The Adaptor Protein Fish Associates with Members of the ADAMs Family and Localizes to Podosomes of Src-transformed Cells*

Clare L. AbramDagger §, Darren F. Seals||, Ian Pass, Daniel Salinsky, Lisa Maurer**, Therese M. RothDaggerDagger, and Sara A. Courtneidge§§

From Dagger  SUGEN Inc., South San Francisco, California 94080 and  Van Andel Research Institute, Grand Rapids, Michigan 49503

Received for publication, January 9, 2003, and in revised form, February 19, 2003

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Fish is a scaffolding protein and Src substrate. It contains an amino-terminal Phox homology (PX) domain and five Src homology 3 (SH3) domains, as well as multiple motifs for binding both SH2 and SH3 domain-containing proteins. We have determined that the PX domain of Fish binds 3-phosphorylated phosphatidylinositols (including phosphatidylinositol 3-phosphate and phosphatidylinositol 3,4-bisphosphate). Consistent with this, a fusion protein of green fluorescent protein and the Fish PX domain localized to punctate structures similar to endosomes in normal fibroblasts. However, the full-length Fish protein was largely cytoplasmic, suggesting that its PX domain may not be able to make intermolecular interactions in unstimulated cells. In Src-transformed cells, we observed a dramatic re-localization of some Fish molecules to actin-rich structures called podosomes; the PX domain was both necessary and sufficient to effect this translocation. We used a phage display screen with the fifth SH3 domain of Fish and isolated ADAM19 as a binding partner. Subsequent analyses in mammalian cells demonstrated that Fish interacts with several members of the ADAMs family, including ADAMs 12, 15, and 19. In Src-transformed cells, ADAM12 co-localized with Fish in podosomes. Because members of the ADAMs family have been implicated in growth factor processing, as well as cell adhesion and motility, Fish could be acting as an adaptor molecule that allows Src to impinge on these processes.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Fish was originally isolated in a screen to identify Src substrates (1). It has a PX1 domain at its amino terminus and five SH3 domains, as well as multiple polyproline-rich motifs that could mediate association with SH3 domains, several possible phosphorylation sites for both serine/threonine and tyrosine kinases, and potentially four alternatively spliced forms. The presence of these domains and motifs in Fish suggests that it might act as a scaffold or docking molecule for both proteins and lipids.

PX domains are independently folding modules of ~120 amino acids, with an overall hydrophobic character but few totally conserved amino acids. They are frequently found in combination with protein interaction domains such as SH3 domains and exist in a diverse array of proteins with wide ranging functions (2). For example, the p40phox and p47phox subunits of the NADPH oxidase system of phagocytes contain PX domains. The enzymes CISK (cytokine-independent survival kinase) and phospholipase D have PX domains, as do several proteins that function in vesicular sorting (for example the sorting nexins), and proteins involved in cytoskeletal organization (including the yeast bud emergence proteins). The binding capabilities of several PX domains have been reported recently. All PX domains tested bind to phosphorylated phosphatidylinositol lipids. The most common binding partner is PtdIns3P, but some PX domains will bind PtdIns(3,4)P2 and other substituted phosphatidylinositol molecules (3). When PX domains were first identified, it was also noted that many of them contained a PXXP motif, suggesting that they might be able to bind SH3 domains (2). Indeed, structural analysis of the PX domain of p47phox by NMR showed that its PXXP motif is on the surface of the domain and is able to bind to the second SH3 domain of p47phox (4). These data suggested that SH3 binding might impact the ability of a PX domain simultaneously to bind PtdIns3P. In the related protein p40phox, which also has a PXXP motif in its PX domain, lipid and SH3 binding to the PX domain appears to be neither cooperative nor antagonistic (5). However, it remains possible that in other proteins, lipid and SH3 domain binding might influence each other. The PX domain of Fish has both a PXXP motif and the conserved residues required for phospholipid binding.

We recently reported that Fish is a Src substrate both in vitro and in vivo in Src-transformed cells (1). Fish is also tyrosine-phosphorylated in a Src-dependent manner in normal cells treated with concentrations of cytochalasin D that result in rearrangement of the cortical actin cytoskeleton. Furthermore, tyrosine phosphorylation of Fish, albeit with slow kinetics, was detected in Rat1 cells in response to treatment with growth factors such as platelet-derived growth factor, lysophosphatidic acid, and bradykinin that are known to promote changes in the actin cytoskeleton (1). These data suggest that Fish may impact, or be impacted by, cytoskeletal regulation.

