The Src substrate Fish binds to members of the ADAMs family

Fish, a Src substrate and ADAMs family binding protein

The adaptor protein Fish associates with members of the ADAMs family

and localizes to podosomes of Src-transformed cells.

Clare L. Abram 1,2, Darren F. Seals 1,3, Ian Pass3, Daniel Salinsky3, Lisa Maurer3,4,

Therese M. Roth3,5, & Sara A. Courtneidge3,6

1these authors contributed equally to this work 2 SUGEN Inc., 230 East Grand Ave, South San Francisco, CA 94080 3 Van Andel Research Institute, 333 Bostwick NE, Grand Rapids, MI 49503 4present address: Kenyon College, Gambier, OH 5present address: Program in Biomedical Sciences, University of Michigan,

Ann Arbor, MI 6to whom correspondence should be addressed:

phone: 616 234 5704

fax: 616 234 5705

email: [email protected]

Keywords: metalloproteases, integrins, invadopodia, SH3 domain, PX domain

Running title: Fish, a Src substrate and ADAMs family binding protein

1

Copyright 2003 by The American Society for Biochemistry and Molecular Biology, Inc.

JBC Papers in Press. Published on March 3, 2003 as Manuscript M300267200 by guest on February 16, 2018

http://ww

w.jbc.org/

Dow

nloaded from

mailto:[email protected]

http://www.jbc.org/


Abbreviations used: PX, phox homology; ADAM, a disintegrin and metalloprotease

protein; GFP, green fluorescent protein; NMR, nuclear magnetic resonance; GST,

glutathione S-transferase.

2

by guest on February 16, 2018http://w

ww

.jbc.org/D

ownloaded from

http://www.jbc.org/


Summary

Fish is a scaffolding protein and Src substrate. It contains an amino terminal PX

domain, five 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 PtdIns3P and PtdIns(3,4)P2).

Consistent with this, a fusion protein of green fluorescent protein (GFP) 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. Since 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.

3


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/


Introduction

Fish was originally isolated in a screen to identify Src substrates (1). It has a PX

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.

Phox homology (PX) domains are independently folding modules of

approximately 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 recently been

reported. All PX domains tested bind to phosphorylated phosphatidylinositol (PtdIns)

lipids. The most common binding partner is PtdIns3P, but some PX domains will bind

PtdIns(3,4)P2 and other substituted PtdIns 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 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

4


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/


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. While 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

5


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/


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, while the 2-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.

In order 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 5th SH3 domain of Fish.

6


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/


Experimental procedures

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) from Upstate Biotechnology, anti-Myc (9E10) from Santa Cruz

Biotechnology and anti-HA (12CA5) 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, 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, UK) and Ulla Wewer (University of Copenhagen, Denmark).

The fragment of ADAM15 was obtained from the ATCC (Clone Id 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 E.

coli DH5 or BL21. Fusions of the green fluorescent protein (GFP) and the PX domain of

Fish (aa 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 2 FYVE domains (which bind PtdIns3P) of the adaptor protein Hrs

(21) was the kind gift of Dr. Ed Skolnik (The Skirball Institute, New York, New York).

Standard molecular biology techniques were used. Point mutations in the fifth

SH3 domain (W1056A) and the PX domain (R42,93A) 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-∆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 (6µg/ml). Immunoprecipitation

7


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/


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 0.2mM IPTG, bacteria

lysed, then the fusion proteins 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 10cm dishes, and incubated 4.5 hrs.in

methionine and cysteine free DMEM containing 0.5mCi of 35S-methionine and 35S-

cysteine, then lysed in NP40 lysis buffer. 150 µg of lysate was incubated with

individual GST fusion proteins bound to glutathione Sepharose for 4 hours at 4oC.

Fusion protein-associated proteins were analyzed by SDS-PAGE and fluorography.

