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INTRODUCTION |
Protein kinases C
(PKCs)1 are a family of
serine/threonine kinases that transduce the myriad of signals
activating cellular functions and proliferation (1, 2). More than 10 members of the PKC family have been identified by molecular cloning.
All PKCs contain an amino-terminal regulatory domain and a
carboxyl-terminal catalytic domain. Based on structural differences in
the regulatory domain, PKCs are generally classified into three groups;
conventional PKC (
, 
I, II, and 
subtypes), novel PKC (
,
, 
, and 
subtypes), and atypical PKC (
and 
subtypes).
The regulatory domain of conventional PKCs is composed of two conserved
membrane-targeting modules, C1 and C2 domains, as well as a
pseudosubstrate region and variable regions. Conventional PKCs are
activated by the Ca2+-dependent translocation
of proteins to the membrane containing phosphatidylserine (PS) and
diacylglycerol (DAG). Structural (3) and mutational (4, 5) studies have
shown that the C2 domain of conventional PKC is responsible for the
Ca2+-dependent translocation of the protein to
membranes. It has also been shown that the C1 domain, which is composed
of a tandem repeat of cysteine-rich zinc-finger domains (C1a and C1b),
is involved in binding of PKC to DAG and its structural analogs,
phorbol esters (6-8). However, the temporal and spatial sequences of
membrane targeting and activation of PKC have not been fully
elucidated. Furthermore, the interplay of the C1 and C2 domains in
these complex processes is poorly understood. Finally, the roles of
individual zinc finger domains in the DAG-dependent
membrane binding and activation of PKC are not well defined. To address
these questions, we functionally expressed the isolated C1 and C2
domains of PKC- and measured their vesicle binding and monolayer
penetration. We also mutated C1a and C1b domain residues of the native
PKC- and measured the effects of mutations on vesicle binding,
enzyme activity, and monolayer penetration. Results indicate that the C2 domain of PKC- is responsible for its initial Ca2+-
and PS-dependent membrane binding, whereas the C1 domain is involved in subsequent membrane penetration and DAG binding, which eventually lead to enzyme activation. These studies also show that the
two C1 domains have distinct roles in membrane binding and activation
of PKC-.
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EXPERIMENTAL PROCEDURES |
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),
N-dansyl-1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (dansyl-PE), and 1,2-sn-dioleoylglycerol (DOG) were
purchased from Avanti Polar Lipids, Inc. (Alabaster, AL) and used
without further purification. Tritiated POPC was prepared from
1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine and
[9,10-3H]oleic acid (American Radiochemical Co., St.
Louis, MO) using rat liver microsomes as described (9, 10).
Phospholipid concentrations were determined by phosphate analysis
(11). Fatty acid-free bovine serum albumin was from Bayer Inc.
(Kankakee, IL). [-32P]ATP (3 Ci/µmol) was
from Amersham Pharmacia Biotech, and cold ATP was from Sigma.
4--[3H]hydroxyphorbol-12,13-dibutyrate (PDBu) was from
American Radiochemical Co. Triton X-100 was obtained from Pierce.
Restriction endonucleases and enzymes for molecular biology were
obtained from either Roche Molecular Biochemicals or New England
Biolabs (Beverly, MA).
Mutagenesis--
Baculovirus transfer vectors encoding the
cDNA of PKC- with appropriate C1 domain mutations were generated
by the overlap extension polymerase chain reaction (PCR) using
pVL1392-PKC- plasmid (12) as a template. Briefly, appropriate
complementary synthetic oligonucleotides introducing the desired
mutation and two other primers at the 5'-end of the PKC- gene
and around NcoI site inside PKC- gene were used as
primers for PCR performed in a DNA thermal cycler (Perkin-Elmer) using
Pfu DNA polymerase (Stratagene). The protocol consists of
two steps. In the first step, two DNA fragments overlapping at the
mutation site were generated and purified on an agarose gel. Then,
these two fragments were annealed and extended to generate a part of
PKC- gene containing a mutated regulatory domain (residues 1-389),
which was further amplified by PCR. The product was subsequently
purified on an agarose gel, digested with NcoI, and
subcloned into the pVL1392-PKC- plasmid. The pVL1392-PKC- plasmid
used for subcloning was first digested with NcoI,
dephosphorylated with alkaline phosphatase to prevent self-ligation,
and finally purified on an agarose gel. The mutagenesis was verified by
DNA sequencing of part of PKC- gene that had been subjected to PCR
mutagenesis using a Sequenase 2.0 kit (Amersham Pharmacia Biotech).
Expression vectors for isolated C1 and C2 domains were generated by PCR
using the pVL1392-PKC- as a template. For the C1 domain expression
vector, a NcoI site was incorporated at the 5'-end so that
the translation started from Met in position 31 of the PKC- coding
sequence. A XhoI site was also introduced after
His155 at the 3'-end. The gene for the C1 domain (residues
31-155) was subcloned into the pET21d vector using the NcoI
and XhoI sites (Novagen, Madison, WI). This vector is
designed to introduce a carboxyl-terminal His6 tag between
the XhoI site and a stop codon for affinity purification of
expressed proteins. For the C2 domain expression vector, a new
NcoI site was created at the 5'-end in order for the
translation to start at the Met153, and residue 154 was
mutated to Ala to ensure the removal of Met153 by bacterial
methionine amino peptidase. A XhoI site was incorporated after residue 284, and the gene for the C2 domain, spanning residues 153-284, was subcloned into pET21d vector. This construct also contains a carboxyl-terminal His6 tag. All constructs were
verified by restriction digestion and DNA sequencing.
Expression of PKC- and Mutants in Baculovirus-infected
Sf9 Cells--
Wild type PKC- and mutants were expressed in
baculovirus-infected Sf9 cells (Invitrogen, La Jolla, CA).
Transfection of Sf9 cells with mutant pVL1392-PKC- constructs
was performed using a BaculoGoldTM transfection kit from
Pharmingen (San Diego, CA). Plasmid DNA for transfection was prepared
by using an EndoFree Plasmid Maxi kit (Qiagen, Valencia, CA) to avoid
potential endotoxin contamination. A detailed protocol for expression
and purification of PKC- from Sf9 cells is described
elsewhere (12). Protein concentration was determined by the
bicinchoninic acid method using bovine serum albumin as standard (Pierce).