The cytoskeleton in Src-transformed cells is grossly abnormal. Very few actin filaments are detected. Instead, much of the F-actin has a ring-like appearance in the cortex of the cells and is found in structures that have been called podosomes (6, 7). Each podosome is a fine, cylindrical, actin-rich structure on the ventral surface of the cell. In Src-transformed fibroblasts, these podosomes cluster to form rings or semi-circles that are called rosettes. Although it was thought that these structures might simply represent remnants of focal adhesions, more recent research has suggested that podosomes are involved in driving locomotion and invasion of Src-transformed cells (8). Podosomes contain a number of cytoskeleton-associated proteins, including N-WASP, cortactin, paxillin, and p190RhoGAP (9-11). Src-transformed cells are not the only cells that contain podosomes; they have also been reported in invasive human breast cancer and melanoma cells (10, 12), raising the possibility that these structures might also be involved in metastatic properties of human tumor cells. Osteoclasts and macrophages also contain structures called podosomes (13). In this case, one large actin ring is formed, from which protrusions emerge that are involved in bone remodeling. It is not yet clear whether the podosomes of osteoclasts and of transformed cells contain the same components.

The metzincin family of metalloproteases contains not just the matrix metalloproteases (some of which co-localize with podosomes) but also ADAMs family proteases (14-17). In addition to a metalloprotease domain, ADAMs proteins have disintegrin, cysteine-rich, and EGF-like domains that are involved in cell adhesion, a membrane spanning sequence, and a cytoplasmic tail. In many members of the ADAMs family, this cytoplasmic tail contains multiple PXXP motifs that can mediate the interaction with SH3 domain-containing proteins. Members of the ADAMs family function as sheddases (by cleaving active growth factors and cytokines from their inactive precursors), as well as mediating cell and matrix interactions.

To isolate proteins that bound to the SH3 domains of Fish, we chose to use a phage display screen. Phage display has been used extensively for the generation of monoclonal antibodies and for screening peptide libraries. Recent advances in technology have allowed larger insert sequences to be expressed on the phage surface, thus facilitating the rapid screening of cDNA libraries against target proteins or peptides (18-20). Phage display does have limitations, such as issues with the production of the target protein in bacterial cells. However, one advantage over other methods is that once reagents are generated, the process is very rapid, with screening taking just a few days. Furthermore, although the two-hybrid system often results in the detection of large numbers of low affinity interactors, phage display screening allows the identification of only the highest affinity interacting molecules.

To determine the role of Fish in signaling pathways downstream of Src, we have begun to look for binding partners of Fish. Given Fish's array of lipid and protein binding domains, we used both lipid binding assays and phage display screens in our analyses. Here we describe the identification of molecules that interact with the PX domain and the fifth SH3 domain of Fish.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cells, Antibodies, Constructs, and General Methodology-- We have described our cell lines, as well as Fish- and Src-specific antibodies before (1). The following antibodies were purchased from commercial sources; anti-phosphotyrosine (4G10) was from Upstate Biotechnology, anti-Myc (9E10) was from Santa Cruz Biotechnology, Inc., and anti-hemagglutinin (12CA5) was from Roche Applied Science. Antibodies specific for GST and ADAM19 were generated by immunizing rabbits with purified GST or GST fused to amino acids 728-807 of ADAM19, respectively. Additional antibodies recognizing ADAM12 and ADAM19 were the kind gifts of Drs. Ulla Wewer (University of Copenhagen, Copenhagen, Denmark) and Anna Zolkiewska (Kansas State University, Manhattan, Kansas). Full-length ADAM19 was cloned by PCR using the cDNA library prepared from NIH3T3 cells for phage display. ADAMs 9 and 12 were the kind gifts of Drs. Deepa Nath (University of East Anglia, East Anglia, United Kingdom) and Ulla Wewer (University of Copenhagen, Copenhagen, Denmark). The fragment of ADAM15 was obtained from the ATCC (clone identification number 592208).

Fragments of Fish containing the PX domain or individual SH3 domains were generated by PCR, subcloned into pGEX-2T or pGEX-4T vectors, and expressed in Escherichia coli DH5 or BL21. Fusions of the green fluorescent protein (GFP) and the PX domain of Fish (amino acids 1-121) were constructed by subcloning a PCR-generated PX domain fragment (XhoI-KpnI ends; contains a Kozak sequence) into pEGFP-N1 (BD Biosciences). The GFP fusion containing two FYVE domains (which bind PtdIns3P) of the adaptor protein Hrs (21) was the kind gift of Dr. Ed Skolnik (The Skirball Institute, New York, NY).

Standard molecular biology techniques were used. Point mutations in the fifth SH3 domain (W1056A) and the PX domain (R42A,R93A) of Fish were generated using the Stratagene Quik Change kit according to manufacturer's instructions. All constructs were confirmed by DNA sequence analysis.

