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INTRODUCTION |
The agonist-induced subcellular targeting of protein is an
important process in cell signaling and regulation. Recently, the membrane targeting of peripheral proteins (e.g.
phospholipases, lipid-dependent protein kinases, lipid
kinases, and lipid phosphatases) by Ca2+ and lipid
mediators, including phosphoinositides, has received much attention as
an important event in cell signaling and membrane trafficking. It has
been shown that the subcellular targeting of peripheral proteins is
driven by a growing number of membrane targeting domains. These domains
include protein kinase C
(PKC)1 conserved 1 (C1)
domain, PKC conserved 2 (C2) domain, pleckstrin homology (PH) domain,
Fab1, YOTB, Vac 1 and EEA1 (FYVE) domain, band four-point-one, ezrin,
radixin and moesin (FERM) domain, epsin amino-terminal homology (ENTH)
domain, and phox (PX) domains (1-5).
The C2 domain has been identified in many cellular proteins involved in
signal transduction or membrane trafficking (5-7). A majority of C2
domains bind the membrane in a Ca2+-dependent
manner and thereby play an important role in
Ca2+-dependent membrane targeting of peripheral
proteins. Structural analyses of Ca2+-dependent
membrane binding C2 domains have demonstrated similar tertiary
structures in which three Ca2+ binding loops are located at
an end of eight-stranded antiparallel
-sandwich (8-14). Despite
similar structural folds, C2 domains exhibit great functional
diversities due to local structural variations, particularly in the
Ca2+ binding loops. Most
Ca2+-dependent membrane binding C2 domains have
higher affinity for anionic membranes than for zwitterionic ones. In
particular, the C2 domains of PKC-
(PKC-
-C2) and phospholipase
C-
1 show selectivity for phosphatidylserine (PS) (15-17). In
contrast, the C2 domains of group IVa cytosolic phospholipase
A2 (cPLA2) (18) and 5-lipoxygenase (19)
strongly favor zwitterionic phosphatidylcholine (PC). Furthermore, C2
domains show distinct subcellular localization patterns. For instance,
the C2 domains of PKC-
(20) and phospholipase C-
1 (17)
translocate to the plasma membrane, whereas the C2 domains of
cPLA2 (cPLA2-C2) (21, 22) and 5-lipoxygenase
(19) translocate to the perinuclear region. Although recent studies
indicate that the Ca2+ binding loops are involved in lipid
selectivity of C2 domains (17, 19, 23, 24), the mechanisms of
differential lipid selectivity and distinct subcellular localization of
C2 domains have not been fully elucidated. To understand these
mechanisms, we performed biophysical studies using purified recombinant
proteins and vesicles whose lipid compositions resemble those of cell
membranes and measured the spatiotemporal dynamics of enhanced green
fluorescent protein (EGFP)-tagged C2 domains and mutants in live cells.
These studies not only identify the residues that are responsible for their distinct lipid selectivity but also demonstrate that in vitro membrane binding properties of C2 domains are quantitatively correlated with their subcellular targeting behaviors.
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EXPERIMENTAL PROCEDURES |
Materials--
1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine
(POPC),1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine
(POPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoinositol
(POPI), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine
(POPS), and cholesterol were purchased from Avanti Polar Lipids, Inc.
(Alabaster, AL) and used without further purification. Phospholipid
concentrations were determined by phosphate analysis (25). The
Liposofast microextruder and 100-nm polycarbonate filters were from
Avestin (Ottawa, Ontario). Fatty acid-free bovine serum albumin was
from Bayer Inc. (Kankakee, IL). 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). CHAPS and octyl
glucoside were from Sigma and Fisher Scientific, respectively. Pioneer
L1 sensor chip was from Biacore AB (Piscataway, NJ).
Mutagenesis and Protein Expression--
The isolated
cPLA2-
-C2 and mutants (26) and the isolated PKC-
-C2
and mutants (15) were expressed and purified as previously described.
Mutants of PKC-
-C2 were generated by the overlap extension PCR (27)
using pVL1392-PKC-
plasmid as a template (28). Briefly, four
primers, including two complementary oligonucleotides introducing a
desired mutation and two additional oligonucleotides complementary to
the 5'-end of the C2 domain (residue 154) and the 3'-end (284) of the
PKC-
gene, respectively, were used for PCR 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 C2
domain gene containing a desired mutation, which was further amplified
by PCR. The product was subsequently purified on an agarose gel,
digested with NcoI and XhoI, and subcloned into
the pET21d vector digested with the same restriction enzymes. The
mutagenesis was verified by DNA sequencing using a T7 Sequenase kit
(U.S. Biochemical, Cleveland, OH).
