From the Departments of Chemistry and
§ Medicinal Chemistry and Pharmacognosy, University of
Illinois at Chicago, Chicago, Illinois 60607 and the ¶ Department
of Microbiology and Immunology, Weill Medical College of Cornell
University, New York, New York 10021
Received for publication, December 10, 2002, and in revised form, January 9, 2003
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ABSTRACT |
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Phox (PX) domains are phosphoinositide
(PI)-binding domains with broad PI specificity. Two cytosolic
components of NADPH oxidase, p40phox and p47phox,
contain PX domains. The PX domain of p40phox specifically binds
phosphatidylinositol 3-phosphate, whereas the PX domain of
p47phox has two lipid binding sites, one specific for
phosphatidylinositol 3,4-bisphosphate and the other with affinity for
phosphatidic acid or phosphatidylserine. To delineate the mechanisms by
which these PX domains interact with PI-containing membranes, we
measured the membrane binding of these domains and respective mutants
by surface plasmon resonance and monolayer techniques and also
calculated the electrostatic potentials of the domains as a function of
PI binding. Results indicate that membrane binding of both PX domains is initiated by nonspecific electrostatic interactions, which is
followed by the membrane penetration of hydrophobic residues. The
membrane penetration of the p40phox PX domain is induced by
phosphatidylinositol 3-phosphate, whereas that of the p47phox
PX domain is triggered by both phosphatidylinositol 3,4-bisphosphate and phosphatidic acid (or phosphatidylserine). Studies of enhanced green fluorescent protein-fused PX domains in HEK293 cells indicate that this specific membrane penetration is also important for subcellular localization of the two PX domains. Further studies on the
full-length p40phox and p47phox proteins showed that an
intramolecular interaction between the C-terminal Src homology 3 domain
and the PX domain prevents the nonspecific monolayer penetration of
p47phox, whereas such an interaction is absent in
p40phox.
Phosphoinositides (PI)1
play pivotal roles in cell signaling and membrane trafficking by
serving as site-specific membrane signals to modulate the intracellular
localization and/or biological properties of effector proteins. Since
the discovery of the pleckstrin homology (PH) domain as an effector for
PIs, many PI-binding domains, including FYVE (Fablp,
YOTB, Vac1p, and EEA1), ENTH
(epsin N-terminal homology), FERM (four-point one,
ezrin, radixin, moesin), and PX
(Phox) domains, with different PI specificities
and affinities have been identified. The PX domain was initially
identified as a conserved structural module of about 130 amino acids in
the p40phox and p47phox subunits of the NADPH oxidase
complex (1). The PX domain has been since found in over 100 eukaryotic
proteins (2-6). As is the case with the PH domain, PX domains show
broad PI specificity. Whereas many PX domains, including those from
p40phox (7, 8), Vam7p (9), and sorting nexin 3 (10), have been
found to be specific for phosphatidylinositol 3-phosphate (PtdIns(3)P),
PX domains from p47phox (11, 12), yeast Bem1p (13), and Class
II PI 3-kinase (14) preferentially bind to phosphatidylinositol
3,4-bisphosphate (PtdIns(3,4)P2), phosphatidylinositol-4-phosphate, and
phosphatidylinositol-4,5-bisphosphate, respectively.
NADPH oxidase, which is activated upon phagocytosis of foreign
particles, plays an instrumental role in the host defense mechanism of
neutrophils and macrophages. NADPH oxidase is a multisubunit protein
complex consisting of cytosolic components p40phox,
p47phox, p67phox, and Rac as well as a membrane-bound
heterodimer of p22phox and gp91phox. When invasive
microorganisms are sequestered, the cytosolic subunits translocate to
the membrane surface and fuse with the membrane-bound subunits,
creating the activated complex (15). Activated NADPH oxidase then
produces superoxide, which in turn generates microbicidal reactive
oxygen species in phagocytic vesicles. Chronic granulomatous disease,
which confers severe susceptibility to infection, is due to naturally
occurring mutations in the NADPH oxidase complex, including some in the
p47phox subunit. p47phox is a prerequisite for assembly
of the entire complex (16, 17), and the p40phox subunit is
required for subunit translocation and proper functioning of the active
complex (18). Both p40phox and p47phox contain PX
domains. The recently determined x-ray crystal structures of these PX
domains (7, 12) have revealed the basis of the stereospecific
recognition of their cognate PIs (i.e. PtdIns(3)P for
p40phox-PX and PtdIns(3,4)P2 for
p47phox-PX). However, the PI-dependent
membrane-binding mechanisms of these PX domains as well as their roles
in NADPH oxidase activation are relatively poorly understood. Evidence
indicates that these PX domains have distinct membrane binding
properties. For instance, the p47phox-PX has a unique secondary
binding site that nonspecifically binds anionic phospholipids,
including phosphatidic acid (PtdOH) and phosphatidylserine (PtdSer).
Furthermore, the p40phox-PX was shown to be localized at
PtdIns(3)P-rich endosomes when expressed ectopically in mammalian cells
(7, 19), whereas the isolated p47phox-PX exhibited cytoplasmic
distribution under similar conditions (19).
This study was undertaken to determine the origin of differential
membrane targeting mechanisms of p40phox-PX and
p47phox-PX, with an emphasis on elucidating the roles of
PtdIns(3)P and PtdIns(3,4)P2 in their membrane binding.
Results from in vitro membrane binding measurements by
monolayer and surface plasmon resonance (SPR) analyses, electrostatic
potential computation, and subcellular localization measurements using
enhanced green fluorescent protein (EGFP)-tagged domains provide new
insight into how differently PtdIns(3)P and PtdIns(3,4)P2
mediate the membrane targeting of p40phox-PX and
p47phox-PX, respectively, which in turn provide an important
clue to the understanding of the membrane recruitment mechanisms of
p40phox and p47phox subunits of NADPH oxidase.
Materials--
1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphate
(POPA), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine
(POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine (POPS), and
1-palmitoyl-2-oleoyl-sn-glycero-phosphoethanolamine (POPE)
were from Avanti Polar Lipids (Alabaster, AL). 1,2-Dipalmitoyl derivatives of PtdIns(3)P, phosphatidylinositol-5-phosphate,
PtdIns(3,4)P2, and phosphatidylinositol-4,5-bisphosphate
were synthesized as described elsewhere (20). These derivatives
were selected over naturally occurring PIs containing
cis-unsaturated sn-2 acyl groups due to
difficulties involved in the chemical synthesis of the latter. Since
the concentration of PIs used in this study was limited to 3 mol %,
the effect of their sn-2 acyl chains on the membrane
structure would not be significant. Phospholipid concentrations were
determined by phosphate analysis. The Liposofast microextruder and
100-nm polycarbonate filters were from Avestin (Ottawa, Canada). Fatty
acid-free bovine serum albumin was from Bayer, Inc. (Kankakee, IL).
Restriction endonucleases and other enzymes for molecular biology were
from New England Biolabs (Beverly, MA).
