1 The Randall Centre for Molecular Mechanisms of Cell Function, New Hunt's
House, King's College London, Guy's Campus, London SE1 1UL, UK
2 INSERM U461, F-92296, France
* Author for correspondence (e-mail: gareth.jones{at}kcl.ac.uk )
Accepted 12 November 2001
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Summary |
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Key words: Cell migration, Chemotaxis, Phosphoinositide 3-kinase, Rho GTPases, Actin, Cytoskeleton
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Introduction |
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The activation of guanine nucleotide exchange factors (GEFs) by 3'
phosphoinositides produced by PI3K (Han et
al., 1998) and attenuation of cytoskeletal rearrangements by
targeted inhibition of specific PI3K p110 catalytic subunits
(Hill et al., 2000
;
Vanhaesebroeck et al., 1999
)
suggest that increased PI3K activity switches the small Rho GTPases into an
active state. In particular, the small Rho GTPases Cdc42, Rac1 and RhoA have
well established roles in regulating the complex cytoskeletal dynamics that
drive cell migration (Kjoller and Hall,
1999
; Ridley et al.,
1999
; Schmitz et al.,
2000
). Cdc42 stimulates microspike formation, polarisation and
controls cell directionality (Allen et al.,
1997
; Allen et al.,
1998
; Kozma et al.,
1995
; Nobes and Hall,
1999
). Rac 1 stimulates lamellipodia formation, membrane ruffling
and increases migratory speed (Allen et
al., 1997
; Allen et al.,
1998
; Nobes and Hall,
1999
; Ridley et al.,
1992
; Rottner et al.,
1999
). RhoA induces formation of stress fibres and focal adhesions
and regulates cell contractility (Machesky
and Hall, 1997
; Nobes and
Hall, 1995
; Nobes and Hall,
1999
; Ridley and Hall,
1992
; Rottner et al.,
1999
). In actively migrating cells, Cdc42 and Rac both regulate
the formation of focal complexes (small integrin clusters at the edge of
lamellipodia that are distinct from the large focal adhesions formed by RhoA
in adherent and stationary cells) (Adams,
2001
; Nobes and Hall,
1995
).
The identification of Wiskott Aldrich syndrome protein (WASP) family
members as downstream molecular binding targets for small Rho GTPases
represents a direct link between these effectors and reorganisation of the
actin cytoskeleton (Millard and Machesky,
2001; Mullins and Machesky,
2000
; Takenawa and Miki,
2001
). WASP expression is restricted to haematopoietic cells, but
its homologue N-WASP is expressed ubiquitously
(Kolluri et al., 1996
;
Miki et al., 1998
;
Rohatgi et al., 1999
;
Symons et al., 1996
). A model
for N-WASP activation by Cdc42 has been conceived from in vitro studies.
Binding of Cdc42 to the GDB/CRIB (GTPase binding domain/Cdc42 and Rac
interactive binding) region disrupts an autoinhibitory interaction between the
N- and C-termini of N-WASP. The newly exposed VCA (Verprolin homology, Cofilin
homology, acidic region) domain binds to actin and the Arp2/3 complex to
promote actin nucleation and formation of protrusive structures, most notably
microspikes/filopodia and lamellipodia
(Kim et al., 2000
;
Millard and Machesky, 2001
;
Takenawa and Miki, 2001
). The
polyproline region of WASP and N-WASP binds to SH3 domains of numerous
signalling molecules (Fawcett and Pawson,
2000
; Takenawa and Miki,
2001
), including the p85
regulatory subunit of PI3K
(Banin et al., 1996
;
Finan et al., 1996
). So far no
function has been assigned to the direct interaction of p85
with
N-WASP; however it is likely that PI3K can promote WASP/N-WASP activation in
vivo via regulation of small Rho GTPase activity.
Epidermal growth factor (EGF) receptor (EGFR) signalling is strongly linked
to breast cancer metastasis (Kim and
Muller, 1999) and has a well defined role in the promotion of cell
motility (Wells et al., 1998
).
Class 1A PI3Ks are key effectors in EGF/EGFR signal transduction
(Moghal and Sternberg, 1999
).
Evidence for an EGF/EGFR signalling pathway for the activation of N-WASP
includes the finding that EGFR associates with N-WASP in fibroblasts via an
interaction with Grb2 (Miki et al.,
1996
; She et al.,
1997
). Furthermore, filopodia production in Cos7 cells is
attenuated by an N-WASP mutant that fails to bind actin
(Miki and Takenawa, 1998
).
Urokinase-type plasminogen activator (uPA) is a serine protease that binds
to the glycosyl phosphatidylinositol (GPI)-anchored uPA receptor (uPAR).
uPA/uPAR expression is strongly correlated with the metastatic potential of
breast carcinomas (Andreasen et al.,
1997; Foekens et al.,
2000
) and represents a viable target for anti-tumour therapy
(Schmitt et al., 2000
). uPA
bound to uPAR exhibits enhanced proteolytic activity and directly activates
plasminogen, matrix metalloproteinases and growth factors at the cell surface,
which gives rise to enhanced extracellular matrix degradation, cell migration
and proliferation (Andreasen et al.,
1997
; Preissner et al.,
2000
). Ligation of uPA with uPAR can activate cellular responses
that are independent of any pericellular proteolytic activity. Non-proteolytic
uPA stimulates chemotaxis/migration through the activation of Src-type kinase
p56/p59hck, protein kinase C and extracellular signal-regulated kinase
(Busso et al., 1994
;
Chiaradonna et al., 1999
;
Degryse et al., 1999
;
Degryse et al., 2001
;
Fibbi et al., 1998
;
Nguyen et al., 1999
;
Nguyen et al., 2000
;
Resnati et al., 1996
). uPAR
lacks a transmembrane domain and must associate with transmembrane adaptor
protein(s) to activate intracellular signalling molecules. Direct interaction
of uPAR with integrins located in the plasma membrane stimulates signal
transduction and regulates molecular changes in integrin-containing complexes
(Bohuslav et al., 1995
;
Carriero et al., 1999
;
Ossowski and Aguirre-Ghiso,
2000
; Priessner et al., 2000;
Simon et al., 2000
;
Sitrin et al., 1996
;
Wei et al., 1996
;
Xue et al., 1997
;
Yebra et al., 1999
). uPAR
overexpression in fibroblast cell lines and its direct binding to vitronectin
induces membrane protrusions and increased migratory speed through activation
of Rac1 (Kjoller and Hall,
2001
). uPAR ligation with uPA promoted cytoskeletal changes and
chemotaxis in vascular smooth muscle cells through a pertussis-toxin-sensitive
mechanism and a pathway involving Tyk2 and PI3K
(Degryse et al., 1999
;
Degryse et al., 2001
;
Kusch et al., 2000
).
We considered whether PI3K was required for chemotaxis and signalling to Rho GTPases and N-WASP in response to uPAR and EGFR ligation. PI3K was essential for Cdc42, Rac1 and N-WASP activation and chemotaxis induced by EGF but was not required for similar responses induced by uPA. A ß1-integrin-dependent mechanism for the activation of N-WASP by uPA that supports PI3K-independent chemotaxis is also proposed.
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Materials and Methods |
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Cell culture
MDA MB 231 cells, a highly invasive and oestrogen-receptor-negative human
breast carcinoma cell line of epithelial origin were from ECACC. Unless
otherwise stated, media and supplements were from Gibco BRL (UK). Cells were
grown and passaged in DMEM containing 10% v/v heat inactivated foetal calf
serum (FCS) (Biopharm, UK), penicillin/streptomycin (100 IU/ml and 100
µg/ml) and 2 mM L-glutamine. Cells were grown at 37°C in T25 tissue
culture flasks (Falcon, UK) and maintained in a humid atmosphere of 5%
CO2. Cells were subcultured when 70-80% confluent by washing twice
in Versene (0.2% v/v EDTA solution), incubated for 1-2 minutes in
non-enzymatic cell disassociation buffer, resuspended in growth medium and
seeded into fresh T25 flasks at a ratio of 1:5. Cells were used from passages
2-15 after recovery of stocks from liquid nitrogen. For culture of cells on
Matrigel®, coverslips or culture dishes were thinly coated with
Matrigel® (0.5 µg/cm2) diluted in DMEM for 1 hour at room
temperature and washed twice with PBS before seeding. Cells were allowed to
adhere for 4 hours in a 37°C and 5% CO2 humidified incubator.