Bacterial Expression and Purification of Isolated C1 and C2
Domains of PKC---
Escherichia coli strain BL21(DE3)
(Novagen) was used as a host for protein expression. One liter of Luria
broth supplemented with 100 µg/ml ampicillin was inoculated with 1 ml
of overnight culture grown at 37 °C. Cells were grown at 37 °C
until their absorbance at 600 nm reached approximately 0.8, and the
protein expression was then induced with 0.5 mM
isopropyl-1-thio--D-galactopyranoside (Research
Products, Mount Prospect, IL). After 4 h, cells were harvested by
centrifugation (5000 × g for 10 min at 4 °C). For C1 domain purification, cells were resuspended in 20 ml of 50 mM Tris-HCl buffer, pH 7.5, containing 50 mM
NaCl, 0.4% (v/v) Triton X-100, 0.4% (w/v) sodium deoxycholate, and 1 mM phenylmethylsulfonyl fluoride. After the suspension was
sonicated, the inclusion body pellet was obtained by centrifugation at
100,000 × g for 15 min at 4 °C. The pellet was
resuspended in 20 ml of 50 mM Tris-HCl buffer, pH 7.5, containing 50 mM NaCl, 0.8% (v/v) Triton X-100, 0.8%
(w/v) sodium deoxycholate, and the suspension was sonicated as
described above. After centrifugation at 100,000 × g
for 15 min at 4 °C, the pellet was resuspended in 20 ml of 50 mM Tris-HCl buffer, pH 7.5, and stirred for 15 min at room
temperature, and the suspension was centrifuged at 100,000 × g for 10 min at 4 °C. The washed inclusion body was
resuspended in 20 ml of 50 mM Tris-HCl buffer, pH 7.5, containing 8 M urea and 5 mM dithiothreitol and
stirred at room temperature for 1-2 h. Insoluble matter was removed by
centrifugation at 100,000 × g for 10 min at 4 °C,
and the supernatant was dialyzed against 50 mM Tris-HCl, pH
7.5, 1.5 M urea, 50 µM ZnCl2, and
0.5 mM dithiothreitol and then against 50 mM
Tris-HCl, pH 7.5. The refolded C1 domain was purified using a Ni-NTA
agarose column (Qiagen) according to the manufacturer's instructions.
For isolated C2 domain purification, harvested cells were resuspended
in 50 mM KH2PO4 buffer, pH 7.5, 300 mM NaCl, and 10 mM imidazole, and the
suspension was sonicated. The supernatant was collected by
centrifugation at 50,000 × g for 45 min at 4 °C.
The C2 domain was purified using a Ni-NTA agarose column according to
the manufacturer's instructions. Purity of protein samples was judged
to be higher than 90% using SDS electrophoresis gels. Aliquots of
purified proteins were stored at 
20 °C.
Determination of PKC Activity--
The activity of PKC was
assayed by measuring the initial rate of [32P]phosphate
incorporation from [-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 100 µM CaCl2 in 50 µl of 20 mM
HEPES, pH 7.0. Protamine sulfate (200 µg/ml) was used to assess 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 (13). Reactions were
started by adding the MgCl2 to the mixture and quenched by adding 50 µl of 1% aqueous phosphoric acid solution after a given period of incubation (e.g. 5 min for histone) at room
temperature. Seventy-five microliters of quenched reaction mixtures was
spotted on P-81 ion-exchange papers (Whatman), and papers were washed four times with 1% 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 dependence of the reaction was checked by
monitoring the degree of phosphorylation at regular intervals.
Vesicle Binding Measurements--
The binding of PKC to
phospholipid vesicles was measured by a centrifugation assay using
large sucrose-loaded unilamellar vesicles (100 nm in diameter) (14).
Sucrose-loaded vesicles were prepared as described elsewhere (4). 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
(approximately 12 nM) was incubated for 15 min with
sucrose-loaded vesicles (0.1 mM), 1 µM bovine serum albumin, and Ca2+ (or EGTA; see under "Results")
in 150 µl of 20 mM HEPES (pH 7.0) containing 100 mM KCl. Bovine albumin 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 a 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 either
the anionic lipid content or the DAG content in vesicles. PS, PG, and
DOG concentrations giving rise to half-maximal vesicle binding and
activity ([PS]1/2, [PG]1/2, and
[DOG]1/2) were estimated graphically from individual plots.
The binding of isolated C1 and C2 domains to phospholipid vesicles was
measured by the same centrifugation assay. For these measurements,
binding assay mixtures containing 0.6 µM protein in 500 µl of 10 mM HEPES buffer, pH 7.0, containing 0.1 M KCl, 0.1 mM Ca2+ (or 0.5 mM EGTA), and 0.5 mM phospholipid were
incubated for 15 min at room temperature and centrifuged at
100,000 × g for 30 min at 25 °C. Supernatants were
decanted, and pellets were resuspended in 15 µl of 10 mM
HEPES buffer, pH 7.0, containing 0.1 M KCl and 0.5 mM EGTA. Resuspended pellets were loaded on 14%
polyacrylamide gels and proteins were separated by SDS-polyacrylamide gel electrophoresis. The amount of protein in each band was quantified using an IS-1000 digital imaging system (Key Scientific, Mt. Prospect, IL). To convert the protein band density to the protein concentration, a standard curve was constructed from density values of varying amounts
of C1 and C2 domain samples (1-5 µg).
Phorbol Ester Binding Assay--
The association of PKC- and
its C1 domain to [3H]PDBu was measured by the method of
Quest and Bell (15) with modifications. Assay mixtures (200 µl)
contained 100 µM POPC:POPS (1:1 mol/mol) large
sucrose-loaded unilamellar vesicles, 1-150 nM
[3H]PDBu, 10 nM protein, 1 µM
bovine serum albumin in 10 mM HEPES, pH 7.0, containing 0.1 M KCl and 0.1 mM CaCl2. The
mixtures were incubated at room temperature for 15 min, and then the
bound and the free [3H]PDBu were separated by vesicle
pelleting at 100,000 × g for 30 min using a Sorvall
RC-M120EX Microultracentrifuge. The nonspecific pelleting of
[3H]PDBu with vesicles was determined from the assay
mixtures minus protein, and these values were used to correct the bound
and free [3H]PDBu concentrations.
