charged residues located on the domain surface interact
non-specifically with anionic phospholipids prior to membrane
penetration of hydrophobic residues (47), and this could be the reason
for the additional 25-30% binding and activation obtained in a
PMA-dependent process.
It is also interesting to note that PtdIns(4,5)P2 is able
to activate the enzyme to a greater extent than POPS, which has always
been defined as the main activator of PKC In relation to phosphoinositide specificity, it seems that
PtdIns(4,5)P2 is the best of the activators studied in this
work, probably because the docking site in full-length PKC It is important to note that although there are no sequence or
structural homologies between the ENTH and C2 domains, they share
important functional traits. Basically, the ENTH domains consist of
nine
by this phosphoinositide. By using two double mutants from two
different sites located in the C2 domain of protein kinase C
, we
have determined and characterized the PtdIns(4,5)P2-binding
site in the protein, which was found to be important for its
activation. Thus, there are two distinct sites in the C2 domain: the
first, the lysine-rich cluster located in the
3- and
4-sheets and
which activates the enzyme through direct binding of
PtdIns(4,5)P2; and the second, the already well described
site formed by the Ca2+-binding region, which also binds
phosphatidylserine and a result of which the enzyme is activated. The
results obtained in this work point to a sequential activation model,
in which protein kinase C
needs Ca2+ before the
PtdIns(4,5)P2-dependent activation of the
enzyme can occur.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
,
I,
II, and
, can be distinguished
from the other groups because its activity is regulated by
diacylglycerol (DAG) and, cooperatively, by Ca2+ and acidic
phospholipids, particularly phosphatidylserine (PS). Members of the
second group are the novel mammalian (
,
,
, and
) and yeast
PKCs that are not regulated by Ca2+. The third group
comprises the atypical PKC isoforms,
,
, and
, whose
regulation has not been clearly established, although it is clear that
they are not regulated by DAG or Ca2+ (8, 10).
-C2 domain, we
observe that some of the Lys/Arg residues described for synaptotagmin are conserved. Previous studies in our laboratory (32)
have suggested that the highly positive charged
3-
4-sheets could
interact electrostatically with the negatively charged phospholipids located at the membrane surface. Whether or not this is significant in
the context of the full-length protein is still not clear.
-C2 domain and
PtdIns(4,5)P2 and the consequent enzyme activation. For
this purpose, we cloned the PKC
C2 domain fused to glutathione
S-transferase (GST) and full-length PKC
fused to a
hemagglutinin (HA) tag, which enabled us to perform binding and
specific activity studies. Site-directed mutagenesis of key residues
located in two areas of the C2 domain shed light on the interaction of
the classical isoenzyme with PtdIns(4,5)P2 and its
activation mechanism.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
cDNA was a gift from Drs. Nishizuka and Ono (Kobe
University, Kobe, Japan). The cDNA fragment corresponding to
residues 158-285 of the PKC
-C2 domain and mutants was amplified
using PCR (33). Full-length PKC
mutants were generated by PCR
site-directed mutagenesis (14, 34). All constructs, both wild-type and
mutant genes, were subcloned into the mammalian expression vector pCGN (a gift from Dr. Tanaka, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY). This vector contains the cytomegalovirus promoter and the
multicloning sites that allow expression of the genes fused 3' to the
HA epitope (35). All constructs were confirmed by DNA sequencing.
-C2 domain and mutants was amplified using PCR (33).
-C2
-C2 domain was
transformed into HB101 Escherichia coli cells. Proteins were
expressed and purified as described in a previous work (33).
-C2 domain bound to glutathione-Sepharose beads was used.
Lipid vesicles were generated by mixing chloroform lipid solutions in
the desired proportions and then dried from the organic solvent under a
nitrogen stream and further dried under vacuum for 60 min.
1,2-Dipalmitoyl-L-3-phosphatidyl-N-[methyl-3H]choline
(PerkinElmer Life Sciences; specific activity 56 Ci/mmol) was included
in the lipid mixture as a tracer, a concentration of ~3000-6000
cpm/mg phospholipid. Dried phospholipids were resuspended in buffer
containing 50 mM Hepes, pH 7.2, 0.1 M NaCl, and
0.5 mM EGTA by vigorous vortexing and subjected to direct
probe sonication (four cycles of 1 min). Beads with protein bound to
them were prewashed with the respective test solutions and resuspended
in 50 µl of the corresponding lipid solution. The mixture was
incubated at room temperature for 15 min with vigorous shaking and then briefly centrifuged in a tabletop centrifuge. The beads were washed twice with 0.4 ml of the incubation buffer without liposomes. Liposome
binding was then quantified by liquid scintillation counting of the
beads. The data in the paper and figures are expressed in number of
nmol of lipid bound/10 µg of protein.