Mammalian cell transient transfections were carried out in the 293 cell line using LipofectAMINE (Invitrogen) and in NIH3T3 cells using LipofectAMINE 2000 (Invitrogen). For stable expression in NIH3T3 cells, ADAM19 and ADAM19-Delta MP were subcloned into the pBABEpuro3 retroviral vector, transfected using calcium phosphate (CellPhect Transfection kit; Amersham Biosciences) according to manufacturer's instructions, and selected for growth in puromycin (6 µg/ml). Immunoprecipitation analyses, gel electrophoresis, and immunoblotting were carried out according to standard procedures. Detailed protocols are available on request.

GST Fusion Protein Production, Purification, and Use-- Protein expression in bacterial cultures was induced with 0.2 mM isopropyl-1-thio-beta -D-galactopyranoside, bacteria were lysed, and then the fusion proteins were affinity-purified using glutathione-Sepharose, eluted, and, where required, coupled to cyanogen bromide-activated Sepharose (Amersham Biosciences) according to the manufacturer's instructions.

NIH3T3 cells were grown to 50% confluence in 10-cm dishes and incubated for 4.5 h in methionine-and cysteine-free Dulbecco's modified Eagle's medium containing 0.5 mCi of [35S]methionine and [35S]cysteine and then lysed in Nonidet P-40 lysis buffer. 150 µg of lysate was incubated with individual GST fusion proteins bound to glutathione-Sepharose for 4 h at 4 °C. Fusion protein-associated proteins were analyzed by SDS-PAGE and fluorography.

Phage Display-- The methodology was essentially as described in Ref. 18. NIH3T3 cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum (Invitrogen). poly(A)+ RNA was purified from the cells using a FastTrack 2.0 mRNA purification kit (Invitrogen). Randomly primed cDNA was synthesized, ligated with adapters, and uni-directionally cloned into the EcoRI-HindIII-cut T7Select1-1b phage using a T7Select1-1 cloning kit (Novagen) according to the manufacturer's instructions. The cDNA was size-selected by agarose gel electrophoresis to exclude fragments of less than 500 bp. The library contained 1 × 107 primary recombinants with an average insert length of 0.7 to 0.8 kb, as determined by PCR analysis of 24 randomly picked plaques. The library was amplified once in liquid culture before use. To prepare the cDNA library for panning, 15 ml of an overnight culture of E. coli BL5615 cells (Novagen) grown in Terrific Broth-ampicillin (100 µg/ml) was diluted with 45 ml of Terrific Broth-ampicillin and infected with ~1010 plaque-forming unit of library stock and then grown at 37 °C with good aeration until complete lysis. The lysate was cleared by centrifugation (15 min, 18,000 × g), filtered through a 0.45 µM filter, then supplemented with 1% (v/v) of E. coli protease inhibitors mixture (P-8465; Sigma-Aldrich) and 10× Pan Mix (Novagen) at 9:1 (v/v) ratio. The 10× Pan Mix contains 5% Nonidet P-40 (Fluka), 10% nonfat dry milk (Carnation brand; Nestle), 10 mM EGTA, 250 µg/ml of heparin (H-2149; Sigma), 250 µg/ml of boiled, sheared salmon sperm DNA (D8661; Sigma), 0.05% sodium azide, 10 mM sodium vanadate, and 250 mM sodium fluoride in Dulbecco's PBS (14190-144; Invitrogen) base. 1 ml of the lysate prepared as described above was incubated with 15 µl of the Sepharose-coupled GST fusion protein at 4 °C for 30 min (see "GST Fusion Protein Production, Purification, and Use" above for more details). The beads were collected by centrifugation and washed three to four times by complete resuspension in 1.5 ml of PBS wash buffer (PBS supplemented with 0.5% Triton X-100 and 25 µg/ml heparin). After the final wash, the beads were eluted with 100 µl of 1% SDS for 15 min at room temperature. The eluate was separated from beads, added to 1 ml of overnight culture of E. coli BL5615 cells diluted with 3 ml of Terrific Broth-ampicillin (100 µg/ml), and incubated at 37 °C with vigorous shaking until cell lysis was complete. The lysate was clarified by centrifugation (10 min, 10,000 × g) and filtration, supplied with 10× Pan Mix, and subjected to the next panning round. After the third and final panning round, serially diluted phage eluate was used to infect BL5403 cells and plated on LB-ampicillin (100 µg/ml) plates. Plaques appeared after 3-4 h of incubation at 37 °C. Plaques were picked, and the inserts were amplified by PCR with T7-specific primers and sequenced.

Lipid Binding Analysis-- Protein-lipid overlay assays were performed as described by Deak et al. (22). Individual lipids, PIP strips, and PIP arrays were obtained from Echelon Biosciences.