(3-[3-Cholamidopropyl)dimethylammonio]-1-propane sulfonate and octyl
glucoside were from Sigma and Fisher, respectively. The Pioneer L1
sensor chip was from Biacore AB (Piscataway, NJ).
Mutagenesis and Protein Expression--
Mutations of
p40phox-PX were performed by the overlap extension PCR method
(21). Each construct was subcloned into the pET21a vector containing a
C-terminal His6 tag and transformed into DH5 EGFP Constructs--
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. p40phox-PX
was cloned by PCR to remove the stop codon and add two C-terminal glycines and an EcoRI site. The PCR product was digested
with NotI and EcoRI and ligated into pIND/EGFP to
generate a C-terminal EGFP-fused p40phox-PX with spacer
sequence GGNSGG. EGFP-fused constructs of p40phox-PX mutants,
p47phox-PX, and p47phox-PX mutants were generated by
the same method.
Monolayer Measurements--
The penetration of
p40phox-PX and p47phox-PX into the phospholipid
monolayer was measured by monitoring the change in surface pressure ( Kinetic and Equilibrium SPR Experiments--
All SPR
measurements were performed at 23 °C. A detailed protocol for
coating the L1 sensor chip has been described elsewhere (27, 28).
Briefly, after washing the sensor chip surface, 90 µl of vesicles
containing various phospholipids (see Table I) were injected at
5 µl/min to give a response of 4000 resonance units. Similarly, a
control surface was coated with vesicles, typically without the PI of
interest, to give the same resonance unit response as the active
binding surface. Under our experimental conditions, no binding was
detected to this control surface beyond the refractive index change for
the PX domains. Each lipid layer was stabilized by injecting 10 µl of
50 mM NaOH three times at 100 µl/min. Typically, no
decrease in lipid signal was seen after the first injection. Kinetic
SPR measurements were done at the flow rate of 60 µl/min. 90 µl of
protein in 10 mM HEPES, pH 7.4, containing 0.1 M KCl was injected to give an association time of 90 s, whereas the dissociation was monitored for 400 s or more. The
lipid surface was regenerated using 10 µl of 50 mM NaOH.
After sensorgrams were obtained for five different concentrations of each protein within a 10-fold range of Kd, each of
the sensorgrams was corrected for refractive index change by
subtracting the control surface response from it. The association and
dissociation phases of all sensorgrams were globally fit to a 1:1
Langmuir binding model: protein + (protein binding site on vesicle) Electrostatic Potential Computation--
The electrostatic
properties of the PX domain with and without PIs were calculated with a
modified version of the program DelPhi and visualized in the program
GRASP (33), as described previously (34). In the
panels of Fig. 6, the electrostatic potentials are
represented by two-dimensional equipotential contours in 0.1 M KCl: magenta, 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
supplemented with 10% fetal bovine serum 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. For transfection, 80-90% confluent
cells in Lab-TechTM chambered cover glass wells were
exposed to 150 µl of unsupplemented Dulbecco's modified Eagle's
medium 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, the
cells were washed once with fetal bovine serum-supplemented Dulbecco's
modified Eagle's medium and overlaid with fetal bovine
serum-supplemented Dulbecco's modified Eagle's medium containing
ZeocinTM (Invitrogen) and 2 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. EGFP was
excited using the 488-nm line of an argon/krypton laser. A linepass
505-nm filter was used on channel 1 for all experiments. A ×63, 1.2 numerical aperture water immersion objective was used for all
experiments. Immediately before imaging, the induction medium was
removed, and the cells were washed with 150 µl of 1 mM
HEPES buffer, pH 7.4, containing 2.5 mM MgCl2,
1 mM NaCl, 0.6 mM KCl, 0.67 mM
D-glucose, 6.4 mM sucrose and then overlaid
with the same buffer. For p47phox-PX translocation
measurements, PtdIns(3,4)P2 was delivered to HEK293 cells
by the lipid-shuttle method using histone or neomycin as a lipid
carrier as described (36). Briefly, PtdIns(3,4)P2 (20 µM) and histone (or neomycin) (20 µM) were
incubated together for 10 min at 25 °C and then sonicated for 2 min
in a water bath sonicator. Translocation was initiated by treating
cells with the lipid-shuttle complex, and the translocation of
p47phox-PX was monitored by scanning cells every 5-10 s.
Treatment of cells with histone or PtdIns(3,4)P2 alone did
not induce translocation of p47phox-PX after 20 min. In control
experiments, cells were treated with 100 nM wortmannin for
30 min to determine the effects of PI 3-kinase on PX domain subcellular localization.
Effects of Phosphoinositides on the Monolayer Penetration of PX
Domains--
Despite the availability of crystal (7, 12) and
solution (37, 38) structures of several PX domains, exact
roles of PIs in the membrane binding of PX domains have yet to be
elucidated. Recently, it was shown that binding of PtdIns(3)P to the
FYVE domains of Vps27p and Hrs induces their membrane penetration by causing changes in their local conformations and electrostatic potentials (30). To determine whether or not PIs play similar roles
in the membrane binding of p40phox-PX and p47phox-PX,
we measured the interactions of the PX domains with lipid monolayers of
different compositions.
We first measured the effects of PtdIns(3)P on the monolayer
penetration of the p40phox-PX. Specifically, a POPC/POPE
(80:20) or a POPC/POPE/PtdIns(3)P (77:20:3) monolayer of a given
Our recent study showed that the p47phox-PX has a
PtdIns(3,4)P2-specific site as well as a secondary site
that nonspecifically bind anionic phospholipids with smaller
headgroups, including PtdOH and PtdSer (12). This would suggest that
membrane penetration of the p47phox-PX might be influenced by
anionic phospholipids other than PtdIns(3,4)P2. To test
this notion, we measured the penetration of the p47phox-PX into
monolayers of various lipid compositions. We first measured the
monolayer penetration of p47phox-PX to a POPC/POPE (80:20)
monolayer. When compared with the p40phox-PX, the
p47phox-PX showed a higher degree of penetration into the
POPC/POPE (8:2) monolayer (i.e. Effects of Mutations on the Monolayer Penetration of PX
Domains--
To better understand the mechanisms by which PIs and
other anionic phospholipids induce the membrane penetration of
p40phox-PX and p47phox-PX, we measured the monolayer
penetration of selected mutants of the two PX domains. For
p40phox-PX, we mutated arginine residues (Arg58,
Arg60, and Arg105) essential for specific
recognition of PtdIns(3)P (7) and hydrophobic residues
(Phe35, Tyr94, and Val95) in the
loop regions surrounding the PtdIns(3)P binding pocket. All three
arginine mutants (R58A, R60A, and R105A) exhibited reduced penetration
into the POPC/POPE/PtdIns(3)P (77:20:3) monolayer (Fig.
2A), supporting the notion
that PtdIns(3)P binding specifically induces the membrane penetration
of p40phox-PX. Likewise, F35A, Y94A, and V95A had low monolayer
penetration both with (Fig. 2B) and without (data not shown)
PtdIns(3)P in the POPC/POPE monolayer, confirming the notion that these
loop regions are involved in PtdIns(3)P-induced membrane penetration. In contrast, the mutation of Leu82 located in the other end
of the membrane binding surface (see Fig. 8) had little effect on the
monolayer penetration, showing that this part of p40phox-PX
does not participate in membrane penetration (Fig. 2B).
