1 Ludwig Institute for Cancer Research, PO Box 2008, Royal Melbourne Hospital,
Victoria 3050, Australia
2 The Australian Centre for Blood Diseases, Monash Department of Medicine, Box
Hill Hospital, Victoria 3128, Australia
3 Queensland Institute for Medical Research, 300 Herston Rd, Herston, Queensland
4029, Australia
4 Centre for Drug Design and Development, IMB, St Lucia, Queensland 4068,
Australia
Author for correspondence (e-mail:
martin.lackmann{at}ludwig.edu.au
)
Accepted 3 December 2001
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Summary |
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Key words: Cell adhesion, Actin cytoskeleton, Eph receptor protein-tyrosine kinases, Melanoma, Metastasis
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Introduction |
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Previously we showed that Eph signalling is essential for directing the
movement of cells during zebrafish embryogenesis, disruption of the EphA3
function leading to severe defects in gastrulation
(Lackmann et al., 1998;
Oates et al., 1999
). The
nature of these gastrulation defects and loss of cell adhesion in
pre-gastrulation Xenopus embryos by activated EphA4 or ectopically
expressed ephrin-B (Jones et al.,
1998
; Winning et al.,
1996
) suggests that Eph receptors and ephrins function by
modulating cell adhesion and motility
(Bruckner and Klein, 1998
;
Flanagan and Vanderhaeghen,
1998
; Xu et al.,
2000
). This notion is supported by accumulating evidence
suggesting that stimulated Eph receptors and ephrins modulate integrin
function (Becker et al., 2000
;
Davy et al., 1999
;
Huynh-Do et al., 1999
;
Jones et al., 1998
;
Wahl et al., 2000
;
Winning et al., 1996
;
Zisch et al., 2000
;
Zou et al., 1999
).
Eph receptor activation leads to tyrosine phosphorylation of three major
autophosphorylation sites, two of which are located in the highly conserved
juxtamembrane region, and a third in the activation loop of the kinase domain.
Together, these residues function to regulate kinase activity, their
phosphorylation being required for full intrinsic enzyme activity
(Binns et al., 2000;
Zisch et al., 2000
). The
juxtamembrane tyrosines are also likely to be important for signal transfer,
and were suggested as docking sites for known signalling molecules including
Fyn, Src, Ras GTPase-activating protein (RasGAP), SHEP-1 and Nck (reviewed by
Mellitzer et al., 2000
). The
involvement of most of these molecules in the organisation of the cytoskeleton
(Schoenwaelder and Burridge,
1999
; Schlaepfer and Hunter,
1998
) supports an emerging concept from functional experiments of
a direct communication between activated Eph receptors and signalling pathways
regulating cell adhesion and plasticity of the actin cytoskeleton (Schmucker,
2001).
The reorganisation of cell morphology during adhesion and detachment
involves changes in focal adhesion complexes and in the actin/myosin
cytoskeleton and is regulated principally by signals emanating from clustered
cell adhesion receptors. Members of the Ras/Rho families of GTPases are
essential in these pathways (reviewed by
Hall, 1998;
Schoenwaelder and Burridge,
1999
), as they relay signals from various growth factor or
integrin-derived pathways to effect characteristic changes to the cell
morphology. The Crk family of adapter proteins
(Matsuda et al., 1992
) are
also intimately involved in signal-transduction processes regulating
integrin-dependent cell adhesion (Vuori et
al., 1996
; Zvara et al.,
2001
) and migration (Arai et
al., 1999
; Klemke et al.,
1998
; Petit et al.,
2000
). Via its src homology, SH2 and SH3 domains, Crk interacts
with downstream signalling molecules including c-Abl,
p130CAS (Crk-associated substrate), paxillin, Cbl, C3G and
DOCK 180 (reviewed by Feller et al.,
1998
).
Although the integrin-mediated interaction with the extracellular matrix is
essential for migration and survival of adherent cells, the deregulated
adhesion or loss of adhesion dependence is a hallmark of tumour progression
and metastasis (Giancotti and Ruoslahti,
1999). It is now well established that tumour progression in human
cutaneous melanoma correlates with integrin expression and function
(Johnson, 1999
;
Seftor et al., 1999
).
Intriguingly, several Eph receptors and ephrins are overexpressed in tumour
specimens and tumour cell lines (reviewed by
Dodelet and Pasquale, 2000
).
Moreover, increased expression levels of EphA2, EphA3, ephrin-A1 and ephrin-B2
correlate with melanoma tumorigenesis and metastatic progression
(Chiari et al., 2000
;
Easty et al., 1995
;
Easty et al., 1999
;
Vogt et al., 1998
). Given
their essential function in regulating cell motility and adhesion during
normal development, it is possible that the prometastatic effects of
Eph/ephrin overexpression in tumours are due to changes in cell adhesion and
motility that facilitate tumour cell dislodgement and invasion. To test this
notion we studied the responses of EphA3-expressing melanoma cell lines and
EphA3-transfected 293T cells (human kidney epithelial 293T cells) to ephrin-A5
stimulation and its impact on changes in cell adhesion and actin cytoskeletal
organization.
We show that EphA3 activation induces retraction of cell protrusions, cell rounding, membrane blebbing and de-adhesion, and that ephrin-A5-mediated receptor clustering and the tyrosine kinase activity of EphA3 are essential for this response. By expression cloning we identify CrkII as a cytoplasmic ligand of activated EphA3 and show that CrkII signalling is crucial for ephrin-A5-mediated cell rounding. EphA3 activation also results in transient activation of RhoA, and treatment of cells with the Rho inhibitor C3-transferase or the ROCK inhibitor Y-27623 abrogate ephrin-A5-mediated cell rounding and membrane blebbing, respectively. A dominant-negative, SH3 mutant CrkII abrogates both ephrin-A5-induced cell morphological changes and RhoA activation. Our findings suggest a pathway where ephrin-A5-induced recruitment of CrkII to EphA3 and a rapid, transient increase in activated RhoA result in cell rounding, membrane blebbing and detachment.
