From the Department of Neurology and Neurosurgery, Montreal
Neurological Institute, McGill University, Montreal,
Quebec H3A 2B4, Canada
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INTRODUCTION |
Protein kinase Cs (PKC)1
are a family of lipid-activated enzymes that play important roles in
many cellular processes including regulating the strength of synapses
in the nervous system (1, 2). This role of PKC has been well conserved
over evolution with activation of PKC causing similar changes in
vertebrate and invertebrate synapses (1, 3-6). The structure of PKCs
has also been well conserved. Ca2+-activated PKCs (cPKCs)
in vertebrates (
,
, and
) and invertebrates (PKC Apl I in
Aplysia) have two C1 domains and a C2 domain.
Ca2+-independent or novel PKCs (nPKCs) in vertebrates (
,
,
, and
) and invertebrates (PKC Apl II in Aplysia)
also have two C1 domains and a C2 domain. However, the C2 domain in
these kinases is at the N-terminal side of the C1 domain and lacks
several of the critical aspartic acids that are required for
Ca2+ binding (7).
In cPKCs, the C2 domain confers the ability to bind to acidic lipids in
the presence of Ca2+ ions, while the C1 domain confers the
ability to bind to lipid in a diacylglycerol
(DAG)-dependent manner (8, 9). The
DAG-dependent lipid binding to the C1 domain is specific
for phosphatidylserine (PS) while binding to other lipids is
independent of DAG (10). The two lipid-binding domains act in concert
to activate cPKCs by causing a conformational change leading to the
release of kinase inhibition by the pseudosubstrate. In contrast to
cPKCs, a detailed model for the activation of nPKCs has not been
presented. Removing the C2 domain of PKC
does not affect substrate
specificity or the ability to be activated by PIP2 or
PIP3 (11, 12). However, removing the C2 domain of PKC Apl
II reduces the levels of PS required for enzyme activation, suggesting
that the C2 domain plays an inhibitory role (13). As well, peptides
derived from the C2 domains of both cPKCs and nPKCs act as
isoform-specific dominant negative inhibitors in cells, suggesting that
the physiological function of both kinases requires specific binding of
factors to these domains (14, 15).
C2 domains are thought to be important in the translocation of proteins
to membranes (7, 8, 16). In Aplysia, short-term treatments
with the facilitating transmitter 5-HT can translocate PKC Apl I to
membranes, but not PKC Apl II (17). In contrast, insulin can
persistently translocate PKC Apl II, but not PKC Apl I to membranes
(18). These results suggest that the regulation of enzyme translocation
is different in these two PKC isoforms, perhaps due to their distinct
C2 domains.
One way to determine the roles of C1 and C2 domains is to study these
domains in isolation using fusion proteins containing one or both of
these domains. These studies have illustrated that the C1 domains are
sufficient to bind to phorbol dibutyrate (PDBu, a pharmacological
analog of DAG) in a lipid-dependent manner, and that some
C2 domains bind to lipids in a Ca2+-dependent
manner (16, 19-22). Furthermore, there is some evidence that these two
domains may interact since addition of a C2 domain to a single C1
domain of PKC
leads to a decrease in PDBu affinity (21). Similarly,
a fusion protein containing both the C2 domain and C1 domains of PKC
Apl II has a lower affinity for PDBu than a fusion protein containing
only the C1 domains (23). Small amounts of phosphatidic acid (PA)
increase the affinity of the C2 domain containing construct for PDBu,
suggesting that PA might act specifically to remove the effect of the
C2 domain of PKC Apl II (23).
In this paper we have examined lipid interactions of fusion proteins
containing C1 or C2 domains of the cPKC Apl I and the nPKC Apl II,
using either a direct liposome binding assay or measuring the ability
of lipids to stimulate PDBu binding to the fusion proteins. We have
compared these results to the ability of lipids to stimulate either
PDBu binding or kinase activity of purified preparations of PKC Apl I,
PKC Apl II, and PKC Apl II without its C2 domain. Our results indicate
that the major difference between cPKCs and nPKCs is that lipids act
through the C2 domain to enhance activity of cPKCs but not nPKCs.
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MATERIALS AND METHODS |
Reagents--
4
-Phorbol 12,13-dibutyrate was from LC
Services; [3H]4
-phorbol 12,13-dibutyrate (19.6 Ci/mmol), NEN Life Science Products Inc.;
1,2-dipalmitoyl-L-3-phosphatidyl-[N-methyl-3H]choline
(100 mCi/mg), Amersham, Oakville, ON, Canada; dioleoyl phosphatidylserine (PS), dioleoyl phosphatidylcholine (PC), dioleoyl phosphatidylethanolamine (PE), dioleoyl phosphatidic acid (PA), and
dioctylglycerol (DOG) were from Avanti Polar Lipids Inc., Alabaster,
AL; phosphatidylinositol (PI) (mostly linoleic and palmitic acid) from
Sigma; Triton X-100 (Pierce); Sephacryl S-100 HR from Pharmacia,
Uppsala, Sweden; prestained molecular weight markers, Amersham Corp.,
and trypsin from Worthington Biochemicals, Freehold, NJ. All other
reagents were of the highest grade available.
Construction of Plasmids Encoding Fusion Proteins--
The
generation of GST-Apl II-C1 and GST-Apl II-C2 has been described (23).
