JFC1, a Novel Tandem C2 Domain-containing Protein Associated with the Leukocyte NADPH Oxidase*

Jeanne K. McAdara BerkowitzDagger§, Sergio D. CatzDagger, Jennifer L. Johnson, Julie M. Ruedi, Vicki Thon||, and Bernard M. Babior**

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


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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&cjs1138;2) at the expense of NADPH (1, 2). Free radical production is directly related to the bactericidal capacity of the cell, since patients with chronic granulomatous disease, whose NADPH oxidase is inactive (3, 4), suffer recurrent bacterial infections.

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.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 lambda -ZAP cDNA leukocyte library was kindly provided by Dr. Ernest Beutler (The Scripps Research Institute, La Jolla, CA).

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 lambda -ZAP was screened by standard filter hybridization methods (29), using the [alpha -32P]dCTP-labeled PstI fragment of the yeast two-hybrid clone as a probe. After isolation, the lambda -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).

Sequencing-- The sequences of both the yeast two-hybrid cDNA fragment in pACTII and the full-length JFC1 cDNA in lambda -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.

RNA Blot Analysis-- Leukocyte RNA was obtained as previously described (29). The probe was labeled with [alpha -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.

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, delta  = 1.076 g/ml; step 2, delta  = 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.

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 gamma -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.

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).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 lambda -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.

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).


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Fig. 2.   Distribution of the JFC1 message in human tissues. The full-length cDNA insert was labeled with [alpha -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 [alpha -32P]dCTP (PerkinElmer Life Sciences) using the Prime-It RmT random primer labeling kit from Stratagene (La Jolla, CA).

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.


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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).

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 (alpha ), the specific granules (beta ), the plasma membranes and secretory vesicles (gamma ), and a layer of cytosol. As can be seen in Fig. 4B (upper panel), JFC1 was detected only in the gamma  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 gamma  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.

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.


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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.

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).


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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 C2alpha (residues 281-330) (DocC2alpha ; 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.

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.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 beta  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).

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&cjs1138;2 in PMA-activated neutrophils, leading to the suggestion that PtdIns 3-kinase is not necessarily upstream of protein kinase C in the oxidase signaling pathway (63). Thus, other events might be involved downstream of PtdIns 3-kinase in the signaling pathway that lead to the activation of the oxidase. In this way, it has been indicated that members of the unidentified "renaturable kinases" take action downstream of PtdIns 3-kinase during human neutrophil oxidase activation, while mitogen-activated protein kinase and mitogen-activated protein kinase/extracellular signal-regulated kinase kinase would be involved in the secretory pathway, also downstream of PtdIns(3,4,5)P3 formation (59). In conclusion, while there is no doubt about the essential participation of PtdIns(3,4,5)P3 in the activation of the oxidase, the signaling pathways downstream of PtdIns 3-kinase that direct the activation remain uncertain. We speculate that JFC1 could act as an accessory factor in the regulation of the oxidase, since it seems directly to establish a connection between an early event associated with oxidase activation (i.e. PtdIns(3,4,5)P3 generation) and an oxidase subunit itself.

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.

    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.

Dagger 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.

    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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ABSTRACT
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RESULTS
DISCUSSION
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