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Materials and Methods |
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Expression plasmids encoding fusion proteins in which either the
extracellular domains of ephrin-A5 or EphA3 were fused to the hinge and Fc
region of human IgG1 (gift from D. Cerretti, Immunex Corp., Seattle, WA) were
used to transfect Chinese Hamster Ovary (CHO) cells. EphA3-Fc and Ephrin-A5 Fc
(fusion protein between the Fc portion of human IgG and the C-terminus of
ephrin-A5) were purified from cell culture supernatants by protein-A affinity
chromatography. Flag-tagged monomeric ephrin-A5 was purified to homogeneity
from transfected CHO cell supernatants as described previously
(Lackmann et al., 1997). To
facilitate formation of clustered ephrin-A5, ephrin-A5 Fc was incubated (20
minutes) at 1/10 molar ratio with anti-human Fc antibody (Jackson
Laboratories).
A native EphA3-specific (clone IIIA4) monoclonal antibody (Mab) and
affinity-purified rabbit polyclonal antibodies have been described previously
(Boyd et al., 1992;
Lackmann et al., 1997
).
Anti-myc 9E10 antibodies were a generous gift from E. Stanley (WEHI,
Melbourne, Australia). Additional antibodies and reagents were purchased from
Transduction Laboratories (focal adhesion kinase, Crk and
p130CAS), New England Biolabs and Upstate Biotechnology
(phosphotyrosine), Santa Cruz (RhoA), BD Pharmingen (annexin V-FITC), and
Sigma (
-tubulin), HRP-labelled secondary antibodies from Jackson
laboratories (anti-mouse) and Bio-Rad (anti-rat), Alexa-labelled antibodies
and rhodamine-phalloidin from Molecular Probes.
Cell culture and microscopy
The AO2 and AO9 melanoma cells lines were obtained as clones of adherent
cells derived from mechanically dispersed deposits of secondary metastatic
melanomas (M. D., C. W. Schmidt, M. O'Rourke, unpublished). LiBr secondary
malignant melanoma cells were described previously
(Coates and Crawford, 1977).
Melanoma cells were cultured in RPMI, 10% iron-fortified fetal bovine serum.
Human kidney epithelial (HEK) 293T cells were maintained in DME, 10% fetal
calf serum (FCS). Transient transfection with the various expression
constructs was carried out using Fugene 6 (Roche Biochemicals).
To study stimulation in situ, melanoma or 293T cells were seeded onto
fibronectin-coated round glass coverslips in 6-well dishes. 293T cells were
transfected with the various expression constructs or the w/t EphA3 or
pEGFP-actin, or both, using Fugene. Before examination, the cells were kept in
DME, 1% FCS for at least 3 hours. For analysis on a Bio-Rad 1024 confocal
microscope, coverslips were placed in Sykes Moore chambers
(Sykes and Moore, 1959) and
images taken every minute, 10 minutes before and 50 minutes after the addition
of ligand or control solution.
For immunocytochemistry, cells on coverslips were washed with PBS, fixed with 4% para-formaldehyde in PBS for 10 minutes and permeabilised with 0.5% Triton X100 for 5 minutes before blocking for 30 minutes in PBS/0.2%BSA. Cells were incubated with relevant antibodies as indicated in figure legends, washed with PBS and incubated with Alexa-labelled secondary antibodies. For actin staining, permeabilised cells were stained with rhodamine-labelled phalloidin (0.1 µg/ml).
Adhesion assays
Ephrin-coated surfaces were prepared by immobilising ephrin-A5 Fc onto
`Reacti-Bind' Protein A coated plates (Pierce). Wells were washed with
PBS/0.1%Tween 20 (PBST) before incubating overnight at 4°C with ephrin-A5
Fc in PBST/0.1% BSA at concentrations as indicated. Nonbound ephrin-A5 Fc was
recovered for analysis and the plate was washed extensively before seeding
cells. The density of bound ephrin-A5 Fc was calculated using Biacore analysis
(Biacore AB) by estimating the ephrin-A5 concentration in nonbound fractions
with a sensor chip containing EphA3 extracellular domain
(Lackmann et al., 1997). The
ability of Protein-A-tethered ephrin-A5 to bind EphA3 was confirmed by
applying a constant concentration of the soluble EphA3 extracellular domain to
wells of increasing ephrin-A5 densities and estimating the fraction of
nonbound EphA3 by Biacore analysis as described
(Lackmann et al., 1998
).
Before seeding cells, residual Fc binding sites in wells were blocked by incubation with culture media containing 10% FCS. Cells were seeded (3x104 cells/well) onto the plates and allowed to adhere for 6 hours before fixation in 4% paraformaldehyde for microscopy. Adherent cells were stained with 0.5% crystal violet, solubilised in 0.1% SDS, and quantified by measuring the OD at 570 nm of individual wells in a microtiter plate reader. Each value represents the mean and s.d. from absorbance readings of triplicate wells made in parallel.
Melanoma cells cultured in 96-well plates were stimulated with preclustered ephrin-A5. Following PBS washes, adherent cells were fixed with 4% paraformaldehyde and quantified by crystal violet staining as described above.