Phage display

The methodology was essentially as described in (18). NIH3T3 cells were grown

in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal calf serum

(FCS) (Gibco-BRL). Poly(A)+ RNA was purified from the cells using 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 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 500bp. The library contained 1x107 primary recombinants with an average

insert length 0.7–0.8kb, 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, 15ml of an overnight culture of E. coli BL5615 cells (Novagen)

grown in Terrific Broth (TB)-ampicillin (100µg/ml) was diluted with 45ml of TB-

ampicillin and infected with ~1010 p.f.u. of library stock, then grown at 37°C with

good aeration until complete lysis. The lysate was cleared by centrifugation (15mins,

18,000 g), filtered through a 0.45µM filter, then supplemented with 1% vol/vol of E.

coli protease inhibitors cocktail (Sigma-Aldrich, P-8465) and 10x Pan Mix (Novagen) at

8


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/


9:1 (vol/vol) ratio. The 10x Pan Mix contains 5% NP-40 (Fluka), 10% nonfat dry milk

(Carnation brand, Nestle), 10mM EGTA, 250µg/ml of heparin (Sigma, H-2149),

250µg/ml of boiled, sheared salmon sperm DNA (Sigma, D8661), 0.05% sodium azide,

10mM sodium vanadate, and 250mM sodium fluoride in Dulbecco's PBS (GibcoBRL,

14190-144) base. 1ml of the lysate prepared as described above was incubated with

15µl of the Sepharose-coupled GST fusion protein at 4oC for 30mins (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 1.5ml of PBS wash buffer (PBS supplemented with 0.5% Triton X-100 and 25µg/ml

heparin). After the final wash, the beads were eluted with 100µl of 1% SDS for 15 min

at room temperature. The eluate was separated from beads, added to 1ml of

overnight culture of E. coli BL5615 cells diluted with 3ml of TB-ampicillin (100µg/ml),

and incubated at 37°C with vigorous shaking until cell lysis was complete. The lysate

was clarified by centrifugation (10mins, 10,000 g) and filtration, supplied with 10x

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 (100µg/ml) plates. Plaques appeared after 3–4h incubation at 37°C. Plaques

were picked, the inserts amplified by PCR with T7 specific primers and sequenced.

Lipid binding analysis

Protein-lipid overlay assays were performed as described in 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 hours post-passage or post-transfection

on glass coverslips. Cells were fixed in 3% formaldehyde (Electron Microscopy

Sciences)/PBS for 10 minutes, permeablized in 0.1% TritonX100/PBS for 10 minutes,

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).

9


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/


Results

The PX domain of Fish

It was recently reported that many PX domains have the ability to bind

phosphorylated phosphatidylinositol lipids (23-26). In order 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 (Figure 1). In this analysis also, 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 green fluorescent protein (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 (Figure 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 (Figure 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, since both

domains were fused to GFP, we were unable to compare the localizations of PX and

10


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/


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

(Figure 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 (Figure 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 in Src

transformed cells, and correspond to rosettes of podosomes (6,7). 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 (Figure 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 (Figure 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 (Figure 4A). We then tested a tagged version of Fish lacking

its PX domain (Fish∆PX). This protein, in contrast to the full-length version, was

unable to localize to podosomes, and instead only showed the diffuse cytoplasmic

staining (Figure 4B). Thus the PX domain of Fish is both necessary and sufficient to

target Fish to the podosomes of Src-transformed cells.

11


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/


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-labelled lysates of

NIH3T3 cells (Figure 5). Each SH3 domain bound selected proteins from the lysates,

compared to 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 1). 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 eighteen times in our first screen. In a second, independent screen

we isolated ADAM19 a further six times (data not shown). Figure 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 analyzed by

immunoblotting (Figure 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

12


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/


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 carboxy-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 approximately 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 (Figure

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 (∆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 (Figure 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.

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 (ADAM 9 and 12), or the

cytoplasmic tail (ADAM15), of different ADAMs. We determined that ADAMs 12 and 15,

but not ADAM9, associated with Fish (Figure 7A, middle panel). Figure 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. Due to a lack of suitable reagents, we were only

13


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/


able to examine ADAM12 (Figure 8). In normal NIH3T3 cells (Figure 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 (Figure 8B). Furthermore, co-staining analysis of

Src-transformed cells also showed that Fish and ADAM12 co-localized (Figure 8C).

These results strongly suggest that the association of Fish and ADAMs occurs in vivo,

and co-localizes these proteins to podosomes.

14


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/


Discussion

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). While 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 (unpublished observations).

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 PtdIns(3,4,5)P3 by the action of a 5-

15


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/


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 approximately 0.4 µm in diameter, which 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 to focal adhesions, which are stable for

several hours (8). Podosomes contain several actin-binding proteins (see

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 β1 integrins (38), the

tyrosine kinase Pyk2 (in osteoclasts) (39,40) and Src itself (unpublished observations

and 8,41). 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. Firstly, 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 β1 subunits, and

16


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/


therefore can modulate cell/cell interactions (14,42). Secondly, 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. Thirdly, many of the ADAMs have extended

cytoplasmic tails which 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, PI 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 ADAM

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 TPA-induced HB-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. Fish research in SAC’s laboratory is generously

supported by the Van Andel Research Institute.

