From the Department of Chemistry (M/C 111), University of Illinois at Chicago, Chicago, Illinois 60607-7061
Received for publication, September 15, 2000
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ABSTRACT |
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On the basis of extensive structure-function
studies of protein kinase C- Protein kinases C (PKC)1
are a family of serine/threonine kinases that play crucial roles in
many different signal transduction pathways (1, 2). At least 10 isoforms of mammalian PKCs have been identified to date and they all
contain an amino-terminal regulatory domain linked to a COOH-terminal
kinase domain. Based on structural differences in the regulatory
domain, PKC isoforms have been generally subdivided into three classes;
conventional PKC ( Materials--
1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine
(POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol
(POPG), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine (POPS), and 1,2-sn-dioleoylglycerol (DOG) were purchased
from Avanti Polar Lipids, Inc. (Alabaster, AL) and used without further purification. Tritiated POPC ([3H]POPC) was prepared from
1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine and
[9,10-3H]oleic acid (American Radiochemical Co.) using
rat liver microsomes as described (15, 16). Phospholipid concentrations
were determined by phosphate analysis (17). Fatty acid-free bovine
serum albumin (BSA) was from Bayer Inc. (Kankakee, IL).
[ Mutagenesis--
Baculovirus transfer vectors encoding the
cDNA of PKC- Expression of PKC- Determination of PKC Activity--
Activity of PKC was assayed
by measuring the initial rate of [32P]phosphate
incorporation from [ Vesicle Binding Measurements--
The binding of PKC to
phospholipid vesicles was measured by a centrifugation assay using
large sucrose-loaded unilamellar vesicles (100 nm diameter) (20).
Sucrose-loaded vesicles were prepared as described previously (18). The
final concentration of vesicle solution was determined by measuring the
radioactivity of a trace of [3H]POPC (typically 0.1 mol
%) included in all phospholipid mixtures. For binding experiments, PKC
(~12 nM) was incubated for 15 min with sucrose-loaded
vesicles (0.1 mM), 1 µM BSA, and varying
concentrations of Ca2+ in 150 µl of 20 mM
Tris-HCl, pH 7.5, containing 0.1 M KCl. BSA was added to
minimize the loss of protein due to nonspecific adsorption to tube
walls. Vesicles were pelleted at 100,000 × g for 30 min using Sorvall RC-M120EX Microultracentrifuge. Aliquots of
supernatants were used for protein determination by PKC activity assay
using protamine sulfate as a substrate. The fraction of bound enzyme was plotted against the anionic lipid composition (mol %) of mixed vesicles. Mol % values of PS and PG giving rise to half-maximal vesicle binding and activity ([PS]1/2 and [PG]1/2)
were estimated graphically from individual plots.
Monolayer Measurements--
Surface pressure ( Surface Plasmon Resonance (SPR) Measurements--
400 µg/ml
vesicle solutions were prepared in an appropriate flow buffer solution
(typically 10 mM HEPES, pH 7.4, containing 0.15 M NaCl and varying concentrations of Ca2+).
Before SPR measurements, the Biacore X (Biacore AB) instrument was
allowed to equilibrate with the buffer until the drift in signal was
less than 0.3 resonance units/min. The Pioneer L1 sensor chip was then
coated with the vesicles at a flow rate of 5 µl/min. The immobilized
lipid vesicles were washed with 10 µl of 10 mM NaOH at
100 µl/min flow rate to remove unattached vesicles. In control
experiments, the fluorescence intensity of the flow buffer after
rinsing the sensor chip coated with vesicles incorporating 10 mM 5-carboxyfluorescein (Molecular Probes) was monitored.
Lack of detectable fluorescence signal indicated that the vesicles remained intact on the chip. Next, 25 µl of 0.1 mg/ml BSA was injected at 5 µl/min to block exposed sites on the chip surface, which was once again washed with 10 mM NaOH. All
experiments were performed with a control cell in which a second sensor
surface was coated with 0.1 mg/ml BSA at 5 µl/min and then washed
with 10 mM NaOH. The drift in signal for both sample and
control flow cells was allowed to stabilize below 0.3 resonance
unit/min before any kinetic experiments were performed. All kinetic
experiments were performed at 24 °C and a flow rate of 60 µl/min.
A high flow rate was used to circumvent mass transport effects. The
association was monitored for 90 s (90 µl) and dissociation for
4 min. The immobilized vesicle surface was then regenerated for
subsequent measurements using 10 µl of 10-50 mM NaOH or
3 M NaCl. The regeneration solution was passed over the
immobilized vesicle surface until the SPR signal reached the initial
background value before protein injection. For data acquisition, 5 or
more different concentrations (typically within a 10-fold range around
the Kd) of each protein were used. After each set of
measurements, the entire immobilized vesicles were removed by injection
of 25 µl of 40 mM CHAPS, followed by 25 µl of octyl
glucoside at 5 µl/min, and the sensor chip was re-coated with a fresh
vesicle solution for the next set of measurements. All data were
evaluated using BIAevaluation 3.0 software (Biacore). For each trial,
the control surface response was subtracted out to eliminate any
nonspecific binding and refractive index changes due to buffer change.
