1
A new phosphatidylinositol 4,5-bisphosphate binding site located in the
C2 domain of protein kinase Cαααα.
Senena Corbalan-Garcia, Josefa Garcia-Garcia, Jose A. Rodríguez-Alfaro and Juan C.
Gomez-Fernandez1
Dept. de Bioquímica y Biología Molecular (A). Facultad de Veterinaria, Universidad de
Murcia, Apdo. 4021, E-30100 Murcia, Spain.
1Corresponding author:
Juan C. Gomez-Fernandez
Phone: 34 968 36 47 66
Fax: 34 968 36 47 66
running title: role of the lysine-rich cluster of the C2 domain of PKCα
Copyright 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
JBC Papers in Press. Published on November 7, 2002 as Manuscript M209385200 by guest on A
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SUMMARY
In view of the interest shown in PtdIns(4,5)P2 as a second messenger, we study
the activation of Protein Kinase Cα by this phosphoinositide. Using two double-mutants
from two different sites located in the C2 domain of PKCα, 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 β3 and β4 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 as a results of
which the enzyme is activated. The results obtained in this work point to a sequential
activation model, in which PKCα needs Ca2+ before the PtdIns(4,5)P2-dependent
activation of the enzyme can occur.
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INTRODUCTION
Phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2) 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) comprises 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
mammaliam PKC isotypes have been grouped into smaller subfamilies. The first group,
which includes the classical isoforms, α, βI, βII and γ, 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 (δ, ε, η and θ) and yeast PKCs
which are not regulated by Ca2+. The third group comprises the atypical PKC
isoforms,ζ, ι and λ, whose regulation has not been clearly established, although it is
clear that they are not regulated by DAG or Ca2+ (8, 10).
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
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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
have addressed the activation of PKC isotypes by this class of lipid (11, 18-24).
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
colinear sequences that consist of highly basic aminoacidic 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 aminoacidic sequence of the PKCα-C2 domain, we observe
that some of the K/R residues described for synaptotagmin are conserved. Previous
studies in our laboratory have suggested that the highly positive charged β3-β4 sheets
could interact electrostatically with the negatively charged phospholipids located at the
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membrane surface (32). Whether or not this is significant in the context of the full-
length protein is still not clear.
In this paper, we focus on the characterization of the interaction mechanism
between PKCα-C2 domain and PtdIns(4,5)P2 and the consequent enzyme activation.
For this purpose, we cloned the PKCα C2 domain fused to glutathione-S-transferase
(GST) and full-length PKCα fused to a haemagglutinine (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
isozyme with PtdIns(4,5)P2 and its activation mechanism.
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EXPERIMENTAL PROCEDURES
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
(POPA), and phosphatidylinositol-4,5-bisphosphate (PIP2) were purchased from Avanti
Polar Lipids Inc.(Birmingham, AL). Phosphatidylinositol-3,4,5-trisphosphate (PIP3)
and phosphatidylinositol-3-phosphate (PI(3)P) were purchased from Echelon
Biosciences Inc (Salt Lake City, UT). Phosphatidylinositol (PI) and
phosphatidylglycerol (PG) were purchased from Lipid Products (Nutfield, Surrey, UK)
and phorbol 12-myristate 13-acetate (PMA) from Sigma Chemical Co. (Madrid,
Spain).
Construction of expression plasmids
Rat PKCα cDNA was a gift from Drs Nishizuka and Ono (Kobe University,
Kobe, Japan). The cDNA fragment corresponding to residues 158-285 of the PKCα-C2
domain and mutants was amplified using PCR (33). Full-length PKCα mutants were
generated by PCR site-directed mutagenesis (14, 34). All contructs, 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 haemagglutinine (HA) epitope (35). All constructs were
confirmed by DNA sequencing.
The cDNA fragment corresponding to residues 158-285 of the PKCα-C2
domain and mutants was amplified using PCR (33).
Expression and purification of GST-PKCα-C2
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The pGEX-KG plasmid containing the PKCα-C2 domain was transformed into
HB101 E. coli cells. Proteins were expressed and purified as described in a previous
work (33).
Cell culture, transfection and purification of PKCα
HEK293 cells were grown in Dulbecco´s modified Eagle´s medium (DMEM)
with 10% of fetal calf serum (FCS). Transfection was performed with the Ca2+
phosphate method described by Wigler el al. (36). Protein purification was performed
as described by Conesa-Zamora et al., (14).