Cell viability and apoptosis assays
Cell viability was assessed by vital dye (0.5% trypan blue) exclusion and
apoptotic cells quantified by flow cytometric analysis using established
methods, including annexinV-FITC cell membrane staining and examination of
nuclear condensation as described (Vermes
et al., 2000). Briefly, combined adherent and detached cells from
cultures of adherent cells, treated for 4 hours or as indicated with
preclustered (see below) ephrin-A5 Fc or with staurosporin (Sigma), with
washed once with PBS before incubation either in Ca2+-containing
buffer with 1 µg/ml FITC-annexin and 1 µg/ml propidium iodide (PI), or
after permeabilisation in 0.2% Triton-X100/0.1% sodium acetate, pH 6.0 with 50
µg/ml PI. In the latter assay, apoptotic cells are characterised by a PI
staining intensity of their nuclei that is lower than that of normal cells.
Viable and apoptotic cells were quantified by flow cytometry.
Immunoprecipitation and western blotting
Cells were serum starved for 4 hours in DME/1% FCS. After 10 minutes
stimulation with preclustered ephrin-A5 (1.5 µg/ml or as indicated in
figure legends), cells were lysed in 50 mM Tris, pH 7.4, 150 mM NaCl, 1%
Triton X100, 1 mM NaV04, 10 mM NaF and protease inhibitors
(TBS-Tx100). Lysates were immunoprecipitated with IIIA4 Mab coupled to
Trisacryl (1 hour at room temperature or overnight at 4°C)
(Boyd et al., 1992) or with 5
µg/ml anti-CrkII Mab and protein A Sepharose (Pharmacia). Washed
(TBS-Tx100) immunoprecipitates were analysed by western analysis with
appropriate antibodies. Blots were visualised using an ECL substrate
(Pierce).
Assay for GTP-bound RhoA and ADP-ribosylation of RhoA by
C3-exoenzyme
RBD was expressed as GST fusion proteins in BL21 cells. The levels of
GTP-bound RhoA in cell lysates were measured as previously described
(Schoenwaelder et al., 2000),
according to Ren et al. (Ren et al.,
1999
), except that cells were lysed in TBS-Tx100, and the
NaCl and MgCl2 concentrations were adjusted to 500 mM and 10 mM,
respectively, before the addition of the beads. ADP-ribosylation of RhoA in
cell extracts was analysed as described previously
(Leng et al., 1998
). Briefly,
EphA3-293T cells (60-70% confluent), after 15-20 hours incubation with 10
µg/ml C3 exoenzyme, were rinsed with PBS and lysed on ice in buffer
containing 1.5% Triton X-100, 0.8% DOC, 0.2% SDS, 145 mM NaCl, 20 mM Hepes, pH
7.4, 3 mM EGTA, 2 mM MgCl2, 25 µg/ml PMSF, 5 µg/ml leupeptin,
5 µg/ml aprotinin. Control cells were supplemented with an appropriate
amount of C3 before washing and lysis. In vitro ADP-ribosylation was performed
immediately at 30°C for 60 minutes on 50 µg samples of total cell
lysate by the addition of 200 µM GTP, 10 µM NAD+, 10 µg/ml
C3 exoenzyme and 0.5-2.5 µCi/assay 32P NAD+.
Reactions were terminated by the addition of sample buffer, boiled and
analysed on SDS-PAGE, followed by autoradiography.
Bacterial expression of EphA3 ID and protein expression library
screen
EphA3 ID constructs were transformed into Escherichia coli
BL21(DE3). After overnight induction at 16°C, cell pellets were sonicated
in phosphate buffer pH 8.0, 300 mM NaCl, 10 mM imidazole, pH 8, 10 mM
ß-mercaptoethanol and protease inhibitors (EDTA-free `Complete', Roche
Biochemicals), cleared lysates applied to Ni-NTA Fast Flow resin (Qiagen) and
bound protein eluted with 500 mM imidazole in the above buffer. Thioredoxin
and His Tags were cleaved from the eluted protein in 10 mM Tris, 150 mM NaCl,
2mM CaCl2 with enterokinase (Novagen) for 8 hours. Impurities and
thioredoxin/His tags were removed by re-application to Ni NTA, the EphA3 ID
remaining in the unbound fraction. All experiments were performed with
enterokinase cleaved EphA3 ID. A 16 day mouse embryo cDNA library in
Exlox (Novagen) was used to identify potential signalling
partners of EphA3. Approximately 2x105 independent clones
from the library were screened according to the manufacturer's instructions.
Briefly, cDNA clones were immobilised on nitrocellulose filters (Osmonics),
which were washed and blocked with PBST/1% BSA and incubated with 1 µg/ml
phosphorylated EphA3 ID in PBS/0.1% Triton X100/1mM NaVO4. Clones
that bound EphA3 ID were identified by incubation with 4G10
anti-phosphotyrosine (PY) antibody and HRP-conjugated anti-mouse antibody,
visualised with ECL (Amersham Pharmacia Biotech). Positive clones were plaque
purified and their cDNA inserts rescued by auto-subcloning in pExlox.
The cDNA inserts were sequenced and the proteins identified using BLAST 2.0
(Altschul et al., 1997
).
Mapping of EphA3 derived phosphopeptides and phosphopeptide
synthesis
EphA3 ID was dephosphorylated by the addition of TC45PTP linked to
glutathione Sepharose overnight at 4°C. Phosphorylated and
dephosphorylated EphA3 ID were purified by reverse phase HPLC and recovered
proteins digested with TPCK-treated trypsin (Roche Biochemicals). Tryptic
peptides were fractionated by C18 reverse phase HPLC (2.1x250 mm, Vydac)
with a 90 minute gradient of 60% CH3CN in 0.1% trifluoroacetic acid
and individual peptides recovered manually according to their 214 and 280 nm
absorbances. Tyrosine phosphopeptides, identified by their altered retention
were analysed by MALDI/MS as described
(Reid et al., 1998).