For p47phox-PX, we prepared two sets of mutants in two separate
lipid binding sites (see Fig. 8). First, two Arg residues
(Arg43 and Arg90) involved in
PtdIns(3,4)P2 binding (12) and Trp80 located in
an adjacent loop were mutated to Ala. Also mutated were
Arg70, which is a part of the secondary binding pocket
(12), and Ile65, located in an adjacent loop. Fig.
3A shows that mutations of PtdIns(3,4)P2 binding residues have direct impact on the
PtdIns(3,4)P2-induced monolayer penetration of
p47phox-PX. In contrast, the mutation of Arg70 had
a negligible effect under the same condition. Also, the mutation of
Trp80 had a greater effect on the penetration of
p47phox-PX into the POPC/POPE/PtdIns(3,4)P2
(77:20:3) monolayer than the mutation of Ile65 (Fig.
3B). These results thus suggest that
PtdIns(3,4)P2 binding induces the membrane penetration of
the region containing Trp80 and that the secondary lipid
binding site does not significantly contribute to this event. We then
measured the effects of mutations on the penetration of the
p47phox-PX into non-PtdIns(3,4)P2-containing
monolayers. As shown in Fig. 3C, I65A and R70Q mutations
exerted much greater effects on the penetration of p47phox-PX
into the POPC/POPE/POPS (50:20:30) monolayer than did W80A and R90A
mutations. Similar data were obtained when the POPC/POPE/POPA (77:20:3)
monolayer was used (data not shown). This indicates that the binding of
an anionic phospholipid to the secondary site induces the membrane
penetration of the region surrounding the site, which is independent of
the PtdIns(3,4)P2-induced membrane penetration of the
region surrounding the PtdIns(3,4)P2-binding site. Last, we
measured the penetration of p47phox-PX mutants into the
POPC/POPE (70:30) monolayer to determine the origin of high intrinsic
monolayer penetrating power of the p47phox-PX (Fig.
3D). Interestingly, only I65A exhibits significantly reduced
monolayer penetration, which is comparable with the penetration of the
p40phox-PX into the POPC/POPE (70:30) monolayer. This indicates
that Ile65 is responsible for the high intrinsic monolayer
penetrating activity of the p47phox-PX, which is also
consistent with the above finding that the I65A mutation significantly
impairs the monolayer penetration of p47phox-PX regardless of
the lipid composition of monolayers.
Monolayer Penetration of Full-length p40phox and
p47phox--
Recently, it was shown that the C-terminal Src
homology 3 (SH3) domain of p47phox interacts with the
PXXP motif within the p47phox PX domain to block the
PtdIns(3,4)P2-dependent membrane binding of
p47phox (12). It was also shown that the phosphorylation of
p47phox on up to nine sites in the C-terminal tail, including
Ser303, Ser304, Ser328,
Ser359, and Ser370, untethers the SH3-PX
interaction to facilitate the
PtdIns(3,4)P2-dependent membrane binding of
p47phox (12). To determine the effects of the SH3-PX
interaction and the phosphorylation on the membrane penetration of
p47phox-PX, we first measured the monolayer penetration of the
full-length p47phox. As illustrated in Fig.
4A, the full-length protein
showed greatly reduced monolayer penetration (
It has been shown that the SH3 domain of p40phox does not
interfere with soluble PtdIns(3)P binding (7); however, it is not known
whether or not the SH3 domain hinders membrane binding of the
p40phox-PX. To answer this question, we measured the monolayer
penetration of full-length p40phox. As shown in Fig.
4B, the full-length p40phox penetrated the
POPC/POPE/PtdIns(3)P (77:20:2) monolayer as well as the
p40phox-PX, indicating that the SH3 domain of p40phox
does not interfere with the membrane binding of its PX domain.
SPR Membrane Binding Analysis of PX Domains--
We previously
reported that PtdIns(3,4)P2 enhances the membrane affinity
(Kd) of p47phox-PX mainly by reducing the
membrane dissociation rate constant (kd), whereas
PtdOH or PtdSer increases the membrane affinity by affecting both
ka and kd (12). To determine the
effects of PtdIns(3)P on membrane binding of p40phox-PX, we
varied the concentration of PtdIns(3)P in POPC/POPE/PtdIns(3)P (80
In the absence of PtdIns(3)P, the p40phox-PX had extremely low
affinity for POPC/POPE (80:20) vesicles (Kd > 50 µM). This is consistent with our finding that the
p40phox-PX has extremely low affinity for POPC/POPE (80:20)
monolayer above 30 dyne/cm, which represents the surface pressure value of the large vesicles used in the SPR measurements (42). As summarized
in Table I, PtdIns(3)P greatly enhanced the membrane affinity of
p40phox PX; an increase in PtdIns(3)P concentration from 0.2 to
5 mol % resulted in a >700-fold decrease in Kd. As
was the case with the p47phox-PX, the enhanced affinity by
PtdIns(3)P was mainly attributed to reduced kd;
ka increased only 1.6-fold, whereas kd decreased 112-fold when the PtdIns(3)P
concentration was varied from 0.2 to 5 mol % in the vesicles. This
finding is also consistent with our monolayer data indicating that
PtdIns(3)P facilitates the membrane penetration of
p40phox-PX, thereby allowing the domain to attain a
longer membrane residence time.
The effects of mutations on binding of p40phox-PX to
POPC/POPE/PtdIns(3)P (77:20:3) vesicles are also summarized in Table I. Mutation of Arg58, which was shown to be essential for
interaction with the 3-phosphate group (7), caused the most dramatic
177-fold reduction in affinity, which was attributed to a reduction in
ka and an increase in kd. Also,
mutation of Arg105 that forms hydrogen bonds with the 4- and 5-hydroxyl groups (7) reduced membrane binding by 35-fold, and this
reduced affinity was due to a reduced ka and an
increased kd. Similarly, mutation of
Arg60, which interacts with the 1-phosphate of PtdIns(3)P
(7), resulted in an 18-fold decrease in Kd by
changing both ka and kd.
Since PtdIns(3)P increases the membrane affinity of the
p40phox-PX by decreasing kd (see above),
these results suggest that the basic residues in the binding pocket
might be involved in more than PtdIns(3)P coordination (see below). On
the other hand, mutations of hydrophobic residues in the loop regions
(F35A, Y94A, and V95A) reduced membrane affinity of p40phox-PX
by 33-, 27-, and 5.9-fold, respectively, which was primarily due to
increases in kd. This is consistent with our
monolayer data indicating that these residues are involved in membrane
penetration. Last, L82A behaved like wild type, confirming that the
region near this hydrophobic residue is not directly involved in
membrane interaction.