GST-Apl I C1 domain was made using polymerase chain reaction with VENT
polymerase (New England Biolabs, Beverly, MA) from the plasmid
expressing PKC Apl I. The 5'-primer was
GGGATCCTTATGGAGAAGCGAGTCGCTAGGC and the 3'-primer was
GGAATTCCTTGACAGCTCCTTTGATGA. The amplified product was cut with
BamHI and EcoRI and inserted into pGEX-3X (Pharmacia) which had been cut with the same enzymes. This generated a
fusion protein with PKC sequences beginning from the initiator methionine of PKC Apl I Met-Glu-Lys-Arg and ending with
Gly-Ala-Val-Lys, which included the pseudosubstrate of PKC Apl I at the
5'-terminal and strand 1 of the C2 domain at the 3'-terminal. The
pseudosubstrate in PKC Apl I is immediately after the initiator
methionine and there is no V1 region in this kinase. Earlier constructs
made with shorter boundaries did not produce stable fusion proteins. We
had previously made a GST-Apl II-C1 construct including the pseudosubstrate and its behavior was identical to that of the construct
without this domain (23). For GST-Apl I-C2, an Eco47III fragment was excised from the PKC Apl I plasmid and inserted into pGEX-3X after cutting the vector with EcoRI and filling in
with Klenow. The fusion protein contains PKC sequences beginning with Cys-Glu-Lys-Asn from near the end of the C1 domain to Ser-Ser-Glu-Lys in the hinge region of PKC Apl I, 30 amino acids after the end of
strand 8 from the crystal structure. Expression of fusion proteins was
as described (23). For each fusion protein preparation, gels were
scanned and analysis performed using the public domain NIH Image
program (developed at the U. S. National Institutes of Health and
available on the Internet at http://rsb.info.nih.gov/nih-image/) to
determine the percentage of purified fusion protein at the correct
molecular weight and these values (Apl I-C1, 19 ± 3; Apl I-C2,
81 ± 10; Apl II-C1, 26 ± 11; Apl II-C2, 82 ± 20;
errors are S.D., n = 3) were used to calculate the
stochiometries for fusion protein binding. Much of this degradation
occurred during or after elution of the proteins from the glutathione
beads as the percentage of purified fusion protein at the correct
molecular weight was higher for experiments which used fusion proteins
still attached to beads (Apl I-C1, 63 ± 6; Apl I-C2, 90 ± 2; Apl II-C1, 89 ± 10; Apl II-C2, 96 ± 3; errors are S.D.,
n = 2). These values were used to standardize the
amount of fusion protein in liposome-binding experiments.
Purification of PKC Apl I and PKC Apl II from
Baculovirus--
We purified the kinases after expression in
baculovirus as described (13). The purification was done in 1 day
leading to less autonomous activity and higher specific activity than
previously. Aliquots of kinase were stored in 10% glycerol at
70 °C. Maximal activities of preparations of PKC Apl I ranged from
100 to 400 pmol/min/mg and ranged from 20 to 100 pmol/min/mg for PKC
Apl II. The kinases were both approximately 50% pure based on
Coomassie staining of the purified proteins (13) and this value was
used to estimate the stochiometry of PDBu binding.
[3H]PDBu Binding Assay--
Assays for
[3H]PDBu binding used the mixed micelle method (24, 25).
The 50-µl reaction mixture consisted of 20 µM Tris, pH
7.5, 200 µM CaCl2 or MgCl2, mixed
micelles (0-40 mol %) of phospholipids in a final concentration of
0.6% Triton X-100, [3H]PDBu from 1 to 200 nM, fusion protein ranging from 0.02 to 1.6 µg, and
purified kinase from 0.25 to 0.35 µg. Mixed micelles were prepared by
drying the appropriate volume of each lipid under nitrogen and
resuspending in 3% Triton X-100 with vortexing for 1 min followed by
incubation at 30 °C for 10 min. Reactions were started by addition
of protein and were allowed to proceed for 10 min at room temperature.
Reaction tubes were then placed on ice and bound PDBu was separated
from free PDBu on Sephacryl S-100 HR gel filtration columns (2-ml
column volume) at 4 °C by washing with equilibration buffer (20 µM Tris, pH 7.5, 0.015% Triton X-100, and 200 µM CaCl2 or MgCl2). The
nonspecific binding component for all experiments was measured in the
presence of 10 µM unlabeled PDBu and was subtracted from
the total binding to yield the specific binding. No differences were
seen in experiments using 200 µM CaCl2 or
MgCl2.
Liposome Binding Assay--
Phospholipids were prepared as
described (22). Liposome mixtures consisted of either 1.75 mg of PC or
1.4 mg of PC and 0.35 mg of PA, PS, PI, or PE. Phospholipids were mixed
in a 15-ml tube and dried under a stream of nitrogen. All experiments
measuring phospholipid binding used liposome mixtures containing 5 µCi of 1,2-dipalmitoyl-L-3-phosphatidyl-[N-methyl-3H]choline
(specific activity 108 mCi/mg) as a tracer. Dried phospholipid mixtures
were resuspended in 5 ml of binding buffer (50 mM HEPES, pH
7.2, 100 m NaCl) by vortexing for 3 min. The phospholipid
suspensions were then sonicated for 30 s using a probe sonicator
(Vibracell) and centrifuged before use to pellet aggregates. The
liposome suspensions were stored at 4 °C and used within 1 month.
Recombinant proteins (5 or 30 µg of protein) bound to
glutathione-Sepharose beads (standardized to 15 µl wet volume) were prewashed with binding buffer and then added to a reaction mixture containing either 1 mM CaCl2 or 1 mM MgCl2 and 1 mM EGTA, 50 mM HEPES, pH 7.2, 100 mM NaCl and
3H-labeled liposomes (approximately 200,000 cpm) in a total
assay volume of 400 µl. The mixture was incubated at room temperature for 15 min with vigorous shaking and then briefly centrifuged in a
tabletop centrifuge. The bound beads were then washed three times with
1 ml of the binding buffer and liposome binding was quantified by
liquid scintillation counting of the beads. We confirmed that equal
amounts of fusion proteins were used in the assay by visual inspection
of Coomassie-stained gels.
