From the Department of Molecular and Experimental Medicine, Division of Biochemistry, The Scripps Research Institute, La Jolla, California 92037
Received for publication, December 12, 2000, and in revised form, March 6, 2001
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ABSTRACT |
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We have employed a yeast two-hybrid system to
screen a B lymphoblast-derived cDNA library, searching for
regulatory components of the NADPH oxidase. Using as bait the
C-terminal half of p67phox, which contains both Src homology
3 domains, we have cloned JFC1, a novel human 62-kDa protein.
JFC1 possesses two C2 domains in tandem. The C2A domain shows homology
with the C2B domain of synaptotagmins. JFC1 mRNA was abundantly
expressed in bone marrow and leukocytes. The expression of JFC1 in
neutrophils was restricted to the plasma membrane/secretory vesicle
fraction. We confirmed JFC1-p67phox association by affinity
chromatography. JFC1-containing beads pulled down both p67phox
and p47phox subunits from neutrophil cytosol, but when the
recombinant proteins were used, only p67phox bound to JFC1,
indicating that JFC1 binds to the cytosolic complex via p67phox
without affecting the interaction between p67phox and
p47phox. In contrast to synaptotagmins, JFC1 was unable to bind
to inositol 1,3,4,5-tetrakisphosphate but did bind to
phosphatidylinositol 3,4,5-trisphosphate and to a lesser extent to
phosphatidylinositol 3,4-diphosphate. From the data presented here, it
is proposed that JFC1 is acting as an adaptor protein between
phosphatidylinositol 3-kinase products and the oxidase cytosolic complex.
The NADPH oxidase, a multisubunit enzymatic complex that is
present in neutrophils and B lymphocytes, is responsible for the monoelectronic reduction of oxygen to produce superoxide anion (O In resting cells, the components of the oxidase are distributed between
different subcellular compartments and thus remain unassembled while
the oxidase is inactive. The main membrane component is cytochrome
b558, an integral membrane protein containing
one subunit of gp91phox and one of p22phox that is
located in the secretory vesicles and specific granules (5). Meanwhile,
the p47phox and p67phox, components that
are known to be essential for the oxidase activation in
vivo, remain in the cytosol in a complex that also includes p40phox (6, 7), a protein that is reported to regulate the
activity of the oxidase. Two other factors, the small GTPases Rac2 (8) and Rap1a (9, 10), are also known to participate in the regulation of
the oxidase. The exact mechanism of the activation process, however,
remains obscure. In the presence of adequate stimuli, the cytosolic
factor p47phox is phosphorylated and translocated, together
with p67phox (11, 12), to the particulate fraction, where the
cytosolic complex interacts with cytochrome
b558. The subunit gp91phox is a
flavohemoprotein that contains two hemes necessary to transfer electrons from NADPH to molecular oxygen (13, 14). The C terminus of
p22phox binds to tandem
SH31 domains present in
p47phox (15). Because p47phox binds to p67phox
(16, 17), it is considered to be responsible for assembling the oxidase
in vivo. On the other hand, recent studies showed that
p47phox is not required for the reconstitution of NADPH oxidase
in a cell-free system when high concentrations of p67phox and
Rac are present (18, 19). Therefore, these studies suggest that
p67phox is the essential cytosolic factor for enzyme activity
(19, 20).
Major structural features of p67phox are an acidic C terminus,
two SH3 domains located in the middle (residues 245-295) and
C-terminal region (residues 462-512), a Rac binding domain in its
amino-terminal fragment, and two proline-rich regions in residues
226-234 and 317-329. Our group has previously reported that
p67phox catalyzes pyridine nucleotide dehydrogenation,
suggesting an active role of this factor during electron transfer (21).
Moreover, we have recently shown that p67phox has a NADPH
binding site located in its amino-terminal fragment (22). In agreement
with this, it has been previously reported that the amino-terminal
portion of p67phox lacking both SH3 domains was active in a
cell-free system (23). However, it has been shown in the same study
that complete restoration of NADPH oxidase in p67phox-deficient
Epstein-Barr virus-B cells derived from chronic granulomatous disease
patients was only achieved with full-length p67phox cDNA
expression. Moreover deletions of either SH3 domain dramatically reduced NADPH oxidase activity in this system, findings that correlated with decreased membrane binding (23). It is well known that SH3 domains
play an important role in protein-protein interactions regulating
cellular localization of interacting factors, and, although the
importance of these domains in the p67phox-p47phox
interaction has been described (16, 17), it is not unlikely that
p67phox-SH3 domains interact with other accessory proteins that
could take part in the still unclear process of NADPH oxidase assembly or activation.
We used the yeast two-hybrid system to search for additional components
of the phagocyte antimicrobial machinery. Using as bait the C-terminal
half of p67phox, including both SH3 domains, we isolated a
protein that interacts with oxidase components. In this communication,
we describe the cloning and some of the properties of this protein,
which we have designated JFC1.
Materials--
Reagents for the yeast two-hybrid assay,
including vectors, yeast strains, the yeast two-hybrid cDNA
library, and control vectors, were the generous gifts of Stephen
Elledge (Baylor College of Medicine, Houston, TX). The
Phosphatidylcholine, phosphatidylinositol, phosphatidylserine, and
phosphatidylinositol 4-phosphate (PtdIns(4)P), were purchased from
Sigma; phosphatidylinositol 4,5-diphosphate (PtdIns(4,5)P2) was obtained from Calbiochem; and phosphatidylinositol 3-phosphate (PtdIns(3)P), phosphatidylinositol 3,4-diphosphate
(PtdIns(3,4)P2), and phosphatidylinositol
3,4,5-trisphosphate (PtdIns (3,4,5)P3) were obtained from
Matreya, Inc. (Pleasant Gap, PA). Inositol 1,3,4,5-tetrakisphosphate
(InsP4) was purchased from Sigma, and [3H]InsP4 was from PerkinElmer Life Sciences.
