Cancer Research UK Laboratories, Beatson Institute for Cancer Research, Switchback Road, Bearsden, Glasgow, G61 1BD, UK
* Author for correspondence (e-mail: d.black{at}beatson.gla.ac.uk)
Accepted 18 November 2002
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Summary |
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Key words: TES, Focal adhesion, LIM domain, Cell spreading
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Introduction |
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TES is widely expressed in normal tissues and is predicted to
encode a highly conserved protein of 421 amino acids containing three
C-terminal LIM domains. LIM domains are approximately 55 amino-acid
zinc-binding double finger motifs, with the consensus amino-acid sequence
CX2CX17-19HX2(C/H)X2CX2CX17-19CX2(C/H/D)
(Dawid et al., 1998). LIM
domains were first identified in three developmentally important transcription
factors Lin-11, Isl-1 and Mec-3
(Freyd et al., 1990
; Karlsson,
1990; Way and Chalfie, 1988
).
Subsequently, many LIM-domain-containing proteins have been identified that
play key roles in a diverse range of biological processes including
differentiation, cytoskeletal organisation and oncogenesis
(Arber et al., 1994
;
Brown et al., 1996
;
Fisch et al., 1992
;
Sattler et al., 2000
). Several
studies have demonstrated that LIM domains function as protein-protein
interaction motifs that are involved in both intramolecular and intermolecular
interactions (Feuerstein et al.,
1994
; Nagata et al.,
1999
). In some instances, LIM domain proteins are thought to act
as scaffolds on which multiprotein complexes are formed
(Arber and Caroni, 1996
).
To elucidate the function of TES, we examined the subcellular localisation
of the TES protein and performed yeast two-hybrid screens to identify
TES-interacting proteins. Here we report that TES localises to focal adhesions
and areas of cell-cell contact. In addition, we show that TES is a novel
interacting partner of the known focal adhesion proteins mena, zyxin and
talin. Focal adhesions are multiprotein complexes linking the cytoskeleton and
signal transduction pathways with integrins at the extracellular matrix (ECM)
(Burridge and Chrzanowska-Wodnicka,
1996; Critchley,
2000
). Focal-adhesion-associated proteins may function to modulate
cell adhesion, migration or cell signalling events
(Burridge and Chrzanowska-Wodnicka,
1996
; Critchley,
2000
). The localisation of TES to regions of cell-cell and
cell-substratum contact suggests that TES has a role in cell adherence, cell
communication and, possibly, cell motility.
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Materials and Methods |
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Cell culture and stable transfections
Rat-1 fibroblast and HeLa cervical carcinoma cells were routinely grown at
37°C under a humidified atmosphere of 5% CO2 in DMEM
supplemented with 1% glutamine and 10% FBS. Cells were routinely passaged at
70-80% confluency using 10% trypsin.
For stable transfections, 1x106 cells were seeded into 100 mm dishes and transfected with either 1 µg of pEGFP-C1 vector or GFP-tagged TES constructs using Effectene reagent (Qiagen) as recommended by the manufacturer. Stable colonies were selected using G418 (600 µg/ml), and pools of stable colonies were sorted on the basis of fluorescence using a Becton Dickinson FACS vantage SE.
Plasmid construction
Plasmids used to express TES proteins were created by sub-cloning
PCR-amplified human TES cDNA into the appropriate expression vector.
Constructs were created that encoded full-length TES (amino acids 1-421 in
pEGFP-C1, pGEX-2T, pAS2-1 and pACT2), LIM-less TES (amino acids 1-241 in
pEGFP-C1 and 1-233 in pGEX-2T), LIM-only TES (amino acids 231-421 in
pEGFP-C1), LIM 1 TES (amino acids 230-297 in pGEX-2T), LIM 1 and 2 TES (amino
acids 230-360 in pGEX-2T) and LIM 3 TES (amino acids 351-421 in pGEX-2T) (see
Fig. 1A). Human zyxin cDNA was
obtained by restriction digest of pEGFP-N1/zxyin (gift from J. Wehland and K.