Tyrosine phosphorylated (P) peptides corresponding to
Lys589-Glu611 of the juxtamembrane domain and to
Asp773-Ile788 of the activation loop of EphA3 were
synthesised by solid-phase chemistry, and purity (>98%) and integrity
confirmed by electro-spray mass spectrometry
(Meutermans and Alewood,
1996):
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Results |
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Ephrin-A5-mediated morphological changes during de-adhesion are
differentially affected by mutation of tyrosines within the EphA3
juxtamembrane and activation loop domains
To examine the molecular mechanism of ephrin-A5-mediated cell-morphological
changes in more detail, we expressed w/t or mutant EphA3 in 293T cells.
Although we could not detect endogenous ephrin-A in these cells
(Fig. 1Af), they do contain
some endogenous EphA3 (Fig.
1Ag) (Dottori et al.,
1999), suggesting them as an appropriate model system for our
studies. 293T cells do not display distinct actin stress fibres, but staining
of permeabilised cells with rhodamine-phalloidin and an anti-
-tubulin
antibody displays complex actin (Fig.
3A-C) and microtubule networks
(Fig. 3E), with distinct fibre
bundles extending into cell protrusions and lamellipodia. Following exposure
to ephrin-A5, nontransfected 293T cells do not change their shape or gross
morphology, and changes to the actin cytoskeleton are unremarkable
(Fig. 3B). By contrast,
treatment of w/t EphA3-transfected 293T cells with ephrin-A5 results in a
dramatic contraction of the actin and microtubule cytoskeleton into dense
fibre bundles surrounding cell nuclei (Fig.
3D,F). Parallel staining of permeabilised cells with an
anti-phosphotyrosine Mab to outline focal adhesion complexes suggests that
cell contraction is accompanied by loss of focal contacts and focal adhesions
from the cell periphery (Fig.
3G,H). Furthermore, examination of vertical sections suggests that
rounding and contraction of the cell body is accompanied by a substantial gain
in cell height and is not due to a net loss in cell volume
(Fig. 3I,J).
|
We addressed the dependence of these cellular effects on EphA3 kinase
activity and tyrosine phosphorylation within the EphA3 cytoplasmic domain by
examining the effects of phenylalanine mutants of the conserved juxtamembrane
tyrosines, Y596 and Y602, and of Y779, which is located within the activation
loop of the kinase. Immunoblot analysis of parallel cell lysates indicated
that these three tyrosine residues correspond to the major EphA3
phosphorylation sites of the receptor and that w/t and mutant receptors were
expressed at comparable levels in transfected cells (see below,
Fig. 6E). Cotransfection with
an enhanced green fluorescent actin fusion protein (EGFP-actin) enabled the
examination, by time-lapse fluoresence microscopy, of cell morphology changes
resulting from ephrin-A5 stimulation of w/t or mutant EphA3-transfected cells.
Expression of EGFP-actin on its own had no effect on the cell morphology or
the expression levels of endogenous actin in transfected cells
(Ballestrem et al., 1998). In
agreement, our 293T cells did not change their shape or actin cytoskeleton
upon ligand stimulation (Fig.
4B, EGFP-actin). In the absence of ephrin-A5 stimulation w/t
EphA3-transfected cells were flat, and spread out on the fibronectin-coated
glass slide (Fig. 4, w/t, 0
minutes). They started to retract their extensions and lamellipodia 5-10
minutes after ligand addition (Fig.
4, w/t, 10). Concurrently, dynamic blebbing of the cell membrane
occurred with increasing intensity and by 50 minutes many of the cells
revealed membrane blebs as indicated by globular, actin-containing membrane
protrusions surrounding the cells (Fig.
4A, w/t, 50). At this time, transfected cells started to detach,
and by 50 minutes more than 50% of cells within the selected, representative
field had moved out of the focal plane. Again, this response was not due to
apoptosis (Fig. 2D).
Importantly, addition of soluble, monomeric ephrin-A5 blocks the response to
ephrin-A5 Fc and cells did not change their morphology throughout the time
course of the experiment (Fig.
4B, w/t + Inhibitor).
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Mutation of either of the juxtamembrane tyrosines, Y559 or Y602, retarded the onset of cell rounding to about 20 minutes after stimulation. Although pronounced membrane blebbing was still apparent in Y559 EphA3-expressing cells after 50 minutes (Fig. 4A, Y559F, 50), blebbing was abrogated in Y602 EphA3-expressing cells (Fig. 4B, Y602F, 50). Mutation of both tyrosines together, or of the EphA3 activation loop tyrosine, Y779, profoundly affected the cellular responses to ephrin-A5 (Fig. 4B), the cells showing no alterations to their shape or actin distribution throughout the experiment. Transfection of cells with EphA3 harbouring mutations in all three tyrosines (3xYF) affected the cell morphology independent of ephrin-A5 stimulation (Fig. 4B, 3YF). The cells, which display thin, dendrite-like processes remained adherent and did not significantly change their morphology during the time course of the experiment.
Surface-bound Ephrin-A5 Fc impairs adhesion and spreading in 293T
cells expressing wild-type but not mutant EphA3
We sought to examine whether EphA3 activation by surface-bound ephrin-A5
would affect the re-adhesion of detached 293T cells. To mimic activation of
EphA3 by its cell membrane-bound ligand in vitro, chimeric ephrin-A5 Fc was
coupled to protein-A-coated 96-well plates via its C-terminal Fc domain.