Furthermore, the derivative plot was used to monitor potential mass
transport effects. Once these factors were checked for each set of
data, the association and dissociation phases of data were globally fit
to a 1:1 Langmuir binding model: [protein·vesicle] Roles of Cationic C1 Domain Residues
in PKC Activation--
Figs. 1 and 2
illustrate amino acid sequences and model structures of C1a and C1b
domains of PKC-
We systematically analyzed the effects of the above mutations by
measuring the anionic phospholipid dependence of vesicle binding and
enzyme activity for wild type and mutants. First, we measured the PS
dependence of binding to POPC/POPS mixed vesicles containing 1 mol % of DOG. As shown in Fig. 3, two C1a
domain mutants, K62A and R77A, required significantly higher mol % of PS for vesicle binding ([PS]1/2 = 20 and 30 mol %,
respectively) whereas another C1a mutant K76A and all C1b mutants
behaved essentially the same as wild type ([PS]1/2 = 16 to 18 mol %). We then measured the kinase activity of wild type and mutants
toward histone under the same conditions (i.e. in the
presence of POPC/POPS/DOG mixed vesicles). In general, C1a domain
mutants exhibited lower activity than did C1b domain mutants at a given
PS concentration (Fig. 4). For instance,
at 40 mol % PS wild type PKC-
We have previously shown that the isolated C1 domain (i.e.
C1a + C1b) has essentially the same affinity for PS and
phosphatidylglycerol (PG)-containing vesicles, indicating lack of a
specific PS-binding site in the domain (14). This, in turn, suggests
that the role of cationic residues in the C1 domains is to interact
nonspecifically with anionic phospholipids. If this is the
case, the mutations of the cationic residues of the C1 domains of
PKC- Roles of C1 Domain Aspartates--
Our model structures of C1a and
C1b domains of PKC- The Origin of PS Specificity--
Since PS specifically allows the
membrane penetration and DAG binding of C1a domain, we reasoned that PS
with a carboxylic group in the head group might be able to release the
hypothetical C1a domain tethering by competing with Asp55.
If so, D55A (and D55K) should lose PS specificity in vesicle binding
and activation due to lack of C1a domain tethering. To explore this
possibility, we measured the vesicle binding and kinase activity of
wild type and mutants as a function of anionic phospholipid composition
in two different mixed vesicles, including POPC/POPS/DOG and
POPC/POPG/DOG. As shown in Fig. 10,
wild type PKC- Monolayer Measurements--
We have shown that PS specifically
induces the penetration of C1a domain into the membrane (18). To
corroborate the notion that PS specifically disrupts the tethering of
C1a domain mediated by Asp55, we measured the penetration
of PKC- SPR Measurements--
The SPR analysis of membrane-protein
interactions offers an advantage over other methods in that the effects
of the mutations of membrane-binding residues on membrane association
(ka) and dissociation (kd) rate
constants can be directly determined (25, 26). In our recent study on
the membrane binding of phospholipase A2, we showed that
electrostatic interactions driven by ionic residues mainly affect
ka whereas hydrophobic interactions resulting from
the membrane penetration of hydrophobic residues largely influence
kd.2 By
means of the SPR analysis, we determined the values of
ka and kd for PKC- Differential Roles of C1a and C1b Domains--
Both conventional
and novel PKCs contain a tandem repeat of C1 domains, which serve as a
binding site for DAG and phorbol esters (8-13). Irie et al.
(27) recently reported that despite high sequence homology, isolated C1
domains of conventional and novel PKCs have different phorbol ester
affinity, with dissociation constants ranging from 1 nM to
>3 µM. Oancea et al. (28) also reported that
isolated C1a and C1b domains of PKC- The Role of C1 Domain Aspartates and the Origin of PS
Specificity--
A consensus model of conventional PKC activation
holds that the Ca2+-dependent binding of PKC to
PS and DAG (or phorbol esters) triggers conformational changes of PKC,
resulting in the removal of the pseudosubstrate from the active site
and PKC activation (2). Our previous studies provided specific
mechanistic details for this general model: Ca2+ and PS
induce the membrane binding of protein and the specific membrane
penetration of the C1a domain of PKC-
The understanding of the exact chemical nature of the C1a domain
tethering and the identification of residues that interact with
Asp55 would require high-resolution structural information
of the full-length PKC molecule. Based on several lines of evidence
supporting the importance of C1-C2 interdomain interactions in PKC
regulation, we speculate that Asp55 interacts with a C2
domain residue in the calcium-binding loop. Although either the C1 or
C2 domain alone is capable of recruiting conventional PKC to the
membrane, a concerted action of both domains is absolutely required for
full activation of the enzyme and, in particular, for its PS
specificity. For instance, our previous study with the isolated C1 and
C2 domains of PKC- (PKC-
), we have proposed an
activation mechanism for conventional PKCs in which the C2 domain and
the C1 domain interact sequentially with membranes (Medkova, M., and
Cho, W. (1999) J. Biol. Chem. 274, 19852-19861). To
further elucidate the interactions between the C1 and C2 domains during
PKC activation and the origin of phosphatidylserine specificity, we
mutated several charged residues in two C1 domains (C1a and C1b) of
PKC-
. We then measured the membrane binding affinities, activities,
and monolayer penetration of these mutants. Results indicate that cationic residues of the C1a domain, most notably Arg77,
interact nonspecifically with anionic phospholipids prior to the
membrane penetration of hydrophobic residues. The mutation of a single
aspartate (Asp55) in the C1a domain to Ala or Lys resulted
in dramatically reduced phosphatidylserine specificity in vesicle
binding, activity, and monolayer penetration. In particular, D55A
showed much higher vesicle affinity, activity, and monolayer
penetration power than wild type under nonactivating conditions,
i.e. with phosphatidylglycerol and in the absence of
Ca2+, indicating that Asp55 is involved in the
tethering of the C1a domain to another part of PKC-
, which keeps it
in an inactive conformation at the resting state. Based on these
results, we propose a refined model for the activation of conventional
PKC, in which phosphatidylserine specifically disrupts the C1a domain
tethering by competing with Asp55, which then leads to
membrane penetration and diacylglycerol binding of the C1a domain and
PKC activation.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
,
I,
II, and
isoforms), novel PKC (
,
,
, and
isoforms), and atypical
PKC (
and
/
isoforms). Conventional PKCs are activated by the
Ca2+-dependent translocation of proteins to the
membrane containing anionic phospholipids, preferably
phosphatidylserine (PS) and diacylglycerol (DAG). The membrane
translocation is mediated by two types of membrane-targeting domains
(C1 and C2 domains) in the regulatory region of conventional PKC (3).