Rottner, Braunschweig, Germany) with HindIII and BamHI and
subcloned into the HindIII and BamHI sites of pcDNA3.1/HisC
for in vitro transcription/translation using T7. All constructs were verified
by sequencing.
|
Immunofluorescence
Fixation and staining of cells for immunofluorescence were performed as
described previously (Gonzalez et al.,
1993). Briefly, the coverslips were fixed with 3.7% formaldehyde
and, where necessary, permeabilised with 0.5% (v/v) TritonX-100 and incubated
with the appropriate primary antibody overnight at 4°C at the following
dilutions: anti-paxillin 1/200, anti-zyxin hybridoma supernatant (164D4),
phalloidin-TRITC 1/500 and anti-mena 1/50. Coverslips were incubated with the
appropriate TRITC-labelled secondary antibody where necessary, mounted in
vectashield (Vector Laboratories) and immunofluorescence performed using a
Leica SP2 confocal microscope at 63x magnification.
Glutathione S-transferase (GST) pull-down assays and western
blotting
Whole cell extracts (WCE) were obtained after incubating cells in lysis
buffer (0.5% (v/v) NP40, 250 mM NaCl, 50 mM HEPES pH 7.4, 5 mM EDTA, 50 mM
NaF, 200 µM sodium-orthovanadate, 50 mM ß-glycerophosphate, 1 mM PMSF,
10 µg/ml aprotinin, 10 µg/ml leupeptin) for 30 minutes on ice. Extracts
were clarified by centrifugation at 17,500 g for 10 minutes at
4°C. pGEX-2T vector containing the cDNA sequence encoding full-length TES
(Fig. 1) was expressed in
E. coli as a GST-fusion protein. Bacterial cultures were induced
using isopropyl-ß-D-thiogalactopyranoside (IPTG), and lysates were
incubated with glutathione sepharose 4B for 30 minutes at room temperature. 2
µg of GST-fusion protein coupled to glutathione sepharose or 2 µg of GST
alone were incubated with 500 µg of WCE in cell lysis buffer overnight at
4°C. GST complexes were centrifuged at 1000 g for 1
minute, and the pellet was washed four times with cell lysis buffer before
resuspending it in 20 µl of Laemmli SDS-loading buffer. GST-pull downs
using in-vitro-translated zyxin were performed as above using 10 µl of
rabbit reticulocyte lysate reaction.
For western blotting and immune detection, a 20 µl sample was run on a
10% SDS-PAGE gel according to the method of Laemmli
(Laemmli, 1970). Blots were
transferred to a PVDF membrane (Millipore) using a semi-dry transfer apparatus
and membranes blocked in 5% skimmed milk TBS-T [Tris-buffered saline
containing 0.2% (v/v) Tween-20] for 1 hour at room temperature. Blots were
incubated overnight with mouse anti-talin (1/1000), anti-mena (1/250),
anti-zyxin (1/5000) and anti-VASP (1/1000) antibodies in 5% skimmed milk TBS-T
at 4°C. Blots were washed before incubation with anti-mouse HRP (1/3000 in
5% skimmed-milk TBS-T) for 1 hour at room temperature. Blots were then washed
with TBS-T and protein detected using ECL reagent (Amersham) according to the
manufacturer's instructions.
Yeast two-hybrid screens
Yeast two-hybrid screens and controls were performed as described in the
Clontech Matchmaker II literature (BD Clontech). The full-length TES
open reading frame cloned into the yeast two-hybrid GAL4 DNA-binding domain
(DBD) vector, pAS2-1, was used as a bait to screen both a human mammary gland
cDNA library and a pre-transformed mouse testis cDNA library cloned into the
GAL4 activation domain (AD) vector, pACT-2. Full-length TES-expressing S.
cerevisiae Y190 cells were either transformed with a human mammary gland
cDNA library or mated with S. cerevisiae Y187 cells containing a
pre-transformed mouse testis cDNA library and plated onto selective medium as
described in the manufacturer's protocol. 1.1x106
transformants from the human mammary gland cDNA library and
5.6x107 transformants from the mouse testis cDNA library were
tested for lacZ reporter gene expression by a colony lift filter
assay using 5-bromo-4-chlor-3-indolyl-ß-D-galactopyranoside (X-gal) as
the substrate. Plasmid DNA was isolated from positive colonies (Nucleon yeast
DNA isolation kit, Tepnel Life Sciences, UK), amplified with pACT2-specific
primers and sequenced on an ABI 3700 automated sequencer. Clone identities
were determined by non-redundant and dbEST searches using the BlastN and
BlastX programs at the NCBI web site
(http://www.ncbi.nlm.nih.gov/BLAST/).