Biacore analysis of the nonbound fractions with an EphA3-coated sensor chip
suggested a surface capacity of 0.2 µg ephrin-A5 Fc/well. We also applied
the soluble EphA3 extracellular domain to the ephrin-A5-coated surface, and by
measuring the nonbound EphA3 in the supernatant confirmed that the immobilised
ephrin-A5 is in a biologically active form, capable of interacting with the
receptor (Fig. 5A). Although
exposure of 293T cells, transfected with EGFP-actin, to ephrin-A5 had no
measurable effect on adhesion, w/t EphA3-expressing cells remained round and
failed to adhere to surfaces of increasing coating density of ephrin-A5
(Fig. 5B,C). The effect was
noticeable even at the lowest ephrin-A5 density tested (8 pg/mm2).
At a density of 0.18 ng/mm2, corresponding to an ephrin-A5 Fc
concentration of 2 nM, only approximately 50% of the cells were able to
attach.
|
Mutation to phenylalanine of single EphA3 juxtamembrane tyrosines Y596 or Y602 reduced the number of nonadherent cells. The effect of the Y602F mutation was more potent than the Y596 mutation, with more cells adhering to the ephrin-A5 surface. Mutation of both juxtamembrane tyrosines or the activation loop tyrosine Y779 or of all three tyrosines (Y596, Y602 and Y779) essentially ablated the response to ephrin-A5 surfaces and no significant effect of ephrin-A5 on 293T cell adhesion was observed (Fig. 5C). Thus, at an ephrin-A5 Fc surface density of 1.5 ng/mm2, only 30-40% of w/t EphA3-expressing cells attached to the wells, whereas 90-95% of the mutant Y779 EphA3 or Y596/Y602 EphA3-expressing cells were adherent at this ephrin-A5 concentration.
Taken together, these observations suggest that ephrin-A5-triggered mechanisms, causing cell detachment or repulsion, are dependent on EphA3 kinase activity and tyrosine phosphorylation, but are independent from prior engagement of cell adhesion mechanisms.
Identification of Crk as an interaction partner for activated
EphA3
To identify cytosolic proteins downstream from activated EphA3, we screened
a 16 day mouse embryo cDNA expression library using recombinant phosphorylated
EphA3 intracellular domain (ID) as the probe. The soluble, tyrosine
phosphorylated fusion protein was isolated from E. coli after
overexpression of a cDNA encoding the intracellular portion of EphA3 fused to
a cleavable thioredoxin and His tag. Following dephosphorylation with TC45 PTP
(Tiganis et al., 1999), the
purified EphA3 ID was able to rephosphorylate tyrosine residues upon the
addition of ATP and Mg2+, indicating that the kinase was functional
(Fig. 6A). Analysis by mass
spectrometry of tryptic peptides of the phosphorylated and rephosphorylated
EphA3 ID unambiguously confirmed that the juxtamembrane tyrosines Y596, Y602
and the kinase activation loop tyrosine 779 were phosphorylated in the
recombinant protein (M. L., unpublished data). Screening of approximately
2x105 plaques yielded a single clone corresponding to the
adapter protein CrkII (Matsuda et al.,
1992
).
Ephrin-A5-induced recruitment of Crk in 293T cells depends on EphA3
kinase activity
In lysates from LiBr melanoma cells and from EphA3-transfected 293T cells,
Crk is associated with tyrosine-phosphorylated EphA3
(Fig. 6B-F). Immunoprecipitation of whole-cell lysates suggested that LiBr melanoma cells
contain discernible levels of endogenous Crk and EphA3
(Fig. 6B, bottom panels).
Endogenous EphA3 was apparent in anti-Crk immunoprecipitates from
ephrin-A5-stimulated LiBr cells, but was undetectable in lysates of
nonstimulated cells (Fig. 6C,
top panel). Furthermore, exposure of EphA3-expressing 293T cells to increasing
concentrations of surface-bound ephrin-A5 resulted in association of CrkII
with the receptor, readily noticeable at surface concentrations above 0.18
ng/mm2, equivalent to 2 nM ephrin-A5. By comparison, a slightly
reduced level of EphA3 was detected in anti-Crk immunoprecipitates of cells
that had been stimulated with a comparable concentration (10 nM) of
preclustered ephrin-A5 Fc in solution (Fig.
6C). The association of EphA3 with Crk was apparent in stably
transfected 293T cells within 1 minute of ephrin-A5 Fc addition and increased
to maximal levels 30 minutes after stimulation
(Fig. 6D). In agreement with
previous studies indicating ligand-independent autophosphorylation of EphA3 at
high receptor density (Lackmann et al.,
1998), anti-PY western blots of anti-EphA3 immunoprecipitates from
transiently transfected 293T cells revealed substantial receptor
autophosphorylation in the absence of ligand
(Fig. 6E, middle panel).
Phenylalanine mutation of the EphA3 tyrosine Y779 (activation loop) reduced
the phosphotyrosine level, whereas mutation of the juxtamembrane tyrosines
Y596 and Y602 had little effect. Mutation of both (2xYF) or of all three
tyrosines (3xYF) reduced the anti-PY signal to background levels
(Fig. 6E, middle panel).
Western blot analysis of anti-Crk immunoprecipitates from 293T cell lysates
suggested association of EphA3 with CrkII in ephrin-A5-stimulated,
EphA3-transfected cells. Interestingly, a comparable amount of EphA3
coprecipitated in Y596 and Y602 mutant EphA3-expressing cells was, in both
cases, at higher levels than in nonstimulated cells
(Fig. 6E). By contrast, Crk
association with the receptor was not evident in samples from Y779F,
2xYF or 3xYF mutant EphA3-expressing cells. The sequence motifs
encompassing the EphA3 juxtamembrane tyrosines accommodate potential YXXP Crk
SH2 domain docking sites (Birge et al.,
1993
). We further examined whether these motifs are involved in
CrkII binding: A Sepharose-tethered 2xY phosphopeptide, but not the
nonphosphorylated counterpart, was able to bind CrkII from EphA3-transfected
293T lysates (Fig. 6F, left
panel). However, no significant effect was observed when phosphopeptides
corresponding to the juxtamembrane or activation loop sequences were added to
CrkII immunoprecipitation reactions (Fig.