Phospholipid binding measurements
a) Standard assay. The procedure described by Davletov and Südhof (37), was
used with minor modifications. A total of 10 µg of PKCα-C2 domain bound to
gluthatione-Sepharose beads were 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 minutes. 1,2-
dipalmitoyl-L-3-phosphatidyl N-[methyl-3H]choline (Dupont, Boston, MA; specific
activity 56 Ci/mmol) was included in the lipid mixture as a tracer, a concentration of
approximately 3000-6000 cpm/mg of 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
minute). 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, 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
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scintillation counting of the beads. The data in the paper and figures are expressed in
number of nmol lipid bound/10 µg protein.
b) PKCα membrane binding assay. PKCα 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 13000xg for 30 min at 20ºC. Aliquots from the supernatants and pellets were
separated by SDS-PAGE (12.5% separating gel). The proteins were transferred on to 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 chemiluminiscence reagents (NEN Life Science, Inc., Boston, MA). The proteins
were analyzed by densitometry.
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 20µl 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 [γ-32P]ATP (300,000 cpm/nmol), 5 mM MgCl2,
and 200 µM CaCl2. The reaction was started by addition of 5 µl of the PKCα purified
from transfected HEK293 cells. After 10 or 30 min, the reaction was stopped with 1 ml
of ice-cold 25% trichloroacetic acid (TCA) 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% TCA. The
amount of 32Pi incorporated into histone was measured by liquid scintillation counting.
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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 PKCα and non-specific activities, which
represented less than 1% of the total enzyme activity measured.
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 Glaxo Wellcome Experimental Research (N. Guex, A.
Diemand, T. Schwede and M.C. Peitsch) was used to visualize the structures.
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RESULTS
Characterization of the PtdIns(4,5)P2 and PKCαααα-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 PKCα. For this, a recombinant fusion
protein was used, in which the C2 domain of PKCα was N-terminally fused to GST
(PKCα-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 (Figure 1A). The results
show that in the presence of Ca2+, PtdIns(4,5)P2 slightly inhibited the PKCα-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+ since, in this case, the PKCα-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% (Figure 1A). These
results suggest that the C2 domain of PKCα 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 (Figure 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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In order to study whether Ca2+ can affect the PKCα-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 (Figure 1B). In the absence of Ca2+, lipid binding reached a maximum of 7
nmol and [PIP2]1/2=1.2 mol%, while 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.
Binding mechanism of PtdIns(4,5)P2 to PKCαααα-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 behaviours could still
not be answered at this point. To address this question, we made use of two PKCα-C2
domain mutants. One of them (PKCα-C2-D246/248N) has been demonstrated to be
affected in the Ca2+ binding site (33, 34) (Figure 2A) and does not bind Ca2+ or PS. The
second (PKCα-C2-K209/211A) has been demonstrated to be affected in an area
corresponding to a lysine-rich cluster located in the β3-β4 sheets (Figure 2C) and
partially lacks its ability to bind PS or PA in the absence of Ca2+ (32).
Figure 2B shows the results obtained when PKCα-C2-D246/248N 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 wild-type C2 domain was used in the assay (Figure 1B and
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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.
When the second mutant, PKCα-C2-K209/211A, was included in the binding
assay under the same conditions as described above (Figure 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 PKCα-C2-K209/211A 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 (Figure 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.
Characterization of the PtdIns(4,5)P2-PKCαααα (full-length) interaction.
As described in the introduction, PKCα activation is regulated by multiple
factors (10). Besides, several of the domains included in the protein are involved in the
full activation of the enzyme, which further complicates the situation. To investigate
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the role of this new PtdIns(4,5)P2 binding site in PKCα 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 esther (PMA) as a C1 domain-dependent activator (Figure 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.
Strikingly, very different results were obtained when these experiments were
performed in the absence of Ca2+ (Figure 3B). No PKCα 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 further test this, a
PKCα 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.
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PtdIns(4,5)P2-dependent activation of PKCαααα.
To study whether protein translocation to the phospholipid vesicles correlates
with its activation, PKCα specific activity was measured in conditions similar to that
used in the binding assays. Figure 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,
since 5 mol% PtdIns(4,5)P2 exhibited a similar binding and activation capacity to 20
mol% POPS.
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 (Figure 4B). These data correlated well with those obtained in
the binding assay, since 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 PKCα 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.