Fluorescence Microscopy-- Cells were grown for a minimum of 48 h post-passage or post-transfection on glass coverslips. Cells were fixed in 3% formaldehyde (Electron Microscopy Sciences)/PBS for 10 min, permeabilized in 0.1% Triton X-100/PBS for 10 min, and processed with various antibody applications in 5% donkey serum (Jackson ImmunoResearch Laboratories)/PBS. Slides were analyzed using an Axioplan 2 fluorescent microscope (Zeiss) using the appropriate filters, and images were captured with Axiovision 3.0 software (Zeiss).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The PX Domain of Fish-- It was reported recently (23-26) that many PX domains have the ability to bind phosphorylated phosphatidylinositol lipids. To test whether the Fish PX domain bound lipids, we prepared GST fusion proteins of the wild-type Fish PX domain and a mutant PX domain (PXdA) with arginines 42 and 93 mutated to alanines. These residues are conserved between different PX domains and have been shown to disrupt lipid binding function in other PX domains (27, 28). We prepared nitrocellulose membranes spotted with several different lipids, as well as using commercially prepared PIP strips, to show that the Fish PX domain bound to PtdIns3P and, less strongly, to PtdIns(3,4)P2. Much weaker binding was seen to other phospholipids (data not shown). To confirm these findings, we used a PIP array spotted with phosphoinositides of different concentrations (Fig. 1). In this analysis the Fish PX domain bound most strongly to PtdIns3P and PtdIns(3,4)P2 with less binding detected to other phosphoinositides. The PXdA mutant was unable to bind to lipids.


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Fig. 1.   Phosphoinositide binding specificity of the Fish PX domain. Phosphatidylinositol (PtdIns) and the indicated phosphorylated derivatives were spotted onto nitrocellulose membranes at, from left to right, doubling dilutions of lipids ranging from 100 to 1.6 pmol/spot. Membranes were incubated with purified fusion proteins of GST and either the wild-type (FishPXwt) or mutated (FishPXdA; R42A,R93A) PX domain of Fish. Bound fusion proteins were detected by blotting with a GST antibody.

The Subcellular Localization of Fish-- We next began to explore the subcellular localization of Fish. We first generated a protein consisting of GFP fused to the PX domain of Fish. We transiently transfected this construct into NIH3T3 cells and determined its subcellular localization by fluorescence analysis. We noted a punctate distribution of the protein (Fig. 2A). This punctate staining was not seen with GFP alone or with a fusion protein of GFP and the PXdA mutant (data not shown). FYVE domains also bind PtdIns3P, and it has been shown that proteins containing FYVE domains are found associated with early endosomal membranes (21). Indeed, cells transfected with a fusion protein of GFP and two copies of the FYVE domain of the adaptor protein Hrs (GFP-FYVE) showed a punctate distribution typical of early endosomes (Fig. 2B). The similarities in punctate staining seen with both GFP-PX and GFP-FYVE domain localization suggest that the isolated Fish PX domain may also be able to associate with PtdIns3P in endosomal membranes. However, because both domains were fused to GFP, we were unable to compare the localizations of PX and FYVE domains in the same cell. Thus it remains possible that the Fish PX domain can also associate with other subcellular structures.


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Fig. 2.   The subcellular localization of Fish in normal cells. A, NIH3T3 cells were transfected with a fusion of GFP with the PX domain of Fish, and the localization of the protein was detected by fluorescence. B, NIH3T3 cells were transfected with a fusion of GFP with tandom FYVE domains of the adaptor protein Hrs, and the localization of the protein was detected by fluorescence. C, the localization of Fish in NIH3T3 cells was determined by immunocytochemistry with a Fish-specific antibody.

We went on to examine the localization of the full-length Fish protein by immunofluorescence analysis of fixed NIH3T3 cells using Fish-specific antibodies (Fig. 2C). In contrast to the isolated PX domain, Fish showed a more generalized cytoplasmic distribution. The same result was obtained with several different Fish antibodies (data not shown). The lack of detection of punctate staining in these experiments suggests that, in the context of the full-length protein, the PX domain of Fish may be unable to associate intermolecularly with lipid and/or protein targets.

We next examined the subcellular localization of Fish in Src-transformed NIH3T3 cells (Fig. 3A). We noticed a striking re-localization of some fraction of Fish from the cytoplasm to the cell periphery, where it was co-localized with F-actin. These rings or semi-circles of intense actin staining have been observed before (6, 7) in Src-transformed cells and correspond to rosettes of podosomes. To determine whether the PX domain might be involved in the podosomal localization of Fish, we first transfected Src-transformed NIH3T3 cells with GFP-PX (Fig. 3B). Some of this construct did indeed localize to podosomes. In contrast, a GFP-FYVE protein gave typical punctate early endosomal staining in the Src-transformed cells (Fig. 2C). These data suggest that, in Src-transformed cells, the PX domain of Fish may be preferentially interacting with a lipid or protein target in podosomes.