Calculation of the Electrostatic Potential--
Our monolayer data
demonstrate distinct membrane binding properties of the two PX domains
(i.e. PtdIns(3)P specifically triggers the monolayer
penetration of p40phox-PX, whereas PtdIns(3,4)P2
and other anionic lipids can induce the monolayer penetration of
p47phox-PX). Furthermore, the p47phox-PX has higher
intrinsic monolayer penetration activity than p40phox-PX in the
absence of PtdIns(3,4)P2 and/or other anionic lipids. To
account for these differences, we calculated the electrostatic potentials for p40phox-PX and p47phox-PX in the absence
and presence of PtdIns(3)P (for p40phox-PX) and of
PtdIns(3,4)P2 and PtdSer (for p47phox-PX). The
results are illustrated in Fig. 6. In the
absence of PtdIns(3)P, the loop regions surrounding the PtdIns(3)P
binding pocket of p40phox-PX that interact with the membrane
have strong positive electrostatic potential (see Fig. 6A).
This positive potential is largely due to basic residues in the
PtdIns(3)P binding pocket (i.e. Arg58,
Arg60, and Arg105) and may initially contribute
to recruiting the domain to the anionic membrane surface through
nonspecific electrostatic attraction, which then leads to productive
PtdIns(3)P binding. This is consistent with the finding that mutations
of the basic residues in the PtdIns(3)P binding pocket reduce
ka (due to reduced nonspecific electrostatic
interactions) as well as increase kd (due to lower
PtdIns(3)P binding) (Table I). However, this region of high positive
potential also surrounds the hydrophobic residues (i.e.
Phe35, Tyr94, and Val95) and is
thus expected to produce an energetic barrier opposing the penetration
of these residues into the low dielectric membrane interface. This is
because the desolvation that is prerequisite for their membrane
penetration would disrupt favorable interactions between water
molecules and charged and polar groups on both the protein and
membrane. Interestingly, the positive potential is dramatically reduced
when PtdIns(3)P binds to the domain (see Fig. 6B). Fig.
6C shows quantitatively that the electrostatic potential surrounding the hydrophobic residues are significantly less
positive when PtdIns(3)P is bound. This suggests that PtdIns(3)P may function as an electrostatic switch to decrease the highly positive
potential surrounding the hydrophobic residues, thereby facilitating their membrane penetration.
Our electrostatic potential calculations on the p47phox-PX also
provide insight into the origin of the unique membrane binding properties of p47phox-PX. As shown in Fig. 6D, the
tip of the Ile65 side chain is in a region of very low
electrostatic potential even in the absence of
PtdIns(3,4)P2 and PtdSer, which explains how this
hydrophobic residue can penetrate the POPC/POPE (80:20) monolayer to
some extent. In agreement with our monolayer penetration data,
PtdIns(3,4)P2 reduces the electrostatic potential of the loop region containing Trp80 (Fig. 6E), which
would cause favorable partitioning of this region into the monolayer
due to reduced desolvation penalty associated with the process.
Calculations with PtdSer (Fig. 6H) also show that the
electrostatic potential of the secondary lipid binding pocket is
reduced when PtdSer binds to this site, which is consistent with the
ability of PtdSer to promote the monolayer penetration of
p47phox-PX. Last, we calculated the reduction in electrostatic
potential of p47phox-PX in the presence of both
PtdIns(3,4)P2 and PtdSer. As shown in Fig. 6K,
simultaneous docking of both ligands reduces the potential of
p47phox-PX most dramatically, consistent with the reported
synergistic effect of the two ligands.
Subcellular Localization of PX Domains--
It has been shown that
the p40phox-PX is localized to PtdIns(3)P-rich endosomes in
resting porcine aortic endothelial cells (7) and COS cells (19),
whereas the p47phox-PX is dispersed in the cytosol of COS cells
(19). To assess the importance of membrane binding residues of
p40phox-PX and p47phox-PX in their cellular membrane
targeting, we monitored the subcellular localization of EGFP-tagged
constructs of p40phox-PX, p47phox-PX, and respective
mutants in HEK293 cells. In agreement with previous reports, the
p40phox-PX showed distinct endosomal localization pattern (see
Fig. 7A). The prelocalization
of p40phox-PX at endosomal membranes was a
PtdIns(3)P-dependent process, since treatment of the cells
with PI 3-kinase inhibitor wortmannin (100 nM) resulted in
a homogenous distribution of the protein in the cytosol (Fig.
7B). This notion is further supported by the finding that
R58Q with greatly reduced PtdIns(3)P affinity exhibited a homogenous
cytosolic distribution (Fig. 7C) under the conditions in
which the wild type showed the endosomal localization. Furthermore,
mutations of loop residues (F35A and Y94A) also caused cytoplasmic
distribution of proteins in HEK293 cells (Fig. 7, D and
E), underscoring the physiological significance of the
membrane penetration of these residues in subcellular targeting of the p40phox-PX domain.
In contrast to p40phox-PX, the p47phox-PX was evenly
distributed in the cytosol of HEK293 cells (Fig. 7F). It was
reported that the p47phox-PX could be localized at membrane
ruffles when COS cells were stimulated with insulin-like growth
factor-1 (19). In HEK293 cells, however, insulin-like growth factor-1
failed to induce the membrane translocation of the p47phox-PX.
When HEK293 cells were fed with 20 µM
PtdIns(3,4)P2-histone complex, the p47phox-PX
translocated to the plasma membrane in less than 1 min (Fig. 7G). This translocation was a
PtdIns(3,4)P2-specific process, since R90A did not migrate
to the plasma membrane even after 20 min under the same conditions
(Fig. 7H). Interestingly, R70Q with reduced affinity for
PtdOH or PtdSer did not translocate to the plasma membrane after 20 min
(Fig. 7I), suggesting that binding of PtdSer that is
abundant in the inner plasma membrane to the secondary site binding
also contributes significantly to the cellular membrane targeting of
p47phox-PX. Last, neither W80A (Fig. 7J) nor I65A
(data not shown) showed translocation under these conditions,
indicating that membrane penetration of these residues is also
important for the subcellular targeting of the p47phox-PX domain.
The recent finding that PX domains interact with PtdIns(3)P and
other PIs suggests a new mechanism by which PIs can regulate multiple
cellular processes via a spectrum of PX domain-containing peripheral
proteins. The first crystal structure of p40phox-PX liganded
with a water-soluble PtdIns(3)P illustrated how the domain achieves the
stereospecific recognition of PtdIns(3)P (7). More recently, the
crystal structure of p47phox-PX revealed the presence of two
lipid binding pockets, one specific for PtdIns(3,4)P2 and
the other with high affinity for anionic phospholipids with smaller
headgroups, such as PtdOH (12). Despite this structural information,
less is known as to how various PIs affect the in vitro and
cellular membrane targeting of different PX domains.