Demonstrating the existence of the new PtdIns(4,5)P2 site in full-length PKCαααα.
To address this question the same C2 domain mutants as above were used in the
context of full-length PKCα, namely, PKCα-D246/248N and PKCα-K209/211A to
measure the effect of increasing concentrations of PtdIns(4,5)P2 and POPS on the
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specific enzyme activity. The results were compared to those obtained with wild-type
protein. Figure 5A shows the activation of PKCα-D246/248N and PKCα-K209/211A
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 PKCα-K209/211A activation represents
only 34% and PKCα-D246/248N 60% of the total activity exhibited by wild-type
protein (table 1).
In contrast, when POPS was included in the lipid vesicles instead of
PtdIns(4,5)P2 (Figure 5B), PKCα-D246/248N activation was only 28%, while PKCα-
K209/211A 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+ (Figure 6), no differences were found with
wild-type PKCα, 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.
It is also interesting to note that the maximal specific activity obtained when the
PKCα-K209/211A mutant was assayed in the presence and in the absence of Ca2+ was
279 and 235 nmol Pi/min/mg, respectively (Table 1), representing small differences in
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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 PKCα-
D246/248N 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 while 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.
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 PKCα 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, PKCα-D246/248N mutant did not activate
more than 45% of the total activity exhibited by wild-type protein (Figure 7 and Table
1). The activation capacity of PKCα-K209/211A 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.
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 (Figure 8). In a similar
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way to what happened in the activation assays, when PtdIns(4,5)P2 was included in the
membranes, 78% of wild-type PKCα, 45% of PKCα-D246/248N and 18% of PKCα-
K209/211A bound to them. When PMA was included into the membrane vesicles, up
to 100% of wild-type protein was bound, and only 70 and 28% of PKCα-D246/248N
and PKCα-K209/211A 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.
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 PKCα
and, while 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.
Specificity of phosphoinositides for PKCαααα 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
PKCα 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 (Figure 9).
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, while 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% of that obtained with
PtdIns(4,5)P2, demonstrating that PKCα exhibits a higher specificity for PtdIns(4,5)P2
than for other phosphoinositides.
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DISCUSSION
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 PKCα formed by the β3 and β4 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.
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: one, 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 the second located in the crevice formed by β3-β4 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 DAPS and DCPA, mainly
through electrostatic interactions (32). Strikingly, binding inhibitions of about 50-70%
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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, since the lysine-rich cluster has
higher affinity for PtdIns(4,5)P2 than PS and this interaction is Ca2+-independent.
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 are necessary to promote the liberation of the
pseudosubstrate, leading to an “open conformation” and full-activation of the enzyme
(10, 44, 45).
The use of PKCα-D246/248N and PKCα-K209/211A 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
PKCα-K209/211A mutant was completely abolished and, taking into account that the
Ca2+ binding region was intact in this case, it is clear that K209 and K211 are key
residues in the PtdIns(4,5)P2-driven activation process. However, the experiments
performed with the PKCα-D246/248N mutant also revealed that some contribution
from this site is needed to obtain full activation of the enzyme since the lysine-rich
cluster is intact in this case and the catalytic activity recovers to reach only 45% (Figure
7). Note that this mutant (D246 substituted by N) has been described as the most
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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.
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 (Figure 5). The
D246/248N substitution was more critical in the PS-dependent activation, whereas the
K209/211A 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
PKCα. These findings have to be taken carefully since 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,
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nascent phagosomes of macrophages or membrane ruffles (see reference 31 for a
review).
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
PKCα 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
PLDβ (48), the epsin NH2-terminal (ENTH) domains of epsin (49) and clathrin
assembly lymphoid myeloid leukemia protein (CALM) (39).
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 α helices forming a solenoid structure (39,
49) while C2 domains consist of an eight-stranded β-sandwich (50). However, recent
studies 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 (39). 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 PKCα, they exhibited a very high degree
of similarity (Figure 10a 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
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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 (Figure
10 c).
Biological implications
Recent studies have indicated that there are many sources of PtdIns(4,5)P2 in the
cell, which appear to involve different enzymes (4, 41). 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). 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 which 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 that the formation of a ternary complex
of PtdIns(4,5)P2, sydecan-4 and PKCα, could be the key event in the regulation of focal
adhesion and stress fiber formation (51-53). There is another nice example which shows
that PKCα 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
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phosphorylate substrate protein such as α6β4 integrin and MARCKS 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 (57).