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Fig. 3.   Localization of the Fish PX domain in Src-transformed cells. A, the localization of Fish in Src-transformed cells was determined by immunocytochemistry with a Fish-specific antibody (left). The localization of actin in the same cells was determined by co-staining with TRITC-conjugated phalloidin (right). B, Src-transformed NIH3T3 cells were transfected with a fusion of GFP and the PX domain of Fish. The localization of GFP-PX was determined by GFP fluorescence (left), and the localization of actin in the same cells was determined by co-staining with TRITC-conjugated phalloidin (right). C, Src-transformed NIH3T3 cells were transfected with a fusion of GFP and tandom FYVE domains of Hrs. The localization of GFP-FYVE was determined by GFP fluorescence (left), and the localization of actin in the same cells was determined by co-staining with TRITC-conjugated phalloidin (right).

We asked whether the PX domain of Fish is necessary for localization of the full-length molecule to podosomes. As a positive control, we first transfected Src-transformed cells with a tagged, full-length version of Fish and determined that it was present in podosomes (Fig. 4A). We then tested a tagged version of Fish lacking its PX domain (FishDelta PX). This protein, in contrast to the full-length version, was unable to localize to podosomes and instead only showed the diffuse cytoplasmic staining (Fig. 4B). Thus the PX domain of Fish is both necessary and sufficient to target Fish to the podosomes of Src-transformed cells.


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Fig. 4.   Localization of Fish in Src-transformed cells. A, Src-transformed NIH3T3 cells were transfected with Fish-myc. The left panel shows the localization of Fish by immunocytochemistry using the myc epitope antibody, and the right panel shows the localization of F-actin using TRITC-conjugated phalloidin. B, Src-transformed NIH3T3 cells were transfected with FishDelta PX-myc. The left panel shows the localization of FishDelta PX by immunocytochemistry using the myc epitope antibody, and the right panel shows the localization of F-actin using TRITC-conjugated phalloidin.

The Association of Fish with Members of the ADAMs Family-- To determine whether Fish has protein binding partners, we incubated GST-SH3 domain fusion proteins with [35S]methionine- and [35S]cysteine-labeled lysates of NIH3T3 cells (Fig. 5). Each SH3 domain bound selected proteins from the lysates compared with the GST control. However, SH3#1, SH3#2, and SH3#4 each had a limited number of binding partners (visible on longer exposures), whereas SH3#3 and SH3#5 showed broader binding capacities. Full characterization and comparison of the binding capacity of each SH3 domain will require the identification of the molecular nature of each of these protein bands. We began this analysis by isolating and characterizing the binding partners of SH3#5.


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Fig. 5.   Fish-associated proteins. Purified fusion proteins of GST and the various SH3 domains of Fish and GST alone were bound to glutathione-Sepharose beads prior to the addition of NIH3T3 cell lysates metabolically labeled with [35S]methionine/[35S]cysteine. Bound proteins were eluted, separated by SDS-PAGE, and analyzed by fluorography. Molecular mass markers (in kDa) are shown on the left.

We chose to use phage display to isolate proteins binding to the fifth SH3 domain of Fish. Briefly, a cDNA library was made from NIH3T3 cells, size-selected, and cloned into the appropriate phage vector (18). We immobilized GST-SH3#5 on glutathione-Sepharose beads and conducted three successive rounds of phage panning. A total of 24 plaques were obtained. Phage inserts were subcloned and analyzed by sequencing (Table I). Most clones isolated expressed proteins with PXXP domains, suggesting that the method was able to detect SH3 domain-interacting sequences. However some clones encoded secreted proteins (TIMP-1 and SPARC) that are unlikely to associate with Fish in a physiological setting. Strikingly, however, we independently isolated a region corresponding to a portion of the cytoplasmic tail of ADAM19 a total of 18 times in our first screen. In a second, independent screen we isolated ADAM19 a further six times (data not shown). Fig. 6A shows the topography of ADAM19 and the region isolated in the phage display screen.


                              
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Table I
Phage display results
A phage display screen was conducted as described under "Experimental Procedures." At the end of three rounds of panning, 24 phage were picked, and the inserts were cloned and sequenced. The results of the sequence analysis for each phage are shown in the first column. The second column denotes how many of the 24 phage isolates contained the sequence, and the third column provides some information on the identity of the sequences obtained.