Yeast Two-hybrid Assay and Retrieval of a Full-length
Clone--
For the yeast two-hybrid assay "bait" constructs, the
C-terminal portion of p67phox, including the two SH3 domains
and the intervening sequence (residues 245-512), was amplified from
cDNA by polymerase chain reaction using a 5' primer
(GAATTCGCTCACCGTGTGCTATTT) that contained an EcoRI site (underlined) and a sequence that annealed to
nucleotides 730-747 and a 3' antisense primer
(GAATTCCCCTTCAACAAAAACTTTGGGGAA) that also contained an
EcoRI site (underlined) and a sequence that annealed to
nucleotides 1516-1536. The resulting fragment was then ligated into
pAS1 so as to be in frame with the upstream GAL4 coding sequences. This
construct was sequenced to verify that it was in frame as planned and
that no mutations had been introduced during polymerase chain reaction.
The construct was used to screen a yeast two-hybrid cDNA library
derived from Epstein-Barr virus-transformed B lymphoblasts cloned in
the vector pACTII, whose inserts are expressed as fusion proteins
consisting of the activation domain of GAL4 attached to the polypeptide
encoded in the insert. The constructs were transfected by the
method of Gietz et al. (24) in Saccharomyces
cerevisiae strain Y190, and the screening was performed
following procedures and using negative controls that have been
described previously (25, 26). From 4.2 million transformants, 46 positive colonies were detected. Twenty-four of these were selected for
sequencing. Of these 24 clones, four corresponded to a single protein
that showed a strong homology to C2 domains in synaptotagmins (27) and
rabphilin 3 (28), two proteins that are involved in lipid binding,
secretion, and protein-protein interactions. One of these clones was
chosen for further study.
To obtain a full-length clone, a peripheral blood leukocyte cDNA
library in Sequencing--
The sequences of both the yeast two-hybrid
cDNA fragment in pACTII and the full-length JFC1 cDNA in
RNA Blot Analysis--
Leukocyte RNA was obtained as previously
described (29). The probe was labeled with [ Preparation and Fractionation of Neutrophils--
Whole blood
was obtained from the anonymous donor program at The Scripps Research
Institute General Clinical Research Center. Neutrophils
(polymorphonuclear leukocytes (PMNs)) were isolated by dextran
sedimentation, hypotonic lysis, and Ficoll density centrifugation as
previously described (30). Fractionation was carried out by a published
protocol (31). Briefly, PMNs were resuspended in PIPES buffer (10 mM), pH 7.3 containing 100 mM KCl, 3 mM NaCl, 3.5 mM MgCl2, and 1 mM ATP (buffer A) supplemented with protease inhibitors
(diisopropylfluorophosphate, 0.5 mM (Sigma) and Complete
protease inhibitor mixture (Roche Molecular Biochemicals)) and then
disrupted by nitrogen cavitation at 400 p.s.i. EGTA was added to a
final concentration of 1.25 mM, and nuclei and unbroken cells were removed by centrifugation at 500 × g. The
supernatant from this spin (postnuclear supernatant (PNS)) was either
centrifuged at 200,000 × g for 30 min to separate
cytosol from particulate matter or fractionated on a two-step
discontinuous Percoll gradient (step 1, Stimulation of PMN with Phorbol 12-Myristate 13-Acetate
(PMA)--
PMNs prepared as described above were washed once in
Ca2+- and Mg2+-free phosphate-buffered saline
(Life Technologies, Inc.) and then resuspended in PBS containing 0.5 mM CaCl2 and 1.5 mM
MgCl2 and warmed to 37 °C for 10 min. PMA (Sigma) was
added to a final concentration of 1 µg/ml and the cells were
incubated for an additional 6 min at 37 °C. The cells were then
washed once in ice-cold PBS and subjected to the
cavitation/fractionation protocol.
In Vitro Binding Studies--
Recombinant JFC1 was expressed in
E. coli as the glutathione S-transferase fusion
protein and purified using glutathione-Sepharose beads (Amersham
Pharmacia Biotech) as recommended by the manufacturer. Sepharose
beads (100 µl) containing 10 µg of the recombinant fusion protein
or GST alone were washed twice in RIPA buffer (10 mM
Tris/HCl, pH 7.5, 140 mM NaCl, 1% Triton X-100, 1% sodium
deoxycholate, 0.1% SDS, 0.025% NaN3) and incubated
overnight at 4 °C in the presence of 1.8 × 107 or
9 × 107 cell equivalent of neutrophil-derived cytosol
or buffer. Beads were washed five times during 10 min with buffer RIPA
at 4 °C, spun down, and boiled in Laemmli SDS sample buffer (31).
Samples were resolved by SDS-PAGE, and proteins were transferred to
nitrocellulose and probed with primary antibodies directed against
p47phox or p67phox. Detection was performed using
alkaline phosphatase-conjugated goat anti-rabbit secondary antibody.
The bound alkaline phosphatase activity was detected using the
5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium
colorimetric reagent (Bio-Rad).