Cell spreading
Rat-1 fibroblasts stably overexpressing pEGFP-C1 or GFP-tagged full-length
TES were trypsinised, washed and pelleted. 1x105 cells in
serum-free DMEM containing 1% (w/v) bovine serum albumin and 600 µg/ml G418
were re-seeded onto six-well plates containing fibronectin-coated coverslips
(25 µg/ml) according to the manufacturer's instructions (Calbiochem). The
plates were incubated at 37°C in a 5% CO2/95% air atmosphere
for the time periods specified in the results. Cells were fixed using 3.7%
formaldehyde and where necessary stained with TRITC-labelled phalloidin. They
were observed using a Leica SP2 confocal microscope at 63x
magnification.
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Results |
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To confirm that TES was in fact localised to focal adhesion structures, cells stably overexpressing GFP-tagged full-length TES were stained with an antibody to the well characterised focal adhesion protein paxillin. As expected, endogenous paxillin strongly localised to distinct focal adhesions (Fig. 1Bviii), and full-length TES often colocalised with paxillin at focal adhesions (Fig. 1Bix), confirming that TES is a novel component of focal adhesions.
Additionally, the intracellular localisation of LIM-only TES (Fig. 1A) was determined. We observed that the LIM-only region of TES stably overexpressed in Rat-1 cells strongly localised to focal adhesions (Fig. 2A,D) where it colocalised with paxillin (Fig. 2F). The LIM domains alone localised to areas of cell-cell contact (Fig. 2B,C); however, we did not observe localisation along actin stress fibres (Fig. 2A-D). By contrast, removing the LIM-domains from TES (Fig. 1) resulted in the loss of the majority of focal adhesion localisation (Fig. 2G-I) with little colocalisation with paxillin at focal adhesions (Fig. 2M-O). The majority of LIM-less TES localised throughout the cell along actin stress fibres (Fig. 2G,H,J,M) where it colocalised with actin (Fig. 2J-L). There was also some localisation of LIM-less TES at areas of cell-cell contact (Fig. 2I). Similar results were seen with Rat-1 cells transiently and stably expressing His-tagged LIM-less TES (data not shown).
|
Together, these data demonstrate that the LIM-domains of TES contain information important for targeting full-length TES to focal adhesions, and both the LIM domains and the LIM-less region of TES can be targeted to regions of cell-cell contact. Note that there were no gross qualitative differences in focal adhesion appearance in any of the TES stable cell lines that we have seen. In addition, our data suggest that the LIM-less region of TES contains sequences that may be involved in targeting TES to actin stress fibres. Co-staining Rat-1 cells stably overexpressing GFP-tagged full-length TES with phalloidin demonstrated that TES localised predominantly to the tips of actin stress fibres found at both focal adhesions and sites of cell-cell contact (Fig. 3A). Treatment of Rat-1 cells with the actin-disrupting agent latrunculin B prevents the formation of stress fibres and prevents localisation of TES to focal adhesions and regions of cell-cell contact (Fig. 3B), which suggests that the localisation of TES is actin dependent. Similar results were seen with cytochalasin B treatment (data not shown). Furthermore, GST-tagged TES can precipitate endogenous actin from HeLa whole cell extracts, suggesting that TES may interact with actin (Fig. 4H).
|
|
Yeast two-hybrid screens to identify potential TES interacting
proteins
In order to identify TES-interacting proteins, yeast two-hybrid screens
were performed using full-length human TES as bait to screen both a human
mammary gland and a mouse testis cDNA library.
Table 1 summarises some of the
proteins identified after screening approximately 7x106
transformants from the two cDNA libraries as described in the Materials and
Methods. Most of the proteins identified in the screens were focal-adhesion-
and/or cytoskeleton-related proteins, supporting our data on the localisation
of TES. Interestingly, two of the TES-interacting clones from the human
library encoded TES from amino acid 25 onwards
(Table 1A) and one of the
TES-interacting clones from the mouse library encoded mouse Tes from amino
acid 189 onwards (Table 1B).
The mouse Tes gene has previously been cloned
(Divecha and Charleston,
1995), but no function has thus far been described. The
interaction between full-length TES and TES in yeast suggests that it may
interact with itself, and data we have obtained from yeast mating experiments
and in vitro pull-down assays suggest that the LIM domains of TES are able to
interact with the LIM-less region of TES (data not shown).