Fluorescence Resonance Energy Transfer--
The association of
the C2 domain of PKC- to vesicles was also measured by fluorescence
resonance energy transfer from tryptophans in the protein to a dansyl
group of dansyl-PE as a function of increasing Ca2+
concentration. Large unilamellar vesicles with 10 mol % of dansyl-PE were prepared by multiple extrusion through a 0.1-µm polycarbonate filter. The fluorescence measurements were performed in a Hitachi F4500
fluorescence spectrometer with the excitation and emission wavelength
set at 281 and 511 nm, respectively. First, lipid vesicles containing
10% dansyl-PE were added to 2.0 ml of 10 mM HEPES buffer, pH 7.0, containing 0.1 M KCl and 0.5 µM C2
domain, and the initial fluorescence intensity was recorded. Then, the
solution was titrated with Ca2+. Increase in fluorescence
(
I) after each addition of Ca2+ was measured, and the
relative fluorescence increase (I/Imax), where
Imax is a maximal fluorescence change, was plotted as a function of Ca2+ concentration. The binding of
Ca2+ ions to the isolated C2 domain was shown to be
consistent with the cooperative Hill model. (16). The concentration of
Ca2+ giving rise to half-maximal binding (or activity)
([Ca2+]1/2) was thus determined from curve
fitting of data to a Hill equation,
![y=a<FENCE><FR><NU>[<UP>Ca<SUP>2+</SUP></UP>]<SUP>h</SUP></NU><DE>[<UP>Ca<SUP>2+</SUP></UP>]<SUP>h</SUP><SUB>1/2</SUB>+[<UP>Ca<SUP>2+</SUP></UP>]<SUP>h</SUP></DE></FR></FENCE>](/content/vol274/issue28/fulltext/19852/img001.gif)
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(Eq. 1)
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where y, a, h, and [Ca2+] are relative
binding, arbitrary normalization constant, Hill coefficient, and free
Ca2+ concentration, respectively.
Monolayer Measurements--
Surface pressure (
) of solution
in a circular Teflon trough was measured using a du Nouy ring attached
to a computer-controlled Cahn electrobalance (Model C-32) as described
previously (12, 17). The trough (4 cm in diameter × 1 cm deep)
has a 0.5-cm-deep well for a magnetic stir bar and a small hole drilled
at an angle through the wall to allow addition of protein solution.
Five to 10 µl of phospholipid solution in ethanol/hexane (1:9 (v/v))
or chloroform was spread onto 10 ml of subphase (20 mM
HEPES, pH 7.0 containing either 0.1 or 0.5 mM of free
Ca2+) to form a monolayer with a given initial surface
pressure (o). The subphase was continuously stirred at
60 rpm with a magnetic stir bar. Once the surface pressure reading of
monolayer had been stabilized (after approximately 5 min), the protein
solution (typically 50 µl) was injected into the subphase, and the
change in surface pressure () was measured as a function of time
at 23 °C. Typically, the value reached a maximum after 20 min. The maximal value depended on the protein concentration and
reached a saturation when the protein concentration was above a certain
value. 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
o plot to the x axis.
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RESULTS |
Vesicle and Monolayer Binding of Isolated C1 and C2
Domains--
To determine the roles of the C1 and C2 domains in
Ca2+-, PS-, and DAG-dependent membrane binding
and activation of PKC-, we expressed the isolated C1 domain
(residues 32-155) and the isolated C2 domain (residues 154-284) and
measured their properties. The isolated C1 domain containing both C1a
and C1b domains was expressed in E. coli as an inclusion
body, which was dissolved in an 8 M urea solution and
refolded. On the other hand, the C2 domain was expressed mainly as a
soluble protein. Because both proteins contain a carboxyl-terminal
His6 tag, they were readily purified by affinity chromatography to near homogeneity. Protein yields after purification were typically 5-10 mg/liter of medium. Although we did not determine the tertiary structures of these isolated domains, they appear to be
correctly folded based on their vesicle binding and monolayer penetration behaviors that are consistent with their putative functions
(see below).
We first measured the PDBu binding of the wild type PKC- and the
isolated C1 domain to test if the C1 domain is correctly folded. Fig.
1 shows that the two proteins have
similar binding isotherms. The stoichiometry (n) and the
dissociation constant (Kd) of the PKC-PDBu complex
were determined using the equation [PDBu]bound/[total
protein] = n/(1 + Kd/[PDBu]free), assuming the presence
of n independent and identical PDBu-binding sites with a
dissociation constant of Kd in the protein. Both
n and Kd values (see the legend to Fig.
1) are comparable for both proteins and compare well with the reported
values (18), indicating that the isolated C1 domain of PKC- is
correctly folded and fully functional. We then measured the binding of
the isolated C1 domain to mixed vesicles with different compositions.
As shown in Fig. 2, the vesicle binding
of the C1 domain depended sharply on the concentrations of anionic
phospholipids and DOG in vesicles. [PS]1/2 and
[PG]1/2 values estimated from these plots are summarized in Table I. As indicated by these
parameters, the C1 domain did not distinguish PS and PG regardless of
the presence of 1 mol % DOG in mixed vesicles, indicating the lack of
specific PS-binding sites in this domain. The binding was also
independent of Ca2+ in solution (data not shown).
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Fig. 1.
Binding of PKC-
( ) and the isolated C1 domain ( ) to PDBu. Assay
mixtures contained 100 µM POPC:POPS (1:1) vesicles,
1-150 nM total [3H]PDBu, 10 nM
protein, 1 µM bovine serum albumin in 10 mM
HEPES, pH 7.0, containing 0.1 M KCl and 0.1 mM
CaCl2. Each data point represents the average of
triplicate measurements. Binding isotherms were analyzed using the
following equation: [PDBu]bound/[total protein] = n/(1 + Kd/[PDBu]free).
n = 1.3 ± 0.3 for PKC- and 1.3 ± 0.2 for
the C1 domain, and Kd = 60 ± 15 nM
for PKC- and 80 ± 30 nM for the C1 domain. The
theoretical curves (solid lines) were constructed using
these parameters.