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Fig. 6.   Association of SH3#5 with ADAM19. A, the domain structure of ADAMs family metalloproteases. The double-headed arrow marks the region of ADAM19 that was identified in the Fish SH3#5 domain interaction screen. SS, signal sequence. TM, transmembrane domain. B, purified fusion proteins of GST alone and GST fused to the various SH3 domains of Fish were bound to glutathione-Sepharose beads prior to the addition of 293 cell line lysates expressing a portion of the cytoplasmic tail region of ADAM19 tagged with the myc epitope (A, PD19-myc in text) or pcDNA3 vector alone (V). mSH3#5 refers to a mutated SH3#5 domain (W1056A). Immunoblots of whole cell lysates are shown at the far left. Interactions between PD19-myc and the GST fusion proteins were detected with the myc epitope antibody. The arrow marks the position of PD19-myc. C, the 293 cell line was transfected with the indicated combinations of pcDNA3 vector, ADAM19-myc, Fish, and/or Fish carrying a mutation in SH3#5 (W1056A). Lysates (WCL) were either assayed directly for ADAM19-myc using an ADAM19 antibody (upper blot) or were immunoprecipitated (IP) with a Fish antibody and blotted with either antibodies to Fish (middle blot) or ADAM19 (lower blot). The arrow in the lower panel marks the position of ADAM19 that was co-immunoprecipitated with Fish. D, lysates (WCL) prepared from stable NIH3T3 cell lines harboring pBABEpuro3 vector alone (V2, V7) or vector containing either ADAM19-myc (M8, M11) or ADAM19-myc lacking its metalloprotease domain (Delta 2, Delta 8) were assayed directly for ADAM19-myc expression with an ADAM19 antibody (upper blot). Immunoprecipitation of these lysates with either myc (middle blot) or Fish (lower blot) antibodies were used to assay ADAM19-myc/Fish interactions and relative Fish expression, respectively, by blotting with a Fish antibody. The arrow in the middle panel marks the position of Fish that was co-immunoprecipitated with ADAM19. Note that in these cells, Fish is detected as a closely migrating triplet of bands.

To determine whether the interaction between SH3#5 and ADAM19 could be detected in vitro, we engineered a myc tag at the 5' end of the ADAM19 fragment obtained in the screen (PD19) and subcloned it into a mammalian expression vector. The 293 cell line was transiently transfected with either empty vector or vector containing myc-tagged PD19. Lysates from these cells were passed over glutathione-Sepharose to which was bound GST-SH3 domains, and bound proteins were analyzed by immunoblotting (Fig. 6B). SH3#5 was able to bind to myc-tagged PD19 in this assay, whereas a point-mutated version in which the ligand binding surface is disrupted by mutation of tryptophan 1056 to alanine (mSH3#5) was not. Furthermore, none of the other SH3 domains of Fish were able to interact with myc-tagged PD19, demonstrating the specificity of the interaction. In separate experiments, we showed that myc-tagged PD19 also associated with full-length Fish protein but not with a full-length Fish molecule containing the mSH3#5 (data not shown).

To test whether full-length ADAM19 bound to Fish, ADAM19 was cloned by PCR from NIH3T3 cell cDNA, engineered to contain a carboxyl-terminal myc tag, and subcloned into an expression vector. We also generated an antibody specific for the amino-terminal portion of the cytoplasmic tail of ADAM19, which detected a specific band of ~115 kDa in lysates from the 293 cell line that had been transfected with the myc-tagged ADAM19 construct (data not shown). Co-transfection of ADAM19 with either wild-type Fish or Fish containing the mSH3#5 mutant showed that ADAM19 bound to wild-type Fish in cells but not to the mSH3#5 mutant (Fig. 6C, bottom panel). In these transfected cells, the interaction between Fish and ADAM19 was constitutive and did not appear to require tyrosine phosphorylation of Fish (data not shown). A form of ADAM19 that lacks the protease domain (Delta MP) also interacted with Fish, implying that protease activity is also not required for the interaction to occur (data not shown). The available antibodies were not of sufficiently high affinity to precipitate endogenous ADAM19 from cells. We therefore generated stable NIH3T3 cell clones expressing modest levels of myc-tagged ADAM19 or myc-tagged ADAM19Delta MP (Fig. 6D). We could co-precipitate endogenous Fish with ADAM19 in these cell lines (middle panel). Again, the interaction was independent of the protease activity of ADAM19, as the Delta MP mutant also bound to Fish.

Several ADAMs family proteins contain proline-rich regions in their cytoplasmic tails (14) and might therefore also be able to bind Fish. To test this, we transfected cells with tagged constructs expressing either full-length (ADAM9 and ADAM12), or the cytoplasmic tail (ADAM15), of different ADAMs. We determined that ADAMs 12 and 15, but not ADAM9, associated with Fish (Fig. 7A, middle panel). Fig. 7B shows PXXP motif(s) in ADAMs 12 and 15 that may mediate the association with Fish.