The C2 domain of conventional PKC is responsible for the
Ca2+-dependent binding of protein to anionic
membranes (4-7). The conventional PKC also contains a tandem repeat of
cystein-rich, C1 domains (C1a and C1b) that provide a binding site for
DAG and phorbol esters (8-13). Based on extensive structure-function
studies on PKC-
, we have recently proposed a mechanism for the
in vitro membrane binding and activation of PKC-
(14). In
this mechanism, PKC-
initially binds to the membrane surface via the
Ca2+-dependent membrane binding of the C2
domain. Once membrane-bound, PS specifically induces the insertion of
the hydrophobic residues of the C1a domain into the membrane. The
membrane penetration allows optimal DAG binding and drives the release
of pseudo-substrate region from the active site, hence the PKC
activation. Although this mechanism accounts for much of the temporal
and spatial sequences of in vitro activation of conventional
PKC, questions still remain as to how the C1 and C2 domains of PKC-
interact with each other during PKC activation and how PS specifically
induces the membrane penetration of the C1a domain. To address these
questions, we performed further structure-function studies on the C1a
and C1b domains of PKC-
, with an emphasis on surface ionic residues. Results from these studies provide an important new clue to the understanding of the origin of PS specificity.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]ATP (3 Ci/µmol) was from Amersham Pharmacia
Biotech and cold ATP was from Sigma. Triton X-100 was obtained from
Pierce Chemical Co. (Rockford, IL). Restriction endonucleases and
enzymes for molecular biology were obtained from either Roche Molecular
Biochemicals or New England Biolabs (Beverly, MA).
with appropriate C1 domain mutations were generated
by the overlap extension polymerase chain reaction using
pVL1392-PKC-
plasmid as a template (18). Briefly, four primers,
including two complementary oligonucleotides introducing a desired
mutation and two additional oligonucleotides complementary to the
5'-end and 3'-end of the PKC-
gene, respectively, were used for
polymerase chain reaction performed in a DNA thermal cycler
(PerkinElmer Life Sciences) using Pfu DNA polymerase (Stratagene). Two
DNA fragments overlapping at the mutation site were first generated and
purified on an agarose gel. These two fragments were then annealed and
extended to generate an entire PKC-
gene containing a desired
mutation, which was further amplified by polymerase chain reaction. The
product was subsequently purified on an agarose gel, digested with
NotI and EcoRI, and subcloned into the pVL1392
plasmid digested with the same restriction enzymes. The mutagenesis was
verified by DNA sequencing using a Sequenase 2.0 kit (Amersham
Pharmacia Biotech).
and Mutants in Baculovirus-infected
Sf9 Cells--
Wild type PKC-
and mutants were expressed in
baculovirus-infected Sf9 cells (Invitrogen, La Jolla, CA) and
purified as described previously (5, 18). The transfection of
Sf9 cells with mutant pVL1392-PKC-
constructs was performed
using a BaculoGoldTM transfection kit from Pharmingen (San
Diego, CA). The plasmid DNA for transfection was prepared by using an
EndoFree Plasmid Maxi kit (Qiagen, Valencia, CA) to avoid potential
endotoxin contamination.
-32P]ATP (50 µM, 0.6 µCi/tube) into the histone III-SS (400 µg/ml) (Sigma). The reaction
mixture contained large unilamellar vesicles (0.1 mM), 5 mM MgCl2, 12 nM PKC, and 0.1 mM CaCl2 in 50 µl of 20 mM HEPES,
pH 7.0. Protamine sulfate (200 µg/ml) was used to determine the free
enzyme concentration in vesicle binding measurements (see below). Free
calcium concentration was adjusted using a mixture of EGTA and
CaCl2 according to the method of Bers (19). Reactions were
initiated by adding MgCl2 to the mixture and quenched by adding 50 µl of 5% aqueous phosphoric acid solution after a given period of incubation (e.g. 10 min for histone) at room
temperature. Seventy-five microliters of quenched reaction mixtures
were spotted on P-81 ion-exchange papers (Whatman) and papers were
washed 4 times with 5% aqueous phosphoric acid solution and washed
once with 95% aqueous ethanol. Papers were then transferred into
scintillation vials containing 4 ml of scintillation fluid (Sigma) and
radioactivity was measured by liquid scintillation counting. The
linearity of the time course of the reaction was checked by monitoring
the degree of phosphorylation at regular intervals (e.g. 5 min).
) of solution
in a circular Teflon trough (4 cm diameter × 1 cm deep) was
measured using a Wilhelmy plate attached to a computer-controlled Cahn
electrobalance (Model C-32) as described previously (18). Five to ten
microliters of phospholipid solution in ethanol/hexane (1:9 (v/v)) or
chloroform was spread onto 10 ml of subphase (20 mM
Tris-HCl, pH 7.5, containing 0.1 M KCl and 0.1 mM free Ca2+) to form a monolayer with a given
initial surface pressure (
0). The subphase was
continuously stirred at 60 rpm with a magnetic stir bar. Once the
surface pressure reading of monolayer had been stabilized (after about
5 min), the protein solution (typically 40 µl) was injected into the
subphase through a small hole drilled at an angle through the wall of
the trough and the change in surface pressure (
) was measured as
a function of time. Typically, the
value reached a maximum after
30 min. The maximal
value depended on the protein concentration
and reached a saturation value (e.g. at [PKC-
]
1 µg/ml). Protein concentrations in the subphase were therefore
maintained above such values to ensure that the observed
represented a maximal value. The critical surface pressure
(
c) was determined by extrapolating the
versus
0 plot to the x axis
(21).
protein + vesicle. The dissociation phase was fit to the integrated rate
equation, R = R0ekd(t
t0,
where kd is the dissociation rate constant,
R0 is the response at the start of fit data, and
t0 is the time at start of fit data. The
association phase is fit to the integrated rate equation:
R = Req(1
e
(kaC+kd)(t0)) + RI, where Req = [kaC/(kaC + kd)] Rmax, RI = refractive index change, Rmax is the theoretical
binding capacity, C is analyte concentration, and
ka is the association rate constant. The curve
fitting efficiency was checked by residual plots and
2.