6F, right panel).
A dominant-negative Crk SH3 domain mutant abrogates
ephrin-A5-mediated cell rounding and actin cytoskeletal changes
To assess the functional relevance of ephrin-A5-induced recruitment of Crk
to EphA3, we monitored actin-cytoskeletal changes in EphA3-overexpressing 293T
cells, which were transfected with either w/t Crk, a dominant-negative Crk SH3
domain mutant (SH3* Crk), or both expression constructs.
Overexpression of w/t Crk induces membrane spreading and membrane ruffling
(Dolfi et al., 1998) by
recruitment of DOCK180 to the Crk/p130CAS complex
(Kiyokawa et al., 1998
),
whereas SH3 domain mutated Crk causes abrogation of membrane ruffling and
spreading (Nakashima et al.,
1999
). In agreement, untreated w/t Crk-transfected EphA3-293T
cells reveal extensive spreading and ruffling, whereas SH3*
Crk-transfected cells lack any signs of ruffling
(Fig. 7, -) and display
distinct, dendrite-like actin-rich cell processes. Treatment with preclustered
ephrin-A5 Fc leads to rounding, contraction of the actin cytoskeleton and loss
of membrane ruffles at comparable levels
(Fig. 7, +) in w/t-Crk
overexpressing and control cells. By contrast, SH3*Crk-expressing
cells appear not to respond to ephrin-A5 treatment and do not change their
morphology during the experiment. Cotransfection of SH3* Crk
together with w/t CrkII rescues the SH3* phenotype, and membrane
ruffling and spreading of the untreated cells resembles that of w/t
CrkII-transfected cells. Importantly, these cells regain their reactivity to
ephrin-A5 stimulation and respond with pronounced cell rounding and
contraction of the actin cytoskeleton. Staining of transiently transfected
cells with anti-myc Mab confirms the expression of myc-tagged Crk constructs
and suggests that, following ephrin-A5 stimulation, Crk colocalises with the
cortical actin ring. By contrast, nonstimulated (not shown) or SH3*
Crk-transfected cells display diffuse cytoplasmic anti-myc (Crk) staining
(Fig. 7, +, anti-myc).
|
Modulation of Rho activity following EphA3 activation relies on a
functional Crk SH3 domain
The distinctive effects of ephrin stimulation on the cytoskeleton prompted
us to examine the involvement of RhoA as a downstream effector molecule. We
examined cell lysates of parental or EphA3 293T cells for levels of activated
Rho, precipitated with the recombinant RhoA binding domain (RBD) of rhotekin
(Ren et al., 1999). Ephrin-A5
Fc stimulation consistently resulted in a transient increase in RBD-bound,
active Rho to maximal levels within 5 minutes
(Fig. 8A). No significant
changes in the level of RBD-bound Rho were observed in the parental 293T cells
(Fig. 8B). Activation in
suspended EphA3 293T cells occurred with very similar kinetics, although peak
levels of active RhoA were observed slightly earlier than in adherent cells, 3
minutes after stimulation (Fig.
8C). The rise in active Rho followed a decline to basal levels 10
minutes after ephrin-A5 addition. Importantly, the rise in active Rho was not
seen in ephrin-A5-stimulated EphA3 293T cells expressing dominant-negative
SH3* Crk (Fig. 8E),
whereas expression of w/t Crk did not affect the kinetics of Rho activation
(Fig. 8D).
|
To assess the involvement of RhoA functionally, we treated EphA3-293T cells
either with recombinant Clostridium botulinum C3 toxin
(Fig. 9A-E), which ribosylates
and specifically inhibits Rho activity
(Leng et al., 1998), or with
Y-27632 (Fig. 9F), a specific
Rho kinase inhibitor (Uehata et al.,
1997
), previously shown to inhibit ROCK-dependent membrane
blebbing (Coleman et al.,
2001
). We adjusted the concentration of C3 to produce modest actin
cytoskeletal changes without causing EphA3-293T cell rounding
(Fig. 9C). Exposure of cells to
this concentration of C3 reduced the level of active Rho significantly, as
indicated by 70% reduced levels of Rho available for radioactive ADP
ribosylation (Fig. 9E). C3
treatment under these conditions completely abrogated ephrin-A5-mediated cell
rounding (Fig. 9D), thus
supporting the involvement of Rho in this response. To examine the role of
ROCK, a downstream effector of active RhoA
(Watanabe et al., 1999
), we
exploited time-lapse microscopy to monitor ephrin-A5-induced membrane blebbing
in parental or EphA3-293T cells that were treated with the ROCK inhibitor
Y-27632. Treatment of EphA3 293T cells with the Rho kinase inhibitor Y-27632
also completely abrogated ephrin-A5-induced membrane blebbing
(Fig. 9F). Interestingly, this
inhibition of ROCK activity had little effect on cell rounding (see
http://www.ludwig.edu.au/addinfo/Figure9Finhib.mpg). For comparison we treated
EphA3 293T cells with LY 294002, to inhibit phosphoinositide-3-kinase, which
is involved in signalling pathways that effect cytoskeletal changes
(Hall, 1998
;
Rodriguez-Viciana et al.,
1997
), and recently shown to modulate integrin activity downstream
of EphA8 (Gu and Park, 2001
).