17


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/


References

1. Lock, P., Abram, C. L., Gibson, T., and Courtneidge, S. A. (1998) Embo J 17,

4346-4357

2. Ponting, C. P. (1996) Protein Sci 5, 2353-2357

3. Sato, T. K., Overduin, M., and Emr, S. D. (2001) Science 294, 1881-1885

4. Hiroaki, H., Ago, T., Ito, T., Sumimoto, H., and Kohda, D. (2001) Nat Struct

Biol 8, 526-530

5. Bravo, J., Karathanassis, D., Pacold, C. M., Pacold, M. E., Ellson, C. D.,

Anderson, K. E., Butler, P. J., Lavenir, I., Perisic, O., Hawkins, P. T., Stephens,

L., and Williams, R. L. (2001) Mol Cell 8, 829-839

6. David-Pfeuty, T., and Singer, S. J. (1980) Proc Natl Acad Sci U S A 77, 6687-

6691

7. Tarone, G., Cirillo, D., Giancotti, F. G., Comoglio, P. M., and Marchisio, P. C.

(1985) Exp Cell Res 159, 141-157

8. Chen, W. T. (1989) J Exp Zool 251, 167-185

9. Mizutani, K., Miki, H., He, H., Maruta, H., and Takenawa, T. (2002) Cancer Res

62, 669-674

10. Bowden, E. T., Barth, M., Thomas, D., Glazer, R. I., and Mueller, S. C. (1999)

Oncogene 18, 4440-4449

11. Nakahara, H., Mueller, S. C., Nomizu, M., Yamada, Y., Yeh, Y., and Chen, W.

T. (1998) J Biol Chem 273, 9-12

12. Monsky, W. L., Lin, C. Y., Aoyama, A., Kelly, T., Akiyama, S. K., Mueller, S. C.,

and Chen, W. T. (1994) Cancer Res 54, 5702-5710

13. Teti, A., Marchisio, P. C., and Zallone, A. Z. (1991) Am J Physiol 261, C1-7

14. Seals, D. F., and Courtneidge, S. A. (2003) Genes Dev 17, 7-30

15. Primakoff, P., and Myles, D. G. (2000) Trends Genet 16, 83-87

16. Black, R. A., and White, J. M. (1998) Curr Opin Cell Biol 10, 654-659

17. Schlondorff, J., and Blobel, C. P. (1999) J Cell Sci 112 ( Pt 21), 3603-3617

18. Zozulya, S., Lioubin, M., Hill, R. J., Abram, C., and Gishizky, M. L. (1999) Nat

Biotechnol 17, 1193-1198

19. Crameri, R., and Suter, M. (1993) Gene 137, 69-75

18


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/


20. Crameri, R., Jaussi, R., Menz, G., and Blaser, K. (1994) Eur J Biochem 226, 53-

58

21. Gillooly, D. J., Morrow, I. C., Lindsay, M., Gould, R., Bryant, N. J., Gaullier, J.

M., Parton, R. G., and Stenmark, H. (2000) Embo J 19, 4577-4588

22. Deak, M., Casamayor, A., Currie, R. A., Downes, C. P., and Alessi, D. R. (1999)

FEBS Lett 451, 220-226

23. Kanai, F., Liu, H., Field, S. J., Akbary, H., Matsuo, T., Brown, G. E., Cantley,

L. C., and Yaffe, M. B. (2001) Nat Cell Biol 3, 675-678

24. Ellson, C. D., Gobert-Gosse, S., Anderson, K. E., Davidson, K., Erdjument-

Bromage, H., Tempst, P., Thuring, J. W., Cooper, M. A., Lim, Z. Y., Holmes, A.