To obtain serum-starved cultures, cells were washed twice with
phosphate-buffered saline (PBS) and maintained for 24 hours in
serum-starvation medium (DMEM as above containing 0.1% FCS).
Immunocytochemistry
Cells seeded on Matrigel®-coated coverslips (13 mm diameter) at a
density of 2.5x103 cells/coverslip were serum starved for 24
hours. Cells were fixed for 10 minutes in 4% w/v paraformaldehyde/3% w/v
sucrose in PBS, warmed to 37°C, washed three times with PBS, permeabilised
with 0.5% v/v Triton X-100 in PBS for 5 minutes and blocked with 2% v/v bovine
serum albumin (BSA) in PBS for 20 minutes at room temperature. Actin filaments
in cells were localised by incubation with 0.1 µg/ml Alexa-568-conjugated
phalloidin for 1 hour at room temperature. ß1-integrins,
vinculin or N-WASP cells were visualised by incubation with 1/500 dilution of
anti-ß1-integrin (clone 8E3), 1/200 dilution of anti-vinculin
or 1/200 dilution of anti N-WASP in PBS containing 1% w/v BSA for 1 hour at
room temperature, followed by incubation with 1/200 Alexa-488-conjugated
anti-mouse/rabbit IgG for 45 minutes. Coverslips were mounted on slides using
10% w/v Mowiol in PBS containing 0.1% w/v P-phenylenediamine and visualised
using a Leica TCS NT confocal laser scanning head attached to a Leica DM RXA
optical microscope (Leica Inc., St. Gallen, Switzerland). Leica TCS scanning
software was used to transpose four sequential images from four separate
optical sections taken at equal distances from the bottom to the top of the
cell. The same software was used to merge confocal images. Cytoskeletal
features were quantified in fields of view blindly selected at random.
Chemotaxis assays
Chemotaxis was assessed by direct observation and recording of cell
behaviour in stable concentration gradients of wild-type or mut C uPA or EGF
using the Dunn chamber chemotaxis chamber (Weber Scientific International
Ltd., Teddington, UK). This apparatus permits speed, direction of migration
and displacement of individual cells to be measured in relation to the
direction of a gradient (Zicha et al.,
1991). Details of the construction and calibration of Dunn
chambers is found elsewhere (Allen et al.,
1998
; Webb et al.,
1996
; Zicha et al.,
1991
). The chamber was filled with serum starvation medium (with
or without inhibitory agents) and a Matrigel®-coated coverslip (18
mmx18 mm) with cells seeded at a density of 5x103 cells
per coverslip was inverted and fixed onto the chamber. Serum starvation medium
containing wild-type or mut C uPA or EGF (with or without inhibitory agents)
was placed into the outer well of the chamber. Chambers were maintained at
37°C and frame grabbing time-lapse recording of cells located on the
bridge of the Dunn chamber was started within 15 minutes of assembly. Images
of cells were digitally recorded at a time-lapse interval of 10 minutes for 5
hours using the Kinetic Imaging AQM System (Kinetic Imaging, Liverpool, UK).
Cells were tracked and analysed as described previously
(Allen et al., 1998
;
Vanhaesebroeck et al., 1999
).
Scatter plots of cell trajectories for the 5 hour duration of each assay were
constructed on x,y axis plots, where the start point is coordinate (0,0) and
the end point of each cell marked with a closed circle. 10 median cell
trajectories from the total cell population were selected using a customised
MathematicaTM notebook (Graham A. Dunn, King's College London, London,
UK). Circular histograms were constructed using MathematicaTM 3.0 for
Windows 95 (Wolfram Research Inc., Champaign, IL, USA) as described previously
(Allen et al., 1998
;
Vanhaesebroeck et al., 1999
).
A virtual horizon of 20 µm was applied to MDA MB 231 cells owing to their
larger size. The Rayleigh test for unimodal clustering of directions
(Mardia, 1972
) was used to
assess whether the cell population pooled from several separate experiments
displayed directionality in the chemotactic gradient. Average speed and cell
displacement were calculated. Displacement equals the % cells that migrate
further than 20 µm (Jones et al.,
2000
).
Total cell lysates
Cells were washed in ice-cold PBS before addition of ice-cold RIPA buffer
(50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 5 mM sodium
molybdate, 1% v/v sodium deoxycholate, 1% v/v Nonidet P-40, 1% Triton X-100,
0.1% SDS) containing 50 mM sodium fluoride, 1 mM sodium orthovanadate, 20 mM
sodium phenylphosphate, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml
aprotonin, 5 µg/ml leupeptin and 2 µg/ml pepstatin A. Cells were
incubated for 15 minutes at 4°C, scraped and passed through a 25 gauge
needle five times before clarifying by centrifugation (16,000
g, 4°C, 10 minutes). Protein content was measured (Bio Rad
protein assay kit).
Akt phosphorylation assay
Total cell lysates (15 µg protein per well) were resolved on 10% SDS
PAGE gels. Protein was electrotransferred onto polyvinylidinefluoride (PVDF)
membranes (Immobilon-P; Millipore, UK). Membranes were incubated in blocking
buffer (2% BSA and 0.1% v/v Tween 20 in Tris buffered saline (TBS): 20 mM Tris
HCl pH 7.5, 0.5 M NaCl) for 1 hour at room temperature and probed with 1/1000
dilution anti-phospho-Akt or anti-total Akt in blocking buffer at 4°C for
16 hours. Membranes were washed five times for 5 minutes, incubated for 1 hour
at room temperature with 1/2000 dilution of HRP-conjugated anti-mouse IgG in
blocking buffer, washed five times with TBS containing 0.1% Tween 20.
Antibody-reactive proteins were detected using chemiluminescent substrate (ECL
Plus; Amersham UK). Equivalent samples of cell lysates were loaded in
duplicate on separate gels to allow analysis of both phospho-Akt and total
Akt.
Cdc42 and Rac1 activity assay
Cells grown on Matrigel®-coated 60 mm dishes were used at 50%
confluence. Cells washed once with ice-cold PBS and 550 µl ice-cold
GST-PAK-CRIB assay lysis buffer (50 mM Tris-HCl pH 7.4, 100 mM NaCl, 1 mM
MgCl2, 1% v/v Nonidet P-40; 10% v/v glycerol, 10 mM sodium
fluoride, 1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 10
µg/ml aprotonin, 5 µg/ml leupeptin and 2 µg/ml pepstatin A) were
added. Cells were scraped and cleared by centrifugation (16,000
g, 4°C, 1 minute). 50 µl of cleared lysate was retained
for analysis of total Rac1/Cdc42 by western blotting. 500 µl of cleared
lysate was mixed by inversion for 1 hour at 4°C with 25 µl of freshly
prepared slurry containing 20 µg GST-PAK-CRIB fusion protein coupled to
glutathione-conjugated agarose beads (Amersham, UK). Preparation of beads is
described elsewhere (Sander et al.,
1998). Beads were pelleted by pulse centrifugation (16,000
g, 4°C, 1 minute), washed three times in GST-PAK-CRIB
assay lysis buffer and split in two before a final pulse centrifugation.
Duplicate samples of pelleted beads were analysed for Rac1/Cdc42 content.
PAK-CRIB precipitated GTP-bound active Rac1/Cdc42, and total Rac1/Cdc42 from
corresponding unprecipitated cell lysates were resolved on 12% SDS PAGE gels.