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Fig. 2.
Binding of the isolated C1 domain to mixed
vesicles as a function of anionic lipid content. Vesicles used
were POPC/POPS (), POPC/POPG ( ), POPC/POPS/DOG (), and
POPC/POPG/DOG ( ). Protein and lipid concentrations were 0.6 µM and 0.5 mM, respectively. DOG content was
1 mol %. Each data point represents the average of
triplicate measurements.
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Table I
Properties of isolated C1 and C2 domains of PKC-
See under "Experimental Procedures" for experimental conditions and
methods to determine [PS]1/2, [PG]1/2,
[Ca2+]1/2, and c values.
[Ca2+]1/2 values are best-fit values ± S.D.
determined from nonlinear least squares analyses using Equation 1.
Numbers in parentheses indicate parameters determined in the presence
of 1 mol % DOG.
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We also measured the binding of the isolated C2 domain to mixed
vesicles with different compositions. Membrane binding properties of
isolated C2 domains of cytosolic phospholipase A2 (16,
19-21) and synaptotagmin (22) have been extensively characterized. However, properties of an isolated C2 domain of conventional PKCs have
not been measured. We first determined the Ca2+ dependence
of binding of the C2 domain to vesicles containing either PG or PS by
the fluorescence resonance energy transfer from tryptophans of the C2
domain to dansyl-PE in vesicles. The presence of four tryptophans in
the C2 domain allowed for sensitive measurement of this
Ca2+-dependent binding. As shown in Fig.
3, Ca2+ was essential for the
vesicle binding of the C2 domain, but DOG had no effect on binding. In
contrast to the C1 domain, which showed no PS selectivity, the C2
domain demonstrated significant PS preference. Over the wide
Ca2+ concentration range, the C2 domain bound more tightly
to POPC/POPS/dansyl-PE (7:2:1) vesicles than to POPC/POPG/dansyl-PE
(7:2:1) vesicles. Because dansyl-PE was shown to promote the membrane
binding and activation of conventional PKCs as well as PS (23), a
larger separation of the two curves would be expected if the binding was measured in the absence of dansyl-PE by, e.g. the
centrifugation method. As listed in Table I, [Ca2+
]1/2 values were 10 µM for PS-containing
vesicles and 60 µM for PG-containing vesicles. To compare
this PS selectivity with that of the native PKC-, we measured the
Ca2+ dependence of binding of PKC- to the same vesicles
(Fig. 4). As reported previously (12), PS
selectivity of PKC- was more pronounced in the presence of DOG in
the vesicles. Importantly, the PS selectivity of the isolated C2
domain, as indicated by, e.g. the ratio of
[Ca2+ ]1/2 values for PS and PG-containing
vesicles, is only modestly higher than that of the native PKC-
measured in the absence of DAG under the same conditions (see Table I).
This indicates that the intrinsic (i.e. DAG-independent) PS
selectivity of PKC- stems from the C2 domain. We also measured the
PS and PG dependence of C2 domain vesicle binding (Fig.
5). In this case, we used the centrifugation method to avoid the use of dansyl-PE, which would complicate the interpretation of PS and PG dependence. Again, PS
preference of the C2 domain was manifest, and DOG had no effect. As
shown in Table II, half-maximal vesicle
binding was achieved with 15 mol % of PS and 30 mol % of PG.
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Fig. 3.
Ca2+ dependence of vesicle
binding of the isolated C2 domain measured by fluorescence resonance
energy transfer. Vesicles used (100 µM) were
POPC/POPS/dansyl-PE (70:20:10) () POPC/POPG/dansyl-PE (70:20:10)
(), POPC/POPS/dansyl-PE/DOG (69:20:10:1) (), and
POPC/POPG/dansyl-PE/DOG (69:20:10:1) (). Protein concentration was
0.5 µM. Solid lines represent theoretical
curves constructed from parameters determined from the nonlinear least
squares fit using Equation 1.
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Fig. 4.
Ca2+ dependence of vesicle
binding of PKC-. Vesicles used (100 µM) were POPC/POPS/dansyl-PE (70:20:10) (),
POPC/POPG/dansyl-PE (70:20:10) (), POPC/POPS/dansyl-PE/DOG
(69:20:10:1) (), and POPC/POPG/dansyl-PE/DOG (69:20:10:1) ().
Protein concentration was 12 nM. The binding was measured
by the centrifugation method as described under "Experimental
Procedures." Solid lines represent theoretical curves
constructed from parameters determined from the nonlinear least squares
fit using Equation 1.
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Fig. 5.
Binding of isolated C2 domain to mixed
vesicles as a function of anionic lipid content. Vesicles used
were POPC/POPS (), POPC/POPG (), POPC/POPS/DOG (), and
POPC/POPG/DOG (). Protein and lipid concentrations were 0.6 µM and 0.5 mM, respectively. DOG content was
1 mol %, and calcium concentration was 0.1 mM. Each
data point represents the average of triplicate
measurements.
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Table II
Properties of C1 domain mutants of PKC-
See under "Experimental Procedures" for experimental conditions and
methods to determine [DOG]1/2, [PS]1/2, and
c values.
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We then measured the monolayer penetration of the isolated C1 and C2
domains. Our recent study showed that PS specifically induces the
Ca2+-dependent membrane penetration of PKC-
whereas other anionic phospholipids such as PG cannot (12). Lipid
monolayers have proven to be a sensitive tool for measuring
lipid-protein interactions (24, 25), including PKC-membrane
interactions (12, 26, 27). In these studies, a phospholipid monolayer
of a given initial surface pressure o was spread at
constant area, and the change in surface pressure () was
monitored after the injection of the protein into the subphase. Those
proteins the actions of which involve the partial or full penetration
of membranes have an ability to penetrate into the phospholipid
monolayer with o comparable to or higher than that of
biological membranes (approximately 31 dyne/cm) (28-31), and
vice versa. The versus o
plots for PKC-, C1 and C2 domains are shown in Fig.