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TES interacts with the adhesion proteins mena, zyxin and talin
TES interacted with several well characterised focal adhesion proteins in
yeast two-hybrid assays (Table
1), which supports our findings that TES is localised to focal
adhesions. One of the TES-interacting clones encodes zyxin from amino acid 381
onwards (Table 1A). Zyxin is a
572 amino acid, low abundance focal adhesion protein that, like TES, contains
three C-terminal LIM domains (Macalma et
al., 1996). GFP-tagged TES colocalised with endogenous zyxin at
focal adhesions and areas of cell-cell contact in Rat-1 cells
(Fig. 4A). In addition, strong
colocalisation was seen with endogenous zyxin in primary CEF cells stably
expressing myc-tagged full-length TES (data not shown). Despite a positive
interaction in yeast and colocalisation of TES and zyxin in vivo, an
interaction of the zyxin with full-length TES in either immunoprecipitation or
GST pull-down assays was not consistently observed despite exhaustive efforts.
Interestingly, we found that endogenous zyxin from HeLa cell extracts
interacted most strongly with the isolated GST-tagged LIM domain 1 of TES
(Fig. 4C). This interaction
appears to be direct as in-vitro-translated zyxin interacts with GST-tagged
LIM 1 (Fig. 4D). Given the fact
that the LIM domains of TES can interact with the N-terminal LIM-less region
(Table 1; data not shown), it
is likely that either an intramolecular interaction or conformational change
in TES accounts for the difficulties that we have observed in identifying an
interaction between full-length TES and zyxin.
Several TES-interacting clones were identified that encoded amino acid 2
onwards of mena (Table 1B).
Mena [mammalian Enabled (Ena)] belongs to the Ena/VASP (vasodilator-stimulated
phosphoprotein) protein family (Gertler et
al., 1996) and localises to focal adhesions and adherens junctions
(Gertler et al., 1996
;
Vasioukhin et al., 2000
).
Immunofluorescence studies demonstrated that mena colocalised with GFP-tagged
full-length TES in Rat-1 fibroblasts at focal adhesions and at areas of
cell-cell contact (Fig. 4B).
GST-tagged full-length TES precipitated endogenous mena and VASP from HeLa
cell extracts (Fig. 4E,F). The
fact that mena interacts directly with zyxin
(Drees et al., 2000
;
Gertler et al., 1996
) suggests
that TES may form part of a complex containing both zyxin and mena.
Furthermore, a number of TES-interacting clones from the mouse testis
library encoded talin, a 225 kDa phosphoprotein found in adhesion complexes
(Critchley et al., 1999).
Talin was present in GST-TES complexes obtained after incubating TES fusion
proteins with HeLa cell extracts (Fig.
4G). Currently, we are delineating the regions of TES that are
important for these interactions as well as investigating whether these
interactions are direct.
TES affects cell spreading
Rat-1 fibroblasts stably overexpressing GFP-tagged TES constructs
(Fig. 1A) were generated to
identify the cellular function of TES. Although cells stably expressing TES
were obtained, we found that initially fewer full-length TES-expressing clones
grow in comparison with vector controls, suggesting that overexpression of TES
has a detrimental effect on the cell. Indeed, previously we showed that TES
can have a growth inhibitory effect, as measured by colony forming assays
(Tobias et al., 2001). Despite
this, once stably overexpressing cells were obtained there appeared to be no
obvious differences in growth parameters as measured by doubling times and
cell cycle analysis (data not shown) nor any obvious phenotypic differences or
differences in the ability of transfected cells to invade matrigel (data not
shown).