Western blotting for Rac1/Cdc42 was carried out as described for Akt
phosphorylation assays except the blocking buffer contained 5% non-fat dry
milk (Blocker; Bio Rad, UK). 1/2000 dilution of mouse anti-Rac1 monoclonal
antibody (clone 23A8) was used with 1/3000 dilution of HRP-conjugated
anti-mouse IgG or 1/500 dilution of rabbit anti-Cdc42 polyclonal antibody was
used with 1/2000 HRP-conjugated anti-rabbit IgG. The ratio of active
Rac1/Cdc42 to total Rac1/Cdc42 was calculated by scanning densitometry of
immunoreactive bands (Imaging software, Kinetic Imaging, UK).
Immunoprecipitation
Cells grown on Matrigel®-coated 60 mm dishes were used at 50%
confluence. Cells were washed once with ice-cold microtubule stabilisation
buffer (0.1 M PIPES HCl pH 6.92, 2 M glycerol, 1 mM EGTA, 1 mM magnesium
acetate) and incubated with Triton X-100 lysis buffer (50 mM Tris-HCl pH 7.5,
150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 0.2% Triton X-100; 50 mM sodium fluoride, 1
mM sodium orthovanadate, 20 mM sodium phenylphosphate, 1 mM
phenylmethylsulfonyl fluoride, 10 µg/ml aprotonin, 5 µg/ml leupeptin and
2 µg/ml pepstatin A) for 15 minutes at 4°C. Lysed cells were scraped
and cleared by centrifugation (16,000 g, 4°C, 10 minutes).
Lysates with equal protein content (0.5 mg/ml) were mixed by inversion with 25
µl of slurry containing protein A agarose for 30 minutes at 4°C to
pre-clear. Beads were pelleted by pulse centrifugation and the supernatant
mixed by inversion with 4 µg/ml of antibody (anti-EGFR;
anti-ß1-integrin (clone 8E3)) overnight at 4°C.
Immunocomplexed proteins were precipitated by adding 25 µl of slurry
containing protein A agarose for 1 hour at 4°C followed by pulse
centrifugation. Immunocomplexed proteins in protein A agarose bead pellets
were resolved on 12% SDS PAGE gels. Western blot analysis was carried out as
described for Akt phosphorylation except the blocking buffer contained 5%
non-fat dry milk. 1/1000 dilution of mouse ß1-integrin
monoclonal antibody (Upstate Biotechnology) was used with 1/2000 dilution of
HRP-conjugated anti-mouse IgG. 1/1000 dilution of rabbit anti-EGFR or 1/4000
dilution of rabbit anti-N-WASP polyclonal antibodies was used with 1/2000
HRP-conjugated anti-rabbit IgG.
Cytoskeletal N-WASP assay
The method of Wei et al. was used to separate the cytosolic and
cytoskeletal fractions of MDA MB 231 cells
(Wei et al., 1996). Cells
grown on Matrigel®-coated 60 mm dishes were used at 50% confluence. Cells
were washed with ice-cold microtubule stabilisation buffer, incubated in 500
µl Triton X-100 lysis buffer (as used for immunoprecipitation) at 4°C
for 15 minutes, scraped and cleared by centrifugation (16,000
g, 4°C, 10 minutes). The supernatant was retained for
analysis of the N-WASP content of Triton X-100 soluble (cytosolic) fraction.
Pelleted Triton X-100 insoluble material was washed three times in 1 ml Triton
X-100 lysis buffer containing 0.1% v/v Triton X-100. Pelleted material was
resuspended in 250 µl RIPA buffer (as used for total cell lysates) by
passing through a 25-gauge needle five times, incubated at 4°C for 15
minutes, cleared by centrifugation (16,000 g, 4°C, 10
minutes) and analysed for N-WASP content of Triton X-100 insoluble
(cytoskeletal) fraction. Lysates of cytosolic and cytoskeletal fractions (15
µg per well) were resolved on 10% SDS PAGE gels and analysed for N-WASP by
western blot.
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Results |
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Cells maintained in growth medium (DMEM + 10% FCS) exhibited a variety of
morphological structures consistent with a motile phenotype, such as
polarisation, abundant membrane ruffles, lamellipodia and protrusions (data
not shown). For this reason the effect of uPA on the morphology of growing
cells could not be clearly assessed. Under conditions of serum starvation
(DMEM + 0.1% FCS for 24 hours), the majority of cells were not polarised,
having very few lamellipodial protrusions and membrane ruffles; 60% of cells
had peripheral actin rings (Fig.
1A) and 50% of cells had stress fibres/actin cables
(Fig. 1C). Abundant membrane
ruffles, lamellipodial protrusions and polarisation were seen in
uPA-stimulated cells after 30 minutes (Fig.
1B,D). uPA stimulation also caused rearrangement and
redistribution of focal adhesion complexes containing vinculin and
ß1-integrins. In unstimulated cells, small punctate
ß1-integrin rich complexes were concentrated around the cell
periphery and sparsely dotted throughout the cell body
(Fig. 1A). In uPA-stimulated
cells these small focal adhesions disappeared and were replaced by much larger
structures containing ß1-integrins that localised to the
leading edge and rear of the cell (Fig.
1B). The large vinculin rich focal adhesions, which colocalised to
the ends of actin stress fibres in unstimulated cells,
(Fig. 1C) were absent in
uPA-stimulated cells; instead smaller punctate and more diffuse
vinculin-containing structures were seen
(Fig. 1D). Similar turnover of
vinculin and ß1-integrin subunit containing structures has
been observed in various migratory cells
(Adams, 2001). For this reason
we decided to assess the effect of uPA on the migratory response of MDA MB 231
cells.
|
uPA and EGF induce chemotaxis that is dependent on
ß1-integrins
As reported above, MDA MB 231 cells maintained in 10% FCS on a
Matrigel® substratum polarised in various orientations. Cells assayed in
Dunn chambers under similar conditions displayed fast (>30 µm/h) random
migration and maximum levels of displacement, with nearly 100% cells migrating
beyond a 20 µm horizon within the 5 hour duration of the assay
(Fig. 2A). In contrast, cells
maintained in DMEM + 0.1% FCS for 24 hours exhibited markedly decreased
migratory speeds and displacement (Fig.
2B). We compared the chemotactic response of MDA MB 231 cells to
uPA with that to EGF, a known chemotactic factor in fibroblasts and breast
cancer cells (Wells et al.,
1998). MDA MB 231 cells express high levels of EGFR, and we
confirmed that EGF increased phosphorylation of EGFR in this cell type (C.
Sawyer, unpublished). Serum-starved cells assayed in isotropic concentrations
of EGF, wild-type uPA or mut C uPA displayed increased speed with random
directionality (data not shown), similar to cells maintained in growth medium.
When serum-starved cells were placed in linear gradients of EGF
(Fig. 2C,E), mut C or wild type
uPA (Fig. 2DF), speed and
displacement were increased. Moreover, cells displayed unimodal upgradient
polarisation and positive chemotaxis towards the source of EGF or uPA. The
concentration of chemoattractant used was found to be critical for the
migratory and chemotactic response. Increased migratory speed and chemotaxis
was only observed at 10-25 ng/ml EGF (Fig.
2E) and 5-25 nM uPA (Fig.
2F). Optimal chemotaxis occurred in gradients generated with 15
ng/ml EGF or 5 nM uPA in the outer well. Mut C uPA stimulated higher migratory
speeds than wt uPA over the same concentration range
(Fig. 2F).
|
The chemotactic responses of cells to EGF and uPA required ß1-integrins. Incubation of cells with anti-ß1-integrin subunit neutralising antibody inhibited increases in cell speed and chemotaxis stimulated by both EGF (data not shown) and uPA (Fig. 2G), whereas anti-ß3-integrin subunit antibody had no effect on the same parameters (data not shown).
uPA stimulates chemotaxis in a PI3K-independent manner
Class 1A PI3Ks have roles in signalling to the actin cytoskeleton and
driving the chemotactic response
(Arrieumerlou et al., 1998;
Hill et al., 2000
;
Hirsch et al., 2000
;
Jones, 2000
;
Li et al., 2000
;
Reif et al., 1996
;
Vanhaesebroeck et al., 1999
).