Kinase Assays--
The kinase assay using mixed micelles and an
Apl II-derived pseudosubstrate peptide was as described (13).
Trypsin Digestion--
The 50-µl proteolysis reaction
contained 10 µl of either purified PKC Apl II-GST-C2 domain fusion
protein derived from PKC Apl II, or GST alone. The protein component
was then incubated with 5 µl of 1% Triton X-100 micelles and 25 µl
of 2 × proteolysis buffer (40 mM Tris, pH 7.5, 40 mM MgCl2, 4 mM 2-mercaptoethanol) for 3 min at 30 °C. The digestion was initiated by addition of 10 µl of trypsin diluted in proteolysis buffer and the reaction was
allowed to proceed at 30 °C for 5 min. The final reaction contained
either 50 or 200 ng of protein, 0.1% Triton X-100, 20 mM
Tris-HCl, pH 7.5, 20 mM MgCl2, 2 mM
2-mercaptoethanol, and 0, 0.4, 1, 2, 5, or 10 µg/ml
N-tosyl-L-phenylalanine chloromethyl ketone-treated trypsin. Proteolysis was quenched by addition of 20 µl
of Laemmli sample buffer. Samples were analyzed by SDS-polyacrylamide gel electrophoresis.
Antibody Production and Immunoblotting--
The MBP-Apl II-C2
fusion protein was injected intramuscularly into rabbits along with
TitreMax (Cedar Lane) three times at 3-4-week intervals. Serum from
the animals was passed over an affinity column consisting of the
GST-Apl II-C2 fusion protein (13) coupled to Affi-Gel (Bio-Rad) and
specifically retained antibodies were eluted and concentrated in a
Centriplus-10 (Amicon Inc., Beverly, MA). Western blots were performed
as described (18) with this antibody at a 1:1250 dilution using a goat
anti-rabbit horseradish peroxidase-conjugated secondary antibody
(Pierce, Rockford, IL). Results were visualized by enhanced
chemiluminescence (NEN Life Science Products Inc.).
Quantitation of Data--
The dependence of phorbol ester
binding or kinase activity on the mole % of micelles was analyzed by a
nonlinear least squares fit to a modified Hill equation (26) using
Systat 5.0, where y is the value (PDBu binding or kinase
activity), a is the maximum value, × is the mole %,
k is the mole % resulting in half-maximal binding, and
n is the Hill coefficient, y = a(xn/(kn + xn)).
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RESULTS |
Lipid Specificity for Enzyme Activation of PKC Apl I and PKC Apl
II--
PKC Apl II requires more PS for activation than does PKC Apl I
(13; Table I). To examine the ability of
other lipids to activate the two enzymes, we determined the lipid
specificity of enzyme activation of PKC Apl I and PKC Apl II in the
presence of 1 mol % DOG using the mixed micelle kinase assay (27). PS
was the most effective lipid for both kinases (Fig.
1A). PI activated PKC Apl I
better than PKC Apl II, although the concentration of PI required was
significantly higher than that of PS (Fig. 1B). PA only
stimulated kinase activity of PKC Apl I and PKC Apl II at high
concentrations (Fig. 1, B and C). Neither PE nor
PC could stimulate kinase activity.
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Table I
Lipid dependence of PKC activation
Values for the Hill coefficient and for half-maximal binding were
calculated by nonlinear regression (see "Materials and Methods")
for each individual experiment, and the means ± S.D. are
presented. n refers to the number of independent
experiments.
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Fig. 1.
Lipid specificity of enzyme activation.
A, activation of PKC Apl I (white bars) and PKC
Apl II (dark bars) in the presence of 1 mol % DOG and 30 mol % of PS, PC, PI, PE, or PA. Activity in the absence of DOG was
subtracted. Values are standardized to activity in the presence of PS.
Values are mean ± S.E., n = 3. Activation curves
of PKC Apl I (B) or PKC Apl II (C) in the
presence of 1 mol % DOG and various concentrations of PS (open
circles), PI (open squares), or PA (closed
circles). Activity in the absence of DOG was subtracted. Values
are mean ± S.E., n = 2-4. D,
activation of PKC Apl I (open circles) and PKC Apl II
(closed circles) in the presence of 10 mol % DOG and
increasing amounts of PS. Activity in the absence of PS was subtracted.
Values are mean ± S.E., n = 3. Activation curve
of PKC Apl I (E) in the presence of 1 mol % DOG, 5 mol % PS or PKC Apl II (F) in the presence of 1 mol % DOG, 10 mol
% PS and increasing amounts of either PS (open circles), PI
(open squares), PA (closed circles), PE
(closed squares), and PC (open diamonds). The
amount of additional lipid added is indicated on the x axis
and is different for PKC Apl I and PKC Apl II. The levels of PA added
were different than the other lipids, but were the same for PKC Apl I
and PKC Apl II. Values are means ± S.E., n = 3-5.
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One possible explanation for the difference between PKC Apl I and PKC
Apl II in the amount of PS required for activity could be that PKC Apl
I has a higher affinity for DOG than PKC Apl II, since the
concentration of DOG affects the amount of PS required for maximal
activity (Table I). However, in the presence of saturating amounts of
DOG, or using PDBu (150 nM) instead of DOG, the difference in lipid required by the two enzymes is not diminished (Fig.
1D and data not shown).
