From the Department of Immunology, The Scripps Research Institute,
La Jolla, California 92037
Received for publication, November 21, 2002, and in revised form, February 3, 2003
Nonmotile cells extend and retract
pseudopodia-like structures in a random manner, whereas motile cells
establish a single dominant pseudopodium in the direction of movement.
This is a critical step necessary for cell migration and occurs prior
to cell body translocation, yet little is known about how this process is regulated. Here we show that myosin II light chain (MLC)
phosphorylation at its regulatory serine 19 is elevated in growing and
retracting pseudopodia. MLC phosphorylation in the extending
pseudopodium was associated with strong and persistent amplification of
extracellular-regulated signal kinase (ERK) and MLC kinase activity,
which specifically localized to the leading pseudopodium.
Interestingly, inhibition of ERK or MLC kinase activity prevented MLC
phosphorylation and pseudopodia extension but not
retraction. In contrast, inhibition of RhoA activity specifically
decreased pseudopodia retraction but not extension. Importantly,
inhibition of RhoA activity specifically blocked MLC phosphorylation
associated with retracting pseudopodia. Inhibition of either ERK or
RhoA signals prevents chemotaxis, indicating that both pathways
contribute to the establishment of cell polarity and migration.
Together, these findings demonstrate that ERK and RhoA are distinct
pathways that control pseudopodia extension and retraction,
respectively, through differential modulation of MLC phosphorylation
and contractile processes.
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INTRODUCTION |
Directional cell migration, or chemotaxis, is initiated when cells
sense the direction and proximity of a chemoattractant gradient (1-3).
Although significant progress has been made in linking various signals
to the general process of chemotaxis, little is known about how these
signals facilitate morphological polarization, which is characterized
by establishment of a leading pseudopodium (lamellipodium), cell body,
and trailing tail region (1, 4). Recent evidence indicates that when
cells encounter a directional cue such as a chemoattractant gradient,
they respond by local activation and amplification of signals on the
side facing the stimulus (1-3, 5). For example, pleckstrin homology
domain-containing proteins strongly and persistently localize to the
plasma membrane facing the gradient (2, 3), indicating that
intracellular signals are spatio-temporally organized within
chemotactic cells. This suggests that cells sense gradients and
establish morphological polarity through the formation of an
intracellular gradient of signals.
Although it is not well defined, signals organized on the gradient side
of cells presumably facilitate localized actin polymerization leading
to membrane protrusion in the direction of the chemoattractant (6). The
extension of a single dominant leading pseudopodium marks the first
sign of morphological polarity prior to cell movement (1, 4). Despite
its inherent importance to chemotaxis, little is known about
gradient-sensing mechanisms and how these responses are transmitted to
the actin-myosin cytoskeleton to achieve cell polarity and pseudopodia extension.
Extracellular signal-regulated kinase
(ERK)1 and RhoA are key
signals that have been linked to cell migration and chemotaxis. However, their precise roles in mediating cell polarity and their spatio-temporal organization within the cell are still undefined. Recent evidence has shown that ERK mediates cell migration via regulation of myosin light chain kinase (MLCK) and myosin light chain
(MLC) phosphorylation (7). Interestingly, MLCK activity and MLC
phosphorylation have been shown to localize to the front of migrating
cells, suggesting a potential role for ERK and MLCK in mediating
contractile processes in the pseudopodium (8, 9). MLCK controls
contractility through phosphorylation of the regulatory serine 19 on
MLC. This phosphorylation event facilitates assembly of a fully
functional actin-myosin motor unit capable of generating tension and
contractile forces (10-13). However, RhoA also regulates MLC
phosphorylation and contractility through inhibition of myosin
phosphatase activity (14, 15). Although Rho, ERK, and MLCK are
necessary for proper migration, it is not known whether these signals
separately or coordinately regulate this process. Moreover, the
spatio-temporal organization of these signals in chemotactic cells is
not known.
We recently reported a novel method of purifying differentially the
pseudopodium and cell body of cells morphologically polarized toward a
chemoattractant gradient (16). Using this model, we show here that ERK
and RhoA are two separate pathways that differentially regulate myosin
function to control pseudopodial dynamics leading to cell polarity and chemotaxis.