Construction of Gene Constructs of cPLA2 and PKC-
Fused with EGFP--
EGFP in pEGFP vector
(Clontech) was modified by PCR to remove the first
methionine and to add two amino-terminal glycines and an
EcoRI site. The modified EGFP gene was inserted into a modified pIND vector (Invitrogen) between the EcoRI and
XhoI sites to yield a plasmid, pIND/EGFP. PKC-
was cloned
by PCR to remove the stop codon and add two carboxyl-terminal glycines
and an EcoRI site. The PCR product was digested with
NotI and EcoRI and ligated into pIND/EGFP to
generate a carboxyl-terminal EGFP fusion PKC-
with spacer sequence
GGNSGG. PKC-
-C2 mutants and cPLA2-C2 constructs were
generated by the same method. Two chimera proteins were prepared as
follows: The C2 domain of PKC-
was cloned out using PCR to introduce
a 5' NotI site and a 3' SmaI site. The catalytic
domain of cPLA2 was cloned out to have a 5' SmaI
site and a 3' BglII site. The catalytic domain of
cPLA2 was first digested and ligated into the pUC18 vector.
After this sequence was confirmed, the C2 domain of PKC-
was
digested and ligated into the pUC18 vector between NotI and
SmaI sites. After confirming this sequence, it was cloned
using PCR to contain a 5' NotI site and a 3'
BglII site and subsequently ligated into the modified
pIND/EGFP vector to possess the EGFP at the N terminus. The catalytic
domain of PKC-
was cloned using PCR to contain a 5' PstI
site and a 3' EcoRI site and to remove the stop codon. The
C2 domain of cPLA2 was then cloned to possess a 5'
SmaI site and a 3' PstI site while the regulatory
domain preceding the C2 domain of PKC-
was cloned to have a 5'
NotI site and a 3' SmaI site. All were digested
with restriction enzymes for their respective recognition end sequences and then ligated stepwise into the pUC18 vector cut with the same restriction enzymes. Each fragment was verified for correct positioning within the vector before the next fragment was ligated in
(i.e. the catalytic domain was checked for its correctness
before the C2 domain was ligated into this vector). After sequence
confirmation, this chimera was cloned using PCR to have a 5'
NotI site and a 3' EcoRI site and ligated into
modified pIND/EGFP vector to possess a carboxyl-terminal EFGP.
SPR Measurements--
The preparation of vesicle-coated Pioneer
L1 sensor chip (Biacore) was described in detail elsewhere (29, 30). In
control experiments, the sensor chip was coated with vesicles
(e.g. POPC/POPS (7:3)) incorporating 10 mM
5-carboxyfluorescein (Molecular Probes), and the fluorescence intensity
of the flow buffer was monitored to confirm that the vesicles remained
intact on the chip. All experiments for cPLA2-C2 were
performed with a control cell in which a second sensor surface was
coated with bovine serum albumin. For all PKC experiments, the control
sensor surface was coated with 100% POPC for which PKC-
-C2 has
extremely low affinity. 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 and dissociation for 4 min.
The immobilized vesicle surface was then regenerated for subsequent
measurements using 10 µl of 50 mM NaOH. The regeneration solution was passed over the immobilized vesicle surface until the SPR
signal reached the initial background value before next protein
injection. For data acquisition, five or more different concentrations
(typically within a 10-fold range above or below 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).
After sensorgrams were corrected for refractive index changes, the
association and dissociation phases of all sensorgrams were globally
fit to a 1:1 Langmuir binding model: protein + (protein binding site on
the vesicle)
(complex) using BIAevaluation 3.0 software (Biacore).
The association phase was analyzed using an equation, R = [kaC/(kaC + kd)]Rmax(1
e
(kaC + kd)(t
t0)) + RI, where
RI is the refractive index change,
Rmax is the theoretical binding capacity,
C is the protein concentration, ka is the
association rate constant, and t0 is the initial
time. The dissociation phase was analyzed using an equation,
R = R0e
kd(t
t0), where
kd is the dissociation rate constant and
R0 is the initial response. The dissociation
constant (Kd) was then calculated from the
equation, Kd = kd/ka. It should be noted that in
our SPR analysis Kd is defined in terms not of the
molarity of phospholipids but of the molarity of protein binding sites
on the vesicle. Thus, if each protein binding site on the vesicle is
composed of n lipids, nKd is the
dissociation constant in terms of molarity of lipid monomer (31). Due
to difficulty involved in accurate determination of the concentration
of lipids coated on the sensor chip, only Kd was
determined in our SPR analysis, and the relative affinity was
calculated as a ratio of Kd values assuming that n values are essentially the same for wild type and mutants.
Mass transport (32, 33) was not a limiting factor in our experiments, as change in flow rate did not affect kinetics of association and dissociation.
Cell Culture--
A stable HEK293 cell line expressing the
ecdysone receptor (Invitrogen) was used for all experiments. Briefly,
cells were cultured in Dulbecco's modified Eagle's medium (DMEM)
supplemented with 10% fetal bovine serum (FBS) at 37 °C in 5%
CO2 and 98% humidity until 90% confluent. Cells were
passaged into eight wells of a Lab-TechTM chambered cover
glass for later transfection and visualization. Only cells between the
5th and 20th passages were used.
Transfection and Protein Production--
80-90% confluent
cells in Lab-TechTM chambered cover glass wells were
exposed to 150 µl of unsupplemented DMEM containing 0.5 µg of
endotoxin-free DNA and 1 µl of LipofectAMINETM reagent
(Invitrogen) for 7-8 h at 37 °C. After exposure, the transfection
medium was removed, and the cells were washed once with
FBS-supplemented DMEM then overlaid with FBS-supplemented DMEM
containing ZeocinTM (Invitrogen) and 5 µg/ml
ponasterone A (Invitrogen) to induce protein production.