In Vitro Translated JFC1 and p67phox--
Wild type
JFC1 cDNA and p67phox cDNA were cloned into pBK-CMV
under the control of the T7 promoter. In vitro translated
proteins labeled with [35S]methionine were produced using
the TNT coupled transcription and translation system from Promega
(Madison, WI) as recommended by the manufacturer. Translational grade
[35S]methionine was purchased from Amersham Pharmacia
Biotech. In order to normalize the amount of radioactive protein added
to the experiments, serial dilutions of each in vitro
translation reaction were electrophoresed on SDS-PAGE, and the specific
protein bands were quantified using a PhosphorImager (Molecular
Dynamics, Inc., Sunnyvale, CA).
Binding of [35S]JFC1 to p47phox or
p67phox in Vitro--
The binding of [35S]JFC1
to GST-p47phox or GST-p67phox was evaluated as
described above under "In Vitro Binding Studies" except
that 5 µg of GST-p47phox, GST-p67phox, or GST were
used in the assays. The reactions were performed either in RIPA buffer
or in a buffer that contained 50 mM Tris/HCl, pH 7.5, 50 mM NaCl, and 0.1% Triton X-100. Serial dilutions of the
in vitro translation reactions containing
[35S]JFC1, [35S]p67phox as a
positive control, or [35S]luciferase as a negative
control were used in the assay with a 200-µl final volume. Samples
were rotated at 4 °C during 1 h, washed four times for 15 min
with the reaction buffer, spun down, and boiled in Laemmli SDS sample
buffer. Samples were resolved by SDS-PAGE, and the specific protein
bands were quantified using a PhosphorImager (Molecular Dynamics).
Binding of JFC1 to Phosphoinositides: Dot Blot Assay--
The
binding of in vitro translated [35S]JFC1 to
several phospholipids and phosphoinositides was evaluated by a dot
blot assay as previously described (32) with minor modifications.
Phospholipids at 2 mg/ml in 1:1 chloroform/methanol solution containing
0.1% HCl were spotted (4 µg) onto nitrocellulose sheets. After
drying, nitrocellulose was blocked overnight at 4 °C in
Tris-buffered saline plus 3% bovine serum albumin. In vitro
translated [35S]JFC1 (0.1-0.5 µCi) in Tris-buffered
saline containing 1.5% bovine serum albumin and 300 µM
L-methionine was then used to probe the phosphoinositide-containing nitrocellulose for 30 min at room temperature. Filters were washed five times with Tris-buffered saline
and dried, and bound radioactivity was visualized by autoradiography. Samples were eluted from the nitrocellulose spots containing the radioactive probe and resolved by SDS-PAGE. Radioactivity was visualized using a PhosphorImager (Molecular Dynamics). Negative controls using in vitro translated
[35S]luciferase were performed in parallel.
Binding of JFC1 to
[3H]InsP4--
[3H]InsP4
binding to JFC1 was evaluated as previously described (33) with minor
modifications. Briefly, the reactions, containing 3 µg of GST-JFC1
and 2 nM [3H]InsP4 (10,000 cpm/assay) were carried out in a medium containing 50 mM
Tris-HCl, pH 7.5, and 2 mM EDTA for 10, 20, or 30 min at 4, 30, or 37 °C. The final volume was 200 µl. Free and bound
InsP4 were resolved either by protein precipitation or by
filtration through nitrocellulose. In the first case, protein was
precipitated (15 min on ice) by adding 300 µg of Antibodies--
The antibody against p67phox was raised
against recombinant p67phox purified from baculovirus and was
the generous gift of Robert M. Smith (University of California, San
Diego, La Jolla, CA). The anti-JFC1 antibody was raised by inoculating
rabbits with the N-terminal peptide
NH2-MAHGPKPETEGLLDLS-COOH conjugated to keyhole
limpet hemocyanin (Chiron Mimotopes, San Diego, CA). The antibody was
affinity-purified over a column of Sulfolink-activated Sepharose
(Amersham Pharmacia Biotech) on which a peptide identical except for
the addition of a C-terminal cysteine had been immobilized. The
antibody raised against p47phox has been previously described
(12).
A C2 Domain-containing Protein Interacts with the C-terminal Half
of p67phox--
We were interested in identifying proteins
that might be involved in the modification and regulation of the
cytosolic subunits of the NADPH oxidase. For this purpose, we
constructed a yeast two-hybrid system "bait" vector containing a
fragment of p67phox comprising residues 245-512, spanning both
SH3 domains and the intervening sequence, and used it to screen a
cDNA library derived from Epstein-Barr virus-transformed human B
lymphoblasts, cells known to contain a fully functional NADPH oxidase.
Among the library clones that were found to interact strongly and
specifically with the C-terminal half of p67phox was a sequence
encoding p47phox, which served as a convenient internal
control, and four copies of a sequence that encoded the C-terminal part
of a previously undescribed protein. This protein fragment contained a
region of homology to the C2 domains of a number of proteins. C2
domains have been found in proteins that function in protein
phosphorylation, lipid modification, GTPase regulation, and membrane
trafficking (34). These domains also serve as lipid-binding domains in
various isoforms of protein kinase C (35), the synaptotagmins (36, 37),
rabphilin 3 (38), and the class II PtdIns 3-kinase (39). Because all of
these processes occur in concert with NADPH oxidase activation, we
decided to pursue this clone as a potential regulator of oxidase activity.