Although the results above suggested that for both PKC Apl I and PKC
Apl II only PS could effectively synergize with DOG to stimulate enzyme
activity, it is possible that in the presence of some PS, other lipids
would become more effective. We used levels of PS (5 mol % for PKC Apl
I and 10 mol % for PKC Apl II) that gave 10-20% of the maximal
activity with 1 mol % DOG and compared addition of more PS to addition
of other lipids (Fig. 1, E and F). For PKC Apl I,
addition of PA was more effective than addition of PS, despite the poor
ability of PA to activate PKC Apl I on its own. PI could replace PS at
an equimolar level, while PC and PE were ineffective (Fig.
1E). For PKC Apl II, PA was equivalent to PS, PI was poorer
than PS, and PC and PE had no ability to replace PS (Fig.
1F).
These experiments demonstrate several similarities between the
isoforms. For both enzymes, PS was the most effective lipid at
increasing PKC activity, while PC and PE had no effects on either
enzyme. These experiments also revealed several differences between the
enzymes: 1) PI was more effective at stimulating PKC Apl I kinase
activity than PKC Apl II; 2) PA was more effective at stimulating
kinase activity in the presence of small amounts of PS in PKC Apl I
than PKC Apl II; and 3) PKC Apl I required less lipid for activation
than PKC Apl II.
PKC Apl I and PKC Apl II Require Similar Levels of Lipid to Induce
PDBu Binding--
To determine the lipid specificity for PKC binding,
we compared the ability of lipids to induce PDBu binding to PKC Apl I and PKC Apl II using the mixed micelle PDBu binding assay (24), since
this assay allows us to use the same lipid compositions used to measure
kinase activity. In this assay bound [3H]PDBu is
separated from free [3H]PDBu by gel filtration since
micelles bound to protein and PDBu are larger than micelles bound to
PDBu alone. This assay will only detect protein bound to PDBu that is
also bound to micelles and thus PDBu binding in this assay is directly
related to the ability of lipids to support enzyme binding to the mixed
micelles (25, 26, 28).
We found that the lipid specificity for PDBu binding differed from that
for kinase activation. In contrast to the superiority of PS in
stimulating kinase activity, PS and PA were equally effective at
inducing PDBu binding (Fig.
2A). PI supported PDBu binding to a greater extent for PKC Apl II than PKC Apl I, while the reverse was true for enzyme activity. Neither PC nor PE could support PDBu
binding.

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Fig. 2.
Phospholipid dependence of PDBu binding to
PKC Apl I and PKC Apl II. A, binding of
[3H]PDBu (150 nM) to PKC Apl I (white
bars) or PKC Apl II (dark bars) in the presence of 20 mol % of PS, PC, PI, PE, or PA. Values are standardized to binding in
the presence of PS. Values are mean ± S.D., n = 3. B, binding of [3H]PDBu (150 nM)
to PKC Apl I (open circles) or PKC Apl II (closed
circles) at different mole % PS. Values are mean ± S.E.,
n = 3.
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There was no difference in the K1/2 for PS to
stimulate PDBu binding between the two isoforms, as opposed to the
large difference in the K1/2 for PS measured for
enzyme activity (Fig. 2B; Tables I and
II). Thus, the difference in the amount
of PS needed to stimulate enzyme activity of PKCs Apl I and Apl II was
not reflected in the ability of PS to stimulate PDBu binding to the two
enzymes.
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Table II
Lipid dependence of PDBu binding
Values for the Hill coefficient and for half-maximal binding were
calculated by nonlinear regression (see "Materials and Methods")
for each individual experiment, and the means ± S.D. are
presented. n refers to the number of independent
experiments.
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Lipid Specificity for PDBu Binding Can be Attributed to Lipid
Specificity of the C1 Domain--
In order to determine whether the
lipid specificity of the enzymes could be explained by the lipid
specificity of the isolated regulatory domains, we measured PDBu
binding to fusion proteins containing the C1 or C2 domains of PKC Apl I
and PKC Apl II. Proteins of the correct molecular weight were observed
for all constructs (Fig. 3), along with
several degradation products the size of GST. Since GST does not bind
to PDBu (data not shown), these proteins should not interfere with the
PDBu binding assays.

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Fig. 3.
Expression of C1 and C2 domain fusion
proteins. A, Coomassie-stained gel of fusion proteins
after elution from glutathione beads. 6 µg of each purified protein
preparation was run in each lane. Lane 1 GST-Apl I C1;
Lane 2, GST-Apl II C1; Lane 3, GST-Apl I C2;
Lane 4, GST-Apl II C2. B, Coomassie-stained gel
of fusion proteins before elution from glutathione beads. 10 µg of
each purified protein preparation was run in each lane. Lane
1, GST; Lane 2, GST-Apl I C1; Lane 3, GST-Apl II C1; Lane 4, GST-Apl I C2; Lane 5, GST-Apl II C2.
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The lipid specificity for PDBu binding to the C1 domains was very
similar to that of the purified enzymes (Fig.
4A). The only exception was
that PDBu binding to the C1 domains of PKC Apl II was induced more
strongly by PA than PS compared with the full-length enzyme. The GST-C2
domain fusion proteins did not bind to PDBu (data not shown). Similar
amounts of PS were required for PDBu binding to the C1 domains of PKC
Apl I and PKC Apl II (Fig. 4B) as to the full-length enzymes
(Fig. 2B and Table II). These results suggest that the lipid
specificity for PDBu binding to the kinases can be largely explained by
the lipid specificity of the C1 domains, and that PS interacts with the
C1 domains of PKC Apl I and PKC Apl II in a similar manner.

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Fig. 4.