Several isoforms of PKC are found in the nucleus or, at least, can translocate there. For
example, PKCα translocates to the nucleus and perinuclear region of NIH-3T3 cells
following PMA treatment (58). 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
PKCα totally distinct from the PS/Ca2+ binding region (Figure 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 (Figure 11).
In general, differential engagement by the two lipid binding pockets of the
PKCα-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.
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REFERENCES
1. Divecha, N., Banfic, H. And Irvine, R.F. (1991) EMBO J. 10, 3207-3214. 2. Tran D, Gascard P, Berthon B, Fukami K, Takenawa T, Giraud F, Claret M. (1993) Cell Signal. 5, 565-81. 3. Martin, T.F.J. (1998) Annu. Rev. Cell Dev. Biol. 14, 231-264. 4. Toker, A. (1998) Curr. Opin. Cell Biol. 10:254-261. 5. Anderson, R.A., Boronenkov I.V., Doughman S.D., Kunz, J. and Loijens, J.C. (1999) J. Biol. Chem. 274, 9907-9910. 6. Toker, A. and Cantley, L.C. (1997) Nature 387, 673-676. 7. Nishizuka, Y. (1992) Science 258, 607-614. 8. Dekker, L.V. and Parker, P.J. (1994) Trends Biochem. Sci. 19, 73-77. 9. Toker, A. (1998) Front Biosci. Nov 1;3:D1134-47. 10. Newton, A.C. (1997) Curr. Opin. Cell Biol. 9, 161-167. 11. Lee, M. H.; and Bell, R. M. (1991). Biochemistry 30, 1041-9. 12. Newton, A. C.; and Keranen, L. M. (1994) Biochemistry 33, 6651-6658. 13. Johnson JE, Zimmerman ML, Daleke DL and Newton AC (1998) Biochemistry 37, 12020-5. 14. Conesa-Zamora, P., Lopez-Andreo, M. J., Gómez-Fernández, J. C. & Corbalán-García, S. (2001). Biochemistry 40, 13898-13905. 15. Newton, A. & Johnson, J.E. (1998). Biochim. Bioph. Acta 1376, 155-172. 16. Toker, A. (2000) Mol. Pharmacol. 57, 652-658. 17. Khan, W.A., Blobe, G.C., and Hannun, Y.A. (1995) Cell Signal 7, 171-184. 18. Chauhan, V. P.; and Brockerhoff, H. (1988). Biochem. Biophys. Res. Commun. 155, 18-23. 19. Singh, S. S.; Chauhan, A.; Brockerhoff, H.; and Chauhan, V. P. (1993) Biochem. Biophys. Res. Commun. 195, 104-12. 20. Kochs, G.; Hummel, R.; Fiebich, B.; Sarre, T. F.; Marme, D.; and Hug, H. (1993) Biochem. J. 291, 627-33.
by guest on April 10, 2018
http://ww
w.jbc.org/
Dow
nloaded from
26
21. Nakanishi, H.; Brewer, K. A.; and Exton, J. H. (1993) J. Biol. Chem. 268, 13-6. 22. Toker, A.; Meyer, M.; Reddy, K. K.; Falck, J. R.; Aneja, R.; Aneja, S.; Parra, A.; Burns, D. J.; Ballas, L. M.; Cantley, L. C. (1994) J. Biol. Chem. 269, 32358-67. 23. Palmer, R.H., Dekker, L.V., Woscholski, R., Le Good, J.A., Gigg, R. and Parker, P. (1995) J. Biol. Chem. 270, 22412-22416. 24. Oh, E. S.; Woods, A.; Lim, S. T.; Theibert, A. W.; Couchman, J. R. (1998) J. Biol. Chem. 273, 10624-9. 25. Fukuda, M.; Aruga, J.; Niinobe, M.; Aimoto, S.; and Mikoshiba, K. (1994) J. Biol. Chem. 269, 29206-29211. 26. Schiavo G, Gu QM, Prestwich GD, Sollner TH, Rothman JE. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 13327-32. 27. Zhang, X., Rizo, J. and Sudhof, t.C. (1998) Biochemistry 37, 12395-12403. 28. Davletov, B.; Perisic, O.; and Williams, R. L. (1998) J. Biol. Chem. 273, 19093-6. 