Our previous study on the FYVE domains indicated that PtdIns(3)P
binding specifically induces the membrane penetration of surface
hydrophobic residues of FYVE domains, presumably by causing local
conformational changes of proteins and by neutralizing the positive
electrostatic potential surrounding hydrophobic loop residues that
interferes with the membrane penetration of domains (30). Based on
these data, we proposed a membrane binding mechanism for the FYVE
domain in which initial membrane adsorption of the domain driven by
nonspecific electrostatic interactions between the cationic protein
surface and the anionic membrane surface is followed by specific
PtdIns(3)P-triggered membrane penetration of the domain. The present
study on the p40phox-PX indicates that its PtdIns(3)P-mediated
membrane binding follows essentially the same mechanism. Our monolayer
data clearly indicate that PtdIns(3)P binding is not a consequence of
but a prerequisite for the penetration of the domain into the
monolayer, the packing density of which is comparable with that of
cellular membranes and large unilamellar vesicles (40-42). The
penetration of hydrophobic residues (Phe35,
Tyr94, and Val95) located in the loops
surrounding the PtdIns(3)P-binding pocket of p40phox-PX is a
specific PtdIns(3)P-dependent process, since it was
abrogated either by removal of PtdIns(3)P in the monolayer or by
mutation of the PtdIns(3)P-coordinating residues, Arg58,
Arg60, or Arg105. The electrostatic potential
calculation suggests that PtdIns(3)P binding serves as a electrostatic
switch that greatly reduces the positive electrostatic potential
surrounding the hydrophobic residues. As is the case with the FYVE
domain, this reduction in electrostatic potential is necessary for
circumventing the high desolvation penalty associated with the membrane
penetration of the hydrophobic residues. Neither the present study nor
the previous structural study on the p40phox-PX-PtdIns(3)P
complex (7) provides direct information as to whether or not PtdIns(3)P
binding causes conformational changes of the p40phox-PX. Other
studies have indicated, however, that binding of a ligand to a membrane
targeting domain causes local conformational changes to reorient the
hydrophobic residues for more productive membrane penetration (22, 29,
30, 43). Furthermore, a recent NMR study on the PX domain of Vam7p (9)
revealed that residues in its putative membrane binding loops underwent
large changes in chemical shift when dodecylphosphocholine micelles were added to the PX domain-dibutanoyl PtdIns(3)P complex. Thus, it is
likely that PtdIns(3)P binding also induces the reorientation of
hydrophobic side chains of the p40phox-PX for their better
partitioning into the lipid bilayer. Our SPR data show that the
PtdIns(3)P-induced membrane penetration of these residues enhances the
membrane affinity of the p40phox-PX by lowering the
kd value. Also, our SPR and electrostatic potential
computation data suggest that clustered cationic residues (Arg58, Arg60, Lys92, and
Arg105) in the PtdIns(3)P-binding pocket are involved not
only in PtdIns(3)P coordination (hence a decrease in
kd) but also in initial nonspecific electrostatic
adsorption to the anionic membrane surface (hence an increase in
ka).
As expected from the presence of two lipid binding sites, the membrane
binding mechanism of the p47phox-PX is more complex than that
of the p40phox-PX. Membrane binding of p47phox-PX has
two salient features. First, it has higher intrinsic monolayer penetration activity than p40phox-PX. Second, not only
PtdIns(3,4)P2 but also PtdOH (or PtdSer) can induce the
monolayer penetration of the p47phox-PX into the monolayer, the
packing density of which is comparable with that of cell membranes. The
higher intrinsic membrane-penetrating activity of p47phox-PX is
attributed to a single hydrophobic residue Ile65. This
residue can nonspecifically penetrate the neutral monolayers to some
extent, because a part of its side chain is not surrounded by the
positive electrostatic potential, and therefore its membrane penetration involves a much lesser degree of desolvation. It should be
noted, however, that PtdIns(3,4)P2 and PtdOH (or PtdSer)
are still required for p47phox-PX to effectively interact with
the monolayer or the bilayer whose lipid packing density is comparable
with that of cell membranes. Thus, the physiological significance of
the higher intrinsic membrane-penetrating activity of
p47phox-PX is not clear. Interestingly,
PtdIns(3,4)P2 and PtdOH (or PtdSer) induce the membrane
penetration of hydrophobic residues located near their binding pockets,
respectively; i.e. PtdIns(3,4)P2 primarily influences Trp80, and PtdOH (or PtdSer) mainly affects
Ile65. This selective effect is readily accounted for by
our electrostatic potential calculations, which show that
PtdIns(3,4)P2 coordination by Arg43 and
Arg90 dramatically reduces the positive potential near
Trp80, whereas PtdSer binding lowers the electrostatic
potential near Ile65. As is suggested for
p40phox-PX, it is also possible that ligand binding to each
pocket induces the local conformational changes, especially in the loop
regions containing hydrophobic residues, Trp80 and
Ile65. We previously showed that PtdIns(3,4)P2
and PtdOH (or PtdSer) synergistically increase the membrane affinity
(12). Our monolayer data as well as electrostatic potential computation
indicate that this synergistic effect is due in part to a dramatic
reduction in electrostatic potential when both binding pockets are
occupied by ligands, which in turn promotes more effective membrane
penetration of hydrophobic side chains. It would seem that the
simultaneous binding of PtdIns(3,4)P2 and PtdOH (or PtdSer)
to the two separate sites (i.e. simultaneous membrane
penetration of two hydrophobic regions) is necessary for effective
membrane targeting of the p47phox-PX, since its affinity for
POPC/POPE/PtdIns(3,4)P2 (77:20:3) vesicles is about 30 times lower than that of p40phox-PX for POPC/POPE/PtdIns(3)P
(77:20:3) vesicles under the same conditions (see Table I). Only in the
presence of both PtdIns(3,4)P2 and PtdOH (or PtdSer) in the
vesicles (i.e. POPC/POPE/POPS/PtdIns(3,4)P2 (74:20:3:3)), the p47phox-PX shows an affinity comparable with
that of the p40phox-PX for POPC/POPE/PtdIns(3)P (77:20:3)
vesicles (see Table I).
Taken together, we propose membrane-binding modes for
p40phox-PX and p47phox-PX (see Fig.