Membrane Binding Assay--
PKC
was incubated with
multilamellar vesicles (500 µM total lipid in 0.15 ml) in
a buffer containing 20 mM Tris/HCl, pH 7.5, 5 mM MgCl2, and 0.5 mM EGTA or 0.2 mM CaCl2 at room temperature for 15 min.
Vesicle-bound enzyme was separated from the free enzyme by centrifuging
the mixture at 13,000 × g for 30 min at 20 °C. Aliquots from the supernatants and pellets were separated by SDS-PAGE (12.5% separating gel). The proteins were transferred onto a
nitrocellulose membrane after electrophoresis. Immunoblot analysis of
the epitope tag fused to the protein was performed by using anti-HA
antibody 12CA5 and developed with chemiluminescence reagents
(PerkinElmer Life Sciences). The proteins were analyzed by densitometry.
-32P]ATP
(300,000 cpm/nmol), 5 mM MgCl2, and 200 µM CaCl2. The reaction was started by
addition of 5 µl of the PKC
purified from transfected HEK293
cells. After 10 or 30 min, the reaction was stopped with 1 ml of
ice-cold 25% trichloroacetic acid and 1 ml of ice-cold 0.05% bovine
serum albumin. After precipitation on ice for 30 min, the protein
precipitate was collected on a 2.5-cm glass fiber filter (Sartorius)
and washed with 10 ml of ice-cold 10% trichloroacetic acid. The amount
of 32Pi incorporated into histone was measured
by liquid scintillation counting. The linearity of the assay was
confirmed from the time course of histone phosphorylation during 30 min. Additional control experiments were performed with mock cell
lysates to estimate the endogenous PKC
and nonspecific activities,
which represented less than 1% of the total enzyme activity measured.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-C2
Domain Interaction--
Our first aim was to characterize the
biochemical properties of the interaction between
PtdIns(4,5)P2 and the C2 domain of PKC
. For this, a
recombinant fusion protein was used, in which the C2 domain of PKC
was NH2-terminally fused to GST (PKC
-C2 domain) (33).
The dependence of phospholipid binding on the PtdIns(4,5)P2
concentration was studied by using phospholipid vesicles containing
POPC/POPS (4:1, mol/mol) in the presence and in the absence of 100 µM CaCl2 (Fig.
1A). The results show that in
the presence of Ca2+, PtdIns(4,5)P2 slightly
inhibited the PKC
-C2 domain phospholipid binding activity, which
amounted to 8 nmol regardless of the PtdIns(4,5)P2 concentration in the lipid vesicles. More interesting were the results
obtained in the absence of Ca2+ because, in this case, the
PKC
-C2 domain bound to a similar phospholipid vesicle composition in
a PtdIns(4,5)P2-dependent manner, showing a
maximal binding of 7 nmol and a [PIP2]1/2 value
of 1 mol % (Fig. 1A). These results suggest that the C2 domain of PKC
may interact with PtdIns(4,5)P2
independently of Ca2+. To explore whether this mechanism is
specific to PtdIns(4,5)P2 or is related to the net negative
charges present in the membrane, binding assays were performed by
substituting PtdIns(4,5)P2 as supplier of net negative
charges to the phospholipid vesicles (POPC/POPS/PIP2,
75:20:5 mol/mol) for POPS (POPC/POPS, 70:30). It was observed that 7 nmol of lipid were bound in the former case and only 1.3 nmol in the
latter case (Fig. 1A, inset), suggesting that there is a
certain specificity for PtdIns(4,5)P2 and that the
increased binding observed under these conditions is not only dependent
on the net negative charges present at the membrane vesicles.
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Fig. 1.