Confocal Microscopy--
Images were obtained using a
four-channel Zeiss LSM 510 laser scanning confocal microscope. All
experiments were carried out at the same laser power, which was found
to induce minimal photobleaching over 30 scans, and at the same gain
and offset settings on the photomultiplier tube. Also, a 63×, 1.2 numerical aperture water immersion objective was used for all
experiments. EGFP was excited using the 488-nm line of an argon/krypton
laser and a line pass 505-nm filter was used to monitor EGFP emission
on channel 1. We used mag-indo-1 for real-time monitoring of changes in
intracellular calcium concentration
([Ca2+]i) in the micromolar range. For
simultaneous monitoring of EGFP and [Ca2+]i,
mag-indo-1 was excited using the 340-nm line of a UV laser, and the
emitted light was collected using band pass filters (395-425 and
450-490 nm) on a second channel. The LSM 510 imaging software was used
to control the time intervals for imaging.
Ca2+-dependent translocation of C2 domains was
monitored as follows: Thirty minutes before imaging, cells were treated
with 2 µl of mag-indo-1 (10 µM final concentration)
(Molecular Probes). Immediately before imaging, the induction media was
removed and the cells were washed with 150 µl of 2 mM
EGTA and then overlaid with 150 µl of HEK buffer (1 mM
HEPES, pH 7.4, containing 2.5 mM MgCl2, 1 mM NaCl, 0.6 mM KCl, 0.67 mM
D-glucose, 6.4 mM sucrose). After initially
imaging cells, 150 µl of HEK buffer containing 10 µM ionomycin and 1 mM CaCl2 was added to the
cells. Protein translocation was monitored by time scanning at 4-s
intervals for PKC-
-C2 and at 15-s intervals for
cPLA2-C2. [Ca2+]i in the cytoplasm
was determined at 4- to 15-s intervals using the equation,
[Ca2+]i = Kca × Q × (R
Rmin)/(Rmax
R) (34), where R represents the fluorescence
intensity ratio F410/F470
that was determined from the average background-corrected pixel values of mag-indo-1 fluorescence from the cytoplasmic area of individual cells at 410 and 470 nm, respectively; Rmin and
Rmax correspond to minimum and maximum
fluorescence intensity ratios at no Ca2+ and saturating
calcium levels (i.e. 1 mM free
Ca2+), respectively. Q is the ratio of
the minimum fluorescence intensity at zero free calcium
(Fmin) to the maximum fluorescence intensity at
1 mM saturating calcium (Fmax) at
470 nm, and Kca is the calcium dissociation
constant for mag-indo-1. Rmin,
Rmax, Q, and
Kca values were determined by in
vitro calcium calibration using 1 µM mag-indo-1 and
a set of standard calcium buffers (Molecular Probes) containing 0-1
mM free Ca2+. The calculated
Kca (34 ± 1 µM) was the same
as the reported value of 35 µM (35).
Cell Imaging Data Analysis--
Images were analyzed using the
analysis tools provided in the Zeiss biophysical software package.
Using these tools, regions of interest in the cytosol were defined, and
the average intensity in a square (1 µM × 1 µM) was obtained with time. Membrane intensities were
determined for each frame in individual cells by extending a line from
the cytosol to the outside of the cell and recording the intensity with
distance along the line. By cross-checking markers on the
diagram with a table of intensity data, four intensity values
corresponding to the place on the line indicating the edge of the cell
were averaged. Lines were drawn in at least three places in each cell,
and membrane intensity values were determined and averaged. Intensity
values outside the photomultiplier tube's linear range were discarded.
The resultant intensity values were converted to a ratio of intensity
at membrane to the sum of intensities at membrane and at cytosol for
each time frame. Membrane translocation rates of individual C2 domains
were then calculated from the slopes of these plots by linear
regression. Each experiment was repeated at least twice on a given day,
and was repeated on at least two different days with different
transfected cells. Cellular distribution of EGFP intensity throughout
the cell was obtained using the profile function on the software
package. A line was drawn through the cell from an arbitrary point, and
the EFGP intensity from each point along the line was plotted
versus arbitrary distance to correlate the difference in
EGFP intensities at different subcellular locations.
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RESULTS |
PS Selectivity of PKC-
-C2--
The C2 domain of PKC-
has
been shown to have PS selectivity with extremely low affinity for
zwitterionic phospholipids (i.e. PS > PG
PC)
(15). Our structure-function analysis of PKC-
(15) and its isolated
C2 domain (36) and the crystal structure of PKC-
-C2 (12) suggested
that the PS selectivity derive from the specific recognition of PS by a
C2 domain-bound calcium ion and several residues located in the calcium
binding loops (Fig. 1). In particular, a
recent mutation study on PKC-
showed that Asn189,
Arg216, Arg249, and Thr251 in the
calcium binding loops are involved in PS binding (24). However, it is
still unclear whether these residues are involved in specific headgroup
recognition or in binding to other parts of phospholipid molecule. We
therefore measured the effect of the mutations of these residues on the
lipid headgroup selectivity. We also mutated another cationic residue
(Arg252) that has been implicated in membrane binding (28).