It has recently been suggested that uPA can signal through the interaction of
Tyk2 and PI3K to promote cytoskeletal changes and migration in vascular smooth
muscle cells (Kusch et al.,
2000
). Therefore, we considered whether uPA could signal through
PI3K in MDA MB 231 cells. In our studies we used EGF stimulation as a positive
control for PI3K-dependent signalling. Immunoblot analysis of whole cell
lysates indicated that both EGF- and mut C uPA-stimulation increased the
phosphorylation of Akt (Fig.
3A), which is a well defined downstream effector of PI3K
(Vanhaesebroeck and Waterfield,
1999
). In addition, LY 294002 totally negated EGF and uPA induced
Akt phosphorylation (Fig. 3B)
at concentrations (1-10 µM) that exclusively inhibit class 1A PI3Ks
(Vanhaesebroeck and Waterfield,
1999
). LY 294002 vehicle alone (0.005-0.05% v/v DMSO) had no
effect on Akt phosphorylation stimulated by either EGF or mut C uPA (data not
shown). These findings confirm that both EGF and uPA are capable of signalling
through PI3K.
|
We next considered whether signalling through PI3K was required for the chemotactic and/or migratory response of MDA MB 231 cells in a gradient of uPA. EGF gradients were used as a comparative control. Cells were analysed in EGF and uPA gradients using LY 294002 at the same concentrations that inhibited Akt phosphorylation. Observation of MDA MB 231 cells using time-lapse recording indicated that LY 294002 did not affect the random migration of serum starved cells at concentrations below 20 µM. The chemotactic response of cells in an EGF gradient was sensitive to inhibition by 1 µM LY 294002, with no effect on random cell movement and speed (Fig. 4A,B). Addition of 5 µM LY 294002 completely attenuated the increase in speed and chemotaxis stimulated by EGF. In contrast, the chemotaxis of cells in linear gradients of wild-type uPA or mut C uPA was not affected by the presence of LY 294002 (1-10 µM) (Fig. 4C,D). In conclusion our data strongly suggested that uPA does not require PI3K to promote chemotactic and migratory responses in MDA MB 231 cells despite being activated (as judged by increased Akt phosphorylation).
|
The majority of cytoskeletal rearrangements induced by uPA are PI3K
independent
The effect of inhibition of PI3K on actin cytoskeleton rearrangements
stimulated by uPA and EGF was investigated. The most notable difference in
cytoskeletal changes induced by the two chemoattractants was the strong and
rapid induction of microspike extensions by uPA but not EGF. Microspike
extensions were not observed in unstimulated cells; 37% of cells produced
microspikes after 3 minutes of uPA stimulation
(Fig. 5A,G), and 5% of cells
produced microspikes after 3 minutes EGF stimulation
(Fig. 5G). Microspikes formed
in response to uPA with and without addition of LY 294002 were similar in
length and structure (Fig.
5A,D). However, LY 294002 decreased the number of cells that
formed microspikes in response to uPA by 60%
(Fig. 5G). In contrast there
was no inhibitory effect of LY 294002 on uPA-induced membrane ruffles,
lamellipodia or cell polarisation (Fig.
5B,E). In fact the number of cells with lamellipodial extensions
stimulated by uPA was increased by
40% in the presence of LY 294002. In
contrast, LY 294002 almost totally negated membrane ruffles, lamellipodia and
polarisation stimulated by EGF (Fig.
5C,F). Most notably, the number of EGF-stimulated cells that
formed lamellipodia was decreased by
65% in the presence of LY 294002
(Fig. 5G). In the presence of
LY 294002 there was a modest increase in the proportion of EGF-stimulated
cells that produced microspikes (an increase from 5% to 10% of total cell
population) (Fig. 5G). However,
it should be noted that the microspikes stimulated by EGF were not a major
morphological feature of this small fraction of cells, as was the case in uPA
stimulated cells.
|
Cdc42 and Rac1 activation by uPA requires ß1-integrins but not
PI3K
The cytoskeletal changes and migratory responses induced by uPA in MDA MB
231 cells were similar to those induced by active small Rho GTPase proteins in
macrophages and fibroblasts. That is active GTP-bound Cdc42 induces microspike
formation and cell polarisation, and active GTP-bound Rac1 induces membrane
ruffles, lamellipodia formation and sustained migration
(Allen et al., 1997;
Allen et al., 1998
;
Kozma et al., 1995
;
Ridley et al., 1992
).
To investigate whether the differences in cytoskeletal structures induced
by uPA and EGF pertained to differences in Cdc42 and Rac1 activation, the
levels of GTP-bound Cdc42 and Rac1 were determined. Active Cdc42 and Rac1 were
precipitated from lysates of cells treated with EGF or mut C uPA using the
Cdc42/Rac1-binding domain of p21-activated kinase (PAK) fused to GST
(Sander et al., 1998). First,
we determined whether the same concentrations of uPA and EGF that stimulated
chemotaxis also activated Rac1 and Cdc42. 10 nM mut C uPA and 10 ng/ml EGF
stimulated maximal levels of Rac1 and Cdc42 activation (data not shown), which
correlated with the stimulation of chemotaxis and increased migratory speed
(Fig. 2). uPA stimulated a
transient but substantial increase in active Cdc42 without affecting total
cellular Cdc42 (Fig. 6A).
Maximum Cdc42 activation occurred after 2 minutes of stimulation, and basal
levels were restored by 30 minutes. In contrast, the increase in Cdc42
activation stimulated by EGF was sustained, albeit at a modest level, for at
least 30 minutes. Both uPA and EGF activated Rac1 within 2 minutes, and this
level of activation was maintained for at least 30 minutes
(Fig. 6B).
|
We next determined whether activation of Rac1 and Cdc42 by EGF and uPA occurred downstream of ß1-integrins and PI3K. LY 294002 inhibited activation of Rac1 and Cdc42 by EGF at concentrations that blocked chemotaxis (Fig. 7A-D). In contrast, LY 294002 had no effect on Rac1 and Cdc42 activation stimulated by uPA (Fig. 7A-D). The requirement of ß1-integrins for chemotaxis in response to EGF and uPA correlated with their involvement in EGF and uPA-induced activation of small Rho GTPases. The same concentrations of anti-ß1-integrin neutralising antibody that inhibited chemotaxis (Fig. 2G) also inhibited Rac1 and Cdc42 activation in response to EGF (data not shown) and uPA (Fig. 7E). Irrelevant mouse IgG did not effect Rac1/Cdc42 activation in response to EGF-uPA (data not shown). This confirms that signalling from uPA-uPAR and EGF-EGFR to the small Rho GTPases requires fully functional ß1-integrins but has divergent requirement for PI3K activity.
|
Divergent requirement of PI3K for N-WASP activation by uPA and EGF
occurs through a mechanism involving ß1-integrins
The existence of an EGFR/PI3K/N-WASP pathway in MDA MB 231 cells was
supported by several findings. (a) N-WASP was detected in EGFR
immunoprecipitates and this association was increased upon EGF stimulation
(Fig. 8A). (b) N-WASP content
increased in the cytoskeletal fraction and decreased in the cytosolic fraction
after 10-30 minutes stimulation with EGF
(Fig. 8B), an effect that was
totally inhibited by LY 294002 (Fig.
8C). (c) N-WASP colocalised with F-actin rich membrane ruffles
upon EGF stimulation (Fig. 9E), an effect that was totally inhibited by LY 294002
(Fig. 9F). LY 294002 alone had
no effect on N-WASP localisation to F-actin in unstimulated cells
(Fig. 9A,B). These findings are
in accordance with previous reports of EGFR signalling to N-WASP and induction
of actin cytoskeletal rearrangements (Miki
et al., 1996; Miki et al.,
1998
; She et al.,
1997
) and provide support to a regulatory role for PI3K in N-WASP
activation downstream of EGFR.
|
|
The dramatic induction of microspikes suggested activation of N-WASP by uPA. N-WASP content increased in the cytoskeletal fraction and decreased in the cytosolic fraction of uPA-stimulated cells (Fig. 8B). The translocation of N-WASP stimulated by uPA occurred after stimulation for 3 minutes (compared with 10 minutes after EGF stimulation) and lasted for at least 30 minutes. Translocation of N-WASP in response to EGF and uPA also differed in sensitivity to LY 294002; the response induced by EGF was totally inhibited whereas that induced by uPA was partially inhibited by the PI3K inhibitor (Fig. 8C). This partial inhibitory effect may explain the decreased number of cells producing microspikes in response to uPA in the presence of LY 294002 (Fig. 5G). Although we could not visualise N-WASP within, or at the base of, uPA-induced microspikes by immunofluorescent staining (data not shown), it colocalised with F-actin at the leading edge of uPA-stimulated cells (Fig. 9C), and this localisation was not affected by PI3K inhibition (Fig. 9D). Taken together these data suggest that both PI3K-dependent and independent activation of N-WASP occurs upon uPAR ligation with uPA.