Although LY 294002 treatment inhibited ephrin-triggered membrane blebbing, the
effect was less pronounced than inhibition by Y-27632
(Fig. 9F).
|
![]() |
Discussion |
---|
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---|
It is well established that signals from within the cells and cross-talk
with RTKs, including Eph receptors (Schmucker, 2001), modulate the activity
and avidity of adhesion receptors and thus the adhesive properties of cultured
cells (Gumbiner, 1996). We
show that, in EphA3-expressing adherent melanoma cell lines and in
EphA3-overexpressing 293T cells, ephrin-A5-induced receptor activation
promotes sequential retraction of cell processes, cell rounding, membrane
blebbing and detachment. Functional and biochemical analysis of the underlying
mechanisms reveals that these morphological changes are mediated through
recruitment of CrkII to ligand-activated EphA3 and a concurrent, transient
activation of RhoA. A rapid condensation of the actin cytoskeleton and
microtubule network and a concurrent loss or re-distribution, or both, of
focal contacts from the cell periphery
(Fig. 3D-J) suggests that, like
other Eph-like RTKs (Miao et al.,
2000
; Schmucker, 2001), ephrin-A5-activated EphA3 modulates focal
adhesion signalling pathways. Although these cellular responses are
reminiscent of the morphological events occurring during apoptosis
(Jacobson et al., 1997
), our
experiments show that activation of EphA3 does not affect cell viability
(Fig. 2D). In contrast to
apoptotic blebbs, which are static and often detach from the cell surface,
ephrin-A5 triggers highly dynamic, spherical membrane protrusions that do not
lead to cell fragmentation. Membrane blebbing is observed frequently in living
cells during mitosis, cell spreading and migration and results from increases
in the intracellular pressure and extensive changes to the actin cytoskeleton
(Hagmann et al., 1999
).
Several recent reports suggest that increased contractility of the actomyosin
network, due to increased phosphorylation of myosin light chains by myosin
light chain kinase (Mills et al.,
1998
) and ROCK (Coleman et
al., 2001
), are essential in both apoptotic and nonapoptotic
blebbing (Hagmann et al.,
1999
).
Tissue culture surfaces decorated with ephrin-A5 at defined densities
allowed us to examine how activation of EphA3 effects the adhesion of detached
cells. At 0.18 ng ephrin-A5/mm2 (2 nM), 50% of the cells
remained in suspension (Fig.
5A,B). This concentration agrees well with the low nanomolar
affinity of the ephrin-A5/EphA3 interaction
(Lackmann et al., 1997
),
showing that these surfaces provide a physiologically relevant model of EphA3
stimulation. With decreasing ephrin-A5 density, w/t cells were able to adhere
and gradually spread out (Fig.
5B). Importantly, Hattori et al. showed recently that the
high-affinity ephrin-A2/EphA3 interaction triggers a specific ADAM
metalloprotease-catalysed ephrin-cleavage at sites of cell-cell contact, which
allows cell repulsion to occur (Hattori et
al., 2000
). Increased adhesion of several cell lines to ephrin-B
Fc surfaces, prepared by passive adsorption onto nitrocellulose, has been
linked to EphB1-mediated integrin activation
(Becker et al., 2000
;
Stein et al., 1998a
), but this
may result from mechanical tethering through EphB/ephrinB interactions
(Miao et al., 2000
). Some of
the apparent differences in these reports may also relate to the different
ephrin-coupling strategies yielding different densities of functional
ephrin.
Two conserved tyrosines in the juxtamembrane region of Eph receptors
reportedly function as SH2-domain docking sites for known signalling molecules
including Fyn (Ellis et al.,
1996), Src (Zisch et al.,
2000
), RasGAP, (Holland et
al., 1997
), SHEP-1 (Dodelet et
al., 1999
) and Nck (Stein et
al., 1998b
), but little is known about the functional consequences
of these interactions. Together with the activation-loop tyrosine, these
residues also function to regulate kinase activity, and their phosphorylation
is required for full enzyme activity (Binns
et al., 2000
; Zisch et al.,
2000
). In agreement, we assigned these tyrosines (EphA3 Y596, Y602
and Y779) as the prominent autophosphorylation sites of EphA3 (M. L.,
unpublished). Exposure of EphA3-expressing w/t or mutant cells (Y596F, Y602F,
Y779F, 2xYF and 3xYF) to either surface-tethered or to clustered
ephrin-A5 in solution confirmed that the cellular response to EphA3 activation
is critically dependent on their phosphorylation and a resultant functional
EphA3 kinase (Figs 3,
4;
Fig. 5C;
Fig. 6E). Phenylalanine
substitutions of individual juxtamembrane tyrosines reduced ephrin-A5 mediated
repulsion (Fig. 5C) and delayed
cell rounding, whereas mutation of the activation-loop tyrosine, of both
juxtamembrane tyrosines, or of all three tyrosines together ablated
ephrin-A5-induced cell rounding and repulsion and reduced the kinase activity
to background.
Analysis of the signalling events following EphA3 activation resulted in
two key observations: a dose-dependent recruitment of Crk to ligand-stimulated
EphA3 and a rapid modulation of Rho activity. We initially identified Crk as a
binding partner for phosphorylated EphA3, by probing a cDNA expression library
with recombinant EphA3 ID. We confirmed this interaction by coprecipitation of
EphA3 together with endogeneous Crk in immunoprecipitates from
ephrin-A5-stimulated LiBr melanoma cells
(Fig. 6B), and from
ligand-stimulated EphA3 293T cells (Fig.