PS selectivity of wild type and mutants was determined by comparing
their binding affinity for immobilized POPC/POPS (7:3) and POPC/POPG
(7:3) vesicles by SPR analysis. PKC-
-C2 shows extremely low affinity
for POPC vesicles. This allowed the use of the physiologically more
relevant mixed vesicles in lieu of 100% POPS or POPG vesicles for
identifying the residues directly involved in specific lipid headgroup
recognition (i.e. serine versus glycerol
headgroup).

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Fig. 1.
Structures of calcium binding loops of
PKC- -C2 and cPLA2-C2.
PKC- -C2 (aqua) and cPLA2-C2
(orange) are shown in ribbon diagrams with two
Ca2+ ions (magenta) bound to the domains. Side
chains of mutated residues are shown and labeled. The coordinates for
PKC- -C2 and cPLA2-C2 were taken from the crystal
structures solved by Verdaguer et al. (12) and by Perisic
et al. (13), respectively. PKC- -C2 achieves its PS
selectivity by specific headgroup recognition by Asn189 and
other interactions involving a Ca2+ ion,
Arg216, and Thr251. For cPLA2-C2,
Phe35, Leu39, Tyr96, and
Val97 are all involved in PC selectivity.
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We have shown that the SPR analysis allows direct determination of
membrane association (ka) and dissociation
(kd) rate constants for peripheral proteins (29,
31). As summarized in Table I, PKC-
-C2
has higher affinity (in terms of Kd) for POPC/POPS
(7:3) than for POPC/POPG (7:3), and the PS selectivity of PKC-
-C2
expressed in term of the ratio of 1/Kd for POPC/POPS
(7:3) to 1/Kd for POPC/POPG (7:3) is about 11. Interestingly, the higher affinity for PS is almost entirely ascribed
to the lower kd value. Our previous study indicated that nonspecific electrostatic interactions primarily accelerate the
association of protein to anionic membrane surfaces, whereas hydrophobic interactions and short-range specific interactions (electrostatic or hydrogen bonds) mainly slow the membrane dissociation (29). Thus, the present data support the notion that PS forms specific
interactions with the PKC-
-C2.
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Table I
Binding parameters for PKC- -C2 and mutants determined from SPR
analysis
Values represent the mean ± S.D. from five determinations. All
measurements were performed in 10 mM HEPES, pH 7.4, containing 0.1 M NaCl and 0.1 mM Ca2+.
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The previous study showed that the T251A mutation had the most
detrimental effect on binding affinity for PS-containing vesicles and
micelles, whereas N189A, R216A, and R249A exhibited lesser effects
(24). Our SPR data also showed that T251A reduced the affinity for
POPC/POPS (7:3) vesicles more significantly than N189A, R216A, and
R249A/R252A. T251A has 18-fold lower affinity than wild type, whereas
N189A, R216A, and R249A/R252A have 5- to 6-fold lower affinity. Most
important, T251A, R216A, and R249A/R252A have essentially the same PS
selectivity as the wild type, because the mutations reduced the
affinity of PKC-
-C2 for POPC/POPS (7:3) and POPC/POPG (7:3) vesicles
to comparable degrees. In contrast, the N189A mutation reduced the PS
affinity of PKC-
-C2 5-fold without affecting its PG affinity. As a
result, N189A exhibited much reduced 2-fold PS selectivity. Together,
these data indicated that Asn189 is involved in specific PS
headgroup binding, whereas other mutated residues are involved in
either binding to a non-headgroup part of an anionic phospholipid, such
as sn-1 or -2 acyl group, or nonspecific binding to the
anionic membrane surface.
Examination of effects of mutations on ka and
kd provided a further insight into their roles. For
binding to POPC/POPS (7:3) vesicles, the N189A, R216A, and T251A
mutations changed kd more than
ka, whereas the R249A/R252A mutation primarily
reduced the ka value (see Table I). This indicates
that Arg216 and Thr251 are involved in specific
short-range interactions with a non-headgroup part of PS molecule and
that Arg249 and Arg252 participate in
nonspecific electrostatic interactions with the anionic membrane surface.
PC Selectivity of cPLA2-C2--
The C2 domain of
cPLA2 contains multiple aliphatic and aromatic residues,
including Phe35, Leu39, Tyr96, and
Val97, in its calcium binding loops (see Fig. 1) that have
been shown to partially penetrate the membrane during the membrane
binding of cPLA2-C2 (26). It has been also known that
cPLA2-C2 has PC selectivity (18). However, the origin of
this selectivity has yet to be elucidated. To identify the residues in
the cPLA2-C2 that are responsible for its PC selectivity,
we mutated aromatic and aliphatic residues in the Ca2+
binding loops and measured their membrane binding by SPR analysis. In
this case, we measured the affinities of wild type and mutants for
100% POPC and POPS vesicles. As listed in Table
II, cPLA2-C2 prefers POPC to
POPS by a factor of 11 in terms of Kd, confirming PC
selectivity of cPLA2-C2. The mutations of all four hydrophobic residues reduced the affinity of cPLA2-C2 for
PC membrane by more than two orders of magnitude. Furthermore, the
mutations of two aromatic residues (Phe35 and
Tyr96) affected ka and
kd to comparable degree, whereas those of two
aliphatic residues (Leu39 and Val97) primarily
increased kd. This again underscores the difference
between aliphatic and aromatic residues in their interfacial interactions. Most importantly, all mutants showed dramatically reduced
PC selectivity: their PC selectivity expressed in terms of the ratio of
(1/Kd) for POPC to (1/Kd) for POPS ranges from 0.6 to 1.1, indicating that all four residues in the
calcium binding loops are involved in PC selectivity.