6, and c values,
determined by extrapolating the plots to the x axis, are
summarized in Table I. As reported previously, PKC- could penetrate
into the POPC/POPS (7:3) monolayer in a
Ca2+-dependent manner even when
o > 35 dyne/cm, demonstrating its ability to penetrate
into biological membranes (12). Toward this monolayer, the isolated C1
domain showed much higher penetrating power (c = 41 dyne/cm) than did PKC- (c = 37 dyne/cm) and the isolated C2 domain (c = 28 dyne/cm). This indicates that
the C1 domain is involved in membrane penetration in the course of membrane binding of PKC-. Unlike the native PKC-, the monolayer penetration of which depends on the presence of Ca2+ in the
subphase and PS in the monolayer (12), the isolated C1 domain exhibited
high penetrating power regardless of the nature of anionic
phospholipids in the monolayer and also in the absence of
Ca2+ in the subphase. The penetration of the isolated C1
domain, however, required the presence of anionic phospholipid in the
monolayer, as it showed much lower penetration into the pure POPC
monolayer (data not shown). As is the case with the native PKC-
(12), DOG in the monolayer did not affect the monolayer penetration of
the C1 domain (data not shown). When compared with the C1 domain, the
C2 domain in general showed a lower penetration power but had a
significant degree of PS preference. Also, the monolayer penetration of
the C2 domain was dependent upon Ca2+ in the subphase.
These results indicate that the C1 domain of PKC- is primarily
responsible for its membrane penetration, whereas the C2 domain is
involved in Ca2+-dependent PS-specific membrane
binding and partial membrane penetration.
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Fig. 6.
Effect of the initial surface pressure of
monolayers on the penetration of PKC- ()
and isolated C1 ( and  ) and C2 domains ( and
). Monolayers contained either POPC/POPS (7:3) (open
symbols) or POPC:POPG (7:3) mixed monolayers (filled
symbols). The protein concentration in the subphase was 1.5 µg/ml for PKC- and 4 µg/ml for C1 and C2 domains. The subphase
contained 20 mM HEPES buffer, pH 7.0, with 0.1 mM free Ca2+. The penetration of C1 () and
C2 ( ) domains into POPC:POPS (7:3) was also measured in the presence
of 0.1 mM EGTA.
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Vesicle Binding and Monolayer Penetration of C1 Domain
Mutants--
An x-ray crystallographic study of a single zinc finger
domain (C1b) of PKC- has shown that this 50-amino acid region adopts a globular, compact / fold with two Zn2+ atoms as an
integral part of the structure (32) (Fig.
7A). This zinc finger domain
has an uneven distribution of hydrophobic and polar residues. The upper
part of molecule, where the DAG/phorbol ester binding pocket is
located, contains a few hydrophobic residues, whereas the middle part
includes a number of cationic residues. Based on this structure and the
NMR structure of micelle-bound C1b domain of PKC- (33), it has been
proposed that the upper part of this domain is inserted into membranes
in the course of membrane binding of PKC, whereas the cationic residues
in the middle make contact with phospholipid head groups (32). Our monolayer penetration data indeed demonstrate that the C1 domain has
high membrane penetration power. Because only one DAG/phorbol ester
molecule is required for activating a conventional PKC molecule (15,
34-36), the roles of the two C1 domains in the membrane binding and
activation of conventional PKC remain unclear. High sequence homology
between C1a and C1b domains (see Fig. 7B) predicts that the
two domains would have similar tertiary structures. To determine the
roles of individual zinc finger domains in the membrane binding and
activation of PKC-, we mutated prominent hydrophobic residues in the
putative membrane-penetrating regions of the two C1 domains:
i.e. Trp58 and Phe60 in C1a domain
and Tyr123 and Leu125 in C1b domain were
replaced by glycine. Because all mutated residues are located in the
loop regions, the above mutations were not expected to cause
deleterious conformational changes. Indeed, all four mutants were
expressed in baculovirus-infected insect cells as well as wild type,
suggesting comparable thermodynamic stability and a lack of gross
conformational changes. Furthermore, all mutants exhibited full
membrane binding affinity and enzymatic activity at saturating
Ca2+, PS, or DOG concentrations (see below), again
indicating that the mutations primarily affected the membrane binding
and activation of PKC- without interfering with its tertiary
structural fold.
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Fig. 7.
A, tertiary structure of the C1b domain
of PKC-. The x-ray structure of the C1b domain of PKC- (32) is
shown in a space-filling representation. The molecule is oriented with
its DAG-binding pocket pointing upward. Two mutated hydrophobic
residues are shown in green, and their residue type and
number in the C1a and C1b domains of PKC- are labeled. The color
code for other groups is as follows: yellow, hydrophobic
side chains; blue, cationic side chains; red,
anionic side chains; white, polar side chains and the
peptide backbone; pink, phorbol ester-bound;
cyan, zinc ions. B, amino acid sequences of C1a
and C1b domains of PKC-. The mutated hydrophobic residues are shown
in boxes, and conserved zinc ligands are shown as
outlined characters.
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To systematically analyze the effects of C1 domain mutations on
Ca2+ and DAG-dependent vesicle binding and
activation of PKC-, we measured the following properties; DOG
dependence of vesicle binding, PS dependence of vesicle binding, DOG
dependence of enzyme activity, PS dependence of enzyme activity, and
monolayer penetration. We first measured the binding of wild type and
mutants to POPC/POPS/DOG [(80 x):20:x in mole
ratio] vesicles as a function of increasing DOG concentration
(x) in the presence of 0.1 mM Ca2+.
The DOG dependence is shown Fig. 8, and
[DOG]1/2 values are summarized in Table II. Evidently,
mutations of C1a domain hydrophobic residues (W58G and F60G) had much
more pronounced effects on the vesicle binding than did those of C1b
domain hydrophobic residues (Y123G and L125G). W58G and F60G required
significantly higher DOG concentrations for vesicle binding than did
the wild type, whereas Y123G and L125G mutants behaved essentially the
same as the wild type did. This suggests that the C1a domain might play a more important role than C1b in the DAG-dependent vesicle
binding of PKC-. To see whether reduced DAG binding affinity of the
C1a domain mutants is directly translated into lower enzyme activity, we then measured the PKC activity of wild type and mutants as a
function of DOG content. When the kinase activity of wild type and
mutants toward histone was measured in the presence of the same
vesicles used for binding measurements, C1a domain mutations exhibited
much larger effects than did C1b domain mutations (Fig. 9). In this case, the differential
effects were more pronounced. Again, Y123G and L125G behaved similarly
to wild type PKC-. However, F60G and W58G showed only 40 and 10% of
the wild type activity, respectively, with 5 mol % DOG, which allowed
full and 70% vesicle binding of F60G and W58G, respectively (see Fig.