Phospholipid dependence of PDBu binding to
GST-C1 fusion proteins. A, binding of
[3H]PDBu (150 nM) to GST-Apl I-C1
(white bars) or GST-Apl II-C1 (dark bars) in the
presence of 20 mol % of PS, PC, PI, PE, or PA. Values for GST-Apl
II-C1 are from Pepio and Sossin (23). Values are mean ± S.D.,
n = 3. Binding of [3H]PDBu to GST-Apl
I-C1 (open circles) and GST-Apl II-C1 (closed
circles) at various concentrations of PS (B). Values
are mean ± S.E., n = 3.
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The C2 Domain of PKC Apl I But Not PKC Apl II Binds Directly to
Lipid--
Since the C2 domains do not bind to PDBu, we determined
their lipid specificity by measuring the ability of liposomes to bind to fusion proteins that were still attached to glutathione beads. This
is a well characterized assay that has been used previously to measure
Ca2+-dependent lipid binding to the
synaptotagmin C2 domain and the C2 domain of PKC
(22, 29). The
liposomes are made up of 80% PC and 20% of each of the other lipids.
The C2 domain of PKC Apl I bound liposomes containing PS or PA, but not
other lipids, while the C2 domain of PKC Apl II bound no liposomes
better than the GST control (Fig.
5A). The ability of the PKC
Apl I C2 domain to bind to liposomes did not require Ca2+,
but could be increased by Ca2+ ions (Fig. 5A).
However, the concentration of Ca2+ ions required to
stimulate additional liposome binding to the C2 domain was extremely
high (1-2 mM Ca2+) (Fig. 5B).
Nevertheless, liposome binding to the GST control was not affected by
this concentration of Ca2+ (Fig. 5B).

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Fig. 5.
Phospholipid dependence of liposome binding
by the C1 and C2 domains of PKC Apl I and PKC Apl II.
A, GST (open bars), GST-Apl I-C2 (hatched
bars), or GST-Apl II-C2 (dark bars) were incubated with
3H-labeled liposomes composed of PC only or PC mixed with
20% PS, PA, PI, or PE in the presence of 1 mM
MgCl2 ( ) or 1 mM CaCl2 (+).
Values are mean ± S.E. (n = 2-4). B,
binding of the GST-Apl I-C2 domain at different concentrations of
Ca2+ to the liposomes was measured. Note that in the
presence of EGTA (0 Ca2+) there is higher background
binding to GST and higher binding to GST-Apl I-C2 compared with binding
in the presence of 1 mM MgCl2 as seen in
A. Values are mean ± S.E., n = 3. C, binding of GST (open bars), GST-Apl I-C1
(hatched bars), and GST-Apl II-C1 (dark bars) to
3H-labeled liposomes composed of PC only or PC mixed with
20% PS, PA, PI, or PE. There were no differences in binding using 1 mM MgCl2 or 1 mM CaCl2
and results were pooled. Values are mean ± S.E.
(n = 4).
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We also compared the ability of the C1 domains to bind liposomes using
this assay. Both GST-C1 domain fusion proteins had higher background
binding to PC liposomes than did the GST-C2 domain fusion proteins
(Fig. 5C). However, the ability of the C1 domains to bind
the different liposomes broadly agreed with the specificity of lipids
to stimulate PDBu binding to the C1 domains. Liposomes containing PI
and PA bound better to the PKC Apl II C1 domains than to the PKC Apl I
C1 domains, similar to results with PDBu binding. Liposomes containing
PS bound equally well to PKC Apl I and PKC Apl II and PS was also
equipotent at inducing PDBu binding to the two C1 domain fusion
proteins.
The studies of fusion protein-lipid interactions can be summarized as
follows. First, the C1 domains of PKC Apl I and PKC Apl II had similar
affinities for PS, suggesting that differences in the C1 domains could
not explain the differential activation of the two enzymes by PS.
Second, PS bound to the C2 domain of PKC Apl I, but not of PKC Apl II,
possibly explaining why less PS is required to stimulate PKC Apl I
activity than PKC Apl II.
The Role of C2 Domains in PKC Apl I and PKC Apl II--
Binding of
lipids to the C2 domain could remove an inhibitory effect of the C2
domain, facilitate the effects of PS acting through the C1 domain, or
do both. To differentiate between these models we compared the amount
of PS required to activate PKC Apl I, PKC Apl II, and a PKC Apl II
construct with the C2 domain removed (Apl II
C2) (13). If the C2
domain is simply an inhibitory domain, activation of PKC Apl II
C2
should require the same amount of PS as that of PKC Apl I, since the C1
domains of PKC Apl I and PKC Apl II have similar interactions with PS.
In fact, PKC Apl II
C2 required an intermediate amount of PS, less
than that of PKC Apl II, but more than that of PKC Apl I, both at low
and high concentrations of DOG (Table I). This finding suggests that
the C2 domain has both effects, inhibiting PKC Apl II but also
facilitating activation of PKC Apl I. This analysis was complicated,
however, by the fact that PS is acting both at the C1 domain and the C2 domain of PKC Apl I.
PA specifically reduces the inhibitory effect of the C2 domain in PDBu
binding experiments with a fusion protein containing both the C1 and C2
domains of PKC Apl II (23), but our data suggest that PA does not
interact with the C2 domain directly (Fig. 5A). To
investigate whether PA binding to the C1 domain of PKC Apl II could
specifically remove the inhibition of enzyme activity mediated by the
C2 domain, we examined the ability of PA to decrease the concentration
of DOG required to activate PKC Apl II and PKC Apl II
C2. Indeed,
deletion of the C2 domain or addition of PA did decrease the
requirement of PKC Apl II for DOG (Fig.