A full-length cDNA was retrieved from a Tissue-specific expression of JFC1 mRNA--
We examined the
tissue-specific expression of JFC1 by probing a semiquantitative
mRNA dot blot with the radiolabeled full-length cDNA JFC-1
fragment (Fig. 2). As expected, the clone
was abundantly expressed in bone marrow and lymphoid tissues, which
have a high leukocyte content. There was also significant expression of
JFC1 in pancreas, trachea, stomach, salivary gland, and prostate.
Northern blot analysis of leukocyte mRNA using the library
cDNA as a probe identified a single band at ~1.8 kilobases,
consistent with the size of the full-length cDNA (Fig.
3).
Subcellular Localization of JFC1--
Many C2 domain-containing
proteins are associated with membranous subcellular structures. We
examined the subcellular localization of JFC1 by immunoblotting (Fig.
4). An antibody raised against an
N-terminal peptide from JFC1 recognized a specific doublet in whole
neutrophils that had been lysed by boiling in SDS-PAGE sample buffer
(Fig. 4A), probably representing either different translation starting points or just phosphorylated and unphosphorylated forms of JFC1. JFC1 has been successfully phosphorylated in
vitro by protein kinase C, mitogen-activated protein kinase, and
Ca2+/calmodulin kinase
II.2 When neutrophils were
lysed by nitrogen cavitation, JFC1 segregated to the PNS.
Ultracentrifugation of the PNS yielded soluble (cytosol) and
particulate (membranes/organelles) fractions. JFC1 was detected exclusively to the particulate fraction of neutrophils by the method
employed.
A more detailed fractionation was carried out using a method previously
described (31). Briefly, the PNS was layered onto a two-step
discontinuous Percoll gradient and centrifuged, resulting in the
separation of azurophil granules ( JFC1 Interacts with NADPH Oxidase Components in Vitro--
Before
functional studies were carried out, we confirmed that JFC1 was able to
interact with the p67phox NADPH oxidase component independent
of the yeast two-hybrid system. This was achieved using an affinity
adsorption technique that tests the ability of proteins from neutrophil
cytosol to bind to immobilized GST-JFC1. As described earlier, JFC1 was
identified through the ability of its C-terminal region to associate
with p67phox in a yeast two-hybrid system. Fig.
5 demonstrates that affinity chromatography confirms this association. p67phox was pulled
down from neutrophil cytosol by JFC1-containing beads (Fig.
5A, lower panel, lanes
3 and 5), while GST alone was not effective (Fig.
5A, lower panel, lanes
1 and 2). It is noteworthy that p47phox,
another cytosolic factor essential for the oxidase activity in
vivo, was also pulled down by JFC1 in the same reaction (Fig. 5A, upper panel, lanes
3 and 5). It is very well established that p67phox exists as a stable complex with p47phox in the
cytosol of nonstimulated neutrophils (6), raising the question whether
JFC1 associated independently with both p47phox and
p67phox or whether it bound to p67phox and took up
p47phox pari passu as a
component of the complex. The fact that we were unable to detect
binding of GST-p47phox attached to glutathione Sepharose beads
to in vitro translated [35S]JFC1 (Fig.
5B, upper panel) although
[35S]JFC1 was pulled down by GST-p67phox (Fig.
5B, lower panel) supports the idea
that JFC1 binds to p67phox but not to p47phox. Binding
of the latter occurs because it is complexed to p67phox.
Moreover, increasing the concentration of cytosol in the reaction augmented the binding of p67phox and p47phox to JFC1
(Fig. 5A, lanes 3-5), again
suggesting that they bind as a complex and indicating that JFC1 binds
to a p67phox site that is different from that involved in the
recognition of p47phox.
JFC1 Binds to PtdIns(3,4,5)P3--
As described above,
JFC1 contains two tandem C2 domains that resemble those present in
synaptotagmins and rabphilin 3. The C2A domain of JFC1 is highly
homologous to the synaptotagmin C2B domain (Fig.
6), suggesting that a functional
correlation may exist. The synaptotagmin C2B domain is known to bind
inositol polyphosphates, mainly InsP4, in the absence of
calcium (43). In Fig. 6, we show a comparison of putative inositol
polyphosphate binding domains of various proteins. It is of special
interest that, despite the homology observed between the JFC1-C2A
domain and the polyinositol binding domain of synaptotagmins, the
former was unable to bind [3H]InsP4 when
evaluated either by a previously described precipitation assay (33) or
by nitrocellulose filter binding assays (Fig. 7B).
To evaluate the ability of JFC1 to bind phospholipids and
polyphosphoinositides, we performed dot blot assays as described under
"Experimental Procedures" (SDS-PAGE analyses of samples eluted from
the nitrocellulose spots are shown here). Fig. 7A shows that
in vitro translated [35S]JFC1 bound
specifically to PtdIns(3,4,5)P3 in these assays. JFC1
binding to PtdIns(3,4)P2, although still significant, was 48% lower than that detected with PtdIns(3,4,5)P3,
indicating that the fully phosphorylated head group is necessary for
maximum binding. JFC1 binding to PtdIns(4,5)P2 was only
16.6% of the maximum observed with PtdIns(3,4,5)P3,
suggesting that 3-phosphorylated phosphoinositides play an important
role in JFC1 recognition. In the same way, JFC1 bound to PtdIns(3)P to
the same extent as that detected for PtdIns(4,5)P2. No
significant binding of JFC1 to phosphatidylcholine, phosphatidylserine,
or PtdIns was detected under these assay conditions.