B., Gaffney, P. R., Coadwell, J., Chilvers, E. R., Hawkins, P. T., and Stephens,

L. R. (2001) Nat Cell Biol 3, 679-682

25. Cheever, M. L., Sato, T. K., de Beer, T., Kutateladze, T. G., Emr, S. D., and

Overduin, M. (2001) Nat Cell Biol 3, 613-618

26. Xu, J., Liu, D., Gill, G., and Songyang, Z. (2001) J Cell Biol 154, 699-705

27. Xu, Y., Seet, L. F., Hanson, B., and Hong, W. (2001) Biochem J 360, 513-530

28. Wishart, M. J., Taylor, G. S., and Dixon, J. E. (2001) Cell 105, 817-820

29. Hougaard, S., Loechel, F., Xu, X., Tajima, R., Albrechtsen, R., and Wewer, U.

M. (2000) Biochem Biophys Res Commun 275, 261-267

30. Yu, J. W., and Lemmon, M. A. (2001) J Biol Chem 276, 44179-44184

31. Ellson, C. D., Andrews, S., Stephens, L. R., and Hawkins, P. T. (2002) J Cell Sci

115, 1099-1105

32. Ago, T., Nunoi, H., Ito, T., and Sumimoto, H. (1999) J Biol Chem 274, 33644-

33653

33. Karathanassis, D., Stahelin, R. V., Bravo, J., Perisic, O., Pacold, C. M., Cho,

W., and Williams, R. L. (2002) Embo J 21, 5057-5068

34. Erneux, C., Govaerts, C., Communi, D., and Pesesse, X. (1998) Biochim Biophys

Acta 1436, 185-199

35. Banfic, H., Tang, X., Batty, I. H., Downes, C. P., Chen, C., and Rittenhouse, S.

E. (1998) J Biol Chem 273, 13-16

19


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/


36. Mueller, S. C., Ghersi, G., Akiyama, S. K., Sang, Q. X., Howard, L., Pineiro-

Sanchez, M., Nakahara, H., Yeh, Y., and Chen, W. T. (1999) J Biol Chem 274,

24947-24952

37. Nakahara, H., Howard, L., Thompson, E. W., Sato, H., Seiki, M., Yeh, Y., and

Chen, W. T. (1997) Proc Natl Acad Sci U S A 94, 7959-7964

38. Mueller, S. C., and Chen, W. T. (1991) J Cell Sci 99 ( Pt 2), 213-225

39. Pfaff, M., and Jurdic, P. (2001) J Cell Sci 114, 2775-2786

40. Sanjay, A., Houghton, A., Neff, L., DiDomenico, E., Bardelay, C., Antoine, E.,

Levy, J., Gailit, J., Bowtell, D., Horne, W. C., and Baron, R. (2001) J Cell Biol

152, 181-195

41. Mueller, S. C., Yeh, Y., and Chen, W. T. (1992) J Cell Biol 119, 1309-1325

42. Evans, J. P. (2001) Bioessays 23, 628-639

43. Kang, Q., Cao, Y., and Zolkiewska, A. (2001) J Biol Chem 276, 24466-24472

44. Suzuki, A., Kadota, N., Hara, T., Nakagami, Y., Izumi, T., Takenawa, T., Sabe,

H., and Endo, T. (2000) Oncogene 19, 5842-5850

45. Howard, L., Nelson, K. K., Maciewicz, R. A., and Blobel, C. P. (1999) J Biol

Chem 274, 31693-31699

46. Poghosyan, Z., Robbins, S. M., Houslay, M. D., Webster, A., Murphy, G., and

Edwards, D. R. (2002) J Biol Chem 277, 4999-5007

47. Galliano, M. F., Huet, C., Frygelius, J., Polgren, A., Wewer, U. M., and Engvall,

E. (2000) J Biol Chem 275, 13933-13939

48. Izumi, Y., Hirata, M., Hasuwa, H., Iwamoto, R., Umata, T., Miyado, K., Tamai,

Y., Kurisaki, T., Sehara-Fujisawa, A., Ohno, S., and Mekada, E. (1998) Embo J

17, 7260-7272

20


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/


Figure Legends

Figure 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;

R42,93A) PX domain of Fish. Bound fusion proteins were detected by blotting with a

GST antibody.

Figure 2. The subcellular localization of Fish in normal cells

Panel 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 immunofluorescence.

Panel B. NIH3T3 cells were transfected with a fusion of GFP with the FYVE domain of

the adaptor protein Hrs, and the localization of the protein was detected by

immunofluorescence.

Panel C. The localization of Fish in NIH3T3 cells was determined by

immunocytochemistry with a Fish-specific antibody.

Figure 3. Localization of the Fish PX domain in Src-transformed cells

Panel 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).