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Table II
Binding parameters for cPLA2-C2 and mutants determined from SPR
analysis
Values represent the mean ± S.D. deviation from five
determinations. All measurements were performed in 10 mM
HEPES, pH 7.4, containing 0.1 M NaCl and 0.1 mM
Ca2+.
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Binding of cPLA2-C2 and PKC-
-C2 to Cell Membrane
Mimic Vesicles--
It has been shown that the C2 domains of
conventional PKCs (
,
I,
II, and
)
translocate to the plasma membrane (20, 37) and cPLA2-C2 to
the perinuclear region in response to Ca2+ import (21, 22).
Also, this subcellular localization pattern of isolated C2 domains
correlates with that of peripheral proteins harboring the C2 domains;
i.e. conventional PKCs translocate to plasma membrane
(38-41) while cPLA2 moves to perinuclear region (42, 43).
Although vesicles used in the preceding measurements serve well for
determining the lipid selectivity of the C2 domains and mutants, they
do not fully represent the cytoplasmic plasma membrane and the
cytoplasmic nuclear envelope that PKC-
-C2 and for
cPLA2-C2, respectively, are targeted to. To better
understand the subcellular targeting behaviors of the C2 domains, we
measured their binding to immobilized vesicles whose lipid compositions recapitulate those of cellular membranes.
The lipid compositions of the cytoplasmic plasma membrane and the
cytoplasmic nuclear envelope were predicted from the reported lipid
compositions of rat liver cell membranes (44) and by making a few
experimentally supported but simplifying assumptions. The assumptions
were: (i) that half of the lipid molecules are present in each leaflet;
(ii) that a large majority of phosphatidylethanolamine (80%),
phosphatidylinositol (80%), and PS (95%) are present in the
cytoplasmic leaflet of the plasma membrane, whereas a majority of PC
(70%) and sphingomyelin (>90%) are in the outer leaflet (45); and
(iii) that all phospholipids are equally distributed between the
cytoplasmic and the nucleoplasmic leaflets of nuclear membranes. This
simple approximation yielded POPC/POPE/POPS/POPI/cholesterol (12:35:22:9:12, molar ratio) as a cytoplasmic plasma membrane mimic and
POPC/POPE/POPS/POPI/cholesterol (61:21:4:7:7, molar ratio) as a
cytoplasmic nuclear membrane mimic, respectively. Table
III summarizes the parameters for binding
of the C2 domains and their respective mutants to these vesicles
determined from SPR analysis.
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Table III
Cell membrane mimic binding parameters for C2 domains and mutants
determined from SPR analysis
Values represent the mean ± S.D. from three determinations. All
measurements were performed in 10 mM HEPES, pH 7.4, containing 0.1 M KCl and 0.1 mM Ca2+.
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PKC-
-C2 showed about 30-fold higher affinity for the plasma membrane
mimic than for POPC/POPS (7:3) vesicles, despite lower PS content in
the former. This suggests that the presence of PE and/or cholesterol
enhances the membrane affinity of PKC-
-C2. Intriguingly, the
mutation of Asn189 that is directly involved in PS
headgroup interaction resulted in a large 34-fold drop in affinity,
whereas the double mutation of Arg249 and
Arg249 led to 5-fold decrease in affinity, which is
comparable to the data obtained with POPC/POPS (7:3) vesicles. Thus, it
would seem that specific PS coordination is critical for interaction of
PKC-
-C2 to the plasma membrane, which was not fully expressed when
binding assay was performed with POPC/POPS (7:3) vesicles. Most
importantly, PKC-
-C2 and R249A/R252A greatly (i.e.
121-fold and 114-fold, see Table III) preferred the plasma membrane
mimic to the nuclear envelope mimic, whereas N189A showed dramatically
reduced 10-fold selectivity for the plasma membrane mimic. This
suggests that N189A might be targeted to the nuclear envelope in
addition to the plasma membrane under certain physiological conditions.
cPLA2-C2 also showed higher (i.e. 5-fold)
affinity for the nuclear envelope mimic than for POPC vesicles. For the
nuclear envelope mimic, L39A, Y96A, and V97A had 100-fold, 310-fold,
and 43-fold lower affinity, respectively, than wild type. Importantly, cPLA2-C2 exhibited 33-fold selectivity for the nuclear
envelope mimic, whereas all mutants of cPLA2-C2, including
L39A, Y96A, and V97A, had much reduced 3- to 5-fold selectivity. This
again suggests that these mutants might be targeted to both nuclear envelope and plasma membrane in the cell. Lastly, affinity of PKC-
-C2 for the plasma membrane mimic is 4.5-fold higher than that
of cPLA2-C2 for the nuclear envelope mimic due in large
part to larger ka for PKC-
-C2. This suggests that
PKC-
-C2 might respond faster than cPLA2-C2 in response
to a rise in [Ca2+]i under the same conditions.