The dissociation constant (Kd) was then calculated from the equation, Kd = kd/ka.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, respectively. In particular, Fig. 2 shows that the
C1 domains of PKC-
have a polarized distribution of hydrophobic and
ionic residues. The upper part of the molecule, where the DAG/phorbol
ester binding pocket is located, contains a few aliphatic and aromatic
residues whereas the middle part has a number of cationic residues. We
have recently performed an extensive structure-function study on the
hydrophobic residues of the C1a and C1b domains of PKC-
, which
revealed their distinct roles in PKC activation (14). Hydrophobic
residues in the C1a domain are essential for the membrane penetration
and DAG-dependent activation of PKC-
, whereas those in
the C1b domain are not directly involved in these processes. To assess
the role of cationic residues of the C1 domains in the membrane binding
and activation of PKC-
, we mutated several cationic residues in the
C1a and C1b domains. Specifically, Lys62,
Lys76, and Arg77 in the C1a domain and
His127, Lys131, and Lys141 in the
C1b domain were replaced by alanine (Fig. 1). Since all mutated
residues are surface exposed (Fig. 2), these mutations were not
expected to cause deleterious conformational changes. Indeed, all six
mutants were expressed in baculovirus-infected insect cells as
efficiently as wild type, suggesting comparable thermodynamic stability
and lack of gross conformational changes.
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Fig. 1.
Sequence alignment of the C1a and C1b motifs
of PKC- . Mutated ionic residues are
marked with asterisks.
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Fig. 2.
A proposed membrane binding mode of C1a and
C1b motifs of PKC- . The model
structures of C1a and C1b domains shown in a ribbon diagram
are built on the backbone of the C1b motif of PKC-
with side chain
replacements using a program Biopolymer (Molecular Simulation). The
side chains of mutated cationic and anionic residues are highlighted in
blue and red, respectively, and
numbered. The upper part of C1a domain that
partially penetrates into the membrane contains hydrophobic and
aromatic side chains (shown in white).
and C1b domain mutants showed full
activity. Under the same conditions, however, K62A and K76A showed only
44 and 55% of the wild type activity, respectively, although they are
fully vesicle-bound (see Fig. 3). Most notably, R77A showed no
detectable activity with up to 80 mol % PS although the protein should
be fully vesicle-bound with 80 mol % PS (see Fig. 3). R77A exhibited
full vesicle-binding affinity and enzymatic activity in the presence of
1 mol % of phorbol 12-myristate 13-acetate in the vesicles (data not
shown), indicating that the extremely low activity of R77A is not due to deleterious conformational changes.
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Fig. 3.
Binding of PKC- and
C1 domain mutants to POPC/POPS/DOG vesicles as a function of POPS
composition. Proteins include wild type (
), K62A (
), K76A
(
), R77A (
), H127A (
), K131A (
), and K141A (
). Total
lipid concentration of POPC/POPS/DOG (99-x:x:1 in
mol %) vesicles and PKC concentration were 0.1 mM and 12 nM, respectively, in 20 mM Tris-HCl buffer, pH
7.5, containing 0.1 M KCl, 0.1 mM
Ca2+, and 1 µM BSA. Each data point
represents an average of duplicate measurements.
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Fig. 4.
Dependence of enzymatic activity of
PKC- and C1 domain mutants on the POPS
composition in POPC/POPS/DOG vesicles. Proteins include wild type
(
), K62A (
), K76A (
), R77A (
), H127A (
), K131A (
),
and K141A (
). Total lipid concentration of POPC/POPS/DOG
(99-x:x:1 in mol %) vesicles and PKC
concentration were 0.1 mM and 12 nM,
respectively, in 20 mM HEPES buffer, pH 7.0, containing 0.1 M KCl, 5 mM MgCl2, histone III-SS
(400 µg/ml), and 0.1 mM Ca2+. Each data point
represents an average of duplicate measurements. The absolute value of
maximal activity is 0.20 ± 0.04 µmol/(mg/min).
should not affect its PS specificity for vesicle binding
affinity and enzyme activity. To test this notion, we measured the PG
dependence of the binding of wild type and mutants to POPC/POPG/DOG
mixed vesicles and compared it with the PS dependence shown in Fig. 3.
As reported previously (18), PKC-
and all mutants required higher
mol % of PG than PS for the same degree of vesicle binding (Fig.
5). As with the PS dependence of vesicle
binding, only two C1a domain mutants, K62A and R77A, showed reduced
binding affinity for PG-containing vesicles, while other mutants
behaved like wild type. We then measured the PG dependence of kinase
activity. As shown in Fig. 6, the PG
dependence of activity compared well with the PS dependence (Fig. 4).
In general, wild type and all mutants had much lower kinase activity in
the presence of PG vesicles, and displayed significant activity only at
high mol % of PG. Even at high mol % of PG, however, K62A and R77A
exhibited much lower activity than wild type. For instance, R77A
exhibited no detectable activity and K62A showed about 50% of wild
type activity at 80 mol % PG. Thus, the PG dependence was
qualitatively similar to corresponding PS dependence, indicating that
the mutations of C1 domain residues affect the PS- and
PG-dependent vesicle binding and activation of PKC-
to
similar extents. Taken together, these results indicate that the
cationic residues in the C1a domain, most notably Arg77,
make important contributions to the membrane binding and activation of
PKC-
by nonspecifically interacting with anionic membrane surfaces.