29. Sutton, R.B., Ernst, J.A., and Brunger, A.T. (1999) J. Cell Biol. 147, 589-598. 30. Chung, S. H.; Song, W. J.; Kim, K.; Bednarski, J. J.; Chen, J.; Prestwich, G. D. and Holz, R. W. (1998). J. Biol. Chem. 273, 10240-8. 31. MacLaughling, S., Wang, J., Gambhir, A. and Murray D. (2002) Annu. Rev. Biophys. Biomol. Struct. 31:151-175. 32. Ochoa, W.F., Corbalan-Garcia, S., Eritja, R., Rodriguez-Alfaro, J.A., Gomez-Fernandez, J.C., Fita, I. And Verdaguer, N. (2002). J. Mol. Biol. 320:277-91. 33. Corbalán-García, S.; Rodríguez-Alfaro, J. A.; and Gómez-Fernández, J. C. (1999) Biochem. J. 337, 513-521. 34. Conesa-Zamora, P., Gomez-Fernandez, J.C. & Corbalan-Garcia, S. (2000). Biochim. Biophys. Acta 1487, 246-254. 35. Tanaka, M., and Herr, W. (1990) Cell 60, 375-386. 36. Wigler, M., Silverstein, S., Lee, L.S., Pellicer, A., Cheng, V.C., and Axel, R. (1977) Cell 11, 223-227. 37. Davletof, B. and Sudhof, T.C. (1993) J. Biol. Chem. 268, 26386-26390. 38. Verdaguer, N.; Corbalán-García, S.; Ochoa, W. F.; Fita, I.; and Gómez-Fernández, J. C. (1999) EMBO J. 18, 6329-6338. 39. Ford, M.G., Pearse, B.M.F., Higgins, M., Vallis, Y., Owen, D.J., Gibson, A., Hopkins, C.R., Evans, P.R. and McMahon, H.T. (2001) Science 291, 1051-1055.
by guest on April 10, 2018
http://ww
w.jbc.org/
Dow
nloaded from
27
40. Murray, D. and Honig, B. (2002). Mol. Cell. 9, 145-154. 41. Czech, M. (2000) Cell 100, 603-606. 42. Honda, A., Nogami, M., Yokozeki, T., Yamazaki, M. and Nakamura (1999) Cell 99,521-532. 43. Raucher, D., Stauffer, T., Chen, W., Shen, K., Guo, S., York, J.D., Sheetz, M.P. and Meyer, T. (2000) Cell 100, 221-228. 44. Oancea, E.; and Meyer, T. (1998) Cell 95, 307-318. 45. Bolsover, S., Gomez-Fernandez, J.C. and Corbalan-Garcia, S. (submitted). 46. Medkova M. and Cho, W. (1998) J. Biol. Chem. 273, 17544-17552. 47. Bittova L, Stahelin RV, Cho W. (2001). J Biol Chem. 276, 4218-26. 48. Zheng, L., Shan, J., Krishnamorrthi, R. and Wang, X. (2002) Biochemistry 41, 4546-4553. 49. Itoh, T., Koshiba, S., Kigawa, T., Kikuchi, A., Yokoyama, S. and Takenawa, T. (2001) Science 291, 1047-1051. 50. Nalefski, E.A. and Falke, J.J. (1996) Protein Sci. 5, 2375-2390. 51. Baciu, P.C., and Goetinck, P.F. (1995) Mol. Biol. Cel 6, 1503-1513. 52. Eok-Soo, O., Woods, A. and Couchman, J.R. (1997) J. Biol. Chem. 272, 8133-8136. 53. Eok-Soo, O., Woods, A., Lim, S.T., Theibert, A.W. and Couchman, J.R. (1998) J. Biol. Chem. 273, 10624-10629. 54. Prevostel, C., Alice, V., Joubert, D. and Parker, P.J. (2000) J. Cell Sci. 113, 2575-2584. 55. Myat, M.M., Anderson, S., Allen, L.A. and Aderem, A. (1997) Curr. Biol. 7, 611-614. 56. Rabinovitz, I., Toker, A. and Mercurio, A.M. (1999) J. Cell Biol. 146, 1147-1159. 57. Cocco, L., Martelli, A.M., Gilmour, R.S., Ognibene, A., Manzoli, F.A., and Irvine, R.F. (1987) Biochem. J. 268, 765-770. 58. Leach, K.L., Powers, E.A., Ruff, V.A., Jaken, S. and Kaufmann, S. (1989) J. Cell Biol. 109, 685-695.
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FOOTNOTES
1This 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.