Subcellular Translocation of cPLA2-C2 and
PKC-
-C2--
It remains unknown whether specific subcellular
localization of C2 domains are due to their different lipid
specificities or the presence of specific adapter proteins for
individual proteins. To address this question, we transiently
transfected HEK293 cells with isolated C2 domains, mutants, full-length
cPLA2, and full-length PKC-
, all tagged with EGFP at
their carboxyl termini, and determined their spatiotemporal dynamics by
time-lapse confocal microscopy. In control experiments, all EGFP-tagged
proteins showed the membrane binding affinities comparable to those of
their non-tagged counterparts when assayed by SPR analysis under the
same conditions (data not shown). Also, transfected cells expressed the
corresponding proteins when tested by Western blotting analysis of
lysed cells (data not shown). As shown in Fig.
2, both cPLA2-C2-EGFP and
PKC-
-C2-EGFP are evenly dispersed in the cytoplasm when cells were
incubated in a Ca2+-depleted medium. When cells were
activated with 2 µM ionomycin that gave rise to ~0.5
µM [Ca2+]i, wild type C2 domains,
PKC-
-C2 in particular, showed rapid membrane translocation, but most
of the mutants with reduced membrane affinity did not migrate even
after 30 min (data not shown). We therefore activated the cells with 10 µM ionomycin and 1 mM CaCl2,
which resulted in sustained [Ca2+]i level at
~70 µM (Fig. 3). It
should be noted that, although this does not reflect a physiological
condition, it allowed quantitative determination of translocation rates
of wild type and mutant proteins under the same experimental
conditions. Upon calcium activation, PKC-
-C2-EGFP rapidly moved to
the plasma membrane, which was synchronized with the
[Ca2+]i change as observed by mag-indo 1 imaging
(Fig. 3). In contrast, cPLA2-C2 moved more slowly to the
perinuclear region that includes the nuclear envelope and the
endoplasmic reticulum, which is consistent with its lower affinity and
smaller ka than PKC-
-C2.

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Fig. 2.
Subcellular translocation of
PKC- -C2 and cPLA2- C2 upon
calcium activation. HEK293 cells treated with Mag-indo-1 were
washed with 2 mM EGTA and then overlaid with HEK buffer
for PKC- -C2 (A) or cPLA2-C2 (B).
Cells were then activated by adding 10 µM ionomycin and 1 mM calcium. Cell images were taken every 4 s for
PKC- -C2 and every 15 s for cPLA2-C2,
respectively.
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Fig. 3.
Change in [Ca2+]i and
subcellular translocation of PKC- -C2 and
cPLA2-C2 upon calcium activation. Experimental
conditions are the same as described for Fig. 2. A, the
time-lapse changes in EGFP intensity ratio at the plasma membrane (=
plasma membrane/[plasma membrane + cytoplasm]) for PKC- -C2 ( )
and R249A/R252A ( ) and the change in [Ca2+]i
in the cytoplasm ( ). B, the time-lapse changes in EGFP
intensity ratio at the perinuclear region (= nuclear membrane/[nuclear
membrane + cytoplasm]) for cPLA2-C2 ( and the change in
[Ca2+]i in the cytoplasm ( ).
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Also, full-length PKC-
and cPLA2 showed the same
subcellular targeting patterns as their respective C2 domains (Fig.
4). Furthermore, the time dependences of
their subcellular localization (data not shown) were reminiscent of
those of respective isolated C2 domains shown in Fig. 2. This suggests
that subcellular targeting of these proteins is driven by their C2
domains under the given conditions. To further verify this notion we
measured the subcellular targeting behaviors of chimera proteins. As
shown in Fig. 4, a chimera of PKC-
containing the C2 domain of
cPLA2 migrated to the perinuclear region, whereas a chimera
of cPLA2 containing the C2 domain of PKC-
translocated
to the plasma membrane in response to calcium influx. As was the case
with wild type PKC-
and cPLA2, the time dependences of
their subcellular localization (data not shown) were similar to those
of respective isolated C2 domains (Fig. 2).

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Fig. 4.
Subcellular translocation of
PKC- , cPLA2, and chimera
proteins. Experimental conditions are the same as described for
Fig. 2. Images represent cells expressing PKC-
(A), cPLA2 (B), a cPLA2
chimera harboring the PKC- -C2 (C), and a PKC- chimera
containing the cPLA2-C2 (D) before (first
column) and 10 min after (second column) the addition
of 10 µM ionomycin and 1 mM calcium. Membrane
translocation kinetic patterns of full-length proteins were similar to
those of the respective isolated C2 domains shown in Fig. 2.