8).
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Fig. 8.
DOG dependence of vesicle binding of
PKC- and C1 domain mutants. Proteins used
(12 nM) include wild type (), W58G (), F60G (),
Y123G (), and L125G (). Total lipid concentration of
POPC/POPS/DOG ((80 x):20:x) vesicles and
Ca2+ concentration were both 0.1 mM.
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Fig. 9.
DOG dependence of enzyme activities of
PKC- and C1 domain mutants. Proteins used
include wild type (), W58G (), F60G (), Y123G (), and L125G
(). Total lipid concentration of POPC/POPS/DOG ((80 x):20:x) vesicles and PKC concentration were 0.1 mM and 12 nM, respectively, in 20 mM HEPES, pH 7.0, containing 0.1 M KCl, 5 mM MgCl2, histone III-SS (400 µg/ml), and 100 µM Ca2+. Each data point
represents an average of duplicate measurements. The absolute value of
maximal activity was 0.2 nmol/(µg·min).
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We then measured the PS dependence of the binding of wild type and
mutants to POPC/POPS vesicles containing 1 mol % of DOG in the
presence of 0.1 mM Ca2+. The PS dependence is
shown in Fig. 10 and
[PS]1/2 values are summarized in Table II. To achieve the
same degree of vesicle binding, W58G and W60G required higher PS
concentrations than did wild type, L123G, or Y123G. With PS content in
vesicles above 40 mol %, however, all mutants, including W58G, were
fully bound to vesicles containing 1 mol % DOG. This suggests that
reduced DAG affinity of C1a domain mutants can be compensated for by
higher PS concentrations, which is also consistent with our previous finding that PKC- can be fully vesicle-bound without DAG in the presence of high PS content in vesicles (12). We also measured the PS
dependence of the enzyme activity of wild type and mutants (Fig.
11). Note that under the conditions
where all protein molecules are vesicle-bound (i.e. [PS] > 40 mol %), F60G and W58G showed only 60 and 10% of the wild type
activity, respectively (see also Table II for [PS]1/2
values). Thus, vesicle binding of the C1 domain mutants driven by
higher PS concentrations did not lead to full PKC activation. Taken
together, these results suggest that hydrophobic interactions between
C1a domain and membranes are essential for the DAG binding and
activation of PKC-. In contrast, the C1b domain is not directly
involved in these processes.
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Fig. 10.
Binding of PKC- and
C1 domain mutants to POPC/POPS/DOG vesicles as a function of POPS
content. Proteins used include wild type (), W58G (), F60G
(), Y123G (), and L125G (). DOG content in vesicles was
maintained at 1 mol %. Experimental conditions were the same as
described for Fig. 8.
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Fig. 11.
Dependence of enzymatic activity of
PKC- and C1 domain mutants toward histone on
the POPS content in POPC/POPS/DOG vesicles. Proteins used include
wild type (), W58G (), F60G (), Y123G (), and L125G ().
DOG content in vesicles was maintained at 1 mol %. Experimental
conditions were the same as described Fig. 9. The absolute value of
maximal activity was 0.36 nmol/(µg·min).
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Finally, we measured the interaction of PKC- and C1 domain mutants
with phospholipid monolayers to test the notion that a part of the C1a
domain, but not that of C1b domain, penetrates into membranes in the
course of its membrane binding. As shown in Fig.
12, the penetrating power of was
greatly reduced compared to wild type PKC-, and its c
value is 30 dyne/cm, suggesting that it could not penetrate into
biological membranes. In contrast, C1b domain mutants, Y123G and L125G,
showed essentially the same monolayer penetration as wild type. These
results thus strongly support the notion that the C1a domain of PKC-
is primarily responsible for membrane penetration, DAG binding, and
enzyme activation.
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Fig. 12.
Effect of the initial surface pressure of
POPC/POPS (5:5) mixed monolayers on the penetration of
PKC- (), W58G (), Y123G (), and L125G
(). The protein concentration in the subphase was 20 nM. The subphase was 20 mM HEPES, pH 7.0, containing 0.5 mM free Ca2+. Each data
point is from a single measurement. The penetration of W60G was
not measured due to difficulty in obtaining sufficient amounts of
purified protein.
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DISCUSSION |
The membrane translocation and activation of conventional PKC
requires Ca2+, PS, and DAG under physiological conditions.
Conventional PKCs have two membrane-targeting modules, the C1 and C2
domains, which are responsible for its membrane binding and activation.
A consensus mechanism of in vitro PKC activation is that the
C1 and C2 domains work in concert to bring the PKC molecule to the
membrane surface, where the protein undergoes conformational changes to
remove the pseudosubstrate region from the active site, resulting in
PKC activation (1, 37). Extensive structural and mutation studies have
helped understand the roles of individual domains in the membrane
targeting and activation of PKCs and identify those amino acids that
are critically involved in these processes. For instance, our
structure-function study of the C2 domain of PKC- defined the role
of C2 domain as a membrane docking unit as well as a module that
triggers conformational changes of protein for its activation (4).
Also, extensive mutagenesis studies on the C1b domain of PKC-
identified the essential amino acids for phorbol ester binding (38,
39). Less is known, however, about the temporal and spatial sequences
of membrane targeting and activation of PKCs. A recent elegant cell
study indicated that the activation of PKC- follows well defined
sequential steps in which the Ca2+-dependent
membrane binding of the C2 domain is followed by the DG/phorbol ester
binding of the C1 domain (40). This report describes systematic
structure-function studies of the two domains of PKC- that provide
first detailed insights into the temporal and spatial sequences of
in vitro membrane targeting and activation of PKC-.