How does uPAR ligation with uPA induce the rapid activation of N-WASP? We predicted that ß1-integrins, which associate with uPAR upon ligation with uPA, are involved in uPAR signalling to N-WASP. ß1-integrins were required for chemotaxis and Rho GTPase activation by EGF and uPA (Fig. 2G; Fig. 7E), and similar F-actin structures were colocalised with ß1-integrins and N-WASP in unstimulated and stimulated cells (Fig. 1B; Fig. 9C). We analysed ß1-integrin subunit immunoprecipitates for N-WASP content and found the two proteins were associated in unstimulated cells (Fig. 10A). This association was almost totally disrupted in cells stimulated with uPA for 1-3 minutes and was returned to basal levels at 30 minutes (Fig. 10A). The disassociation of ß1-integrin and N-WASP stimulated by uPA also occurred in the presence of LY 294002 (Fig. 10A). In contrast, EGF stimulated partial and latent (after 10 minutes stimulation) disassociation of ß1-integrin-N-WASP, which was totally inhibited by LY 294002 (Fig. 10B).
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The stimulatory effects of uPA in MDA MB 231 cells were independent of the
proteolytic activity of uPA since both fully proteolytically active wild-type
uPA and proteolytically inactive mut C uPA induced similar responses. The only
observable difference between the two forms of uPA was that a faster migratory
speed was induced by mut C uPA, an effect that may be explained by its
slightly higher affinity for uPAR than wild-type uPA
(Hamelin et al., 1993). The
relocalisation of focal contacts, formation of abundant new actin rich
structures, increased migratory speed, polarisation and chemotaxis of cells
induced by both EGF and uPA were indicative of their signalling through the
small Rho GTPases and N-WASP (Allen et al.,
1998
; Miki et al.,
1998
; Ridley et al.,
1992
; Rohatgi et al.,
1999
). The concentrations of EGF or uPA that induced maximal
chemotactic and migratory responses also stimulated maximal levels of Rho
GTPase activity. The temporal profiles of Cdc42, Rac1 and N-WASP activation in
uPA and EGF-stimulated cells correlated with the formation of their associated
cytoskeletal structures. uPA and EGF both stimulated the formation of
N-WASP/F-actin rich membrane ruffles and lamellipodia over the same time
course as the initiation of the migratory response and sustained Rac1
activation (after 3-30 minutes stimulation). Cdc42 was rapidly activated by
EGF, sustained at modest levels for up to 30 minutes and associated with
latent translocation of N-WASP to the cytoskeleton (after 10-30 minutes
stimulation) and microspike production in only few cells. Cdc42 activation in
response to uPA was also rapid but transient and occurred directly in concert
with the translocation of N-WASP to the cytoskeleton and formation of abundant
microspikes (after only 1-3 minutes stimulation). Similar microspike
production induced by uPA-uPAR signalling was observed in vascular smooth
muscle cells (Degryse et al.,
1999
). Although uPAR binding to vitronectin stimulated
lamellipodial protrusions through Rac1 activation, microspike formation or
Cdc42 activation was not detected in an uPAR overexpression model
(Kjoller and Hall, 2001
). This
contrary finding may have several explanations. Signal transduction molecules
recruited by activated uPAR could be cell-type specific or different uPAR
ligands could stimulate distinct cytoskeletal changes by preferential
activation of specific signal transduction pathways, as suggested by Degryse
et al. (Degryse et al., 2001
).
It may be that overexpression of uPAR by transfection results in elevated and
sustained Rac1 activation, which suppresses Cdc42 activation and associated
cytoskeletal rearrangements. The later possibility could result from the
ability of small Rho GTPases to positively or negatively regulate each others
activity during cell migration (Kjoller
and Hall, 1999
).
The involvement of PI3K in activation of GEFs for the small Rho GTPases,
cytoskeletal rearrangements and chemotaxis
(Arrieumerlou et al., 1998;
Han et al., 1998
;
Hirsch et al., 2000
;
Li et al., 2000
;
Reif et al., 1996
) led us to
consider whether uPA-uPAR chemotactic signalling utilized PI3K for the
activation of the small Rho GTPases. PI3K is suggested to act as an upstream
effector for Rac1, and in some cases for Cdc42, in several growth factor and
chemoattractant stimulated pathways
(Banyard et al., 2000
;
Benard et al., 1999
;
Hawkins et al., 1995
;
Hooshmand-Jones, 2000; Ma et al.,
1998
). Recent studies by us and others have further confirmed that
activation of specific PI3K p110 catalytic subunit isoforms in breast
carcinoma cells is necessary for EGF-induced cytoskeletal changes
(Hill et al., 2000
) and
chemotaxis (Sawyer et al., submitted for publication). uPA and EGF signalling
through PI3K in MDA MB 231 cells was evident through their ability to
stimulate Akt phosphorylation that was sensitive to inhibition by LY 294002.
As predicted, Cdc42 activation, Rac1 activation, lamellipodial protrusion and
chemotaxis stimulated by EGF were totally dependent on PI3K activity.
Surprisingly we found that PI3K activity was not necessary for the vast
majority of uPA-stimulated responses. Inhibition of PI3K had no effect on
Cdc42 or Rac1 activation, chemotaxis or speed of cell movement.
What are the different mechanism(s) for PI3K-independent and PI3K-dependent
activation of Cdc42 and Rac1 by uPA and EGF? Vav2 seemed a likely candidate
GEF for differential upstream signalling to Cdc42 and Rac1 via uPA and EGF.
Evidence from studies in HEK293 and fibroblast cell lines suggested that
growth factor receptors signal via phosphorylation of Vav2 to activate the Rho
GTPases Cdc42, Rac1 and RhoA (Liu and
Burridge, 2000). However activation of Rho GTPases Cdc42, Rac1 and
RhoA in response to ß1-integrin activation was found to be
independent of Vav2 activation (Liu and
Burridge, 2000
). Despite successful Vav2 immunoprecipitation from
MDA MB 231 cells, we could not detect altered tyrosine phosphorylation in
response to either EGF or uPA (J.S., unpublished). This suggests that neither
EGF nor uPA activates Vav2 in this cell line. However, differential signalling
through other GEFs could underlie PI3K-dependent/independent activation of Rho
GTPases by EGF and uPA.
PI3K-dependent signalling by uPA to the actin cytoskeleton was evident in
MB MDA 231 cells. LY 294002 partially inhibited uPA-induced microspikes, an
effect that correlated with a partial decrease in N-WASP localisation to the
cytoskeletal fraction. Therefore, PI3K-dependent and PI3K-independent
activation of N-WASP can activate microspike production in response to uPA.
However, lamellipodia formation and Rho GTPase activation in response to uPA
was unaffected by LY 294002, which is consistent with the lack of effect of
PI3K inhibition on uPA-induced polarisation and chemotaxis. PI3K-independent
N-WASP activation by uPA probably occurs through PI3K-independent activation
of Cdc42/Rac1, whereas PI3K-dependent activation of this member of the WASP
family may involve direct interaction of p85 with its polyproline
region (Banin et al., 1996
;
Finan et al., 1996
) or through
other effector interactions. Nevertheless, it should be appreciated that our
results indicate `PI3K-dependent' N-WASP activation is not necessary for
uPA-induced chemotaxis, whereas `PI3K-independent' N-WASP activation is
sufficient for uPA-induced chemotaxis.