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Fig. 5.
Binding of PKC- and
C1 domain mutants to POPC/POPG/DOG vesicles as a function of POPG
composition. Proteins include wild type (
), K62A (
), K76A
(
), R77A (
), H127A (
), K131A (
), and K141A (
).
Experimental conditions are the same as described for Fig. 3.
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Fig. 6.
Dependence of enzymatic activity of
PKC- and C1 domain mutants toward histone on
the POPG composition in POPC/POPG/DOG vesicles. Proteins include
wild type (
), K62A (
), K76A (
), R77A (
), H127A (
), K131A
(
), and K141A (
). Experimental conditions are the same as
described for Fig. 4. The absolute value of maximal activity is
0.20 ± 0.04 was µmol/(mg/min), as described for Fig. 4.
reveal the presence of single surface-exposed
anionic residues, Asp55 (C1a) and Asp116 (C1b),
located near the cationic patches (Fig. 2). Residues 55 and 116 are not
perfectly matched in the sequence alignment but their relative location
in the molecule should be similar based on our modeling (see Fig. 1).
Conventional and novel PKCs invariably contain an anionic
residue, predominantly Asp, in these positions. To determine the role
of these unique aspartates, we replaced Asp55 in the C1a
domain and Asp116 in the C1b domain with alanine and
lysine, respectively. We then measured the vesicle binding and kinase
activity of the mutants as a function of PS composition in
POPC/POPS/DOG (99-x:x:1) mixed vesicles and also
as a function of Ca2+. As shown in Fig.
7, D55A showed higher membrane affinity
than wild type at a given PS composition in the range of 0 to 20 mol %. As a result, [PS]1/2 (
10 mol %) for D55A was
significantly lower than that of wild type (
17 mol %). In contrast,
D116A behaved similarly to wild type. A similar trend was seen with
relative activity. In this case, the maximal activity of D55A was
35% higher than that of wild type even when both enzymes were fully
activated. Fig. 8 shows the calcium
dependence of PKC activity of the three proteins in the presence of
POPC/POPS/DOG (69:30:1) vesicles. Again, D55A required less
Ca2+ than wild type and D116A for activation and showed
40% higher maximal activity. Since our previous studies showed that
Ca2+ and PS are required for triggering the membrane
penetration and DAG binding of the C1a domain (14, 18), lower
Ca2+ and PS requirements for D55A activation suggest that
this mutant might have higher intrinsic activity to penetrate the
membrane and bind DAG (see the monolayer penetration data below). This, in turn, implies that Asp55 might be involved in the
specific tethering of C1a domain, which is relieved upon
Ca2+-dependent binding to PS-containing
membranes. The observed properties of D55A were not due to stronger
nonspecific electrostatic interactions between the C1a domain and the
anionic membrane caused by the removal of negative charge on the C1a
domain, because D55K behaved essentially the same as D55A. On the basis
of simple electrostatic effect, the former would have higher affinity
and activity than the latter. To further test the notion that D55A
exists in a more or less preactivated conformation, we measured the
activity of wild type, D55A, and D116A in the presence of POPC/POPG/DAG
(99-x:x:1) vesicles and 0.1 mM EGTA,
which represents a highly nonproductive condition for PKC activation.
As shown in Fig. 9, wild type and D116A
exhibited extremely low PKC activity with POPG composition up to 80 mol
% under these circumstances. In sharp contrast, D55A showed
considerable residual activity: D55A was >10 times more active than
wild type in a wide range of POPG concentration (i.e. 40-80
mol %). These data renders more credence to the notion that Asp55 is involved in the tethering of C1a domain, which
keeps PKC-
in an inactive conformation.
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Fig. 7.
Relative binding affinity and enzyme activity
of PKC- and the C1 domain aspartate mutants as
a function of PS composition. Wild type (
/
), D55A (
/
),
D55K (
/
), and D116A (
/
) were incubated with 0.1 mM POPC/POPS/DOG (99-x:x:1) vesicles.
The relative affinity (open symbols and broken
lines) and activity (filled symbols and solid
lines) were measured as described in the legends to Figs. 3 and 4,
respectively. The maximal activity of wild type toward histone is
0.20 ± 0.04 was µmol/(mg/min). Each data point represents an
average of triplicate measurements.
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Fig. 8.
Dependence of enzyme activity of
PKC- and the C1 domain aspartate mutants on
calcium concentration. Wild type (
), D55A (
), and D116A
(
) (all 12 nM) were incubated with 0.1 mM
POPC/POPS/DOG (69:30:1) vesicles in 20 mM HEPES buffer, pH
7.0, containing 0.1 M KCl, 5 mM
MgCl2, histone III-SS (400 µg/ml), and varying
concentrations of Ca2+. Each data point represents an
average of duplicate measurements. The maximal activity of wild type
toward histone is 0.20 ± 0.04 was µmol/(mg/min).
View larger version (18K):
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Fig. 9.
Dependence of enzyme activity of
PKC- and the C1 domain aspartate mutants on PG
composition. Wild type (
), D55A (
), and D116A (
) were
incubated with 0.1 mM POPC/POPG/DOG
(99-x:x:1) vesicles. The enzyme activity was
measured as described in the legend to Fig. 4, except for 0.1 mM EGTA instead of 0.1 mM Ca2+.