6C-F). Binding of the recombinant Crk SH2 domain to the EphB3
juxtamembrane PY motif containing two consecutive `YXXP' Crk SH2 domain
binding sites has been reported (Hock et
al., 1998). In agreement, we extracted CrkII from 293T cell
lysates using a phosphopeptide comprising the corresponding EphA3 sequence,
and site-directed mutagenesis of both of these consensus Crk binding sites
together ablated the EphA3/Crk interaction. Interestingly, addition of excess
juxtamembrane phosphopeptide to the immunoprecipitation reaction did not
significantly reduce the amount of coprecipitated EphA3. We suggest two
alternative explanations for these observations. Either the inability to
disrupt the CrkII/EphA3 interaction by peptide competition indicates that
stable CrkII recruitment occurs indirectly via an as yet unidentified
component. This would imply that the interaction observed during our
expression cloning strategy and the CrkII `pull down' with Sepharose-tethered
2xY phosphopeptide are due to the fortuitous use of the CrkII consensus
sequence. Alternatively, CrkII binding via an SH2 interaction may require a
defined configuration of ligand-induced EphA3 clusters, which cannot be
disrupted by a short consensus phosphopeptide. Importantly, in
EphA3-transfected 293T cells, CrkII binds prominently only to the ligand
activated, and not to the autophosphorylated receptor
(Fig. 6B-E). Furthermore, the
increase of EphA3-associated CrkII during ephrin-A5 stimulation closely
parallels the cellular response monitored by time-lapse microscopy
(Fig. 4;
Fig. 6D). Perhaps in a cellular
context CrkII is recruited to an EphA3 docking site that is functional only at
a critical density or configuration of the EphA3 aggregate, which, in turn is
determined by the concentration or density of preclustered or
membrane-anchored ephrin-A5. This notion is in agreement with an earlier
report showing that the composition of EphB1 signalling complexes and
resulting cellular responses depend on the oligomeric state of the interacting
ephrin-B1 (Stein et al.,
1998a
). Importantly, our experiments show that recruitment of Crk
to EphA3 is accompanied by a concurrent, transient increase in active RhoA.
Both events are essential in ephrin-A5-induced rounding and detachment of
EphA3-bearing cells and are not reliant on cell adhesion. An increasing body
of evidence implicates CrkII and its association with
p130CAS in signal-transduction processes that regulate
cytoskeletal rearrangements during cell adhesion and migration
(Arai et al., 1999
;
Cary et al., 1998
;
Klemke et al., 1998
). In
support of our observations, a role for the Crk/p130CAS
complex in regulating cell spreading was reported
(Vuori et al., 1996
) and
Crk-induced membrane ruffling, cell spreading and Jun N-terminal kinase
activation is blocked by a dominant-negative form of Rac. This suggests a
linear pathway, where recruitment/activation of Rho-family GTPases to the
Crk/p130CAS complex leads to JNK activation
(Dolfi et al., 1998
).
Increasing evidence implies Eph receptor signalling upstream of this pathway,
including EphB2, which modulates the activity of Erk2 and JNK1
(Zisch et al., 2000
), and
EphB1, which recruits a complex containing Nck, p62dock
and RasGAP and mediates JNK activation
(Holland et al., 1997
;
Stein et al., 1998c
). The
guanine nucleotide exchange factor (GEF) ephexin, which has GEF activity for
Rho, Rac and Cdc42, and in retinal ganglion cells is constitutively associated
with EphA4, provides further evidence for communication from Eph RTKs via RhoA
to the cytoskeleton (Shamah et al.,
2001
). Our observation, that overexpression of SH3-domain mutated,
dominant-negative CrkII affects ephrin-A5 induced RhoA activation, could
indicate that CrkII acts upstream of Rho as a molecular link to effect
EphA3-induced contraction of the cytoskeleton. Supporting this notion, it was
shown recently that activation of Rho and Rho kinase is responsible for
ephrin-A5-induced growth cone collapse
(Wahl et al., 2000
).
Adhesion and de-adhesion of cells to the substratum is regulated by a
tightly controlled interchange of signals from several extracellular and
intracellular origins, and their deregulation or uncoupling has been
correlated with tumourigenesis and metastasis
(Giancotti, 1997). Although an
essential role of Eph RTKs in modulating actin dynamics, in particular in
growing axons, is now well recognised (Schmucker, 2001), our observations
indicate that activated EphA3 triggers CrkII- and Rho-mediated cytoskeletal
changes and detachment, but not apoptosis. As the unscheduled expression of
EphA3 and ephrins correlate with melanoma progression, we speculate that this
response to EphA3 activation may be one of the initiating events leading to
melanoma metastasis. It is noteworthy that, although ephrin-A5-induced
detachment was not observed in EphA3-negative melanoma cells, the intensity of
the response varied between EphA3-positive, strongly adherent and less
strongly adherent melanoma cell lines. Interestingly, some of the less
adherent LiBr cells were routinely rounded before stimulation with exogenous
ephrin, indicating potential autocrine EphA3 activation by endogenous ephrins.
In support of this notion we found comparable cell-surface expression levels
of EphA3 and ephrin-A on these cells.
Both CrkII and RhoA have central roles in the regulation of the dynamic
actin signalling network. Reagents that modulate GTPase activities and affect
the formation of CrkII signalling complexes induce the disassembly of focal
adhesions and loss of cell substratum contact
(Klemke et al., 1998;
Escalante et al., 2000
;
Ren et al., 1999
). Consistent
with this concept, results presented in this study imply that recruitment of
Crk to the EphA3 receptor and transient activation of Rho facilitate
ephrin-A5-induced cell rounding, membrane blebbing and deadhesion. CrkII and
RhoA are candidates for mediating the communication between the
ligand-activated EphA3 receptor and the actin cytoskeleton.
![]() |
Acknowledgments |
---|
![]() |
Footnotes |
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