A recently proposed mechanism for the promotion of vascular smooth muscle
cell migration by uPA involved PI3K activation
(Kusch et al., 2000). A
similar mechanism was not apparent in MDA MB 231 breast carcinoma cells. Kusch
et al. based the requirement of p110 PI3K catalytic activity in the migratory
response to uPA on results from experiments using very high concentrations of
LY 294002 (50 µM) and wortmannin (200 nM)
(Kusch et al., 2000
). Both
inhibitors lose specificity for PI3K and inhibit kinases in other signalling
pathways at such high concentrations
(Vanhaesebroeck and Waterfield,
1999
). In two recent reports, it was suggested that the p85
regulatory subunit of class 1A PI3Ks binds to and activates Cdc42 with no
requirement for p110 catalytic activity
(Hill et al., 2001
;
Jimenez et al., 2000
). A
similar interaction could explain the involvement of Tyk2-p85 in migration
induced by uPA in vascular smooth muscle cells
(Kusch et al., 2000
). A
similar mechanism could also exist in MDA MB 231 cells.
The involvement of ß1-integrins in chemotaxis and cytoskeletal reorganisation in MDA MB 231 cells stimulated by uPA was confirmed by several findings. Firstly, stimulation of cells with uPA resulted in re-distribution of focal complexes containing ß1-integrins. Secondly, a ß1-integrin blocking antibody inhibited chemotaxis towards uPA, and this correlated with inhibition of Cdc42/Rac1 activation. However, the inhibitory effects of anti-ß1-integrin on chemotaxis and activation of small GTPases was apparent in EGF-stimulated cells, and so it was not an uPAR-specific effect. However, an uPAuPAR-specific role for ß1-integrins was identified and appears to be part of the mechanism involved in PI3K-independent activation of N-WASP. An interaction between ß1-integrins and N-WASP was fully disrupted upon uPA stimulation. This effect was rapid and transient (occurring after 1 minute uPA stimulation), in concert with the activation of Cdc42, and was not sensitive to inhibition by LY 294002. In contrast, the effect of EGF on the interaction between ß1-integrins and N-WASP was far less dramatic and entirely PI3K dependent, being a latent and partial response (occurring after 10 minutes stimulation) and sensitive to inhibition by LY 294002. The transient nature of the disassociation of ß1-integrins and N-WASP induced by uPA indicates it may well have a role in the initiation of N-WASP activation. It remains to be determined whether the association of ß1-integrins with N-WASP occurs through a direct interaction between the two molecules or whether intermediate proteins recruit N-WASP to the complex. The exact mechanism of this molecular interaction and its disruption upon uPAR ligation awaits further characterisation. Nevertheless, this potential mechanism for N-WASP activation could endow cells with the ability to respond to specific chemotactic cues in the absence of PI3K activity.
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Acknowledgments |
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References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Adams, J. C. (2001). Cell-matrix contact structures. Cell. Mol. Life Sci. 58,371 -392.[Medline]
Allen, W. E., Jones, G. E., Pollard, J. W. and Ridley, A. J.
(1997). Rho, Rac and Cdc42 regulate actin organization and cell
adhesion in macrophages. J. Cell Sci.
110,707
-720.
Allen, W. E., Zicha, D., Ridley, A. J. and Jones, G. E.
(1998). A role for Cdc42 in macrophage chemotaxis. J.
Cell Biol. 141,1147
-1157.
Andreasen, P. A., Kjoller, L., Christensen, L. and Duffy, M. J. (1997). The urokinase-type plasminogen activator system in cancer metastasis: a review. Int. J. Cancer 72, 1-22.[Medline]
Arrieumerlou, C., Donnadieu, E., Brennan, P., Keryer, G., Bismuth, G., Cantrell, D. and Trautmann, A. (1998). Involvement of phosphoinositide 3-kinase and Rac in membrane ruffling induced by IL-2 in T cells. Eur. J. Immunol. 28,1877 -1885.[Medline]
Banin, S., Truong, O., Katz, D. R., Waterfield, M. D., Bricknell, P. M. and Gout, I. (1996). Wiskott-Aldrich syndrome protein (WASp) is a binding partner for c-Src family protein-tyrosine kinases. Curr. Biol. 6,981 -988.[Medline]
Banyard, J., Anand-Apte, B., Symons, M. and Zetter, B. R. (2000). Motility and invasion are differentially modulated by Rho family GTPases. Oncogene 19,580 -591.[Medline]
Benard, V., Bohl, B. P. and Bokoch, G. M.
(1999). Characterization of Rac and Cdc42 activation in
chemoattractant-stimulated human neutrophils using a novel assay for active
GTPases. J. Biol. Chem.
274,13198
-13204.
Bohuslav, J., Horejsi, V., Hansmann, C., Stockl, J., Weidle, U. H., Majdic, O., Bartke, I., Knapp, W. and Stockinger, H. (1995). Urokinase plasminogen-activator receptor, beta-2-integrins, and src-kinases within a single receptor complex of human monocytes. J. Exp. Med. 181,1381 -1390.[Abstract]
Busso, N., Masur, S. K., Lazega, D., Waxman, S. and Ossowski, L. (1994). Induction of cell-migration by prourokinase binding to its receptor possible mechanism for signal-transduction in human epithelial-cells. J. Cell Biol. 126,259 -270.[Abstract]
Carriero, M. V., Del Vecchio, S., Capozzoli, M., Franco, P.,
Fontana, L., Zannetti, A., Botti, G., D'Aiuto, G., Salvatore, M. and
Stoppelli, M. P. (1999). Urokinase receptor interacts with
alpha(v)beta(5) vitronectin receptor, promoting urokinase-dependent cell
migration in breast cancer. Cancer Res.
59,5307
-5314.
Chiaradonna, F., Fontana, L., Iavarone, C., Carriero, M. V.,
Scholz, G., Barone, M. V. and Stoppelli, M. P. (1999).
Urokinase receptor-dependent and independent p56/59(hck) activation
state is a molecular switch between myelomonocytic cell motility and
adherence. EMBO J. 18,3013
-3023.
Degryse, B., Resnati, M., Rabbani, S. A., Villa, A., Fazioli, F.
and Blasi, F. (1999). Src-dependence and pertussis toxin
sensitivity of urokinase receptor-dependent chemotaxis and cytoskeleton
reorganization in rat smooth muscle cells. Blood
94,649
-662.
Degryse, B., Orlando, S., Resnati, M., Rabbani, S. A. and Blasi, F. (2001). Urokinase/urokinase receptor and vitronectin/alpha(v)beta(3) integrin induce chemotaxis and cytoskeleton reorganization through different signaling pathways. Oncogene 20,2032 -2043.[Medline]
Fawcett, J. and Pawson, T. (2000). Signal
transduction N-WASP regulation the sting in the tail.
Science 290,725
-726.
Fibbi, G., Caldini, R., Chevanne, M., Pucci, M., Schiavone, N., Morbidelli, L., Parenti, A., Granger, H. J., Del Rosso, M. and Ziche, M. (1998). Urokinase-dependent angiogenesis in vitro and diacylglycerol production are blocked by antisense oligonucleotides against the urokinase receptor. Lab. Invest. 78,1109 -1119.[Medline]
Finan, P. M., Soames, C. J., Wilson, L., Nelson, D. L., Stewart,
D. M., Truong, O., Hsuan, J. J. and Kellie, S. (1996).
Identification of regions of the Wiskott-Aldrich syndrome protein responsible
for association with selected Src homology 3 domains. J. Biol.
Chem. 271,26291
-26295.
Foekens, J. A., Peters, H. A., Look, M. P., Portengen, H.,
Schmitt, M., Kramer, M. D., Brunner, N., Janicke, F., Gelder, M. E. M.,
Henzen-Logmans, S. C. et al. (2000). The urokinase system of
plasminogen activation and prognosis in 2780 breast cancer patients.
Cancer Res. 60,636
-643.