6, A and B and
Table III). However, PA also decreased the requirement for DOG in PKC Apl II
C2 (Fig. 6C and
Table III). Thus, although adding PA had the same effect as removing
the C2 domain, the effect of PA was not occluded by removing the C2
domain.

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Fig. 6.
Effect of PA PKC Apl II. Activation of
PKC Apl II (A) or PKC Apl II C2 (B) in the
presence of 16 mol % PS and increasing amounts of DOG in the presence
(closed circles/squares) or absence (open
circles/squares) of 2.5 mol % PA. Values are mean ± S.E.,
n = 2. C, activation of PKC Apl II
(circles) or PKC Apl II C2 (squares) in the
presence of 10 mol % DOG in the presence (closed) or
absence (open) of 0.5 mol % PA. Activity in the absence of
PS was subtracted. Values for PKC Apl II in the absence of PA are the
same as in Fig. 1D. Values are mean ± S.E.,
n = 3.
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Table III
DOG dependence on PKC activity
Values for the Hill coefficient and for half-maximal binding were
calculated by nonlinear regression (see "Materials and Methods")
for each individual experiment and the means ± S.D. are
presented. n refers to the number of independent
experiments.
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A small amount of PA (0.5 mol %) has a large affect on PDBu binding to
both the C1-C2 domain PKC Apl II fusion protein and the purified enzyme
in the presence of PS (23). The same amount of PA also had a large
effect on the amount of PS required for enzyme activity at saturating
concentrations of DOG (Fig. 6C and Table I). However, this
was true in the enzyme with or without the C2 domain (Fig.
6C and Table I). Thus, although small amounts of PA did have
significant effects on PKC Apl II activity, this is not mediated by
removal of C2 domain-mediated inhibition.
Another way of testing the hypothesis that lipid binding to the C2
domain in PKC Apl I facilitates the effects of PS acting through the C1
domain is to examine the role of PA, which interacted with the C2
domain of PKC Apl I, but not PKC Apl II, and does not synergize with
DOG to stimulate enzyme activity through the C1 domain of either enzyme
(Fig. 1). In the presence of saturating levels of DOG and PA, PKC Apl I
required less PS for activation than did PKC Apl II or PKC Apl II
C2
(Fig. 7). This experiment illustrates
both the inhibitory role of the C2 domain in the absence of an effector
at the C2 domain (PKC Apl II versus PKC Apl II
C2) and the
facilitatory role of the C2 domain in the presence of an effector (PA)
at the C2 domain (PKC Apl I versus PKC Apl II
C2). In the
presence of PA, the K1/2 for PS in PKC Apl I was
near 1 mol % (or approximately two molecules in a Triton X-100
micelle) (Table I), suggesting that only a few PS molecules are
specifically required to act at the C1 domain in synergy with DOG in
the presence of lipids interacting with the C2 domain.

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Fig. 7.
Role of the C2 domain in kinase
activation. Activation of PKC Apl I (open circles), PKC
Apl II (closed circles), and PKC Apl II C2 (open
squares) in the presence of 10 mol % DOG, 5 mol % PA, and
increasing amounts of PS. Activity in the absence of PS was subtracted.
Values are mean ± S.E., n = 2.
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Effect of Ca2+ on PKC Apl I--
The C2 domain of PKC
Apl I contains the aspartic acid residues predicted to confer
Ca2+ specificity, while the C2 domain of PKC Apl II does
not. Indeed, Ca2+ activated PKC Apl I, but not PKC Apl II
which was actually inhibited at high concentrations of Ca2+
ions (Fig. 8, A and
B and Ref. 13). Under conditions of limiting PS and DOG, the
K1/2 for Ca2+ was 390 µM
and the Hill coefficient was 2 (Fig. 8A). Although this
concentration is high compared with vertebrate PKCs, it is lower than
the amount of Ca2+ required to increase lipid binding to
the isolated C2 domain (Fig. 4B). As another approach to
examine the effect of the C2 domain in PKC Apl I, we examined the
effects of Ca2+ on the levels of PS and DOG required for
activity. The effect of Ca2+ is to lower the lipid
requirement of PKC Apl I at constant DOG, or to decrease the
requirement for DOG at constant lipid (Fig. 8, C and
D). However, PKC Apl I did not require Ca2+ for
activity and the presence of Ca2+ did not increase the
maximal amount of PKC activity at saturating concentrations of PS and
DOG (Fig. 8C).

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Fig. 8.
Effects of Ca
on PKC Apl I and PKC Apl II. A,
activation of PKC Apl I in the presence of 5 mol % PS and 1 mol % DOG
and increasing concentrations of Ca2+ ions. Value in the
absence of Ca2+ was subtracted. Values are mean ± S.E., n = 3. B, activation of PKC Apl II in
the presence of 20 mol % PS and 1 mol % DOG and increasing amounts of
Ca2+ ions. Values are mean ± S.E., n = 2. C, activation of PKC Apl I with 1 mol % DOG in the
presence (closed circles) or absence (open
circles) of 500 µM Ca2+ and increasing
amounts of PS. Activity in the absence of PS was subtracted. Values are
mean ± S.E., n = 2. D, activation of
PKC Apl I with 5 mol % PS in the presence (closed circles)
or absence (open circles) of 500 µM
Ca2+ and increasing amounts of DOG. Activity in the absence
of DOG was subtracted. Values are mean ± S.E., n = 2.