Since TES localises to areas of actin-based structures we were interested in examining its effects on cell adhesion as measured initially by cell spreading on fibronectin. Fig. 5 demonstrates that in the early stages of spreading on fibronectin, Rat-1 fibroblasts stably expressing TES have a distinctly altered morphology. Full-length TES-overexpressing cells spread with an enhanced rate, appear larger and contain increased numbers and lengths of cell protrusions, an effect that persists for several hours after re-seeding (Fig. 5A). To ensure that the effects we observed were not due to an inability to visualise the entire GFP-vector-expressing cells (due to the differential localisation of GFP and GFP-TES), similar experiments were performed and the cells were stained with TRITC-labelled phalloidin to enable complete visualisation of both the GFP and full-length TES-expressing cells. Again we observed that TES overexpressing fibroblasts appeared larger and more spread on fibronectin compared with the vector-transfected control cells and contained increased numbers of actin-based protrusions (Fig. 5B). TES overexpression appears not to have an effect on the percentage of cells spread (data not shown); instead it appears to affect the degree of cell spreading. The proportion of enlarged TES-expressing Rat-1 fibroblasts was significantly increased compared with GFP-expressing cells (Fig. 5C). Interestingly, LIM-less and LIM-only TES-expressing cells were not as enlarged and protrusive as the full-length TES-expressing cells (data not shown). Similar results were also obtained with CEF cells stably overexpressing myc-tagged TES (data not shown). The effects of TES on cell spreading appear to be transient, as the cells growing normally do not appear to be different in size, have no gross observable differences in the level or distribution of F-actin and do not appear to contain altered focal adhesions (data not shown). Therefore, increased levels of TES appear to increase initial cell spreading on a fibronectin matrix and, together with the localisation of TES, would suggest a role for TES in actin dynamics and/or cell adhesion, a hypothesis that is currently being tested.
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Discussion |
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We have presented data demonstrating that TES is a novel focal adhesion
protein. In addition, several well characterised focal adhesion proteins
interact with and colocalise with TES. One of the TES-interacting proteins we
identified in yeast is zyxin, which, like TES, contains three C-terminal LIM
domains. Zyxin is a low-abundance phosphoprotein that has been localised not
only to focal adhesions but also to adherens junctions, actin stress fibres
and the nucleus (Beckerle,
1986; Nix and Beckerle,
1997
; Vasioukhin et al.,
2000
). Zyxin may play a role in actin assembly
(Golsteyn et al., 1997
), cell
motility (Drees et al., 1999
)
and intracellular communication (Nix and
Beckerle, 1997
). Several zyxin-binding partners have been
previously identified including mena
(Drees et al., 2000
;
Gertler et al., 1996
). Mena is
a member of the mena/VASP protein family; members of this family localise to
regions of dynamic actin formation within cells and are present at focal
adhesions, at the tips of filopodia and the leading edge of lamellipodia, and
at adherens junctions (Gertler et al.,
1996
; Reinhard et al.,
1992
). Mena/VASP proteins interact with actin as well as the
actin-monomer-binding protein profilin and play a role in actin assembly
(Bachmann et al., 1999
;
Bear et al., 2000
;
Drees et al., 2000
;
Gertler et al., 1996
). Studies
have also demonstrated an important role for mena/VASP proteins in cell
motility. Rottner et al. demonstrated that GFP-tagged VASP accumulates at the
lamellipodial front in melanoma cells and this closely correlates with the
protrusion rate (Rottner et al.,
1999
). In addition, fibroblasts lacking endogenous mena/VASP
proteins migrate more slowly than cells re-expressing mena
(Bear et al., 2000
). More
recently, it has been shown that mena/VASP promotes actin filament elongation
within lamellipodia to control cell motility
(Bear et al., 2002
).
The interaction of zyxin with mena/VASP family members also suggests a role
for zyxin in actin dynamics. In fibroblasts, membrane targeting of zyxin
resulted in increased actin-rich surface structures. This effect was believed
to be due to the ability of zyxin to bind mena/VASP proteins, as cells
containing a zyxin mutant that was unable to interact with mena/VASP had a
reduced number of actin-rich surface projections
(Drees et al., 2000). More
recently, Fradelizi et al. have demonstrated that zyxin is capable of inducing
actin polymerisation, which is dependent on VASP, but independent of the
Arp2/3, actin polymerisation complex
(Fradelizi et al., 2001
).
Interestingly, Drees et al. have demonstrated that perturbations of the
zyxin-mena/VASP interaction that result in the mislocalisation of mena/VASP
reduce the degree of fibroblast spreading on fibronectin
(Drees et al., 2000
).
Whether or not TES has a function in actin dynamics remains to be
elucidated, but the fact that fibroblasts overexpressing TES can spread to an
enhanced degree and contain increases in actin-based protrusions suggests that
TES, similar to its binding partners, functions in actin dynamics.
