From 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
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ABSTRACT |
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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.
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.
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- GST Fusion Protein Production, Purification, and
Use--
Protein expression in bacterial cultures was induced with 0.2 mM isopropyl-1-thio-
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).
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.
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.
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.
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 (Fish 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.
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.
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 (
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.
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.
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
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 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.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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.
-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.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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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.
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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.
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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).
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 Fish PX-myc. The left panel shows
the localization of Fish
PX by immunocytochemistry using the myc
epitope antibody, and the right panel shows the localization
of F-actin using TRITC-conjugated phalloidin.
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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.
Phage display results
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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
( 2,
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.
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 ADAM19
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
MP mutant also bound to 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.
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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
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.
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.
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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.
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.
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ABBREVIATIONS |
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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.
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