Hamelin, J., Sarmientos, P., Orsini, G. and Galibert, F. (1993). Implication of cysteine residues in the activity of single-chain urokinase-plasminogen activator. Biochem. Biophys. Res. Commun. 194,978 -985.[Medline]
Han, J. W., Luby-Phelps, K., Das, B., Shu, X. D., Xia, Y.,
Mosteller, R. D., Krishna, U. M., Falck, J. R., White, M. A. and Broek, D.
(1998). Role of substrates and products of PI3-kinase in
regulating activation of Rac-related guanosine triphosphatases by Vav.
Science 279,558
-560.
Haugh, J. M., Codazzi, F., Teruel, M. and Meyer, T.
(2000). Spatial sensing in fibroblasts mediated by 3'
phosphoinositides. J. Cell Biol.
151,1269
-1279.
Hawkins, P. T., Eguinoa, A., Qiu, R. G., Stokoe, D., Cooke, F. T., Walters, R., Wennstrom, S., Claessonwelsh, L., Evans, T., Symons, M. and Stephens, L. (1995). PDGF stimulates an increase in GTP-Rac via activation of phosphoinositide 3-kinase. Curr. Biol. 5,393 -403.[Medline]
Hebert, C. A. and Baker, J. B. (1988). Linkage of extracellular plasminogen-activator to the fibroblast cystoskeleton colocalization of cell-surface urokinase with vinculin. J. Cell Biol. 106,1241 -1247.[Abstract]
Hill, K., Welti, S., Yu, J. H., Murray, J. T., Yip, S. C.,
Condeelis, J. S., Segall, J. E. and Backer, J. M. (2000).
Specific requirement for the p85-p110 alpha phosphatidylinositol 3-kinase
during epidermal growth factor-stimulated actin nucleation in breast cancer
cells. J. Biol. Chem.
275,3741
-3744.
Hill, K. M., Huang, Y., Yip, S. C., Yu, J., Segall, J. E. and
Backer, J. M. (2001). N-terminal domains of the class Ia
phosphoinositide 3-kinase regulatory subunit play a role in cytoskeletal but
not mitogenic signaling. J. Biol. Chem.
276,16374
-16378.
Hirsch, E., Katanaev, V. L., Garlanda, C., Azzolino, O., Pirola,
L., Silengo, L., Sozzani, S., Mantovani, A., Altruda, F. and Wymann, M. P.
(2000). Central role for G protein-coupled phosphoinositide
3-kinase gamma in inflammation. Science
287,1049
-1053.
HooshmandRad, R., ClaessonWelsh, L., Wennstrom, S., Yokote, K., Siegbahn, A. and Heldin, C. H. (1997). Involvement of phosphatidylinositide 3'-kinase and Rac in platelet-derived growth factor-induced actin reorganization and chemotaxis. Exp. Cell Res. 234,434 -441.[Medline]
Jimenez, C., Portela, R. A., Mellado, M., Rodriguez-Frade, J.
M., Collard, J., Serrano, A., Martinez, A., Avila, J. and Carrera, A. C.
(2000). Role of the PI3K regulatory subunit in the control of
actin organization and cell migration. J. Cell Biol.
151,249
-261.
Jones, G. E. (2000). Cellular signaling in
macrophage migration and chemotaxis. J. Leukocyte
Biol. 68,593
-602.
Jones, G. E., Ridley, A. J. and Zicha, D. (2000). Rho GTPases and cell migration: Measurement of macrophage chemotaxis. Regulators and Effectors of Small GTPases, Pt D 325,449 -462.
Kim, A. S., Kakalis, L. T., Abdul-Manan, M., Liu, G. A. and Rosen, M. K. (2000). Autoinhibition and activation mechanisms of the Wiskott-Aldrich syndrome protein. Nature 404,151 -158.[Medline]
Kim, H. and Muller, W. J. (1999). The role of the epidermal growth factor receptor family in mammary tumorigenesis and metastasis. Exp. Cell Res. 253, 78-87.[Medline]
Kjoller, L. and Hall, A. (1999). Signaling to Rho GTPases. Exp. Cell Res. 253,166 -179.[Medline]
Kjoller, L. and Hall, A. (2001). Rac mediates
cytoskeletal rearrangements and increased cell motility induced by
urokinase-type plasminogen activator receptor binding to vitronectin.
J. Cell Biol. 152,1145
-1157.
Kolluri, R., Tolias, K. F., Carpenter, C. L., Rosen, F. S. and
Kirchhausen, T. (1996). Direct interaction of the
Wiskott-Aldrich syndrome protein with the GTPase Cdc42. Proc. Nat.
Acad. Sci. USA. 93,5615
-5618.
Kozma, R., Ahmed, S., Best, A. and Lim, L. (1995). The Ras-related protein Cdc42hs and bradykinin promote formation of peripheral actin microspikes and filopodia in swiss 3T3 fibroblasts. Mol. Cell. Biol. 15,1942 -1952.[Abstract]
Kusch, A., Tkachuk, S., Haller, H., Dietz, R., Gulba, D. C.,
Lipp, M. and Dumler, I. (2000). Urokinase stimulates human
vascular smooth muscle cell migration via a phosphatidylinositol 3-kinase-Tyk2
interaction. J. Biol. Chem.
275,39466
-39473.
Li, Z., Jiang, H. P., Xie, W., Zhang, Z. C., Smrcka, A. V. and
Wu, D. Q. (2000). Roles of PLC-beta 2 and -beta 3 and PI3K
gamma in chemoattractant-mediated signal transduction.
Science 287,1046
-1049.
Liotta, L. A. and Kohn, E. C. (2001). The microenvironment of the tumourhost interface. Nature 411,375 -379.[Medline]
Liu, B. P. and Burridge, K. (2000). Vav2
activates Rac1, Cdc42, and RhoA downstream from growth factor receptors but
not beta 1 integrins. Mol. Cell. Biol.
20,7160
-7169.
Ma, A. D., Metjian, A., Bagrodia, S., Taylor, S. and Abrams, C.
S. (1998). Cytoskeletal reorganization by G protein-coupled
receptors is dependent on phosphoinositide 3-kinase gamma, a Rac guanosine
exchange factor, and Rac. Mol. Cell. Biol.
18,4744
-4751.
Machesky, L. M. and Hall, A. (1997). Role of
actin polymerization and adhesion to extracellular matrix in Rac- and
Rho-induced cytoskeletal reorganization. J. Cell Biol.
138,913
-926.
Mardia, K. V. (1972). Statistics of Directional Data, pp. 1-357. New York, Academic Press.
Miki, H., Miura, K. and Takenawa, T. (1996). N-WASP, a novel actin-depolymerizing protein, regulates the cortical cytoskeletal rearrangement in a PIP2-dependent manner downstream of tyrosine kinases. EMBO J. 15,5326 -5335.[Abstract]
Miki, H., Sasaki, T., Takai, Y. and Takenawa, T. (1998). Induction of filopodium formation by a WASP-related actin-depolymerizing protein N-WASP. Nature 391, 93-96.[Medline]
Miki, H. and Takenawa, T. (1998). Direct binding of the verprolin-homology domain in N-WASP to actin is essential for cytoskeletal reorganization. Biochem. Biophys. Res. Commun. 243,73 -78.[Medline]
Millard, T. H. and Machesky, L. M. (2001). The Wiskott-Aldrich syndrome protein (WASP) family. Trends Biochem. Sci. 26,198 -199.[Medline]
Moghal, N. and Sternberg, P. W. (1999). Multiple positive and negative regulators of signaling by the EGF-receptor. Curr. Opin. Cell Biol. 11,190 -196.[Medline]
Muller, A., Homey, B., Soto, H., Ge, N., Catron, D., Buchanan, M. E., McClanahan, T., Murphy, E., Yuan, W., Wagner, S. N. et al. (2001). Involvement of chemokine receptors in breast cancer metastasis. Nature 410,50 -56.[Medline]
Mullins, R. D. and Machesky, L. M. (2000). Actin assembly mediated by Arp2/3 complex and WASP family proteins. Regulators and Effectors of Small GTPases, Pt D 325,214 -237.