PtdIns(4,5)P2-dependent binding of
PKC -C2 domain. A, binding of
PKC
-C2 domain to small unilamellar vesicles containing 75 mol %
POPC, 20 mol % POPS, and increasing concentrations of
PtdIns(4,5)P2 in the absence (
) and in the presence
(
) of 100 µM CaCl2. The inset
in A represents the lipid bound to the PKC
-C2 domain when
the vesicles contained 5 mol % PtdIns(4,5)P2 and 20%
POPS or 30 mol % POPS. This experiment was performed in the absence
of Ca2+. B, binding of PKC
-C2 domain
to vesicles containing increasing concentrations of
PtdIns(4,5)P2 in the absence (
) and in the presence
(
) of 100 µM CaCl2. Lipid binding was
quantified by scintillation counting of the radioactive PC used as
tracer and expressed as nanomoles of lipid bound to the protein used in
the assay (10 µg). Specific binding was calculated by subtracting the
nonspecific lipid interaction of GST from individual samples at each
particular PtdIns(4,5)P2 concentration.
-C2
domain binding affinity in the absence of POPS in the lipid vesicles, binding assays were performed with POPC and increasing
PtdIns(4,5)P2 concentrations in the absence and in the
presence of 100 µM CaCl2 (Fig.
1B). In the absence of Ca2+, lipid binding
reached a maximum of 7 nmol and [PIP2]1/2 = 1.2 mol %, whereas in the presence of Ca2+, binding reached 9 nmol and [PIP2]1/2 = 0.3 mol %. This
demonstrates that in the absence of POPS, Ca2+ slightly
increases both the affinity of the protein for
PtdIns(4,5)P2-containing vesicles and the maximal binding capacity.
-C2
Domain--
The data described above might be explained by the
existence of two types of binding mechanism, one
Ca2+/PS-dependent and the other mostly
Ca2+-independent and
PtdIns(4,5)P2-dependent. Whether these two
mechanisms corresponded to two different sites in the domain or to just
one site with different biochemical behaviors could still not be
answered at this point. To address this question, we made use of two
PKC
-C2 domain mutants. One of them (PKC
-C2-D246N/D248N)
has been demonstrated to be affected in the Ca2+-binding
site (33, 34) (Fig. 2A) and
does not bind Ca2+ or PS. The second
(PKC
-C2-K209A/K211A) has been demonstrated to be affected in an area
corresponding to a lysine-rich cluster located in the
3-
4-sheets
(Fig. 2C) and partially lacks its ability to bind PS or
phosphatidic acid in the absence of Ca2+ (32).
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Fig. 2.
PtdIns(4,5)P2-dependent binding of
the PKC -C2 domain mutants. A,
three-dimensional model of the PKC
-C2 domain showing the location of
the two aspartate residues mutated to asparagine (D246 and
D248) in the Ca2+ binding region. The two
Ca2+ ions are shown in orange. The
structure corresponds to the PDB 1DSY described by Verdaguer et
al. (38). B, binding of PKC
-C2D246N/D248N domain to
vesicles containing increasing concentrations of
PtdIns(4,5)P2 in the absence of Ca2+ (
). The
dotted line represents the results obtained for wild-type C2
domain in Fig. 1B. C, three-dimensional model of
the PKC
-C2 domain showing the location of the two lysine residues
mutated to alanine (K209 and K211). D,
binding of PKC
-C2K209A/K211A domain to vesicles containing
increasing concentrations of PtdIns(4,5)P2 in the absence
(
) and in the presence (
) of 100 µM
CaCl2. Dotted lines representing the data
obtained for wild-type protein have been included in the figure to
facilitate comparison. The binding assays were performed under the same
conditions stated in Fig. 1.
-C2-D246N/D248N mutant was employed in the binding assays using
vesicles containing POPC and increasing concentrations of
PtdIns(4,5)P2 in the absence of Ca2+. In this
case, maximal binding activity was 8 nmol of lipid and [PIP2]1/2 = 0.5 mol %. These results are
slightly higher than those obtained when the wild-type C2 domain was
used in the assay (Figs. 1B and 2B, dotted
line), suggesting that the substitution of these residues located
at the Ca2+-binding site has no effect on the ability of
the domain to bind to PtdIns(4,5)P2-containing vesicles in
a Ca2+-independent manner. Furthermore, the results
obtained suggest that the neutralization of the
Ca2+-binding sites performed by the mutagenesis strategy
probably helps to reduce the negative electrostatic potential exhibited by this area, in a similar way to the effect brought about by Ca2+ when it binds to this region (40), thus facilitating
the slight increase in the binding affinity observed in both cases.
-C2-K209A/K211A, was included in the
binding assay under the same conditions as described above (Fig.