Using a yeast two-hybrid system, we have identified a protein that
interacts with p67phox, a cytosolic factor of the NADPH
oxidase, opening a new chapter in the regulation of this enzymatic
complex. We confirm that JFC1 specifically binds to p67phox in
an in vitro study, using neutrophil cytosol as a source of the cytosolic factors. The polypeptide p67phox is generally
thought to be complexed with p47phox in the cytosol of resting
PMNs (6). Binding between these two factors has been shown to take
place through interaction between the p67phox N-terminal domain
and the p47phox SH3 domain (17), although association between
C-terminal SH3 domain of p67phox and a proline-rich C-terminal
sequence in p47phox has also been shown by the same group (16).
We observed that JFC1 did not bind to p47phox, although
p47phox and JFC1 interacted in the presence of cytosol, which
implies that their association is through an intermediary factor,
presumably p67phox. The fact that JFC1 binds to p67phox
without altering the association of this cytosolic factor with p47phox has physiological significance and suggests that JFC1
could have an important role in vivo. It should be taken
into account that p47phox is present in molar excess over
p67phox in the cytoplasm and that a large percentage of the
protein actually exists in a dissociated form (6). In this work, we
show that augmenting the cytosol concentration in the reaction medium
increased the binding of p67phox to JFC1 and, consequently, the
detection of p47phox, supporting the idea that free
p47phox does not interfere with the recognition of the complex
by JFC1 and that JFC1 recognizes p67phox at a molecular site
different from that involved in the p67phox-p47phox interaction.
Herein we have demonstrated that JFC1 is associated with a subset of
membranes in the phagocyte, although by our method we were not able to
distinguish whether it localized to the secretory vesicles, the plasma
membrane, or both. Therefore, it is unlikely that JFC1 interacts with
the cytosolic complex in unstimulated cells, the components remaining
in different intracellular compartments. Although little is known about
the molecular basis for the up-regulation of the oxidase in the
phagocytic vesicles or at the phagocyte surface, it is general
knowledge that after stimulation, the cytosolic factors translocate to
the particular fraction, suggesting that, in vivo, the
interaction between JFC1 and the cytosolic factors would take place at
some point after cell activation. In the presence of an appropriate
stimulus, the cytosolic component p47phox becomes sequentially
phosphorylated on serines Ser359 and Ser370
followed by Ser303 and Ser304, and the
cytosolic complex migrates to the particulate fraction where it is
known to interact with cytochrome b558 (44).
p67phox also becomes phosphorylated (45), although the
significance of this phosphorylation in the activation of the oxidase
remains unknown. Although there are still aspects of the oxidase
assembly that are unclear, several studies have shown that
p47phox interacts with the p22phox subunit of the
cytochrome b558 (16). Moreover, it has been previously described that the cytosolic factors fail to translocate in
the absence of cytochrome b558 (46), suggesting
that the latter would serve as a docking site. However, a recent
immunofluorescence study examining phagocytosis indicated that the
cytochrome b558, although required for stable
membrane binding, is insufficient for targeting the cytosolic complex
to the cell periphery and suggested that several other adaptor proteins
may be involved in the process (47). Since JFC1 localizes in the
particular fraction where the assembly of the oxidase takes place and
interacts with the cytosolic subunits of the oxidase, it is conceivable that this protein could play such a role in vivo.
As discussed above, JFC1 possesses tandem type I C2 domains in its
C-terminal end. Comparison of the C2A domain of JFC1 with the sequence
alignment of other C2-containing proteins showed that it is highly
homologous to the C2B domains present in synaptotagmins (Fig. 6).
Several functions have been attributed to the synaptotagmin C2B domains
including dimerization, interaction with clathrin assembly protein-2
(48), a process involved in endocytosis of synaptic vesicles, and
binding to The strong binding of JFC1 to PtdIns(3,4,5)P3, a
phosphoinositide whose polar inositol group is InsP4
with the variation that the 1-phosphate is attached to the
diacylglycerol presented here, appears to be an important observation.
The fact that JFC1 binds to PtdIns(3,4,5)P3 but not to
InsP4 indicates that the lipid moiety of the molecule plays
an essential role in the recognition of the phosphoinositide by JFC1.
This establishes a difference between JFC1 and other putative
PtdIns(3,4,5)P3 receptors like Arf-nucleotide-binding site
opener (54) and Bruton's tyrosine kinase (55), since these have been
described to bind not only to PtdIns(3,4,5)P3 but also to
InsP4 (for a review, see Ref. 52). Moreover, synaptotagmin I should also be included in this group, since it has recently been
shown to possess C2B-mediated phosphoinositide binding activity (37).
This supports the idea that the "lysine stretch" moiety in JFC1
could be responsible for its PtdIns(3,4,5)P3 binding capacity.