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DISCUSSION |
Lipid Specificity of PKC Apl I--
The lipid specificity for
stimulation of cPKCs in vitro has been well studied (8, 10,
28, 30-34). Both Ca2+ and DOG independently act to
increase the affinity of the enzyme for PS independently, presumably
through C1 domains (DOG) and C2 domains (Ca2+) (8). These
results led to a model where the C1 domain and C2 domain act
independently to translocate the enzyme to the membrane, where specific
binding to PS activates the enzyme by inducing a conformational shift
of the pseudosubstrate (8). These studies also distinguished binding of
the enzyme to membranes, which was specific for acidic lipids and not
specific for PS from the synergy with DAG which was specific for PS
(10). Our results with PKC Apl I are in general agreement with these
results. PS stimulates enzyme activity in the presence of DOG much
better than PA despite the ability of PA to bind to both C1 and C2
domains as well as, if not better than, PS. One major difference
between PKC Apl I and vertebrate cPKCs in vertebrates is that
Ca2+ is not necessary for activation of PKC Apl I in the
mixed micelle kinase assay. This may be explained by the ability of the
C2 domain of PKC Apl I, but not of vertebrate cPKCs (16, 29) to bind to
lipids in the absence of Ca2+ ions.
Lipid Specificity of PKC Apl II--
There have been few studies
examining the lipid specificity for activation of nPKCs in
vitro using the mixed micelle assay or comparing cPKCs and nPKCs
in a systematic manner. Our results suggest that the lipid specificity
conferred by the C1 domain is similar but not identical to that of
cPKCs. Similar to cPKCs, PS is the most effective lipid for stimulating
enzyme activation of PKC Apl II and PA is more effective at binding the
enzyme than it is at stimulating enzyme activity. In contrast, PI had
different effects on PKC Apl II and PKC Apl I. PI was better at
stimulating enzyme activity in PKC Apl I than PKC Apl II, but better at
inducing PDBu binding to PKC Apl II than PKC Apl I.
Role of PA in Activation of PKC Apl II--
Previously, we had
suggested that PA may specifically activate PKC Apl II by removing
inhibition mediated by the C2 domain (23). Our results demonstrate that
this is not the case, since effects of PA on enzyme activity are
similar in constructs with or without the C2 domain. Previous studies
also suggested a role for PA in binding of PDBu to PKC Apl II (23). Our
results are consistent with a role for PA in activation of PKC Apl II.
First, PA bound to the C1 domain of PKC Apl II better than to the C1 domain of PKC Apl I (Fig. 4). Second, at saturating levels of DOG, 0.5 mol % of PA (approximately 1 molecule in a Triton X-100 micelle)
significantly reduced the amount of PS required for PKC activity from a
K1/2 of 14 to a K1/2 of 9 mol % (Table I). There was also not much of an additional effect after
raising PA from 0.5 to 5 mol % (Table I). Additionally, PA was very
effective at replacing PS in PKC Apl I, but since PA interacts with the C2 domain of PKC Apl I the results are hard to compare.
There is a specific requirement for PS in enzyme activation for both
PKC Apl I and PKC Apl II. This agrees with the ability of DOG to
increase affinity for PS, but not other lipids in both vertebrate cPKCs
and nPKCs (10). This requirement for PS is not conferred solely by the
C2 domain since DOG increases affinity for PS even when the C2 domain
has been mutated to ablate lipid binding (35) and there is still
specificity for activation by PS in PKC Apl II without the C2 domain
(data not shown). However, our results suggest that other lipid-binding
sites that are not specific for PS are also important for enzyme
activation, and that PA can replace PS at these sites. Furthermore,
since the PS levels in Aplysia are somewhere between 12 and
18 mol % (36), the effect of PA on the C1 domain of PKC Apl II may be
important for physiological enzyme activation. At these levels of PS,
addition of 0.5 mol % PA can double enzyme activity (Fig.
6C).
Role of C2 Domains--
The difference in the amount of PS
required for stimulation of PKC Apl I compared with PKC Apl II can be
explained by lipid binding to the C2 domain of PKC Apl I, but not that
of PKC Apl II. Our data are consistent with the idea that lipid binding
to the C2 domain increases the ability of lipid binding to the C1 domain to induce the conformational shift required for enzyme activity.
Whether binding to the C2 domain is necessary to remove an inhibitory
effect of the C2 domain in cPKCs as in nPKCs was difficult to determine
from our experiments, since the PS required for binding to the C1
domain of PKC Apl I also bound to the C2 domain. However, the
requirement of Ca2+ for PKC activity in cPKCs from
vertebrates is consistent with an inhibitory effect of the C2 domain in
these isoforms. Mutating the Ca2+-binding site in a
vertebrate cPKC greatly increases the amount of lipid required for
enzyme activity (34). It will be interesting to see if a similar amount
of lipid would be required after removal of the C2 domain. Our results
with PKC Apl II suggest that less lipid would be required to activate
cPKCs when the C2 domain is absent compared with when it is present and
cannot bind lipid.
The lipids examined did not bind to the C2 domain of PKC Apl II. While
we cannot rule out the possibility that this was due to incorrect
folding of the fusion protein, it is unlikely given the stability of
this fusion protein (Fig. 3). Furthermore, the pattern of partial
trypsin cleavage of the C2 domain is similar regardless of whether the
C2 domain is derived from purified PKC Apl II or the GST-C2 domain
fusion protein (Fig. 9), suggesting that
the domain is folded similarly in both proteins. Our results examining
kinase activation with and without the C2 domain are also consistent
with the lack of lipid binding to this domain. Removing the C2 domain
allows PKC Apl II to be activated at lower concentrations of PS, but
this difference is not alleviated by increasing DOG concentration or
adding PA since these compounds decrease the amount of PS required in
PKC Apl II without the C2 domain to a similar extent. These experiments
argue against a model where lipids are acting through the C2 domain of
PKC Apl II to facilitate enzyme activity. Moreover, there have been no reports of lipid binding to the related C2 domain of PKC
or PKC
, although it has been speculated that they bind lipids constitutively (8), or that they bind to hydrophobic lipids due to the prevalence of
hydrophobic residues in the loops corresponding to the calcium-binding regions (CBRs) (37). Other lipid activators of PKC like
PIP2, PIP3, or oleic acid stimulate nPKC
activity similarly in the presence or absence of the C2 domain,
suggesting that they also do not interact with the C2 domain (12, 13).