2D), only 1.3 nmol of lipids were bound to the protein, indicating that these two residues are directly involved in the Ca2+-independent and
PtdIns(4,5)P2-dependent binding activity of the domain. Furthermore, when this experiment was performed in the presence
of 100 µM CaCl2, the PKC
-C2-K209A/K211A
mutant was able to bind only 5.3 nmol of lipids at high concentrations
of PtdIns(4,5)P2, which amounted to only 58% of the lipid
bound with wild-type protein under these conditions (Fig.
2D). In summary, these data support a double-site model for
PtdIns(4,5)P2 binding. The first and most important would
be the lysine-rich cluster, which binds PtdIns(4,5)P2 with
no need for Ca2+, and the second would be located in the
Ca2+ binding region. However, this last site exhibits a
relatively low affinity for PtdIns(4,5)P2 even in the
presence of Ca2+, suggesting that this inositide fits
better in the lysine-rich region than in the Ca2+ binding region.
(Full-length) Interaction--
As described in the Introduction,
PKC
activation is regulated by multiple factors (10). Moreover,
several of the domains included in the protein are involved in the full
activation of the enzyme, which further complicates the situation. To
investigate the role of this new PtdIns(4,5)P2-binding site
in PKC
membrane translocation, the binding of the protein to POPC
multilamellar vesicles was studied by including 5 mol %
PtdIns(4,5)P2 or 20 mol % POPS in the presence of
Ca2+ and, in some cases, using a phorbol ester (PMA) as a
C1 domain-dependent activator (Fig.
3A). Both
PtdIns(4,5)P2 and POPS were able to produce 50% protein
translocation to the membranes in the absence of PMA. This
translocation increased to 75% when both phospholipids were included
in the same vesicles (5 mol % PIP2 and 20 mol % POPS).
As expected, the inclusion of a saturating concentration of PMA (0.3 mol %) cooperated and increased the proportion of translocated
protein to 100%, independently of the negatively charged phospholipids
present in the membrane vesicles.
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Fig. 3.
Binding properties of full-length
PKC to membrane models containing POPC and 5 mol % of PIP2, 20 mol % POPS, or both
simultaneously. The assay was performed in the presence of 0.2 mM CaCl2 (A) and 0.5 mM
EGTA (B). Vesicles containing 100 mol % of POPC were used
as control. PMA and histone concentrations were 0.3 mol % (equivalent
to 1.5 µM under the conditions of the assay) and 0.2 mg/ml, respectively.
translocation was detected in the absence of PMA.
Furthermore, when 0.3 mol % PMA was included in the assay, only 10%
of protein translocation was detected in vesicles containing either
POPS or PtdIns(4,5)P2/POPS, suggesting that these binding sites were not functional or accessible to the membrane vesicles under
these conditions. To test this further, a PKC
substrate (histone
III-S) was included in the binding reaction. In this case, protein
translocation was 51, 42, and 45% when PtdIns(4,5)P2, POPS, or POPS/PtdIns(4,5)P2, respectively, were included in
the vesicles, indicating that the presence of substrate enhances the ability of the PtdIns(4,5)P2 and PS sites to interact with
the lipid vesicles. When PMA and histone were included in the model membranes, protein translocation to them was 100%, suggesting that
under these conditions either PtdIns(4,5)P2 or POPS with PMA access their corresponding sites (C2 and C1 domain, respectively), producing full translocation of the enzyme to the membrane vesicles.
--
To study whether protein translocation to the phospholipid
vesicles correlates with its activation, PKC
specific activity was
measured under conditions similar to that used in the binding assays.
Fig. 4A shows the catalytic
activity of the enzyme in the presence of 100 µM
CaCl2, 0.3 mol % PMA, and increasing concentrations of
POPS or PtdIns(4,5)P2 in the phospholipid vesicles. Under
these conditions, 20 mol % PtdIns(4,5)P2 increased enzyme
activity more than 5 times that obtained when using vesicles containing
20 mol % POPS. These data correlate well with the binding assays,
because 5 mol % PtdIns(4,5)P2 exhibited a similar binding
and activation capacity to 20 mol % POPS.
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Fig. 4.
Dependence of enzymatic activity of
PKC on the PtdIns(4,5)P2
(A) and POPS (B) concentrations.
Catalytic activity was measured in the presence of vesicles containing
POPC, 0.3 mol % PMA, 0.2 mM CaCl2 and
increasing mole percentages of the indicated phospholipids. Histone
III-S was used as substrate. Error bars indicate the S.E.
for triplicate determinations.
in the presence of Ca2+ is
almost 3 times the specific activity obtained in the absence of
Ca2+, indicating again that, directly or indirectly,
Ca2+ plays a role in the
PtdIns(4,5)P2-dependent activation of the enzyme.