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Fig. 7.   Fish associates with several ADAMs family proteins. A, the 293 cell line was transfected with the indicated combinations of pcDNA3 vector, Fish, Fish carrying a mutation (W1056A) in SH3#5 (Fishm5), ADAM12-myc, ADAM19-myc, ADAM9-myc, and/or the carboxyl-terminal region of ADAM15 tagged with the myc epitope. Relative Fish and ADAM levels were determined by probing Fish antibody immunoprecipitates (IP) with a Fish antibody (upper blot) or myc antibody immunoprecipitates with the myc epitope antibody (lower blot). ADAM/Fish interactions were determined by probing Fish antibody immunoprecipitates with the myc epitope antibody (middle blot). The closed arrows designate the bands corresponding to ADAM12-myc and ADAM19-myc, and the open arrow marks the position of ADAM15Cterm-myc. B, amino acid sequence comparison of portions of the cytoplasmic tail region of ADAM12, 15, and 19. The boxed residues indicate 100% sequence identity.

Finally, we wanted to determine the subcellular localization of ADAMs in normal and Src-transformed cells. Because of a lack of suitable reagents, we were only able to examine ADAM12 (Fig. 8). In normal NIH3T3 cells (Fig. 8A), ADAM12 showed a diffuse cytoplasmic localization, consistent with previous observations (29). However, in Src-transformed cells, we also noted a co-staining of a fraction of ADAM12 with F-actin in podosomes (Fig. 8B). Furthermore, co-staining analysis of Src-transformed cells also showed that Fish and ADAM12 co-localized (Fig. 8C). These results strongly suggest that the association of Fish and ADAMs occurs in vivo and co-localizes these proteins to podosomes.


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Fig. 8.   ADAM12 co-localizes with Fish in podosomes in Src-transformed cells. A, NIH3T3 cells were probed with an antibody to ADAM12 (left), and F-actin was visualized by co-staining with TRITC-conjugated phalloidin (right). B, NIH3T3 cells transformed with Src were probed with an antibody to ADAM12 (left), and F-actin was visualized by co-staining with TRITC-conjugated phalloidin (right). C, NIH3T3 cells transformed with Src were co-stained with antibodies specific for ADAM12 (left) and Fish (right).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

We have demonstrated that the PX domain of Fish binds predominantly to PtdIns3P and also to PtdIns(3,4)P2, on filters spotted with the lipids. Many PX domains appear to bind PtdIns3P (3, 30, 31), but binding to PtdIns(3,4)P2 is more unusual. Only the PX domain of p47phox, which is most closely related to the Fish PX domain, has also been shown to bind PtdIns(3,4)P2 (23). Although the filter binding assays of the type we used here may not reveal the full specificity or complexity of binding of a given PX domain, the data we have obtained are consistent with our subcellular localization analyses. For example, the isolated PX domain of Fish, when expressed as a GFP fusion protein, was distributed in the cytoplasm in a punctate fashion (that may reflect an endosomal localization) in normal fibroblasts. In contrast to the isolated PX domain, the full-length Fish protein did not show a punctate staining pattern. Rather, it appeared to be more diffusely cytoplasmic. These data suggest that, in the context of the full-length protein, the PX domain of Fish might not be able to make intermolecular contacts. A similar situation has been observed with the related protein, p47phox. In unstimulated neutrophils, p47phox is cytoplasmic, and its PX domain makes an intramolecular contact with its second SH3 domain. Stimulation of the neutrophil results in p47phox adopting an open conformation and associating with membranes containing PtdIns(3,4)P2 (4, 32, 33). A similar mechanism may exist for Fish. Indeed, we have preliminary evidence that the PX domain of Fish makes contact with the third SH3 domain and that this intramolecular interaction is released by Src phosphorylation.2

We observed a striking re-distribution of Fish in Src-transformed cells, with much of the protein found co-localized with actin in structures called podosomes. The PX domain of Fish was both necessary and sufficient to localize to podosomes. Interestingly, in the same cell type, a GFP-FYVE domain fusion protein was localized to the endosomal compartment via interaction with PtdIns3P. These data suggest that, in these Src-transformed cells, the Fish PX domain binds lipids other than PtdIns3P and that this may account for the Fish localization observed. The most likely candidate lipid is PtdIns(3,4)P2, which was able to bind to the PX domain in vitro and which can be produced from phosphatidylinositol 3,4,5-trisphosphate by the action of a 5-phosphatase (34), or by the action of PtdIns3P 4-kinases on PtdIns3P (35). Alternatively, it is also possible that the PX domain of Fish is targeted to podosomes via interaction with a protein. We are currently determining whether podosomes contain PtdIns(3,4)P2 and whether the PX domain must bind either lipid and/or protein to associate with podosomes.