JFC1 showed preference to PtdIns(3,4,5)P3 over
PtdIns(3,4)P2, bound relatively weakly to PtdIns(3)P and
PtdIns(4,5)P2, and showed no binding to PtdIns(4)P in
vitro (Fig. 7A), suggesting a central role for
3-OH-phosphorylated inositol lipids in JFC1 function. It is well
established that phosphorylation of the D-3 position of
phosphoinositides is catalyzed by members of the PtdIns 3-kinase family
(52). Furthermore, there is clear evidence that PtdIns 3-kinase is
involved in the activation of the NADPH oxidase, although the details
of this mechanism remain to be elucidated. It has been previously shown
that PtdIns(3,4,5)P3 accumulates in fMLP-stimulated
neutrophils (56). Moreover, PtdIns 3-kinase inhibitors markedly
inhibited the NADPH oxidase activation in fMLP-stimulated neutrophils
(57-59). On the other hand, extracellular superoxide anion production
by PMA-stimulated neutrophils has been shown to be unaffected by the
same PtdIns 3-kinase inhibitors (58, 59); thus, the suggestion that
PtdIns 3-kinase is involved in the oxidase activation pathway upstream
of the activation of protein kinase C has been proposed (57). It is
well established that p47phox is a substrate for protein kinase
C (11, 60, 61), and as described above, its phosphorylation is an
essential event for the activation of the oxidase in vivo.
It has been demonstrated that the phosphorylation of this cytosolic
factor is inhibited by the PtdIns 3-kinase inhibitor wortmannin in
fMLP-stimulated PMNs (58), which also correlates with the abolition of
the oxidase activity. However, a recent study showed that
p47phox phosphorylation is only moderately reduced in the
presence of wortmannin in opsonized zymosan-activated PMNs, although
complete inhibition of the oxidase activity was observed at
identical inhibitor concentrations (62). These data suggest that
mechanisms other than the protein kinase C-associated p47phox
phosphorylation take place downstream of PtdIns(3,4,5)P3
formation in association with oxidase activation. Moreover, it was
recently observed that wortmannin inhibits intracellular production of O Finally, it is worth mentioning that although NADPH oxidase is
restricted to leukocytes, PtdIns 3-kinase-related events are ubiquitous, and also JFC1 is expressed in several tissues, mostly with
secretory function. Therefore, it is not unlikely that JFC1 plays a
more general role, namely as an adaptor protein, presumably interacting
with other SH3 domain-containing proteins. This function is currently
under investigation in our laboratory.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-ZAP cDNA
leukocyte library was kindly provided by Dr. Ernest Beutler (The
Scripps Research Institute, La Jolla, CA).
-ZAP was screened by standard filter hybridization methods (29), using the [
-32P]dCTP-labeled
PstI fragment of the yeast two-hybrid clone as a probe.
After isolation, the
-ZAP clone containing the full-length JFC1
insert was circularized to the plasmid pBK-CMV-JFC1 by
Cre-lox-mediated recombination using the ExAssist
helper phage and the Escherichia coli strain XLOLR
(Stratagene, La Jolla, CA).
-ZAP were determined in The Scripps Research Institute molecular
biology facility, using an automated fluorescent dye terminator
sequencer. Overlapping oligonucleotide primers (Life Technologies,
Inc.) were designed so as to obtain complete sequence information from
both strands of the cDNA.
-32P]dCTP
(PerkinElmer Life Sciences) using the Prime-It RmT random primer
labeling kit from Stratagene (La Jolla, CA) and probed as described
elsewhere (29). Multiple Tissue Northern blots (CLONTECH, Palo Alto, CA) were probed with the
full-length EcoRI fragment of the JFC1 cDNA, and the
multiple tissue Northern blots were probed as recommended by the manufacturer.
= 1.076 g/ml; step 2,
= 1.11 g/ml, with tonicity adjusted with buffer A containing
1.25 mM EGTA and protease inhibitors) as previously
described (31). All particulate fractions were washed with buffer A
containing 1.25 mM EGTA to remove Percoll.
-globulin and
18% (final concentration) polyethylene glycol. The reactions were
centrifuged at 12,000 rpm at 4 °C for 5 min, and the supernatant was
removed by aspiration. The pellet was solubilized with 500 µl of
tissue solubilizer; 35 µl of glacial acetic acid was added, the
suspension was transferred to 4.0 ml of liquid scintillation fluid, and
radioactivity was determined by scintillation counting. For filtration
assays, samples were vacuum-filtered on 0.45-µm pore size cellulose
nitrate membrane filters (Whatman, Maidstone, England). For this
purpose, a sampling vacuum manifold (Millipore Corp., Bedford, MA) was used. The filters were immediately washed four times with 2 ml of
ice-cold 50 mM Tris-HCl, pH 7.4, containing 2 mM EDTA in less than 20 s. Filters were counted using
a scintillation counter (Beckman LS 6000SC). Negative control assays
were run in parallel using either bovine serum albumin or GST.
Nonspecific binding was determined in the presence of 10 µM InsP4.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-ZAP human peripheral
blood leukocyte library by conventional plaque screening. The sequence
of this clone is shown in Fig. 1.
Translation of the open reading frame disclosed a 562-amino acid
protein (starting from the first methionine) that contained a second C2
domain just upstream of the one found in the original yeast library
clone (Fig. 1). According to a previously described classification
(40), both C2 domains present in JFC1 correspond to topology I. Comparison of the C2 domains from JFC1 with other C2 domain-containing
proteins by sequence alignment identified that the C2A domain has
structural homology with the synaptotagmin-C2B domains and with
rabphilin 3-C2B domain (27, 41).
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Fig. 1.
The nucleotide sequence and deduced amino
acid sequence of JFC1 cDNA. The nucleotide sequence of the
cDNA is shown as determined by sequencing of the human peripheral
leukocyte library clone. Translation of the predicted open reading
frame is presented below the nucleotide sequence. The
correct frame was deduced by analyzing the incomplete clone derived
from the yeast two-hybrid system and applying it to the full-length
cDNA. The nucleotides and amino acids are numbered on
the side. Two possible start codons are shown on a
black background. The asterisk
indicates the stop codon. The regions bearing homology to C2 domains
are shown on a gray background. The beginning of
the partial yeast two-hybrid clone is indicated by an arrow.