--
To address this question the same C2
domain mutants as above were used in the context of full-length PKC
,
namely PKC
-D246N/D248N and PKC
-K209A/K211A to measure the effect
of increasing concentrations of PtdIns(4,5)P2 and POPS on
the specific enzyme activity. The results were compared with those
obtained with wild-type protein. Fig.
5A shows the activation of
PKC
-D246N/D248N and PKC
-K209A/K211A in the presence of 100 µM CaCl2, 0.3 mol % PMA, and increasing concentrations of PtdIns(4,5)P2. It can be clearly observed
that PKC
-K209A/K211A activation represents only 34% and
PKC
-D246N/D248N 60% of the total activity exhibited by wild-type
protein (Table I).
View larger version (14K):
[in a new window]
Fig. 5.
Dependence of enzymatic activity of
PKC -C2D246N/D248N (
) and
PKC
-C2K209A/K211A (
) on the
PtdIns(4,5)P2 (A) and POPS
(B) concentrations. Catalytic activity was
measured in the presence of vesicles containing POPC, 0.3 mol % PMA,
0.2 mM CaCl2 and increasing mole percentages of
the indicated phospholipids. Histone III-S was used as substrate.
Error bars indicate the S.E. for triplicate determinations.
To facilitate comparison the results obtained for wild-type
(WT) protein under the same conditions are represented with
dotted lines (Fig. 4).
Specific activity of wild-type PKC and its mutants (nmol of
Pi/min/mg)
-D246N/D248N
activation was only 28%, whereas PKC
-K209A/K211A activation was
very similar to the maximum activity reached by wild-type protein under
the same conditions. These data suggest that in the activation driven by PtdIns(4,5)P2, lysines 209 and 211 play a more important
role than the aspartate residues located in the
Ca2+-binding site. On the other hand, when both mutants
were tested for activation in the absence of Ca2+ (Fig.
6), no differences were found with
wild-type PKC
, whether vesicles containing PtdIns(4,5)P2
or POPS were used. Nevertheless, the enzyme was increasingly activated
with increasing concentrations of PtdIns(4,5)P2 although
the maximum activation in this case decreased 3-fold. This might be
explained by the fact that in full-length protein there is also a
partial contribution of the C1 domain to the PtdIns(4,5)P2
activation. It is important to note that the maximal activation under
these conditions for wild-type protein only represents 30% of the
maximal enzyme activation obtained in the presence of Ca2+,
suggesting that the greatest contribution to
PtdIns(4,5)P2-dependent activation of the
enzyme resides in the C2 domain.
View larger version (21K):
[in a new window]
Fig. 6.
Dependence of enzymatic activity of
PKC -C2D246N/D248N (
and
) and
PKC
-C2K209A/K211A (
and
) on the
PtdIns(4,5)P2 (
and
) and POPS (
and
)
concentrations in a Ca2+-independent manner. Catalytic
activity was measured in the presence of vesicles containing POPC, 0.3 mol % PMA, 0.5 mM EGTA and increasing mole percentages of
the indicated phospholipids. Dotted lines represent the
results obtained for wild-type protein under the same conditions of
assay (Fig. 4) and are presented for comparison purposes. Error
bars indicate the S.E. for triplicate determinations.
-K209A/K211A mutant was assayed in the presence
and in the absence of Ca2+ was 279 and 235 nmol
Pi/min/mg, respectively (Table I), representing small
differences in the PMA-dependent activation of the mutant. Importantly, the
Ca2+/PtdIns(4,5)P2-dependent
activation was completely abolished in this case, suggesting once again
that the lysine-rich cluster located in the C2 domain plays a very
important role in the activation of the enzyme under these conditions.
Strikingly, when the PKC
-D246N/D248N mutant was assayed, 240 and 485 Pi/min/mg were obtained in the absence and in the presence
of Ca2+, respectively. Note that, even in the presence of
Ca2+, this mutant did not recover full activation although
the lysine-rich cluster remained intact, suggesting again that
Ca2+ access to the Ca2+ binding region is
probably a key event in the
PtdIns(4,5)P2-dependent activation process.
was 1.3-fold less than that obtained in the presence of PMA, suggesting
that the main contribution for this type of activation is supplied by
the C2 domain. Furthermore, when increasing concentrations of
PtdIns(4,5)P2 were used, PKC
-D246N/D248N mutant did not
activate more than 45% of the total activity exhibited by wild-type
protein (Fig. 7 and Table I). The
activation capacity of PKC
-K209A/K211A mutant was even more limited
under these conditions (only 18% of total activation) suggesting that
this site is primarily involved in the
PtdIns(4,5)P2-dependent activation process.