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We then measured the subcellular localization of C2 domain
mutants. For PKC-
-C2, the R249A/R252A mutant translocated to the plasma membrane, whereas N189A with much reduced PS selectivity translocated to both plasma membrane and perinuclear region (Fig. 5). To demonstrate the dual targeting of
N189A, we quantitatively determined the cellular distribution of EGFP
intensity after Ca2+ stimulation (Fig.
6). Clearly, PKC-
-C2 is primarily
localized to the plasma membrane, while N189A is localized both at the
plasma membrane and at the perinuclear region, albeit more abundantly at the plasma membrane. This supports the notion that specific PS
coordination by Asn189 plays a key role in the targeting of
PKC-
-C2 to the plasma membrane. As was the case with PKC-
-C2, the
cPLA2-C2 mutants showed expected cellular behaviors. In
this case, all mutants showed dual targeting to the plasma membrane and
the perinuclear region (see Fig. 5). Fig. 6 demonstrates the difference
in cellular EGFP intensity distribution of cPLA2-C2 and
Y96A upon Ca2+ stimulation.

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Fig. 5.
Subcellular localization of
PKC- -C2, cPLA2-C2, and respective
mutants upon calcium activation. A, PKC- -C2;
B, PKC- -C2 N189A; C, cPLA2-C2;
D, cPLA2-C2 Y96A; and E,
cPLA2-C2 V97A. Experimental conditions are the same as
described for Fig. 2. Images represent the subcellular
localization patterns of indicated C2 domains before (first
column) and 10 min after (second column) the addition
of 10 µM ionomycin and 1 mM calcium. An
arbitrary line was drawn through each cell to determine the EGFP
intensity in each part of the cell. See Fig. 6 for EGFP intensity
quantified in each cell.
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Fig. 6.
Subcellular distribution of EGFP intensity
after calcium activation of PKC- -C2,
cPLA2-C2, and respective mutants. An EGFP intensity
profile was obtained for each cell as described under "Experimental
Procedures" (see also Fig. 5). The differential subcellular
localization is shown for PKC- -C2 (A), PKC- -C2/N189A
(B), cPLA2-C2 (C), and
cPLA2-C2/Y96A (D). PM and NE indicate plasma
membrane and nuclear envelope, respectively.
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Lastly, we determined the membrane translocation rates for the
proteins. Those mutants showing dual membrane targeting were excluded
in this analysis because of ambiguity involved in determining their
membrane targeting rates. As shown in Fig.
7, the R249A/R252A mutant of PKC-
-C2
translocated to the plasma membrane 4.4-fold slower than the wild type,
which is consistent with its 5-fold lower affinity (and 6-fold smaller
ka) for the plasma membrane mimic than the wild
type. Furthermore, cPLA2-C2 migrated to the perinuclear
region about 7 times slower than PKC-
-C2 translocated to the plasma
membrane, which is in reasonable agreement with the finding that the
former has 4.5-fold lower affinity (and 3.7-fold smaller
ka) for the nuclear envelope mimic than the latter has for the plasma membrane mimic. In conjunction with the
altered in vitro lipid selectivity of
cPLA2-C2 and PKC-
-C2 mutants described above, these
results support the notion that subcellular targeting by C2
domains is driven by the forces that govern their in
vitro membrane binding.

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Fig. 7.
Membrane translocation rates for C2 domains
and respective mutants. Relative translocation rates calculated
from the slopes of the EGFP intensity ratio (membrane/[membrane + cytosol]) versus time plots. Rate values indicate means and
standard deviations from a minimum of quadruple determinations.
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DISCUSSION |
The membrane binding of proteins involves different types of
interactions that depend upon the physicochemical properties of both
membrane and protein. Extensive structural and mutational studies of
membrane binding proteins have shown that their membrane binding
surfaces are composed of cationic, aliphatic, and aromatic residues
that drive the membrane binding by electrostatic and non-electrostatic
forces (46). Our recent study by surface plasmon resonance analysis
indicated that cationic residues primarily accelerate the association
of protein to anionic membrane surfaces, whereas aliphatic residues
mainly slow the membrane dissociation by penetrating into the
hydrophobic core of the membrane (29). Aromatic residues, particularly
Trp, play a pivotal role in binding to zwitterionic PC membranes (46,
47) by affecting both membrane association and dissociation steps (29).
The present study systematically and quantitatively addresses the
question as to whether or not these physicochemical principles
governing the in vitro membrane binding of membrane
targeting domains also determine their subcellular targeting behaviors,
using two C2 domains as a model.