8). Both follow essentially the same
sequences for membrane binding: initial membrane adsorption driven by
nonspecific electrostatic interactions between the cationic protein
surface and the anionic membrane surface, followed by PI-induced
membrane penetration of protein. A main difference is that for
p40phox-PX, hydrophobic residues near the PtdIns(3)P-binding
pocket primarily participate in membrane penetration, whereas for
p47phox-PX, hydrophobic residues surrounding both
PtdIns(3,4)P2-binding pocket and the secondary binding site
are involved.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
cells for
plasmid isolation. After verifying the DNA sequence of each construct,
the plasmid was transformed into BL21(DE3) cells for protein
expression. One liter of 2× YT medium containing 100 µg/ml
ampicillin was inoculated with BL21(DE3) cells harboring each construct
and grown at 37 °C until absorbance at 600 nm reached 1.0. At this
time, 1 mM of
isopropyl-1-thio-
-D-galactopyranoside was added, and
cells were then incubated at 25 °C for 5 h. Cells were
harvested for 10 min at 4000 × g, and the resulting
pellet was resuspended in 10 ml of 50 mM
NaH2PO4, pH 8.0, containing 0.3 M
NaCl, 50 µM phenylmethylsulfonyl fluoride, 0.1% Triton
X-100, and 10 mM imidazole. The solution was then sonicated
for 8 min using a 30-s sonication followed by 30-s cooling on ice. This was followed by centrifugation at 48,000 × g to
separate the soluble and insoluble fractions. The supernatant was
filtered into a 50-ml tube, and 1 ml of nickel-nitrilotriacetic acid
solution (Qiagen, Valencia, CA) was added. The mixture was incubated on
ice with gentle stirring (80 rpm) for 1 h. After this time, the
mixture was poured onto a column, which was washed with 20 ml of 50 mM NaH2PO4, pH 8.0, containing 0.3 M NaCl and 15 mM imidazole. Subsequently, the
protein was eluted from the column in six fractions using 0.5 ml of 50 mM NaH2PO4, pH 8.0, containing 0.3 M NaCl and 300 mM imidazole. Purity was checked
on an 18% polyacrylamide gel, and samples were pooled and concentrated
to 1 ml for gel filtration chromatography. The 1-ml sample was loaded
onto a Superdex 200 column (Amersham Biosciences) and eluted with 20 mM Tris, pH 7.5, with 0.1 M NaCl. Fractions
corresponding to the p40phox-PX peak, as checked
electrophoretically, were pooled and concentrated to 1 ml. Protein
concentration was then determined using the BCA method (Pierce). The
full-length human p40phox (residues 2-339) and W207R mutation
were expressed and purified as described (7). Mutants of
p47phox-PX and full-length p47phox were constructed,
expressed, and purified as previously described (12).
) at constant surface area using a 10-ml circular Teflon trough and
Wilhelmy plate connected to a Cahn microbalance as previously described
(22). All our monolayer measurements were performed at 23 °C, since
at higher temperature water evaporation and protein instability caused
significant experimental problems. A lipid monolayer containing various
combinations of phospholipids was spread onto the subphase composed of
10 mM HEPES, pH 7.4, containing 0.1 M NaCl
until the desired initial surface pressure (
0) was reached. After the signal stabilized (~5 min), 30 µg of proteins were injected, and the increase in surface pressure (
) was
monitored for 45 min while stirring the subphase at 60 rpm. Typically,
the
value reached a maximum after 20 min. It has been shown
empirically that
caused by a protein is mainly due to the
penetration of the protein into the lipid monolayer. For example, in
the case of the C2 domain of cytosolic phospholipase A2,
excellent agreement was found between large
caused by several
residues in the calcium binding loops (22) and their actual membrane
penetration measured by fluorescence (23) and electron spin resonance
studies (24, 25). The maximal
value depended on the protein
concentration and reached a saturation value (e.g.
[p40phox-PX]
2.0 µg/ml); therefore, protein
concentration in the subphase was maintained above such values to
ensure that the observed
represented a maximum value. The
resulting
was plotted versus
0, from
which the critical surface pressure (
c) was determined as the x-intercept (26).
(complex) using BIAevalutation 3.0 software (Biacore) as described
previously (28-30). The dissociation constant (Kd)
was then calculated from the equation, Kd = kd/ka. Mass transport (31, 32)
was not a limiting factor in our experiments, since change in flow rate
(from 2 to 60 µl/min) did not affect kinetics of association and
dissociation. After curve fitting, residual plots and
2
values were checked to verify the validity of the binding model. Each
data set was repeated three times to calculate a S.D. value. Also,
Kd values were separately determined from
equilibrium SPR measurements. For these measurements, the flow rate was
reduced to 2 µl/min to allow sufficient time for the association
phase, which in turn allows resonance unit values to reach saturating response values (Req).
Req values were then plotted versus
protein concentrations (C), and the Kd
value was determined by a nonlinear least-squares analysis of the
binding isotherm using an equation: Req = Rmax/(1 + Kd/C),
where Rmax is a maximal Req value.
6 mV; dark
pink,
12 mV; light pink,
25 mV;
dark blue, +25 mV; light
blue, +12 mV. The electrostatic calculations performed used
partial charges taken from the CHARMM27 force field (35) and
spatial coordinates taken from the structures of p40phox-PX
(1H6H) (7) and p47phox-PX (1O7K) (12).
PtdIns(3,4)P2 and PtdSer were docked onto the p47phox-PX structure using the bound sulfate ions as guides in
placing the lipid phosphate groups. Steric clashes were fixed manually.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
0 was spread at constant surface area, and the
was monitored after the injection of the p40phox-PX into the
subphase. As shown in Fig. 1A,
the p40phox-PX has low penetrating power to a POPC/POPE (80:20)
monolayer with a
c value around 20 dyne/cm, implying
that the p40phox-PX has low intrinsic membrane-penetrating
capability. However, incorporation of 3 mol % PtdIns(3)P into the
monolayer greatly elevated the monolayer penetrating capability of the
p40phox-PX, raising the
c value near 31 dyne/cm.
It has been shown that the global cellular concentration of some PIs
can reach up to 3 mol % and that their local concentrations can be
much higher than global concentrations (39). This suggests that
PtdIns(3)P might induce the partial penetration of the
p40phox-PX into cell membranes, since the surface pressure of
cell membranes has been estimated to be near 31 dyne/cm (40-42). The
specific nature of the PtdIns(3)P effect was demonstrated by the lack
of effect by either a stereoisomer of PtdIns(3)P, phosphatidylinositol 5-phosphate, or a bulk anionic phospholipid, PtdSer, on monolayer penetration of the p40phox-PX (Fig. 1A).
View larger version (19K):
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Fig. 1.
versus
o plots for p40phox-PX and
p47phox-PX. A, p40phox-PX was allowed
to interact with POPC/POPE (80:20) (
), POPC/POPE/PtdIns(3)P
(77:20:3) (
), POPC/POPE/PtdIns(5)P (77:20:3) (
), POPC/POPE/POPS
(50:20:30) (
), and POPC/POPE/POPS/PtdIns(3)P (62:20:15:3)
(
). B, p47phox-PX was allowed to interact with
POPC/POPE (80:20) (
), POPC/POPE/PtdIns(3,4)P2 (77:20:3)
(
), POPC/POPE/POPA (77:20:3) (
), and POPC/POPE/POPS (50:20:30)
(
). The subphase was 10 mM HEPES buffer, pH 7.4, with
0.1 M KCl for all measurements.
c
28 dyne/cm) (see Fig. 1B), suggesting that it has higher
intrinsic membrane penetration activity. As was the case with
p40phox-PX, 3 mol % of PtdIns(3,4)P2 in the
POPC/POPE monolayer greatly enhanced the monolayer penetration of
p47phox-PX, increasing the
c value to 37 dyne/cm, indicating that PtdIns(3,4)P2 would also enable
the p47phox-PX to penetrate cell membranes. We then monitored
the penetration of p47phox-PX into monolayers containing 3 mol
% POPA or 30 mol % POPS (note that the secondary site has higher
affinity for PtdOH than for PtdSer) (12). As shown in Fig.