Differential Roles of C1 and C2 Domains in Membrane Binding of
PKC---
For most peripheral membrane binding proteins, both
electrostatic and hydrophobic interactions play roles in their membrane binding, although their relative contributions vary with the type of
proteins (17, 41, 42). We have shown that the membrane binding of
PKC- is also driven by these interactions; electrostatic interactions of the protein-bound calcium ions and other cationic residues of PKC- with anionic phospholipids and hydrophobic
interactions resulting from the Ca2+- and
PS-dependent penetration of PKC into the hydrophobic core of the membrane (4, 12). Thus, Ca2+ and PS are involved not
only in direct electrostatic interaction but also in eliciting
hydrophobic interactions. Extensive in vitro studies have
shown that the activation of conventional PKC requires the binding of
multiple PS molecules (43) and a single DAG (or phorbol ester) molecule
to PKC (34). Also, the PS specificity of conventional PKC is much more
pronounced in the presence of DAG in the membrane, suggesting the
synergism between PS-binding site(s) and a DAG-binding site (44).
Structural (32) and mutation analyses (38, 39) have clearly identified
the DAG-binding site in each of two C1 domains, although it is unclear
which of the two binding sites is actually involved in binding to a
single DAG molecule. The presence and location of PS-specific binding site(s) remains controversial, however, because some synthetic phospholipids, such as dansyl-PE, are also able to simulate the effects
of PS (23).
Our studies indicate that the C2 domain of PKC- has significant PS
selectivity, whereas the C1 domain shows essentially no selectivity.
Both the Ca2+ dependence (Fig. 3) and the anionic lipid
content dependence (Fig. 5) of vesicle binding demonstrate that the
isolated C2 domain prefers PS to PG. It should be noted that the
observed PS selectivity of the isolated C2 domain is comparable to that
of the native PKC- measured in the absence of DAG under the same
conditions (Fig. 4). Taking into account that the pronounced PS
specificity of PKC- results from the synergism between PS- and
DAG-binding sites, these findings provide strong evidence that the C2
domain is largely responsible for the intrinsic PS-selectivity of
PKC-. This also points to the presence of PS-binding sites in the C2 domain. We previously showed that some residues in the C2 domain of
PKC- are involved in Ca2+- and PS-dependent
partial membrane penetration (4). The monolayer penetration properties
of the isolated C2 domain corroborate this notion. When compared with
the C1 domain, however, the C2 domain would play only a minor role in
membrane penetration and hydrophobic interactions. In conjunction with
our previous study on the C2 domain of PKC- (4), these studies thus
show that the C2 domain is mainly responsible for its Ca2+-
and PS-dependent electrostatic binding to membranes.
These studies also show that the C1 domain of PKC- plays a central
role in membrane penetration and resulting hydrophobic interactions.
This notion is consistent with the DAG binding capacity of the C1
domain. Because DAG lacks a large polar head group, it would be
slightly buried from the polar surface of phospholipid membranes, and
as a result, it might not be readily accessible to PKC unless the C1
domain with a DAG-binding site penetrates into membranes. The isolated
C1 domain demonstrates exceptional monolayer penetration power that far
exceeds that of the native PKC-. This suggests that the membrane
penetrating power of the C1 domain in the PKC- molecule is actually
restrained by other parts of the molecule. The high affinity of the
isolated C1 domain for PDBu (see Fig. 1) shows that it is functionally
folded, thereby ruling out the possibility that high monolayer
penetration power of the isolated C1 domain is due to the incomplete
folding of the domain. The vesicle binding and monolayer penetration of
the isolated C1 domain is independent of Ca2+ and PS. Thus,
unlike the case of native PKC-, the membrane penetration of the
isolated C1 domain is not triggered by the specific PS binding and
concomitant conformational changes (12), but takes place spontaneously
due to a propensity of the C1 domain to penetrate into the membrane.
Both the vesicle binding and the monolayer penetration of the C1 domain
require, however, the presence of anionic phospholipids, indicating
that nonspecific electrostatic interactions are a driving force for its
initial membrane binding. A recent monolayer study has shown that the
monolayer penetration of surface active proteins, such as
apolipoproteins, follows a two-step mechanism in which electrostatic
surface adsorption precedes the insertion of hydrophobic side chains
into the hydrophobic moiety of the monolayer (45). Based on the
tertiary structure and monolayer penetration properties of the isolated
C1 domain, it appears that the membrane penetration of the C1 domain
follows a similar mechanism. For the isolated C1 domain, nonspecific
electrostatic interactions between anionic phospholipids and the
cationic cluster in the middle of the C1 domain (see Fig.
7A) would bring the domain to the membrane surface and then
the hydrophobic tip of the domain would penetrate into the hydrophobic
core of the membrane to bind DAG. For the whole PKC- molecule, the
C2 domain would primarily play the role of bringing the C1 domain to
the membrane surface. The contribution of C1 domain cationic residues
to the initial electrostatic membrane interactions of PKC- remains
to be assessed. As is the case with the native PKC- (12), DAG does
not enhance the penetration of the C1 domain, although it greatly
increases the vesicle binding affinity. This indicates that the DAG
binding is the consequence but not the driving force of the membrane
penetration of the domain. In this regard, it is noteworthy that DAG
has been shown to induce local PS-rich membrane domains (46). The
preferential binding of conventional PKCs to such domains would trigger
the membrane penetration and DAG binding. Thus, DAG might indirectly promote the membrane penetration of conventional PKCs via the PS domain
formation. Taken together, these studies show that the C1 domain is
primarily involved in the membrane penetration of PKC-, which is
essential for its DAG binding, hydrophobic membrane interactions, and activation.
Differential Roles of C1a and C1b Domains--
One-to-one
stoichiometry of conventional PKC-DAG (or phorbol ester) binding
indicates that only one of two DAG-binding sites is actually involved
in the DAG binding and PKC activation. A recent binding study using a
fluorescent phorbol ester analog suggested that PKC- has two
discrete phorbol ester-binding sites with different affinity (47). It
was also found that DAG and phorbol esters bind to the two discrete
sites with opposite affinity, so that a high affinity DAG-binding site
is a low affinity phorbol ester-binding site and vice versa
(48). Our structure-function analyses of the two zinc finger domains
show that the C1a domain contains the high affinity DAG-binding site.