Podosomes are interesting structures, found normally in invasive cells such as osteoclasts and macrophages (13) but also present in Src-transformed cells and invasive breast carcinoma and melanoma cell lines (6-8, 10, 12, 36). They are actin-rich protrusions of ~0.4 µm in diameter that extend from underneath the cell body into the extracellular matrix. Podosomes are very dynamic structures, with a half-life in the order of minutes compared with focal adhesions, which are stable for several hours (8). Podosomes contain several actin-binding proteins (see the Introduction), many of which are also Src substrates. In addition, and of interest in regard to the ability of podosomes to degrade the extracellular matrix, the podosomes of Src-transformed cells also contain the matrix metalloprotease MT1-MMP (also known as MMP14), which processes the gelatinases MMP2 and MMP9 to their active forms (37). Other podosome-associated proteins include beta 1 integrins (38), the tyrosine kinase Pyk2 (in osteoclasts) (39, 40), and Src itself (8, 41).3 We now show that, in Src-transformed cells, podosomes contain both Fish and ADAM12.

We have demonstrated that the fifth SH3 domain of the Src substrate, Fish, binds to the cytoplasmic tail of several ADAMs family metalloproteases, particularly ADAMs 12, 15, and 19. Members of the ADAMs family are characterized by the presence of protease and disintegrin domains in the extracellular region, in addition to a pro-domain, a cysteine-rich region, and an EGF-like domain, followed by a transmembrane domain and a cytoplasmic tail of variable length. ADAMs have functions in many different cell processes, including myoblast fusion, fertilization, cell fate determination, and growth factor and cytokine processing (14-17). On a biochemical level, the ADAMs have three distinctive properties. First, the disintegrin domain (probably in conjunction with the cysteine-rich and EGF-like domains) has the ability to associate with integrin receptors, particularly those containing beta 1 subunits, and therefore can modulate cell/cell interactions (14, 42). Second, many of the ADAMs are active proteases that act as sheddases, that is they act to release active growth factors and cytokines from cell surfaces. Third, many of the ADAMs have extended cytoplasmic tails that mediate interactions with several proteins, both with and without SH3 domains.

In this study we identified ADAMs 12, 15, and 19, but not ADAM9, as binding partners for Fish. Each of these ADAMs contains multiple PXXP motifs in their cytoplasmic tails (14), and indeed, some binding partners for these ADAMs have already been reported. These include the association of the SH3 domain-containing proteins Grb2, phosphatidylinositol 3-kinase, and Src with ADAM12 (43, 44) and endophilin 1, Grb2, SH3PX1, and Src family kinases with ADAM15 (45, 46). The cytoplasmic domains of ADAMs 12, 15, and 19 contain polyproline-rich motifs that likely represent the binding site for the Fish SH3 domain. Whether the association of Fish with various ADAMs family members is regulated by Src or Fish acts as an adaptor to allow Src to regulate the ADAMs family remains to be determined.

It has been suggested that the cytoplasmic tails of the ADAMs may connect intracellular signals to the extracellular activity of these proteins. For example, overexpression of the cytoplasmic tail of ADAM12 inhibits myoblast fusion (47), and similar overexpression of the ADAM9 tail inhibits 12-O-tetradecanoylphorbol-13-acetate-induced heparin-binding EGF shedding (48). Presumably this inhibition occurs because the isolated cytoplasmic tails sequester proteins that normally bind to the full-length protein and are required for ADAMs function. Our future analyses will therefore involve testing the effect of Fish binding on ADAMs function in both normal and Src-transformed cells.

    ACKNOWLEDGEMENTS

We thank Ulla Wewer, Anna Zolkiewska, Deepa Nath, and Ed Skolnik for gifts of antibodies and clones and Robert Blake for early immunofluorescence analysis and critical reading of the manuscript.

    FOOTNOTES

* The Fish research in S. A. C.'s laboratory was generously supported by the Van Andel Research Institute.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.

§ Contributed equally to this work.

|| Contributed equally to this work.

** Present address: Kenyon College, Gambier, OH 43022.

Dagger Dagger Present address: Program in Biomedical Sciences, University of Michigan, Ann Arbor, MI.

§§ To whom correspondence should be addressed: Van Andel Research Inst., 333 Bostwick N.E., Grand Rapids, MI 49503. Tel.: 616-234-5704; Fax: 616-234-5705; E-mail: sara.courtneidge@vai.org.

Published, JBC Papers in Press, March 3, 2003, DOI 10.1074/jbc.M300267200

2 D. Salinsky, C. L. Abram, and S. A. Courtneidge, unpublished observations.

3 L. Maurer and S. A. Courtneidge, unpublished observations.

    ABBREVIATIONS

The abbreviations used are: PX, phox homology; GFP, green fluorescent protein; GST, glutathione S-transferase; PtdIns3P, phosphatidylinositol 3-phosphate; PtdIns(3, 4)P2, phosphatidylinositol 3,4-bisphosphate; EGF, epidermal growth factor; SH, Src homology; PBS, phosphate-buffered saline; TRITC, tetramethylrhodamine iso- thiocyanate.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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