Nguyen, D. H. D., Catling, A. D., Webb, D. J., Sankovic, M.,
Walker, L. A., Somlyo, A. V., Weber, M. J. and Gonias, S. L.
(1999). Myosin light chain kinase functions downstream of Ras/ERK
to promote migration of urokinase-type plasminogen activator- stimulated cells
in an integrin-selective manner. J. Cell Biol.
146,149
-164.
Nguyen, D. H. D., Webb, D. J., Catling, A. D., Song, Q.,
Dhakephalkar, A., Weber, M. J., Ravichandran, K. S. and Gonias, S. L.
(2000). Urokinase-type plasminogen activator stimulates the
Ras/extracellular signal-regulated kinase (ERK) signaling pathway and MCF-7
cell migration by a mechanism that requires focal adhesion kinase, Src, and
Shc Rapid dissociation of Grb2/Sos-Shc complex is associated with the
transient phosphorylation of ERK in urokinase-treated cells. J.
Biol. Chem. 275,19382
-19388.
Nobes, C. D. and Hall, A. (1995). Rho, Rac, and Cdc42 GTPases regulate the assembly of multimolecular focal complexes associated with actin stress fibers, lamellipodia, and filopodia. Cell 81,53 -62.[Medline]
Nobes, C. D. and Hall, A. (1999). Rho GTPases
control polarity, protrusion, and adhesion during cell movement. J.
Cell Biol. 144,1235
-1244.
Ossowski, L. and Aguirre-Ghiso, J. A. (2000). Urokinase receptor and integrin partnership: coordination of signaling for cell adhesion, migration and growth. Curr. Opin. Cell Biol. 12,613 -620.[Medline]
Preissner, K. T., Kanse, S. M. and May, A. E. (2000). Urokinase receptor: a molecular organizer in cellular communication. Curr. Opin. Cell Biol. 12,621 -628.[Medline]
Reif, K., Nobes, C. D., Thomas, G., Hall, A. and Cantrell, D. A. (1996). Phosphatidylinositol 3-kinase signals activate a selective subset of Rac/Rho-dependent effector pathways. Curr. Biol. 6,1445 -1455.[Medline]
Resnati, M., Guttinger, M., Valcamonica, S., Sidenius, N., Blasi, F. and Fazioli, F. (1996). Proteolytic cleavage of the urokinase receptor substitutes for the agonist-induced chemotactic effect. EMBO J. 15,1572 -1582.[Abstract]
Ridley, A. J. and Hall, A. (1992). The small GTP-binding protein Rho regulates the assembly of focal adhesions and actin stress fibers in response to growth-factors. Cell 70,389 -399.[Medline]
Ridley, A. J., Paterson, H. F., Johnston, C. L., Diekmann, D., And Hall, A. (1992). The small GTP-binding protein Rac regulates growth-factor induced membrane ruffling. Cell 70,401 -410.[Medline]
Ridley, A. J., Allen, W. E., Peppelenbosch, M. and Jones, G. E. (1999). Rho family proteins and cell migration. Biochem. Soc. Symp. 65,111 -123.[Medline]
Rohatgi, R., Ma, L., Miki, H., Lopez, M., Kirchhausen, T., Takenawa, T. and Kirschner, M. W. (1999). The interaction between N-WASP and the Arp2/3 links Cdc42-dependent signals to actin assembly. Cell 97,221 -231.[Medline]
Rottner, K., Hall, A. and Small, J. V. (1999). Interplay between Rac and Rho in the control of substrate contact dynamics. Curr. Biol. 9,640 -648.[Medline]
Sander, E. E., van Delft, S., ten Klooster, J. P., Reid, T., van
der Kammen, R. A., Michiels, F. and Collard, J. G. (1998).
Matrix-dependent Tiam1/Rac signaling in epithelial cells promotes either
cell-cell adhesion or cell migration and is regulated by phosphatidylinositol
3-kinase. J. Cell Biol.
143,1385
-1398.
Schmitt, M., Wilhelm, O. G., Reuning, U., Kruger, A., Harbeck, N., Lengyel, E., Graeff, H., Gansbacher, B., Kessler, H., Burgle, M., Sturzebecher, J., Sperl, S. and Magdolen, V. (2000). The urokinase plasminogen activator system as a novel target for tumour therapy. Fibrinolysis & Proteolysis 14,114 -132.
Schmitz, A. A. P., Govek, E. E., Bottner, B. and Van Aelst, L. (2000). Rho GTPases: Signaling, migration, and invasion. Exp. Cell Res. 261,1 -12.[Medline]
She, H. Y., Rockow, S., Tang, J., Nishimura, R., Skolnik, E. Y., Chen, M., Margolis, B. and Li, W. (1997). Wiskott-Aldrich syndrome protein is associated with the adapter protein Grb2 and the epidermal growth factor receptor in living cells. Mol. Biol. Cell 8,1709 -1721.[Abstract]
Simon, D. I., Wei, Y., Zhang, L., Rao, N. K., Xu, H., Chen, Z.
P., Liu, Q. M., Rosenberg, S. and Chapman, H. A. (2000).
Identification of a urokinase receptor-integrin interaction site
Promiscuous regulator of integrin function. J. Biol.
Chem. 275,10228
-10234.
Sitrin, R. G., Todd, R. F., Petty, H. R., Brock, T. G.,
Shollenberger, S. B., Albrecht, E. and Gyetko, M. R. (1996).
The urokinase receptor (CD87) facilitates CD11b/CD18-mediated adhesion of
human monocytes. J. Clin. Invest.
97,1942
-1951.
Symons, M., Derry, J. M. J., Karlak, B., Jiang, S., Lemahieu, V., McCormick, F., Francke, U. and Abo, A. (1996). Wiskott-Aldrich syndrome protein, a novel effector for the GTPase CDC42Hs, is implicated in actin polymerization. Cell 84,723 -734.[Medline]
Takenawa, T. and Miki, H. (2001). WASP and WAVE
family proteins: key molecules for rapid rearrangement of cortical actin
filaments and cell movement. J. Cell Sci.
114,1801
-1809.
Vanhaesebroeck, B. and Waterfield, M. D. (1999). Signaling by distinct classes of phosphoinositide 3-kinases. Exp. Cell Res. 253,239 -254.[Medline]
Vanhaesebroeck, B., Jones, G. E., Allen, W. E., Zicha, D., Hooshmand-Rad, R., Sawyer, C., Wells, C., Waterfield, M. D. and Ridley, A. J. (1999). Distinct PI(3)Ks mediate mitogenic signalling and cell migration in macrophages. Nat. Cell Biol. 1, 69-71.[Medline]
Webb, S. E., Pollard, J. W. and Jones, G. E.
(1996). Direct observation and quantification of macrophage
chemoattraction to the growth factor CSF-1. J. Cell
Sci. 109,793
-803.
Wei, Y., Lukashev, M., Simon, D. I., Bodary, S. C., Rosenberg, S., Doyle, M. V. and Chapman, H. A. (1996). Regulation of integrin function by the urokinase receptor. Science 273,1551 -1555.[Abstract]
Wells, A., Gupta, K., Chang, P., Swindle, S., Glading, A. and Shiraha, H. (1998). Epidermal growth factor receptor-mediated motility in fibroblasts. Microscopy Res. Technique 43,395 -411.
Xue, W., Mizukami, I., Todd, R. F., III and Petty, H. R. (1997). Urokinase-type plasminogen activator receptors associate with beta1 and beta3 integrins of fibrosarcoma cells: dependence on extracellular matrix components. Cancer Res. 57,1682 -1689.[Abstract]
Yebra, M., Goretzki, L., Pfeifer, M. and Mueller, B. M. (1999). Urokinase-type plasminogen activator binding to its receptor stimulates tumor cell migration by enhancing integrin-mediated signal transduction. Exp. Cell Res. 250,231 -240.[Medline]
Zicha, D., Dunn, G. A. and Brown, A. F. (1991). A new direct-viewing chemotaxis chamber. J. Cell Sci. 99,769 -775.[Abstract]