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EXPERIMENTAL PROCEDURES |
Antibodies and Reagents--
Phosphospecific antibody to ERK1
and ERK2 (pTpY185/187) was from
BIOSOURCE International. Antibodies to ERK2, RhoA,
and MEK1 were from Santa Cruz Biotechnology, Inc. Antibody to MLC was
kindly provided by Dr. Primal de Lanerolle (University of Illinois at Chicago) or was purchased commercially from Sigma. Phosphospecific antibody to MLC-19 was kindly provided by Dr. Matsumura (9) (Rutgers
University). Goat anti-rabbit and goat anti-mouse horseradish peroxidase antibodies were from Bio-Rad. Rat tail collagen I was from Upstate Biotechnology, Inc., and human fibronectin was from Collaborative Scientific. The MEK1/2 inhibitor PD98059 was obtained from New England BioLabs. LPA was purchased from Sigma. Insulin was
purchased from Roche Molecular Biochemicals. Pertussis toxin (PTX), the
EGF receptor inhibitor AG1478, the myosin light chain kinase inhibitor
ML-7, and the Rho kinase inhibitor Y-27632 were purchased from
Calbiochem. Botulinum C3 exoenzyme (C3 toxin) was purchased from
Upstate Biotechnology, Inc.
Cell Culture and Cell Transfection--
NIH 3T3 fibroblasts were
kindly provided by Dr. Tony Hunter (Salk Institute, La Jolla, CA).
COS-7 cells were from American Tissue Type Culture Collection. In most
instances COS-7 cells were utilized; however, NIH 3T3 fibroblasts were
also tested in some cases, and similar results were obtained. Cells
were maintained in Dulbecco's modified Eagle's medium (Irvine
Scientific) containing 10% fetal bovine serum (Gemini BioProducts),
200 mM L-glutamine, and 50 µg
ml
1 gentamicin (Sigma), and 100 mM sodium
pyruvate (Sigma). Cells were incubated at 37 °C with 5%
CO2. For transfection experiments, 100-mm dishes of
60-80% confluent cells were transfected using LipofectAMINE
(Invitrogen) according to the manufacturer's protocol. Briefly, 20 µl of LipofectAMINE was preincubated with 3.5 µg of total DNA in 1 ml of transfection medium for 45 min. The volume was brought to 6 ml
and layered over cells for 6-8 h at 37 °C. Cells were used for the
appropriate assays within 48 h subsequent to serum starvation
overnight. The HA-tagged kinase-dead Raf (HA-Raf-KD) and wild type Raf
(HA-Raf-WT) constructs were provided by Dr. Michael Karin (University
of California, San Diego). The constitutively active MEK1 mutant (MEK+)
was provided by Dr. Christopher J. Marshall (Chester Beatty
Laboratories, Institute of Cancer Research). The HA-tagged kinase dead
mutant of MLCK (pCMV5-dnMLCK) was provided by Dr. Patricia Gallagher
(Indiana University School of Medicine). The dominant negative Rho
mutant (HA-tagged RhoN19) and wild type Rho construct were kindly
provided by Dr. Martin Schwartz (The Scripps Research Institute).
pcDNA3.1 (Invitrogen) was used as the empty vector to keep the
total amount of DNA to 3.5 µg. Cells used in chemotaxis assays were
cotransfected with 0.5 µg of a reporter construct encoding
-galactosidase (pCMV-SPORT-
-galactosidase; Invitrogen). Cells
analyzed by fluorescence staining were cotransfected with 0.5 µg of
green fluorescent protein reporter construct (pEGFP-C1, Clontech) as a cell transfection marker.
Chemotaxis Assays--
Migration assays were performed using
Boyden chambers containing polycarbonate membranes (tissue
culture-treated 6.5-mm diameter, 10-µm thickness, 8.0-µm pores,
Transwell®; Costar) or QCM kit (Chemicon International) as described
previously (16). Membranes were coated on both sides with either 5 µg/ml human fibronectin or 5 µg/ml collagen I for 2 h at
37 °C. 150,000 cells were allowed to attach to the upper membrane
surface for 2 h and then stimulated to migrate toward 100 ng/ml
LPA in the lower chamber for 3 h. After migration, cells were
fixed and stained with crystal violet (Sigma), or transfected cells
were fixed in
-galactosidase fixative and stained using
5-bromo-4-chloro-3-indolyl-
-D-galactopyranoside as a
substrate according to the manufacturer's recommendations (Promega
Corp.).