1B, both 3 mol % POPA and 30 mol % POPS allow
p47phox-PX to have
c values above 31 dyne/cm,
suggesting that these anionic phospholipids can induce the membrane
penetration of p47phox-PX, presumably through interactions with
the secondary lipid binding pocket (see below). This is in sharp
contrast with p40phox-PX, the monolayer penetration of which
was not affected by 30 mol % POPS under the same conditions (see Fig.
1A).
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Fig. 2.
Effect of mutations on the monolayer
penetration of p40phox-PX. A, penetration of
wild type p40phox-PX ( ), R58Q (
), R60A (
), and R105A
(
) into a POPC/POPE/PtdIns(3)P (77:20:3) monolayer was monitored as
a function of
o. B, penetration of
p40phox-PX (
), F35A (
), L82A (
), Y94A (
),
and V95A (
) into a POPC/POPE/PtdIns(3)P (77:20:3) monolayer was
monitored as a function of
o. The subphase was 10 mM HEPES buffer, pH 7.4, with 0.1 M KCl for all
measurements.
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Fig. 3.
Effect of p47phox-PX mutations on the
monolayer penetration of p47phox-PX to
PtdIns(3,4)P2 and POPS containing monolayers.
A, penetration of p47phox-PX ( ), R43Q (
), R70Q
(
), and R90A (
) into a POPC/POPE/PtdIns(3,4)P2
(77:20:3) monolayer was monitored as a function of
o.
B, penetration of p47phox-PX (
), I65A (
), and
W80A (
) into a POPC/POPE/PtdIns(3,4)P2 (77:20:3)
monolayer was monitored as a function of
o.
C, penetration of p47phox-PX (
), I65A (
), R70Q
(
), W80A (
), and R90A (
) into a POPC/POPE/POPS (50:20:30)
monolayer was monitored as a function of
o.
D, penetration of p47phox-PX (
), I65A (
), R70Q
(
), W80A (
), and R90A (
) into a POPC/POPE (80:20) monolayer
was monitored as a function of
o. The subphase consisted
of 10 mM HEPES buffer, pH 7.4, with 0.1 M KCl
for all measurements.
c = 28 dyne/cm) compared with the p47phox-PX under the same
conditions, suggesting that the C-terminal SH3 domain interferes with
the PtdIns(3,4)P2-dependent monolayer penetration of the p47phox-PX. To further test this notion, we
measured the monolayer penetration of the full-length protein carrying
the W263R mutation, which was shown to disrupt the SH3-PX interaction
(37). Fig. 4A illustrates that this mutant has a monolayer
penetration behavior, which is comparable with that of the
p47phox-PX. We then prepared a quintuple mutant,
S303E/S304E/S328E/S359E/S370E to examine the effects of phosphorylation
of the C-terminal tail on the
PtdIns(3,4)P2-dependent monolayer penetration
of the full-length p47phox. Again, the monolayer penetration
behavior (i.e.
c = 36 dyne/cm) of this
phosphorylation mimic mutant (see Fig. 4A) resembles that of
p47phox-PX, suggesting that the phosphorylation unmasks the PX
domain and thereby leads to its penetration into
PtdIns(3,4)P2-containing membranes.
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Fig. 4.
Effect of initial surface pressure of
monolayer on penetration of p47phox, p47phox mutants
and p40phox. A, penetration of full-length
p47phox ( ), p47phox-PX (
), W263R (
),
R43Q/W263R (
), and 5SE p47phox (
) into a
POPC/POPE/PtdIns(3,4)P2 monolayer (77:20:3) was monitored
as a function of
o. B, penetration of
full-length p40phox (
) and p40phox-PX (
) into a
POPC/POPE/PtdIns(3)P monolayer (77:20:3) was monitored as a function of
o. p40phox (
) and p40phox-PX (
)
were also allowed to interact with a POPC/POPE (80:20) monolayer. The
subphase consisted of 10 mM HEPES buffer, pH 7.4, with 0.1 M KCl for all measurements.
x:20:x) mixed vesicles immobilized to the
sensor surface and measured the kinetics of membrane binding by SPR
analysis. Fig. 5A shows
representative sensorgrams for p40phox-PX binding to
POPC/POPE/PtdIns(3)P vesicles (77:20:3), from which ka and kd values were calculated.
Separate equilibrium SPR measurements yielded the binding isotherms, as
shown for binding of p40phox-PX to POPC/POPE/PtdIns(3)P
vesicles (77:20:3) in Fig. 5B, from which
Kd values were calculated. Typically,
Kd values determined from kinetic measurements
(e.g. Kd = 1.3 ± 0.5 nM
from Fig. 5A) using the relationship, Kd = kd/ka, agree well with
Kd values determined from equilibrium measurements
(e.g. Kd = 0.71 ± 0.02 nM from Fig. 5B). For this reason, kinetically
determined Kd values are used for evaluating the
relative affinity of wild type and mutants (see Table
I).
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Fig. 5.
Kinetic and equilibrium SPR measurements for
binding of p40phox-PX to POPC/POPE/PtdIns(3,4)P2
(77:20:3) vesicles. A, sensorgrams from kinetic
measurements. The p40phox-PX was injected at 60 µl/min at
varying concentrations (0.5, 1, 2, 5, and 10 nM; see the
labels). Solid lines represent the
best-fit theoretical curves. B, equilibrium SPR
measurements. p40phox-PX was injected at 2 µl/min at varying
concentrations (0.5, 1, 2, 5, and 10 nM; from
bottom to top) and Req
values were measured (see inset). A binding isotherm was
then generated from the Req versus
[p40phox-PX] plot. A solid line
represents a theoretical curve constructed from
Rmax (75 ± 1.2) and Kd
(0.71 ± 0.02 nM) values determined by nonlinear
least-squares analysis of the isotherm using an equation:
Req = Rmax/(1 + Kd/C). 10 mM HEPES buffer, pH
7.4, with 0.1 M KCl was used for both measurements.
Binding parameters for p40phax-PX and mutants determined from
SPR analysis
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Fig. 6.
Effects of anionic lipids on the
electrostatic potential of p40phox-PX and
p47phox-PX. The electrostatic potential for the PX domains
was calculated and visualized in the program GRASP by two-dimensional
equipotential contours in 0.1 M KCl. The red
contours are 25 mV; magenta,
12 mV; dark
blue, +25 mV; light blue, +12 mV.
A, p40phox-PX in the absence of PtdIns(3)P;
B, p40phox-PX in the presence of PtdIns(3)P;
C, difference between B and A;
D, p47phox-PX in the absence of
PtdIns(3,4)P2; E, p47phox-PX in the
presence of PtdIns(3,4)P2; F, E
D; G, p47phox-PX in the absence of
PtdSer; H, p47phox-PX in the presence of PtdSer;
I, H
G; J,
p47phox-PX without ligands; K, p47phox-PX in
the presence of both PtdIns(3,4)P2 and PtdSer;
L, K
J. Notice that for
p47phox-PX, the positive potentials in the vicinity of
Trp80 are reduced by PtdIns(3,4)P2 but not by
PtdSer and that the tip of Ile65 side chain has neutral
electrostatic potential with and without lipids. In K, the
effect of two lipids is essentially additive.
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Fig. 7.