Each data point represents an average of triplicate measurements.
and D116A showed similar high PS specificity for
vesicle binding; i.e. [PS]1/2 = 17 mol % and
[PG]1/2 = 30-35 mol %. In contrast, D55A showed a lower
degree of PS specificity; [PS]1/2 = 10 mol % and
[PG]1/2 = 12 mol %. Thus, D55A binds PG-containing vesicles,
for which conventional PKCs are known to have much lower affinity, as
tightly as the wild type PKC-
binds PS-containing vesicles. This
high affinity of D55A for PG vesicles leads to dramatically reduced PS
specificity, when compared with wild type. The relative activities of
the proteins determined in the presence of different mol % of POPS and
POPG further support our model. All three proteins are essentially fully vesicle-bound when the anionic phospholipid composition of mixed
vesicles is above 40 mol % (see Fig. 10). Thus, the relative activity
of the proteins under these conditions should reflect mainly the
effects of mutations on PKC activation. As shown in Fig.
11, the three proteins displayed
distinctly different degrees of PS specificity. As reported previously,
PKC-
showed high PS specificity, i.e. PS
PG. When
compared with wild type, D55A showed a much lower degree of PS
specificity at both 40 and 60 mol % of anionic lipids. At 40 mol % of
anionic lipids, PG was
75% as effective as PS in activating D55A.
At 60 mol % of anionic lipids, activities on PS and PG were
comparable. Also, D55A was more active than wild type under all assay
conditions (up to 140% of wild type activity). On the other hand,
D116A was about 15% less active than wild type in the presence of
PS-containing vesicles but was more active than wild type in the
presence of PG-containing vesicles. As a result, D116A showed
considerable lower PS specificity than did wild type. Taken together,
it is clear that Asp55 in the C1a domain plays an important
role in the PS specificity for membrane binding and activation of
PKC-
. On the other hand, Asp116 had little effect on the
vesicle binding of PKC-
but modestly lowered the PS dependent
activity while enhancing the PG dependent activity. Thus,
Asp116 in the C1b domain might also be involved in PS
specificity in PKC-
activation, albeit indirectly (see
"Discussion").
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Fig. 10.
Binding of PKC- and
the C1 domain aspartate mutants to mixed vesicles as a function of PS
and PG composition. Wild type (
/
), D55A (
/
), and D116A
(
/
) were incubated with 0.1 mM POPC/POPS/DOG
(99-x:x:1) vesicles (open symbols) or
POPC/POPG/DOG (99-x:x:1) vesicles (closed
symbols). Experimental conditions are the same as described for
Fig. 3. Each data point represents an average of triplicate
measurements.
View larger version (28K):
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Fig. 11.
Enzymatic activity of
PKC- and C1 domain aspartate mutants toward
histone at two different anionic lipid compositions. The kinase
activity of wild type, D55A, and D116A was measured in the presence of
POPC/POPS/DOG (open bars) and POPC/POPG/DOG (solid
bars) mixed vesicles. Total lipid concentration and calcium
concentration were 0.1 mM and DOG composition was 1 mol
%.
and two mutants, D55A and D116A, into POPC/POPS (6:4) and
POPC/POPG (6:4) mixed monolayers (Fig.
12). If much reduced PS specificity of
D55A in vesicle binding and activation derives from the loss of the
specific C1a domain tethering, one would expect that D55A should have
high monolayer penetration power regardless the nature of anionic
phospholipids in the monolayer. In these measurements, a phospholipid
monolayer of a given initial surface pressure
0 was
spread at constant area and the change in surface pressure (
) was
monitored after the injection of the protein into the subphase. It has
been shown (18, 22, 23) that those proteins whose actions involve the partial or full penetration of membranes, such as PKC-
, have an
ability to penetrate into the phospholipid monolayer with
0 comparable to or higher than that of biological
membranes (i.e.
c
31 dyne/cm) (24). As
reported previously (18), PS showed the unique ability to induce the
Ca2+-dependent penetration of PKC-
into the
monolayer:
c
33 dyne/cm for the POPC/POPS (6:4)
monolayer and 27 dyne/cm for the POPC/POPG (6:4) monolayer (Fig. 12).
D116A again displayed a similar property with
c
34 dyne/cm for the POPC/POPS monolayer and 29 dyne/cm for the POPC/POPG
monolayer. In contrast, D55A showed no appreciable selectivity for PS
and penetrated equally well into POPC/POPS and POPC/POPG monolayers
(i.e.
c
34 dyne/cm for both PS- and PG-containing monolayers). These results provide strong evidence for
the notion that Asp55 is involved in the C1a domain
tethering and that PS specifically disrupts the tethering and thereby
induces the penetration of the hydrophobic residues of the C1a domain
into the membrane.
View larger version (24K):
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Fig. 12.
Effect of the initial surface pressure of
monolayers on the penetration of PKC- and
mutants. Proteins used were wild type (
/
), D55A (
/
),
and D116A (
/
) and their concentrations in the subphase were 1.5 µg/ml. Monolayers contained either POPC/POPS (6:4) (open
symbols) or POPC/POPG (6:4) mixed monolayers (filled
symbols). The subphase contained 20 mM Tris buffer, pH
7.5, containing 0.1 M KCl and 0.1 mM
Ca2+. Each data point represents an average of duplicate
measurements.
, D55A, and
D116A at varying surface lipid compositions and calcium concentrations.
First, we coated the sensor chip with POPC/POPS/DOG (69:30:1) vesicles
and measured the binding in the presence of 0.1 mM
Ca2+. As summarized in Table
I, little variation of
ka or kd was observed for the
mutants when compared with wild type, hence comparable
Kd values. This is consistent with our vesicle
binding data (see Fig. 10), in which all three proteins exhibited the
maximal binding under these conditions. Since there was a much larger
difference in relative binding affinity with POPC/POPG/DOG (69:30:1)
vesicles and 0.1 mM Ca2+ (see Fig. 10), we then
measured the binding of the three proteins with the sensor chip coated
with POPC/POPG/DOG (69:30:1) vesicles. In agreement with vesicle
binding data, all three proteins showed lower affinity for the
POPG-coated chip than for the POPS-coated chip. Again, D116A mutation
did not significantly influence ka and
kd under these conditions. However, D55A had
2.2-fold lower kd than wild type while having
comparable ka, indicating that the mutation leads to
the enhanced penetration into the POPG-containing vesicles. This is
also consistent with the monolayer penetration data shown in Fig. 12.