Lipid Selectivity of cPLA2-C2 and PKC-
-C2--
A
large degree of structural variations have been found in the
Ca2+ binding loops of C2 domains in terms of both primary
and tertiary structures (8-14,48). Mutational (17, 19, 23, 24, 26, 28), labeling (49, 50), and structural (12-14) studies of C2 domains
have identified the residues in the Ca2+ binding loops that
play a key role in membrane binding. The present study shows that
cationic residues, Arg249 and Arg252, in
Ca2+ binding loops of PKC-
-C2 are involved in its
binding to the anionic membrane surface, whereas Asn189
plays a critical role in PS selectivity. Arg216 and
Thr251 are also involved in PS binding but not specifically
to the lipid headgroup. These results appear to be at odds with the
report by Conesa-Zamora et al. (24) in which
Thr251 was shown to play a much more important role than
Asn189 in binding to PS-containing membranes as well as in
PS-dependent PKC activity. It should be noted, however,
that the study by Conesa-Zamora et al. (24) was performed
only in the presence of PS-containing membranes: no comparison was made
between PS and non-PS membranes. As shown in Table I, we also observed
that T251A had the largest impact on the affinity for POPC/POPS (7:3)
vesicles. Importantly, however, both T251A and R216A mutations reduced
the affinity for POPC/POPS (7:3) and POPC/POPG (7:3) vesicles to
comparable degrees. Only the N189A mutation selectively reduced the
affinity for PS. This is consistent with the structure of a
PKC-
-C2·PS complex, which shows that Arg216 and
Thr251 primarily interact with sn-1 and
sn-2 ester carbonyl moieties, respectively, whereas
Asn189 interacts directly with the serine headgroup (12).
Also, a smaller effect of the N189A mutation on PS membrane affinity in the study by Conesa-Zamora et al. (24) might be due in part to the fact that not isolated C2 domains but full-length PKC-
proteins were used. Presumably, the rest of the PKC-
molecule, including the C1 domains, can compensate for the reduced PS affinity caused by the N189A mutation. In the case of cPLA2-C2, both
aliphatic and aromatic residues in Ca2+ binding loops are
essential for its membrane binding and PC selectivity. Aromatic side
chains have been shown to be involved in PC interactions (46, 47);
however, the involvement of aliphatic side chains in PC selectivity has
not been reported. Further studies are required to understand the
origin of this effect.
Subcellular Targeting of cPLA2-C2 and
PKC-
-C2--
It has been known that conventional PKCs and their C2
domains, including PKC-
and its C2 domain (37), PKC-
(41), and PKC-
-C2 (20), translocate to plasma membrane in response to [Ca2+]i spikes, whereas cPLA2-C2 and
cPLA2 are targeted to the perinuclear region upon
Ca2+ import (21, 22, 43). However, the origin of these
differential subcellular targeting behaviors of PKC C2 domains,
cPLA2-C2, and their host proteins is relatively poorly
understood. Intuitively, one can assume the presence of site-specific
adaptor proteins for these proteins (51, 52). The present study
provides quantitative and structural evidence for an alternative view
that holds that differential subcellular targeting behaviors of the C2
domains derive mainly from their different lipid selectivity.
It has been reported that mammalian cell membranes have different lipid
compositions (44). In particular, the cytoplasmic plasma membrane
contains a high PS content (44, 45, 53, 54), whereas the nuclear
membrane is relatively rich in PC (44, 55). In this study, the
cytoplasmic leaflets of plasma membrane and nuclear envelope were
modeled based on the reported lipid compositions of rat liver cell
membranes (44). Although the lipid compositions of these vesicles might
not reflect the true lipid compositions of cytoplasmic plasma membrane
and nuclear envelope of HEK293 cells, they should serve as good models,
because kidney and liver cell membranes have similar lipid compositions (44). Consistent with their distinct lipid selectivity, PKC-
-C2 and
cPLA2-C2 demonstrate pronounced specificity for the plasma membrane and nuclear envelope mimics, respectively (121-fold and 33-fold preference, respectively, for their favorite membranes). Agreement between this strict in vitro specificity and
highly specific subcellular localization of the C2 domains suggests
that their subcellular targeting is driven primarily by
membrane-protein interactions. This notion is further supported by the
good correlation between altered lipid specificities of PKC-
-C2 and
cPLA2-C2 mutants and their subcellular localization. N189A
of PKC-
-C2 with markedly reduced selectivity for the plasma membrane
mimic showed dual targeting to the plasma membrane and the perinuclear
region. Likewise, cPLA2-C2 mutants with lower selectivity
for the nuclear envelope mimic exhibited the dual targeting behaviors.
Similarly, it was shown that the subcellular targeting patterns of the
C2 domains of phospholipase C-
(17) and 5-lipoxygenase (19) can be
altered by changing their in vitro lipid selectivity.
Lastly, for PKC-
-C2, its R249A/R252A mutant, and
cPLA2-C2 that translocate to a single cellular destination,
the relative affinity (100:20:22, see Table III) for their favorite
membrane mimics agrees reasonably well with their relative membrane
translocation rate (100:25:15, see Fig. 7).
In sum, these studies indicate that the specific subcellular targeting
of PKC-
-C2 and cPLA2-C2 derives from their phospholipid headgroup specificities and that one can control the subcellular localization of a C2 domain by altering its lipid selectivity. The
studies also demonstrate the semi-quantitative correlation between the
in vitro membrane binding of C2 domains and their cellular
membrane targeting. A more detailed study is underway to delineate the
quantitative correlation between in vitro membrane binding
parameters of C2 domains and their cellular responses. Further studies
on other membrane targeting domains will answer the question as to
whether or not their subcellular targeting could be also explained by
biophysical principles that govern their membrane binding.