Subcellular localization of PX domains and
mutants in HEK293 cells. A, p40phox-PX;
B, p40phox-PX 30 min after 100 nM
wortmannin treatment; C, p40phox-PX F35A;
D, p40phox-PX R58Q; E, p40phox-PX
Y94A; F, p47phox-PX; G,
p47phox-PX after PtdIns(3,4)P2 was fed into cells;
H, p47phox-PX R90A; I, p47phox-PX
R70Q; J, p47phox-PX W80A. PtdIns(3,4)P2
was fed into cells for G-J. All proteins are EGFP-tagged at
their C termini. The images in A and C-F were
time-independent. The image in G was taken 1 min after
PtdIns(3,4)P2 treatment, whereas the images in
H-J were taken 20 min after PtdIns(3,4)P2
treatment.
DISCUSSION
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ABSTRACT
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EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
View larger version (49K):
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Fig. 8.
Proposed membrane binding modes of
p47phox-PX (A) and p40phox-PX
(B). The structure of membrane-bound
p47phox-PX (ribbon diagram) with a
PtdIns(3,4)P2 molecule (in space-filling
representation) bound to its binding pocket is modeled based upon
the x-ray structure of p47phox-PX (12). The
structure of membrane-bound p40phox-PX-PtdIns(3)P complex is
derived from the x-ray structure of the complex (7). Mutated residues
are shown in red. Note that two loop regions near
PtdIns(3,4)P2- and PtdOH-binding sites, respectively, are
involved in membrane penetration of p47phox-PX, whereas only
the loop region surrounding the PtdIns(3)P-binding pocket participates
in membrane penetration of p40phox-PX.
Subcellular localization behaviors of the two PX domains and their respective mutants are generally in line with their in vitro membrane binding properties. The p40phox-PX with high affinity for PtdIns(3)P-containing vesicles migrates to PtdIns(3)P-rich endosomes when expressed in HEK293 cells. Also, the lowering cellular level of PtdIns(3)P by wortmannin treatment and mutating the 3-phosphate ligands (Arg58 and Arg105) or membrane-penetrating hydrophobic residues all abrogate the endosomal targeting of the domain. Thus, it is evident that the cellular membrane targeting of the p40phox-PX is governed, at least in part, by the forces that control the in vitro binding of the domain to lipid vesicles. As described above, the p47phox-PX in the presence of both PtdIns(3,4)P2 and PtdOH (or PtdSer) has membrane affinity comparable with that of p40phox-PX in the presence of PtdIns(3)P. This, in conjunction with the fact that all intracellular membranes contain a certain level of anionic lipids, indicates that the lack of membrane localization by the p47phox-PX in HEK293 cells is due to the low level of PtdIns(3,4)P2 in cell membranes. This notion is supported by targeting of the domain to the plasma membrane when PtdIns(3,4)P2 was fed into cells as a histone-lipid complex. Since this method has been shown to effectively deliver PIs to all intracellular membranes (36), the selective translocation of the p47phox-PX to the PtdSer-rich plasma membrane indicates that the secondary lipid-binding site might play a key role in the membrane targeting of p47phox-PX. The secondary site has higher affinity for PtdOH than for PtdSer, and, thus, the spatiotemporal dynamics of PtdOH in conjunction with that of PtdIns(3,4)P2 would drive the translocation of p47phox-PX to the plasma membrane and other membranes, including phagocytic membranes. Our data also show that membrane penetration of hydrophobic residues surrounding the two lipid binding sites is important for subcellular localization of p47phox-PX. It was recently reported that the p47phox-PX could be localized in membrane ruffles of COS cells in response to insulin-like growth factor-1 in a PtdIns(3,4)P2-independent manner (19). However, the driving force for this translocation was not elucidated, and the same kind of translocation was not observed in HEK293 cells. The discrepancy might be simply due to lack of the specific receptor for insulin-like growth factor-1 in HEK293 cells. Further studies are necessary to fully understand the origin of this putative PtdIns(3,4)P2-independent membrane translocation of p47phox-PX.
Many PX domains contain a proline-rich, (R/K)XXPXXP sequence that is characteristic of SH3 domain binding motifs, prompting speculation that they might be interacting partners of SH3 domain proteins (1). Indeed, the PX domain of p47phox has been shown to interact with its C-terminal SH3 domain (37), which inhibits its membrane interactions (12). Our monolayer penetration measurements demonstrate that the membrane penetration of the PX domain in the full-length p47phox is severely interfered with by other parts of the protein. Dramatic effects of W263R and quintuple phosphorylation mimic mutations on the monolayer penetration of the full-length p47phox demonstrate that the C-terminal SH3 domain blocks the PtdIns(3,4)P2 (and PtdOH)-dependent membrane penetration of the PX domain and that the phosphorylation of C-terminal tail relieves this interdomain tethering. This suggests that the full-length p47phox might not spontaneously respond to the spatiotemporal dynamics of PtdIns(3,4)P2. In contrast, the SH3 domain of p40phox plays no role in inhibiting the PtdIns(3)P-dependent membrane penetration of p40phox-PX. Thus, it is expected that the full-length p40phox, when free in cytoplasm, will spontaneously respond to the spatiotemporal dynamics of PtdIns(3)P in the membranes, phagocytic membranes in neutrophils and macrophages in particular.
In summary, the present investigation demonstrates that two different
PIs, PtdIns (3)P and PtdIns(3,4)P2, have a similar effect
on membrane binding for two PX domains. This, in conjunction with our
previous finding that PtdIns(3)P has essentially the same effect on
membrane binding of structurally distinct FYVE domains, suggests that
PI-induced membrane penetration is a common mechanism for many membrane
targeting domains. PX domains have broad PI specificities and variable
amino acid sequences around their putative PI-binding pockets. Thus,
this work would serve as a framework with which one can comprehensively
study the roles of PIs in the membrane targeting of a wide spectrum of
PX domains.
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ACKNOWLEDGEMENTS |
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We thank Drs. Roger L. Williams and Olga Perisic for kindly providing the expression vectors for p40phox, p47phox, p40phox-PX, p47phox-PX, and several mutants.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants GM66147 (to D. M.), GM57568 (to K. S. B.), and GM52598 and GM53987 (to W. C.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Chemistry
(M/C 111), University of Illinois at Chicago, 845 W. Taylor St.,
Chicago, IL 60607-7061. Tel.: 312-996-4883; Fax: 312-996-2183; E-mail:
wcho@uic.edu.
Published, JBC Papers in Press, January 29, 2003, DOI 10.1074/jbc.M212579200
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ABBREVIATIONS |
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The abbreviations used are: PI, phosphoinositide; EGFP, enhanced green fluorescent protein; PtdIns(3)P, phosphatidylinositol 3-phosphate; PtdIns(3, 4)P2, phosphatidylinositol 3,4-bisphosphate; POPA, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphate; POPC, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine; POPE, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoehthanolamine; POPS, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine; PtdOH, phosphatidic acid; PtdSer, phosphatidylserine; SH3, Src homology 3; SPR, surface plasmon resonance.
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