Sequences derived from the cloning process are designated by
lowercase letters.
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Fig. 2.
Distribution of the JFC1 message in human
tissues. The full-length cDNA insert was labeled with
[ -32P]dCTP and used to probe blots of mRNA from
various tissues in a multitissue dot blot as described under
"Experimental Procedures." The corresponding sources of mRNA
dotted onto the membrane are as follows. A1, whole
brain; A2, amygdala; A3, caudate nucleus;
A4 cerebellum; A5, cerebral cortex;
A6, frontal lobe; A7, hippocampus; A8,
medulla oblongata; B1, occipital lobe; B2,
putamen; B3, substantia nigra; B4, temporal lobe;
B5, thalamus; B6, subthalamic nucleus;
B7, spinal cord; C1, heart; C2, aorta;
C3, skeletal muscle; C4, colon;
C5, bladder, C6, uterus; C7, prostate;
C8, stomach; D1, testis; D2, ovary;
D3, pancreas; D4, pituitary gland; D5,
adrenal gland; D6, thyroid gland; D7, salivary
gland; D8, mammary gland; E1, kidney;
E2, liver; E3, small intestine; E4,
spleen; E5, thymus; E6, peripheral leukocyte;
E7, lymph node; E8, bone marrow; F1,
appendix; F2, lung; F3, trachea; F4,
placenta; G1, fetal brain; G2, fetal heart;
G3, fetal kidney; G4, fetal liver; G5,
fetal spleen; G6, fetal thymus; G7, fetal lung;
H1, yeast total RNA 100 ng; H2, yeast tRNA (100 ng); H3, E. coli rRNA (100 ng); H4,
E. coli DNA (100 ng); H5, poly(rA) (100 ng);
H6, human Cot-1 DNA (100 ng); H7, human
DNA (100 ng); H8, human DNA (500 ng).
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Fig. 3.
Leukocyte mRNA Northern blot.
Leukocyte mRNA obtained as described under "Experimental
Procedures" was probed with the full-length EcoRI fragment
of the JFC1 cDNA as described elsewhere (29). The probe was labeled
with [ -32P]dCTP (PerkinElmer Life Sciences) using the
Prime-It RmT random primer labeling kit from Stratagene (La Jolla,
CA).
View larger version (27K):
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Fig. 4.
Subcellular localization of JFC1. Human
neutrophils were disrupted by nitrogen cavitation and fractionated by
centrifugation as described. The presence of JFC1,
p47phox, and p67phox in these fractions was examined by
Western blotting. A, crude fractionation of neutrophil
lysate, probed with anti-JFC1. Lane 1, whole
neutrophils; lane 2, 500 × g
supernatant after nitrogen cavitation (PNS); lane
3, 200,000 × g supernatant; lane
4, 200,000 × g pellet. B, PMA-stimulated
human neutrophils or resting PMNs were disrupted by nitrogen cavitation
and fractionated on a two-step discontinuous Percoll gradient
centrifugation as described under "Experimental Procedures." The
presence of JFC1, p47phox, and p67phox in these
fractions was examined by Western blot. The upper
panel was probed with the antibody raised against
JFC1 described under "Experimental Procedures." The
lower panel was probed with a mixture of
anti-p47phox and anti-p67phox. Lane
1, 500 × g pellet after nitrogen cavitation
(postnuclear pellet (PNP)). Lane 2,
500 × g supernatant (postnuclear supernatant
(PNS)); lane 3, 200,000 × g supernatant; lanes 4-6, fractions
obtained by centrifuging the PNS on a discontinuous Percoll gradient.
C, hydropathy analysis of JFC1. The hydropathy plot was
generated with the OmegaTM 2.0 software (Genetics Computer
Group, Madison, WI) and the method of Kyte and Doolittle with a window
size of nine amino acids (64).
), the specific granules (
),
the plasma membranes and secretory vesicles (
), and a layer of
cytosol. As can be seen in Fig. 4B (upper
panel), JFC1 was detected only in the
fraction of
neutrophils that contain the plasma membranes and secretory vesicles.
JFC1 was not detected in azurophilic or specific granules. Furthermore,
stimulation of the neutrophils by PMA did not have an effect on the
subcellular distribution (Fig. 4B, upper
panel). In contrast, p47phox and p67phox
were found almost exclusively in the cytosol in resting neutrophils, and PMA stimulation induced some of each protein to become associated with the
fraction (Fig. 4B, lower
panel), consistent with earlier findings (42). Despite the
protein's tight association with the membranous structures, a
hydropathy plot of the amino acid sequence did not indicate the
presence of any large hydrophobic regions, suggesting that JFC1 is
peripherally associated with the membrane (Fig. 4C). This
was confirmed by the finding that the protein could be partially eluted
from the membranes by 500 mM NaCl.
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Fig. 5.
Affinity adsorption assays.