View larger version (15K):
[in a new window]
Fig. 7.
Dependence of enzymatic activity of wild-type
PKC (
),
PKC
-C2D246N/D248N (
), and
PKC
-C2K209A/K211A (
) on the
PtdIns(4,5)P2 concentration in the absence of PMA.
Catalytic activity was measured in the presence of vesicles containing
POPC, 0.2 mM CaCl2, and increasing mole
percentages of PtdIns(4,5)P2. Error bars
indicate the S.E. for triplicate determinations.
, 45% of PKC
-D246N/D248N,
and 18% of PKC
-K209A/K211A bound to them. When PMA was included in
the membrane vesicles, up to 100% of wild-type protein was bound, and
only 70 and 28% of PKC
-D246N/D248N and PKC
-K209A/K211A,
respectively, were bound under the same conditions. These results
correlate very well with the specific activity assays, suggesting that
the lack of activity in each case corresponds to a lack of membrane
binding.
View larger version (36K):
[in a new window]
Fig. 8.
Binding of wild-type
PKC ,
PKC
-C2D246N/D248N, and
PKC
-C2K209A/K211A to
PtdIns(4,5)P2-containing vesicles. Results are
represented as a percentage of the protein bound to the lipid vesicles
after centrifugation. Protein was analyzed by immunoblotting with
anti-HA antibody and developed by enhanced chemiluminescence.
and, although contributions of the C2 and C1 domains are necessary for
full activation of the enzyme, the C2 domain plays a more specific role
in this activation mechanism.
Activation--
10 and 20 mol % of PtdIns(3)P and
PtdIns(3,4,5)P3 were included in multilamellar vesicles
containing POPC and 0.3 mol % PMA, and the specific activity of
PKC
was measured in the presence of 100 µM
CaCl2. The effect of each phosphoinositide on specific activity was compared with the activity observed in the presence of 10 and 20 mol % PtdIns(4,5)P2, respectively (Fig.
9).
View larger version (13K):
[in a new window]
Fig. 9.
Inositide specificity in the activation of
PKC . Catalytic activity was measured in the presence
of vesicles containing POPC, 0.2 mM CaCl2, and
10 (black bars) or 20 (gray bars) mol % of
PtdIns, PtdIns(3)P, and PtdIns(3,4,5)P3 and compared with
that obtained using PtdIns(4,5)P2. Error
bars indicate the S.E. for triplicate determinations.
exhibits a
higher specificity for PtdIns(4,5)P2 than for other phosphoinositides.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
formed by the
3- and
4-sheets that is different from the Ca2+
binding region where Ca2+ and PS interact (38, 32).
Furthermore, a different activation mechanism of the enzyme,
specifically triggered by PtdIns(4,5)P2, has been defined.
3- and
4-sheets, which is highly enriched in lysine residues and which binds PtdIns(4,5)P2 in a Ca2+-independent
manner. Recent structural studies in our laboratory have shown that
this lysine-rich cluster can bind negatively charged phospholipids,
such as diacetoyl-sn-glycero-3-phosphoserine and dicaproyl-sn-glycero-3-phosphatidic acid, mainly through
electrostatic interactions (32). Strikingly, binding inhibitions of
about 50-70% were only found when the experiments were performed in the absence of Ca2+, suggesting that these residues might
somehow be involved in the membrane translocation of the protein
independently of Ca2+. The results obtained in the present
work clearly explain the preliminary data obtained, because the
lysine-rich cluster has higher affinity for
PtdIns(4,5)P2 than PS, and this interaction is
Ca2+-independent.
-D246N/D248N and PKC
-K209A/K211A mutants has
enabled us to distinguish between the two different sites and to
discriminate their role in the PtdIns(4,5)P2-dependent
activation. In the absence of PMA, the catalytic activity of the
PKC
-K209A/K211A mutant was completely abolished, and by taking into
account that the Ca2+ binding region was intact in this
case, it is clear that Lys-209 and Lys-211 are key residues in the
PtdIns(4,5)P2-driven activation process. However, the
experiments performed with the PKC
-D246N/D248N mutant also revealed
that some contribution from this site is needed to obtain full
activation of the enzyme because the lysine-rich cluster is intact in
this case and the catalytic activity recovers to reach only 45% (Fig.