We then measured the binding to immobilized POPC/POPS/DOG (69:30:1)
vesicles at the lowest possible Ca2+ concentration that
gave rise to detectable SPR signal under our experimental conditions
(i.e. 7 µM Ca2+). In accordance
with Ca2+ dependence data in Fig. 8, D55A had 15-fold
higher affinity (in terms of Kd) than wild type and
D116A. Interestingly, enhanced affinity of D55A derived from both a
3.2-fold increase in ka and 4.6-fold decrease in
kd. As was the case with binding to POPC/POPG/DOG
(69:30:1) vesicles, the decrease in kd should be due
to enhanced membrane penetration. On the other hand, the increased
ka might originate from the contribution from C1a
cationic residues that can readily interact with anionic membranes due
to the lack of C1a domain tethering. This contribution would become
more important when the C2 domain cannot effectively drive the membrane
association at low calcium concentrations. Together, these data further
supports the notion that Asp55 of PKC-
is involved in
C1a domain tethering, the disruption of which allows the membrane
penetration of the C1a domain, which in turn leads to more favorable
interactions between C1a cationic residues and anionic membrane
surfaces (see Fig. 2 and "Discussion").
Binding parameters for PKC- and mutants determined from SPR analysis
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
showed different translocation
patterns in the cell. A growing body of evidence indicates that C1a and
C1b domains have distinct roles in the full-length PKC molecule. For
instance, Slater et al. (29, 30) have reported that PKC-
contains two distinct binding sites with low and high affinity for
phorbol esters and that DAG and phorbol esters bind to the two discrete
sites with opposite affinity. Although these in vitro and
cell studies have demonstrated distinct properties and roles of the C1a
and C1b domains, no direct correlation between the intrinsic properties
of individual C1 domains and their specific roles in different PKC
isoforms has been established. Our recent study of PKC-
shed new
light on the differential roles of C1a and C1b domain in the activation
of conventional PKC (14). The study showed that differential roles of
the two C1 domains in DAG-induced PKC activation are correlated with
their different membrane penetration behaviors. That is, the C1a domain
plays an essential role because its hydrophobic residues can penetrate into the membrane to bind DAG whereas the C1b domain does not because
of lack of membrane penetration. Differential effects of cationic
residue mutations described in this report corroborate the critical
involvement of C1a domain in the membrane binding and activation of
PKC-
. Among three cationic residues in the C1a domain,
Arg77 is most essential for anionic vesicle binding and
Lys62 also makes considerable contribution to vesicle
binding, suggesting that these residues make immediate contact with
anionic membrane surfaces. Interestingly, an orientation of the
membrane-bound C1a domain that allows the penetration of its
hydrophobic residues into the hydrophobic core of the membrane would
also permit favorable contact of the two cationic residues with anionic
membrane surfaces (see Fig. 2). These interactions are nonspecific
Coulombic interactions, as the mutations reduce the binding to PS- and
PG-containing vesicles to comparable extents. This notion is consistent
with our previous finding that the vesicle binding and monolayer
penetration of the isolated C1 domains (i.e. C1a + C1b)
showed no PS specificity (14). Unlike C1a domain, C1b domain appears to
bind the membrane in an orientation that allows neither the penetration
of its hydrophobic residues nor electrostatic interactions between its
cationic residues with anionic membrane surfaces. Note that C1a domain
mutants display much larger decreases in activity than expected from
their reduced membrane affinity. In particular, R77A shows no activity
even under the conditions where all enzyme molecules are vesicle-bound. Furthermore, R77A showed markedly reduced penetration into the POPC/POPS (6:4) monolayer (
c = 26 dyne/cm) when compared
with wild type (data not shown). This indicates that the electrostatic interactions of the C1a cationic residues with anionic phospholipids are important not only for membrane binding but also for membrane penetration, DAG binding, and activation of PKC-
. It has been shown
that multiple anionic phospholipids, including some PS molecules, are
required for conventional PKC activation (31, 32). It is not likely
that a conventional PKC molecule contains multiple specific binding
sites for these anionic phospholipids. Our results indicate that the
cationic residues in the C1a domain can provide nonspecific
electrostatic interaction sites for the anionic phospholipids.
to allow its interactions with
DAG, which also drives the removal of the pseudosubstrate from the
active site. Based on cellular translocation studies, Oancea and Meyer
(33) proposed that the DAG-binding site of PKC-
(i.e. two
C1 domains) is inaccessible to DAG in the resting state because it is
clamped to the catalytic domain by the pseudosubstrate. The present
study indicates that a single aspartate residue Asp55 is
involved in tethering of the C1a domain of PKC-
to the protein molecule in the resting state, thereby rendering the DAG-binding site
inaccessible to DAG in the membrane. The mutation of Asp55
to alanine (or lysine) dramatically changed the membrane binding, activation, and monolayer penetration of PKC-
. In particular, D55A
shows much higher vesicle affinity, activity, and monolayer penetration
power than wild type under nonactivating conditions, i.e.
with PG and in the absence of (or at low) Ca2+, indicating
that D55A has enhanced conformational flexibility that allows it to be
activated much more easily than wild type. As a result, D55A shows much
reduced PS specificity, which is reminiscent of the isolated C1 domains
(i.e. C1a + C1b) (14), suggesting that its behaviors are
dictated mainly by the C1 domains due to the disruption of C1a domain
tethering. A recent study by Johnson et al. (34) suggested
that a PS-specific binding site is located in the C1 domain of
PKC-
II, based on the finding that the isolated C1b
domain has higher affinity for PS than for other anionic phospholipids.