A, GST-JFC1 fusion protein was immobilized on
glutathione-agarose beads. Beads (100 µl) containing 10 µg of the
recombinant fusion protein or GST alone were washed twice in RIPA
buffer and incubated overnight at 4 °C in the presence of 1.8 × 107 (lanes 2 and 3) or
9 × 107 (lane 5) cell
equivalents of neutrophil-derived cytosol or buffer (lanes
1 and 4). Beads were washed five times during 10 min with RIPA buffer at 4 °C, spun down, and boiled in Laemmli SDS
sample buffer. Samples were resolved by SDS-PAGE, and proteins were
transferred to nitrocellulose and probed with primary antibodies
directed against p47phox (top panel) or
p67phox (bottom panel). The identity of
the immobilized protein in each assay is indicated above the
lanes. B, the binding of in vitro
translated [35S]JFC1 to GST-p47phox
(upper panel) or to GST-p67phox
(lower panel) was evaluated as described under
"Experimental Procedures." In vitro translated
[35S]p67phox was used as positive control, and
in vitro translated [35S]luciferase
(Luc) was used as negative control.
View larger version (18K):
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Fig. 6.
Sequences alignments of C2 domains containing
a "lysine stretch." Sequence similarity of the C2B domains of
synaptotagmin I (residues 303-352) (SytI; NP-005630);
GTPase-activating protein 1m (residues 181-233) (GAP1m;
AAD09821); Doble C2 (residues 281-330) (DocC2
;
NP-003577); rabphilin 3A (residues 590-639) (Rab3A, A8097);
and the C2A domain of JFC1 (residues 295-342). Lysines described to be
essential for InsP4 in synaptotagmins (43) are indicated by
asterisks. Residues that are identical in the five sequences
are darkly shaded; three or more matches are
lightly shaded.
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Fig. 7.
Binding of JFC1 to phosphoinositides.
A, the binding of in vitro translated
[35S]JFC1 to several phospholipids and phosphoinositides
was evaluated by dot blot assay. Phospholipids were spotted (4 µg)
onto nitrocellulose sheets. After drying, nitrocellulose was blocked
with bovine serum albumin. In vitro translated
[35S]JFC1 (0.1-0.5 µCi) was then used to probe the
phosphoinositide-containing nitrocellulose for 30 min at room
temperature. Filters were washed and dried, and bound radioactivity was
visualized by autoradiography. Samples were eluted from the
nitrocellulose spots containing the radioactive probe and were further
resolved by SDS-PAGE. Radioactivity was visualized using a
PhosphorImager (Molecular Dynamics). PC,
phosphatidylcholine; PS, phosphatidylserine;
PI, phosphatidylinositol. Results shown are representative
of three different experiments. B,
[3H]InsP4 binding to JFC1 was evaluated as
described under "Experimental Procedures." The reactions,
containing 3 µg of GST-JFC1 or an equimolar amount of GST and 2 nM [3H]InsP4 (10,000 cpm/assay)
were carried out during 30 min at 30 °C. The final volume was 200 µl. Free and bound InsP4 were resolved by protein
precipitation.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
SNAP (49) and SNAP25 (50). Notably, several members of
the synaptotagmin family have been shown to bind to the second
messenger InsP4 by an InsP4-binding site
present in their C2B domains (43). Despite possessing a close match to
a putative InsP4-binding site, K(K/R)KTXXK(K/R), in its C2A domain, JFC1 did not bind to the second messenger in an
in vitro binding assay (Fig. 7B). A similar lack
of binding to InsP4 has been demonstrated for
synaptotagmins III, V, and X, although their C-terminal truncated forms
have been shown to bind InsP4. In this case, an inhibitory
effect has been proposed for the C-terminal end of these proteins (43).
JFC1, on the other hand, lacks an inhibitory domain homologous to those
found in synaptotagmins III, V, and X. Another example of a protein that possesses a synaptotagmin-like C2 domain unable to bind
InsP4 is the GTPase-activating protein GAP1m. GAP1m binds
InsP4 by its pleckstrin homology domain (51) but
not by its "lysine stretch-like region" located in its C2B domain,
which is homologous to those present in synaptotagmins (52, 53).
![]() |
FOOTNOTES |
---|
* This work was supported in part by United States Public Health Service Grants AI-44434 and AI-24227.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.
These two authors contributed equally to this work.
§ Predoctoral Fellow of the American Heart Association, California Affiliate, during part of this work. Present address: Fischer & Partners, Inc., 4640 Admiralty Way, Marina del Rey, CA 90292.
¶ Postdoctoral Fellow of the American Heart Association, California Affiliate, and recipient of a fellowship from the American Heart Association, western states affiliate.
Present address: Gen-probe Inc., 10210 Genetic Center Dr., San
Diego, CA 92121-1589.
** To whom correspondence should be addressed. Tel.: 858-784-7935; Fax: 858-784-7981; E-mail: babior@scripps.edu.
Published, JBC Papers in Press, March 13, 2001, DOI 10.1074/jbc.M011167200
2 S. D. Catz and B. M. Babior, unpublished observation.
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ABBREVIATIONS |
---|
The abbreviations used are: SH3, Src homology 3; PtdIns, phosphatidylinositol; PtdIns(4)P, phosphatidylinositol 4-phosphate; PtdIns(4, 5)P2, phosphatidylinositol 4,5-diphosphate; PtdIns(3)P, phosphatidylinositol 3-phosphate; PtdIns(3, 4)P2, phosphatidylinositol 3,4-diphosphate; PtdIns(3, 4,5)P3, phosphatidylinositol 3,4,5-trisphosphate; InsP4, inositol 1,3,4,5-tetrakisphosphate; PMN, polymorphonuclear leukocyte; PNS, postnuclear supernatant; PMA, phorbol 12-myristate 13-acetate; RIPA, radioimmune precipitation assay; GST, glutathione S-transferase.
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REFERENCES |
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