7). Note that this mutant (Asp-246 substituted by Asn) has been
described as the most important residue for coordinating the second
Ca2+ bound to the domain and probably for producing a
conformational change in the protein (38, 46). Thus, the results
obtained strongly suggest that Ca2+ binding to its own
region is a preliminary requirement for the PtdIns(4,5)P2
interaction with the lysine-rich cluster in the C2 domain.
. These findings have to be
considered carefully because the exact lateral organization and
effective concentrations of these phospholipids in the plasma membrane
are still not well defined, especially in the case of PtdIns(4,5)P2, which several hypotheses suggest may
accumulate in different metabolic pools existing in the cells, for
example in rafts, nascent phagosomes of macrophages, or membrane
ruffles (see Ref. 31 for a review).
adjusts better to this inositol phosphate than to others. The lesser degree of
activation produced by PtdIns(3,4,5)P3 could be explained
by steric clashes in the area that cannot accept three phosphates. Similar results have been described recently in the literature, and it
is clear that certain regions are more suitable for binding PtdIns(4,5)P2 than PtdIns(3,4,5)P3, for example
a new PtdIns(4,5)P2 binding region described in plant
phospholipase D
(48), the ENTH domains of epsin (49), and clathrin
assembly lymphoid myeloid leukemia protein (CALM) (39).
-helices forming a solenoid structure (39, 49), whereas C2
domains consist of an eight-stranded
-sandwich (50). However, recent
studies (39) on the ENTH domain of CALM have shown an unusual
PtdIns(4,5)P2-binding site, which is located in an exposed
cluster of lysines and appears to be different from the
PtdIns(4,5)P2-binding sites described previously for epsin, another ENTH domain-containing protein. Interestingly, when the tri-dimensional distribution of the side chains of the lysines involved
in PtdIns(4,5)P2 binding in the ENTH domain of CALM was compared with the distribution of the side chains of the lysines involved in the PtdIns(4,5)P2-binding site in the C2 domain
of PKC
, they exhibited a very high degree of similarity (Fig.
10, a and b).
Additionally, when a simulation was performed using Swiss PDB viewer
3.7, the PtdIns(4,5)P2 molecule found in CALM was seen to
fit in the lysine-rich cluster of the C2 domain, and the distances
observed between the lysine residues and the 4- and 5-phosphates of the
inositol ring were compatible with hydrogen bond interactions like
those observed in the ENTH domain of CALM (Fig. 10c).
View larger version (22K):
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Fig. 10.
a, model structure of the 2-
3 of
the ENTH domain of CALM (purple ribbon) complexed to
PtdIns(4,5)P2 (PDB 1HFA). The side chains of lysine residues (Lys-28,
Lys-38, and Lys-40) involved in the interaction with the phosphates 4 and 5 of the inositol ring are represented as a stick model
in yellow. b, model structure of the lysine rich
cluster of the C2 domain of PKC
located in the
3- and
4-sheets
(gray ribbon). Side chains of Lys-197, Lys-209, and Lys-211
are represented as a stick model in blue.
c, overlapping model showing the superimposition of the
lysine side chains of both domains; note how the inositol phosphate
ring originally bound to CALM also fits well in the crevice formed
by the lysine residues of the C2 domain of PKC
. The distances from
the paired side chains of each amino acid to the phosphate groups are
as follows: CALM-Lys-28 to P5 = 4.2 Å and C2-Lys-197 to P5 = 2.8 Å; CALM-Lys-40 to P5 = 2.23 Å and C2-Lys-209 to P5 = 3.7 Å; CALM-Lys-40 to P4 = 2.94 Å and C2-Lys-209 to P4 = 2.1 Å; CALM-Lys-38 to P4 = 3.1 Å and C2-Lys-211 to P4 = 3.5 Å. Note that all the distances calculated for the C2 domain are
compatible with binding. Visualization and calculations of the
structures were performed with Swiss-PDB Viewer 3.7 by GlaxoSmithKline
R&D Geneva (57).2
Biological Implications-- Recent studies (4, 41) have indicated that there are many sources of PtdIns(4,5)P2 in the cell, which appear to involve different enzymes. The recent development of techniques that permit direct visualization of PtdIns(4,5)P2 and PtdIns(3,4,5)P3 has provided important information on the spatio-temporal localization and function of these compounds in living cells (41, 43). By taking into account that the total level of PtdIns(4,5)P2 in a cell does not vary in response to agonist stimulation, it has been postulated that local increases of PtdIns(4,5)P2 at discrete sites, such as caveolae, nuclei, membrane ruffles, and focal contacts, probably mediate the diverse signaling functions of this phosphoinositide. However, the mechanism that could concentrate PtdIns(4,5)P2 in the plane of the plasma membrane is still under debate (3, 5, 31).