Panel 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).

Panel C. Src-transformed NIH3T3 cells were transfected with a fusion of GFP and the

FYVE domain of Hrs. The localization of GFP-FYVE was determined by GFP

21


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/


fluorescence (left), and the localization of actin in the same cells was determined by

co-staining with TRITC-conjugated phalloidin (right).

Figure 4. Localization of Fish in Src-transformed cells

Panel 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 the localization of F-actin using TRITC-conjugated

phalloidin.

Panel 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 the localization of F-actin using TRITC-

conjugated phalloidin.

Figure 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 weight

markers are shown on the left.

Figure 6. Association of SH3#5 with ADAM19

Panel A. The domain structure of ADAMs family metalloproteases. The double-

arrowed line marks the region of ADAM19 that was identified in the Fish SH3#5 domain

interaction screen. SS = signal sequence. TM = transmembrane domain.

Panel 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

22


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/


proteins were detected with the myc epitope antibody. The arrow marks the position

of PD19-myc.

Panel 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 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.

Panel 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.

Figure 7. Fish associates with several ADAMs family proteins

Panel A. The 293 cell line was transfected with the indicated combinations of either

pcDNA3 vector, Fish, Fish carrying a mutation (W1056A) in SH3#5 (Fishm5), ADAM12-

myc, ADAM19-myc, ADAM9-myc, and/or the C-terminal region of ADAM15 tagged with

the myc epitope. Relative Fish and ADAM levels were determined by probing Fish

antibody immunoprecipitates 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 immunoprecipiates with the

myc epitope antibody (middle blot). The closed arrows designate the bands

corresponding to ADAM12-myc, ADAM19-myc, and the open arrow marks the position

of ADAM15Cterm-myc.

Panel B. Amino acid sequence comparison of portions of the cytoplasmic tail region of

ADAM12, 15, and 19. The boxed residues indicate 100% sequence identity.

23


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/


Figure 8. ADAM12 co-localizes with Fish in podosomes in Src-transformed cells.

Panel A. NIH3T3 cells were probed with an antibody to ADAM12 (left), and F-actin was

visualized by co-staining with TRITC-conjugated phalloidin (right).

Panel 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).

Panel C. NIH3T3 cells transformed with Src were co-stained with antibodies specific

for ADAM12 (left) and Fish (right).

24


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/


25

Table 1. Phage display results

Identity of

clone

# of

times

obtained

Comments

ADAM19 18 C-terminal portion of cytoplasmic region, many PxxP

motifs.

TIMP-1 1 C-terminal portion, one PxxP motif. Secreted protein.

SPARC 1 C-terminal portion, one PxxP motif. Secreted protein.

Novel 1 Three PxxP motifs.

- 2 Background. Only a few amino acids fused to phage

capsid protein.

- 1 Sequence failed.

A phage display screen was conducted as described in Experimental Procedures. At

the end of three rounds of panning, twenty four phage were picked, and the inserts

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 twenty four phage

isolates contained the sequence, and the third column provides some information on

the identity of the sequences obtained.


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

and Sara A. CourtneidgeClare L. Abram, Darren F. Seals, Ian Pass, Daniel Salinsky, Lisa Maurer, Therese M. Roth

to podosomes of Src-transformed cellsThe adaptor protein Fish associates with members of the ADAMs family and localizes

published online March 3, 2003J. Biol. Chem.

10.1074/jbc.M300267200Access the most updated version of this article at doi:

Alerts:

When a correction for this article is posted•

When this article is cited•

to choose from all of JBC's e-mail alertsClick here


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/lookup/doi/10.1074/jbc.M300267200

http://www.jbc.org/cgi/alerts?alertType=citedby&addAlert=cited_by&cited_by_criteria_resid=jbc;M300267200v1&saveAlert=no&return-type=article&return_url=http://www.jbc.org/content/early/2003/03/03/jbc.M300267200.citation

http://www.jbc.org/cgi/alerts?alertType=correction&addAlert=correction&correction_criteria_value=early/2003/03/03/jbc&saveAlert=no&return-type=article&return_url=http://www.jbc.org/content/early/2003/03/03/jbc.M300267200.citation

http://www.jbc.org/cgi/alerts/etoc

http://www.jbc.org/

The Src substrate Fish binds to members of the ADAMs family

Documents

Transcript of The Src substrate Fish binds to members of the ADAMs family