Intriguingly, we have also identified an actin-related protein, actin-like 7A
(ACTL7A) (Table 1), as the most
frequent TES-interacting protein from our yeast two-hybrid screens. ACTL7A,
identified by Chadwick et al., is predicted to encode a 48.6 kDa protein with
more than 40% amino-acid identity to members of the actin gene family
(Chadwick et al., 1999), which
suggests that, similar to other actin-related proteins, it may play a role in
actin dynamics.
Talin is thought to play a role in focal adhesion formation and in linking
integrins to the actin cytoskeleton. Several binding partners for talin have
been described, including integrins, focal adhesion kinase, vinculin (which
can interact with VASP) and F-actin (Buck
and Horwitz, 1987; Burridge
and Mangeat, 1984
; Muguruma et
al., 1990
). Studies have demonstrated that downregulation of talin
expression in HeLa cells result in a decreased rate of cell spreading
(Albiges-Rizo et al., 1995
).
Similarly, in undifferentiated mouse embryonic stem cells, disruption of both
copies of the talin gene resulted in cells that were unable to assemble focal
adhesions and had defects in cell spreading
(Priddle et al., 1998
).
Binding of both mena and zyxin to TES suggests that TES may be part of a
complex containing both of these proteins. In addition, the fact that talin
interacts with VASP further links talin to a common complex. Interestingly, an
interaction of zyxin with glutamate-receptor-interacting protein 1 (GRIP1) in
yeast two-hybrid analysis was recently reported; the function of this
interaction remains to be elucidated (Li
and Trueb, 2001). We also identified GRIP1 as a potential
TES-interacting protein in yeast (Table
1) and verified this interaction in biochemical analyses (data not
shown), which further link TES and zyxin to a complex that may contain one or
more common binding partners.
We have demonstrated that GFP-tagged TES localises to focal adhesions and
that TES interacts with the well-described focal adhesion proteins, talin,
zyxin and mena, to provide compelling evidence that TES is a novel component
of focal adhesions. Focal adhesions are specialised structures that connect
the actin cytoskeleton to the extracellular matrix (ECM) via transmembrane
integrins. Integrin receptor engagement triggers multiple signal transduction
pathways that are thought to affect cell motility and spreading as well as
cell proliferation and apoptosis
(Gumbiner, 1996;
Shen and Guan, 2001
). Cell
adhesion and spreading are complex processes involving dynamic rearrangements
of the actin cytoskeleton. How overexpression of TES affects these processes
is not known, but studies are currently ongoing to identify a potential role
for TES in actin dynamics.
Our data also provide evidence that TES, in common with its binding
partners, is a constituent of cell-cell adhesion sites. The cadherin-catenin
adhesion complex plays a role in tissue and organ morphogenesis as well as
cell migration (Gumbiner,
1996; Vasioukhin and Fuchs,
2001
). Vasioukhin et al. have demonstrated that in epithelial
cells cadherin-mediated intercellular adhesion generates a two-rowed zipper of
puncta and that mena, VASP and zyxin are recruited to these adhesion zippers
and this is dependent on
-catenin
(Vasioukhin et al., 2000
).
Adherence zippers are a precursor to adherens junctions
(Vasioukhin et al., 2000
) and
these structures look quite similar to the puncta we see TES, zyxin and mena
localised to in fibroblasts. This suggests that similar structures may occur
in fibroblasts and that TES is a component of these structures. Indeed Ko et
al. demonstrated that adherens junction structures are present in fibroblasts
(Ko et al., 2000
). Transient
expression of TES in epithelial cells does not result in localisation of TES
to adherens junctions (data not shown), which suggests either that the
localisation of TES may be cell-type specific or other proteins not expressed
in the cell lines we examined are involved in the subcellular localisation of
TES.
Initially, we identified TES as a candidate tumour suppressor gene at
7q31.1, a region where LOH is frequently observed in a variety of cancers.
Evidence also suggests that 7q31 harbours a potential tumour metastasis
inhibitor (Ichikawa et al.,
2000), and in prostate cancer increases in LOH at 7q31.1 were seen
in metastatic lesions (Saric et al.,
1999
). In cancerous cells, the breakdown of adhesion structures is
a necessary prerequisite for the invasive phenotype. It is therefore plausible
given the subcellular localisation and protein interactions identified in this
report to suggest that TES is involved in events related to cell motility and
adhesion, and currently we are investigating this possibility.
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Acknowledgments |
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