At the same time, it has been described that PKC can co-localize in
many of these membrane domains, and based on the results obtained in
this work, it could be postulated that PKC-PtdIns(4,5)P2 interaction could be an alternative pathway for translocating and
activating PKC to these particular areas or compartments of the cell.
For example, it has been demonstrated recently (24, 51, 52) that the
formation of a ternary complex of PtdIns(4,5)P2, sydecan-4,
and PKC could be the key event in the regulation of focal adhesion
and stress fiber formation. There is another nice example that shows
that PKC
delivery to the endosome is a caveolae-mediated process
(54), and it is possible that PtdIns(4,5)P2 might
constitute an anchoring point for PKC here. Similarly, it has been
demonstrated that PKC needs to translocate to membrane ruffles in order
to phosphorylate substrate protein such as
6
4 integrin and myristoylated alanine-rich C kinase substrate among others (55, 56). Additionally, many important lipid signaling pathways also occur within the nucleus,
the first components identified being PtdIns(4,5)P2 and its
precursors (45). Several isoforms of PKC are found in the nucleus or,
at least, can translocate there. For example, PKC
translocates to
the nucleus and perinuclear region of NIH-3T3 cells following PMA
treatment (53). This localization could be compatible with a different,
as yet undefined, mechanism of enzyme activation.
In summary, the present work clearly defines a new site in the C2
domain of PKC totally distinct from the PS/Ca2+ binding
region (Fig. 10), located in the lysine-rich cluster, where
specifically binds PtdIns(4,5)P2, leading to the enzyme activation. A sequential mechanism for this particular
PtdIns(4,5)P2-dependent activation would fit the
puzzle posed by the results: in the absence of Ca2+, the
protein is in a "closed conformation" and neither
PtdIns(4,5)P2 nor DAG can access the corresponding sites.
However, Ca2+, when present, binds to the Ca2+
binding region of the C2 domain, presumably leading to a conformational change in the full-length protein that now enables
PtdIns(4,5)P2 to access the lysine-rich cluster and
activate the enzyme (Fig. 11).
|
In general, differential engagement by the two lipid binding pockets of
the PKC-C2 domain could form part of distinct signaling complexes
that may result in the selective activation, inhibition, or
translocation to unique subcellular compartments, thus converting the
enzyme into a multifunctional kinase.
![]() |
FOOTNOTES |
---|
* This work was supported by Grants PB98-0389 from Dirección General de Enseñanza Superior e Investigación Científica (Madrid, Spain), PI-35/00789/ES/01 from Fundación Seneca (Murcia, Spain), and Programa Ramón y Cajal from Ministerio de Ciencia y Tecnologia and Universidad de Murcia (Spain) (to S. C.-G.).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. Tel.: 34-968-36-47-66;
Fax: 34-968-36-47-66; E-mail: jcgomez@um.es.
Published, JBC Papers in Press, November 7, 2002, DOI 10.1074/jbc.M209385200
2 S. Bolsover, J. C. Gomez-Fernandez, and S. Corbalan-Garcia, submitted for publication.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: PtdIns(4, 5)P2, phosphatidylinositol 4,5-bisphosphate; GST, glutathione S-transferase; HA, hemagglutinin; PA, phosphatidic acid; PKC, protein kinase C; PS, phosphatidylserine; PMA, 12-myristate 13-acetate; ENTH, epsin NH2-terminal; POPC, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine; POPS, 1-palmitoyl- 2-oleoyl-sn-glycero-3-phosphoserine; PIP2, phosphatidylinositol 4,5-bisphosphate; DAG, diacylglycerol; PDB, protein data bank; PtdIns(3, 4,5)P3, phosphatidylinositol 3,4,5-trisphosphate; PtdIns(3)P, phosphatidylinositol 3-phosphate; CALM, clathrin assembly lymphoid myeloid leukemia protein.
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