It should be noted that the reduced PS specificity of D55A cannot be
accounted for by this model for at least two reasons. First, D55A has
higher affinity for PS vesicles and higher activity in the
presence of PS vesicles than wild type (see Fig. 7), which precludes
the possibility that Asp55 is directly involved in PS
binding. Second, no mutation of C1a domain cationic residues has a
significant effect on PS specificity of binding and activity, showing
that these residues do not serve as a PS-binding site. Thus, it is also
unlikely that Asp55 indirectly influences PS specificity by
interacting with the PS-binding site in the C1 domain. Furthermore, the
model cannot fully explain the enhanced penetration of D55A into
PG-containing monolayers and its elongated membrane residence time at
PG-containing vesicles and at low Ca2+. These data are more
consistent with the notion that the disruption of
Asp55-mediated tethering leads to the nonspecific membrane
penetration of the C1a domain, which results in dramatically enhanced
binding affinity for non-PS anionic phospholipid aggregates and much
higher activity in the presence of nonspecific lipids. This in turn
suggests that the specific Ca2+- and
PS-dependent membrane penetration and activation of PKC-
involves the disruption of Asp55-mediated C1a domain
tethering. The role of Asp116 of the C1b domain in PKC-
activation is less clearly defined. The wild type-like vesicle affinity
and monolayer penetration of D116A indicate that Asp116 is
not directly involved in PKC activation and PS specificity. D116A,
however, shows consistently lower activity than wild type in the
presence of POPC/POPS/DOG vesicles and has significantly higher
activity under nonspecific conditions, e.g. in the presence of POPC/POPG/DOG vesicles (see Fig. 11). In particular, D116A has >5-fold higher basal activity than wild type in the absence of calcium
and lipid cofactors (data not shown). Thus, Asp116 might be
indirectly involved in PS specificity of PKC activation by suppressing
the level of nonspecific activation. This would supplement the direct
role of Asp55 of the C1a domain in the PS-specific PKC activation.
indicated that a primary determinant of PS
specificity resides in the C2 domain (14), yet the present study shows
that the mutation in the C1a domain has a dramatic effect on the PS
specificity. Furthermore, the full-length PKC-
, which shows much
more pronounced PS specificity than does the isolated C2 domain,
exhibits its full PS specificity only in the presence of C1 domain
ligand, DAG, or phorbol esters. The close interaction of C1 and C2
domains has also been implicated in the regulation of a novel PKC from
Aplysia (35). A recently determined crystal structure of the
C2 domain of PKC-
·calcium·PS complex provides an important clue
to understanding the nature of putative C1-C2 inter-domain interaction
and the origin of PS specificity (7). In this structure, the phosphate
oxygen of a PS molecule specifically coordinates with a calcium ion,
while its carboxylate interacts with the backbone and side chain
nitrogens of Asn189 in the calcium binding pocket (see Fig.
13). The structure raises an intriguing
possibility that Asp55 in the C1a domain specifically
interacts with Asn189 in the Ca2+ binding
pocket of the C2 domain in the resting state to keep the protein in an
inactive conformation, as schematically illustrated in Fig. 13. We
propose that upon membrane binding of PKC, which is driven by
electrostatic interactions involving the C2 domain-bound calcium ions
and cationic residues in the C1a domain, the carboxylate group of PS
(one or more molecules) might unleash the putative tethering by
replacing Asp55. This might allow the C1a domain to
penetrate into the membrane and bind DAG. The molecular motion
accompanying the membrane penetration would then remove the
pseudosubstrate from the active site, hence the activation.
Undoubtedly, the corroboration of this hypothetical model entails
further studies, including the mutation of Asn189 and other
C2 domain residues that might interact with Asp55. Also, it
remains to be seen whether or not the model can account for the
activation of other PKCs. As such, the model provides a basis for
further investigation of the molecular mechanisms underlying the
subcellular targeting and activation of PKC isoforms.
View larger version (34K):
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Fig. 13.
A proposed mechanism of the in
vitro membrane binding and activation of conventional
PKC. In this model, the C1a domain and the C2 domain are
tethered via hydrogen bond between Asp55 and a C2 domain
residue (e.g. Asn189). When the protein binds to
PG-containing membranes (case A), the C1a-C2 tethering
remains intact, and consequently PKC remains largely inactive. When the
protein binds to PS-containing membranes (case B, see the
inset), however, the carboxylate of PS releases
Asp55 of the C1a domain from the tethering, resulting in
the membrane penetration and DAG binding of the C1a domain and PKC
activation.
![]() |
ACKNOWLEDGEMENT |
---|
We thank John Rafter for helpful suggestions and discussions.
![]() |
FOOTNOTES |
---|
* This work was supported in part by National Institutes of Health Grant GM53987.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Established Investigator of the American Heart Association. To
whom correspondence should be addressed. Tel.: 312-996-4883; Fax:
312-996-2183; E-mail: wcho@uic.edu.
Published, JBC Papers in Press, October 11, 2000, DOI 10.1074/jbc.M008491200
2 R. V. Stahelin and W. Cho, submitted for publication.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: PKC, protein kinase C; BSA, bovine serum albumin; DAG, 1,2-sn-diacylglycerol; DOG, 1,2-sn-dioleoylglycerol; PG, phosphatidylglycerol; POPC, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine; POPG, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol; POPS, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine; PS, phosphatidylserine; SPR, surface plasmon resonance; CHAPS, 3-[(3- cholamidopropyl)dimethylammonio]-1-propanesulfonate.
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