2Abbreviations: GST, glutathione-S-transferase; HA, haemagglutinine; PA,
phosphatidic acid; PKC, protein kinase C; PS, phosphatidylserine; PMA, 12-myristate
13-acetate.
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FIGURE LEGENDS
Figure 1. PtdIns(4,5)P2-dependent binding of PKCα-C2 domain. (A) Binding of
PKCα-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
PKCα-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 Ca. (B) Binding of
PKCα-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
nmol of lipid bound to the protein used in the assay (10 µg). Specific binding was
calculated by subtracting the non-specific lipid interaction of GST from individual
samples at each particular PtdIns(4,5)P2 concentration.
Figure 2. PtdIns(4,5)P2-dependent binding of the PKCα-C2 domain mutants. (A) 3D
model of the PKCα-C2 domain showing the location of the two aspartate residues
mutated to asparragine (D246 and D248) in the Ca 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 PKCα-C2D246/248N 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 figure 1B. (C)
3D model of the PKCα-C2 domain showing the location of the two lysine residues
mutated to alanine (K209 and K211). (D) Binding of PKCα-C2K209/211A domain to
vesicles containing increasing concentrations of PtdIns(4,5)P2 in the absence (● ) and in
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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 figure 1.
Figure 3. Binding properties of full-length PKCα 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 in the conditions of the assay) and 0.2 mg/ml,
respectively.
Figure 4. Dependence of enzymatic activity of PKCα 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 SEM for triplicate determinations.
Figure 5. Dependence of enzymatic activity of PKCα-C2D246/248N (● ) and PKCα-
C2K209/211A ( ) 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 SEM for triplicate determinations. To
facilitate comparison the results obtained for wild-type protein under the same
conditions are represented with dotted lines (figure 4).
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Figure 6. Dependence of enzymatic activity of PKCα-C2D246/248N (● , ■ ) and
PKCα-C2K209/211A ( , ❍ ) on the PtdIns(4,5)P2 (● , ) and POPS (■ , ❍ )
concentrations in a Ca-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 (figure 4) and are
presented for comparison purposes. Error bars indicate the SEM for triplicate
determinations.
Figure 7. Dependence of enzymatic activity of wild-type PKCα (● ), PKCα-
C2D246/248N (■ ) and PKCα-C2K209/211A ( ) 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 SEM for triplicate determinations.
Figure 8. Binding of wild-type PKCα, PKCα-C2D246/248N and PKCα-
C2K209/211A 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.
Figure 9. Inositide specificity in the activation of PKCα. Catalytic activity was
measured in the presence of vesicles containing POPC, 0.2 mM CaCl2 and 10 (black
bars) or 20 (grey bars) mol% of PtdIns, PtdIns(3)P and PtdIns(3,4,5)P3 and compared
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with that obtained using PtdIns(4,5)P2. Error bars indicate the SEM for triplicate
determinations.
Figure 10. (a) Model structure of the α2-α3 of the ENTH domain of CALM (purple
ribbon) complexed to PtdIns(4,5)P2 (pdb 1HFA). The side chains of lysine residues
(K28, K38 and K40) 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 PKCα located in the β3-β4 sheets (grey ribbon).
Side chains of K197, K209 and K211 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 PKCα. The distances
from the paired side chains of each aminoacid to the phosphate groups were as follows:
CALM-K28 to P5=4.2 Å, C2-K197 to P5=2.8 Å; CALM-K40 to P5=2.23 Å, C2-K209 to
P5=3.7 Å; CALM-K40 to P4=2.94 Å, C2-K209 to P4=2.1 Å; CALM-K38 to P4=3.1 Å,
C2-K211 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 Glaxo Wellcome Experimental Research (N.
Guex, A. Diemand, T. Schwede and M.C. Peitsch).
Figure 11. Schematic model of the PtdIns(4,5)P2-dependent activation mechanism of
PKCα. (a) represents a state in the absence of Ca2+, similar to that obtained in resting
cells. In this case, the protein adopts a “closed conformation” where the PtdIns(4,5)P2
site is not accessible to the enzyme. (b) shows an intermediate state occurring when the
Ca2+ concentration increases in the cytosol or when high Ca2+ concentrations are used in
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an in vitro model system. Under these conditions, the protein adopts an “open
conformation” state and if PtdIns(4,5)P2 is present in the membrane, it binds to the
lysine-rich cluster (LRC) located in the β3-β4 sheets. (c) represents a fully-active state
when the C1 domain can access the diacylglycerol (DAG) generated in the membrane,
which increases the catalytic activity of the enzyme through a more stable anchorage to
the plasma membrane.