Identifying other lipids or proteins that do interact with its C2
domain will be important in understanding how PKC Apl II is activated
physiologically. One candidate for this interaction is the protein
-COP which binds to the C2 domain of PKC
(38), although it has
not yet been shown that the presence of this protein can increase PKC activity.

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Fig. 9.
Trypsin digests of PKC Apl II and
GST-C2. GST (50 ng), PKC Apl II (200 ng), or GST-Apl II-C2 (50 ng)
were cleaved with increasing concentrations of trypsin (0, 0.4, 1, 2, 5, and 10 µg/ml), separated on a 17% SDS-polyacrylamide gel
electrophoresis gel, and immunoblotted with an antibody specific for
the C2 domain of PKC Apl II. 0.4 µg/ml trypsin was sufficient to
cleave the C2 domain from both PKC Apl II and the GST-C2 domain fusion
protein (arrowhead). Increasing concentrations of trypsin
show a similar pattern of fragmentation whether the C2 domain is
derived from PKC or the GST fusion protein (arrowheads). The
predicted size of the C2 domain core is 17.5 kDa. The antibody
exhibited a cross-reaction with trypsin seen at the highest
concentrations of trypsin, but did not cross-react with GST.
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The Physiological Role of Ca2+ in Stimulating PKC Apl
I--
Although the amount of Ca2+ required for
stimulation of PKC Apl I does not appear to be physiological, it is
still possible that under some conditions increases in Ca2+
concentrations are important for activation of PKC Apl I since the
amount of Ca2+ required is also dependent on levels of PS
and DOG. For example, during pathological events such as axonal damage,
Ca2+ concentrations may reach the required level. Short
treatments of serotonin activate PKC Apl I (17), but do not appear to
raise Ca2+ levels significantly in the absence of action
potentials (39). It is interesting that unlike several vertebrate
isoforms (27), PKC Apl I does not require Ca2+ for activity
in the mixed micelle assay. This parallels the ability of the C2 domain
of PKC Apl I to bind to lipid in the absence of Ca2+, while
isolated C2 domains from enzymes that require Ca2+ do not
bind lipid in the absence of Ca2+ (22). The difference in
the C2 domain that confers this change is not clear. There are three
regions of the C2 domain that are defined as CBRs from crystallographic
studies (7, 37, 40). CBR1 and CBR3 are extremely well conserved between
PKC Apl I and vertebrate cPKCs. In contrast, CBR2 is quite different in
PKC Apl I compared with vertebrate kinases (Fig.
10). The crystal structure of the cPKC
C2 domain has not yet been solved, but CBR2 is important for
Ca2+ coordination in cPLA2 and PLC
1, but not
synaptotagmin. Recently, the autophosphorylation of a serine near the
carboxyl-terminal of a vertebrate cPKC has been shown to alter the
concentration of Ca2+ required for PKC activation,
suggesting that interactions between the C2 domain and the carboxyl
terminus of the enzyme occur (41). This serine is conserved in PKC Apl
I. A role for the carboxyl terminus may explain the large difference in
the amount of Ca2+ required for C2 domain binding alone,
compared with the amount of Ca2+ required to stimulate
enzyme activity.

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Fig. 10.
Comparison of the C2 domain of PKC Apl I and
vertebrate C2 domains. Strand positions and CBRs are as described
(7, 37). Shaded residues are conserved in all isoforms.
D represents conserved aspartic acids involved in
Ca2+ binding. Asterisks emphasize the difference
in CBR2 between PKC Apl I and vertebrate cPKCs.
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The Physiological Role of Lipids in Stimulating PKC Apl II--
In
Aplysia activation of PKC by serotonin leads to the reversal
of synaptic depression, but does not play a role in the augmentation of
synaptic responses also mediated by serotonin (42). In contrast, PDBu
causes both augmentation and the reversal of synaptic depression (43).
Short applications of serotonin activate PKC Apl I, but not PKC Apl II
leading to a model whereby PKC Apl II may lead to augmentation
stimulated by other transmitters (17). Indeed, prolonged incubation
with serotonin can activate both isoforms of PKC (44). Furthermore,
addition of insulin to the bag cell neurons of Aplysia
transiently activates PKC Apl I, but then later activates PKC Apl II
(18). These results suggest that a transient increase in DAG, either
through G-protein-activated phospholipase C-
or tyrosine
kinase-activated phospholipase C-
, is sufficient to activate PKC Apl
I, but not PKC Apl II which appears to require an additional stimulus.
The activation of PKC Apl II by insulin is blocked by wortmannin which
implies a role for either lipids downstream of PI 3-kinase or PA
downstream of phospholipase D (18) in activation of PKC Apl II. Either
lipid may help activate PKC Apl II through binding to the C1 domain,
but perhaps more likely, these stimuli may lead to the production of a
signaling molecule that can interact with the C2 domain of PKC Apl II
and activate the enzyme. Identifying this molecule will be important in
the study of PKC regulation.