We designed our mutations based on the premise that the removal of
hydrophobic residues from the tip of each zinc finger domain that
contains the DAG binding pocket would greatly reduce its ability to
penetrate into the hydrophobic core of the membrane, thereby lowering
DAG affinity. The effects of mutations on the monolayer penetration of
PKC- indicate that the upper part of the C1a domain penetrates into the membrane, whereas its counterpart in the C1b domain does not. Thus,
only the C1a domain would be allowed to interact with DAG. These
studies do not provide the quantitative information about the degree of
membrane penetration by C1a domain. A large decrease in monolayer
penetration by a single W58G mutation suggests that this residue might
fully penetrate into the hydrophobic core of the membrane. Vesicle
binding affinity and enzyme activity of the two groups of mutants
corroborate the differential membrane penetration of the two C1
domains. C1a domain mutants (W58G and F60G) have much lower vesicle
binding affinity and enzyme activity than does the wild type PKC-,
whereas corresponding C1b domain mutants (Y123G and L125G) behave
essentially the same as the wild type does. Both W58G and F60G show
much larger decreases in activity than expected from their reduced
membrane affinity. In particular, W58G show only 10% of the wild type
activity even when it is driven to fully bind to vesicles by
electrostatic interactions (e.g. with high PS concentrations
in vesicles; see Fig. 11). This indicates that the membrane insertion
of the C1a domain is absolutely necessary for PKC activity. However,
our results could not rule out a possibility that C1b domain might also
be able to interact with DAG without penetrating into the membrane. It
has been generally proposed that the activation of conventional PKC
results from the removal of the pseudosubstrate region from the active
site of PKC (49). The C1a domain is immediately linked to the
pseudosubstrate region, and thus conformational changes accompanying
the penetration of C1a domain into the membrane might provide a
mechanical force to remove the pseudosubstrate region from the active site.
The origin of the different membrane penetrating power of the two zinc
finger domains is still unclear. Based on the domain structure of
conventional PKC, however, one can speculate that the difference
derives from a mechanical reason. As described above, the C1 domain in
the intact PKC molecule cannot fully express its intrinsic membrane
penetration power, presumably because other parts of the molecule
interfere with the penetration. The C1a domain is flanked by the
amino-terminal pseudosubstrate region and the C1b domain, whereas the
C1b domain is immediately linked to the C1a domain and the C2 domain
(see Fig. 13). Thus, it is reasonable
to assume that the movement of the C1a domain, which is preceded only
by a short and putatively flexible amino-terminal region, might be
energetically more favorable than that of C1b domain, which might
entail a gross conformational change. It is also possible that the C1b
domain is involved in stabilizing interactions with the C2 domain,
which must be disrupted for the penetration of C1a domain to take
place. Indeed, a recent study suggested that the C1b domain of PKC-
provides several ligands for Ca2+ sites in the C2 domain
(3).
Although our data indicate that a large difference in DAG affinity
between the two C1 domains derives mainly from the difference in their
membrane localization, one cannot rule out the possibility that the two
zinc finger domains actually have different intrinsic DAG/phorbol ester
affinities. Conflicting results have been reported with respect to the
relative DAG/phorbol ester affinity of the two zinc fingers in the C1
domain. On one hand, a cell study using isolated C1a and C1b domains of
PKC- tagged with green fluorescence protein indicated that the C1a
domain is mainly responsible for DAG/phorbol esters binding (50). On
the other hand, mutations of a conserved proline in each C1 domain of
PKC- showed that C1b is responsible for phorbol ester-induced
membrane translocation in vivo (51). Thus, depending on the
PKC isoform and the nature of C1 domain ligand, relative affinity of
C1a and C1b domains might vary to large degrees (52). In any event, our
results underscore that relative affinity of the two C1 domains for a particular ligand depends not only on their intrinsic binding affinity
but also on the spatial arrangement of the binding site and the ligand
in the membrane. The relationship between the relative affinity of the
two C1 domains of PKC- for various phorbol esters and different
membrane localization of these ligands is under investigation.
Proposed Mechanism of in Vitro Membrane Binding and Activation of
PKC---
Based on our previous and present studies, we propose the
following mechanism for the in vitro membrane binding and
activation of conventional PKC (Fig. 13). The protein initially binds
to the membrane surface via the Ca2+-dependent
PS binding of the C2 domain. Once bound to PS-containing membranes, the
protein undergoes conformational changes that include the insertion of
C1a domain into the membrane. This membrane penetration allows for
optimal DAG binding and drives the release of pseudosubstrate region
from the active site. The former results in enhanced hydrophobic interactions and overall membrane affinity, and the latter leads to PKC
activation. The proposed temporal and spatial sequences are further
supported by the following observations. First, dominant negative
mutants of the C2 domain of PKC- (e.g. D246N) containing the intact C1 domain can neither bind to nor penetrate into membranes even in the presence of DAG (4). This indicates that the C2 domain must
first bind to the membrane prior to the membrane penetration of the C1
domain. Second, in the absence of DAG in the membrane, Ca2+- and PS-dependent membrane binding
properties of isolated C2 domain and dominant C1 domain mutants
(e.g. W58G) are essentially the same as the native PKC. This
again indicates that the C2 domain alone can bring the PKC molecule to
the membrane surface. Third, membrane binding properties of the
isolated C1 domain are distinct from those of the native PKC- under
all conditions employed, suggesting that the membrane binding of C1
domain might have to be primed by the membrane binding of C2 domain.
Our proposed mechanism can successfully account for the in
vitro Ca2+, PS- and DAG-dependent membrane
binding, and activation of conventional PKC. However, the mechanism of
phorbol ester-induced activation of PKC might be different from this
mechanism (53, 54). Also, other factors, most notably PKC
phosphorylation (55) and PKC adapter proteins (56), must be taken into
account to understand the in vivo membrane targeting and
activation of PKC. The fact that our proposed mechanism is
in Its
Membrane Binding and Activation*
TOP
ABSTRACT
INTRODUCTION