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TABLE 1
Specific activity of wild-type PKCα and its mutants (nmol Pi/min/mg)a
aSpecific activity of the enzyme was measured in the presence of vesicles containing 20 mol%
PtdIns(4,5)P2 and 80 mol% POPC. Histone III-S was used as a substrate. 0.3 mol% PMA and 0.2 mM
CaCl2 concentrations were used in the assays.
+ PMA - PMA
+ Ca2+ - Ca2+ + Ca2+
PKCα 812 275 620
PKCα-D246/248N 485 240 276
PKCα-K209/211A 279 235 113
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P IP 2 (% )
0 2 4 6 8 10
0
2
4
6
8
10
0
2
4
6
8
10
C orba lan-G arcia e t a l., F igure 1
0
2
4
6
8
P C /3 0 % P S
P C /2 0 % P S /5 % P IP 2
A
B
3 [H]-P
C bou
nd (n
mol)
3 [H]-P
C bou
nd (n
mol)
3 [H]-P
C bou
nd (n
mol)
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P IP 2 (% )
0 2 4 6 8 10
0
2
4
6
8
10
0
2
4
6
8
10Calcium binding sitePKC-C2D246/248Na
Lysine-rich clusterPKC-C2K209/211Aa
A
B
C
D
D246
D248
K209
K211
PKC-C2D246/248Na
PKC-C2K209/211Aa
+Ca 2 +
+Ca 2 +
-Ca 2 +
-Ca 2 +
WT
WT
WT
Corbalan-Garcia et al., Figure 2
3 [H]-P
C bou
nd (n
mol)
3 [H]-P
C bou
nd (n
mol)
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[an io n ic p h o sp h o lip id ] (m o l% )
0 5 10 15 200
50
100
150
200
250
300
0
150
300
450
600
750
900
A
B
C orba lan-G arcia e t a l., F igure 4
Spec
ific ac
tivity
(nmo
l Pi/m
in/mg
)Sp
ecific
activ
ity(n
mol P
i/min/
mg)
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[P IP 2 ] (m ol% )
0 5 10 15 20
0
40
80
120
160
0 5 10 15 20
0
150
300
450
600
750
900
[P O P S ] (m ol% )
W T -P KC a
P KC a - D 246 /248N
P KC a - K209 /211A
W T -P KC a
P KC a - D 246 /248N
P KC a - K209 /211A
+C a 2+
+C a 2+
A
B
C orba lan-G arcia e t a l., F igure 5
Spec
ific ac
tivity
(nmo
l Pi/m
in/mg
)Sp
ecific
activ
ity(n
mol P
i/min/
mg)
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[P IP 2] or [P O P S ] (m ol% )0 5 10 15 20
0
50
100
150
200
250
300
W T -P KC a
P KC a - D 246 /248N
P KC a - K209 /211A
E G T A
C orba lan-G arcia e t a l., F igure 6
Spec
ific ac
tivity
(nmo
l Pi/m
in/mg
)
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- P M A / +C a 2+
[P IP 2 ] (m ol% )
0 5 10 15 20
0
200
400
600
800
C orba lan-G arcia e t a l., F igure 7
Spec
ific ac
tivity
(nmo
l Pi/m
in/mg
)
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0
200
400
600
800
C orba lan-G arcia e t a l., F igure 9
P IP 3P I3P P IP 2
Spec
ific ac
tivity
(nmo
l Pi/m
in/mg
)
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Corbalan-Garcia et al., Figure 11
C1aC1b
C2
PIP2 DAG
cytosol
plasma membrane surface
Cat
inactive stateclosed conformation
active stateopen conformation full active state
+ Ca2+
+ DAG
- Ca2+
a
C1a C1bC2
Cat
bCBR
LRC- - - + + +
PIP2
+ + +
C2Cat
+ + +
c
C1aC1b
PS
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Gómez-FernándezSenena Corbalán-García, Josefa García-García, José A. Rodríguez-Alfaro and Juan C.
protein kinase C alphaA new phosphatidylinositol 4,5-bisphosphate binding site located in the C2 domain of
published online November 7, 2002J. Biol. Chem.
10.1074/jbc.M209385200Access the most updated version of this article at doi:
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