Cell Adhesion/Attachment Assays--
Collagen-coated
cell culture wells were prepared by incubating 5 µg/ml collagen I in
24-well cell culture plates (Costar) for 2 h at 37 °C. The
wells were then blocked with bovine serum albumin for 30 min. Cells
were allowed to adhere for the indicated times at 37 °C. To assess
cellular adhesion, the wells were washed with phosphate-buffered saline
and then fixed with crystal violet in 2% ethanol, and cells on the
underside were counted directly or were eluted with 10% acetic acid
and measured in an enzyme-linked immunosorbent assay plate reader
(A600).
Quantitative Pseudopodia Assay--
Pseudopodia extension and
retraction were monitored using a pseudopodia assay kit (ECM 650;
Chemicon International) as described previously (16). Briefly, 150,000 serum-starved cells were placed in the upper chamber of a 3.0-µm
porous polycarbonate membrane and coated on both sides with the
appropriate ECM protein (5 µg/ml fibronectin for NIH 3T3 cells and 5 µg/ml collagen type I for COS-7 cells). Cells were allowed to attach
and spread on the upper surface of the membrane for 2 h, and then
stimulated with LPA, insulin, or buffer only, which was placed in the
lower chamber to establish a gradient or placed in the upper and lower
chamber to form a uniform concentration. Cells were allowed to extend pseudopodia through the pores toward the direction of the gradient for
various times. To initiate pseudopodia retraction, the chemoattractant was removed, or an equivalent amount of chemoattractant was placed in
the upper chamber to create a uniform concentration. The cell body from
the upper surface was removed manually with a cotton swab, and the
total pseudopodia protein on the underside was determined using BCA and
a microprotein assay system and measured in an enzyme-linked immunosorbent assay plate reader at A562 (Pierce
Chemical Co.). In some instances, pseudopodia were fixed and stained
with crystal violet in 2% ethanol and were eluted with 10% acetic
acid and measured in an enzyme-linked immunosorbent assay plate reader (A600).
Purification of Pseudopodia--
To isolate proteins from
growing pseudopodia specifically, 1.5 × 106 cells
were induced to form pseudopodia toward LPA for 60 min as described
above or not treated and then harvested using a pseudopodia isolation
kit (ECM 660; Chemicon International) as described previously. Briefly,
cells were rinsed in excess cold phosphate-buffered saline and either
rapidly fixed in 100% ice-cold methanol (for immunoblotting of whole
cell lysates) or not fixed (for Rho GTPase activity only). Cell bodies
were removed manually with a cotton swab from the upper membrane
surface and pseudopodia from the undersurface scraped into lysis buffer
(100 mM Tris, pH 7.4, 5 mM EDTA, 150 mM NaCl, 1 mM sodium orthovanadate, protease
inhibitors (mixture tablet; Roche Molecular Biochemicals Corp.))
containing the appropriate detergent for immunoblotting of whole cell
lysates (1% SDS) or Rho GTPase activity assay (Triton X-100 according
to the manufacturer's recommendation; Upstate Biotechnology, Inc.).
Cell bodies were purified in a similar manner except that pseudopodia
on the undersurface were removed, and the cell body on the upper
surface was scraped into lysis buffer and detergent. Retracting
pseudopodia were induced for various times by removing or placing a
chemoattractant in the upper chamber to create uniform concentration
and then harvested as described above.
Haptotaxis Pseudopodia Purification--
For haptotaxis
pseudopodia, Transwell® Boyden chambers (3.0-µm pores) containing
polycarbonate membranes were coated with 10 µg/ml collagen I on the
underside of the membrane only for 2 h at 37 °C. 150,000 cells
were added to the top of each chamber and allowed to extend pseudopodia
to the underside for the indicated times, and lysates were harvested as
described above.
Immunoblotting and Rho GTPase Activity Assay--
Cell bodies,
growing or retracting pseudopodia were purified as described above in
the appropriate lysis buffer and then boiled for 10 min (for
immunoblotting) or placed on ice for 30 min (for Rho GTPase activity
assay). Lysates on ice were clarified by centrifugation for 10 min at
4 °C and protein concentration of both lysates determined using BCA
and a microprotein assay system (Pierce Chemical Co.). Briefly, 10-20
µg of whole cell lysate was separated by one-dimensional SDS-PAGE and
immunoblotted according to standard protocols. Horseradish peroxidase-conjugated goat anti-mouse or goat anti-rabbit secondary antibodies and the enhanced chemiluminescence detection system (Pierce
Chemical Co.) were used to visualize the blotted proteins of interest.
Rho activity GTPase assay was performed as described in the
manufacturer's protocol (Upstate Biotechnology, Inc.) using 100 µg
of total protein.
 |
RESULTS |
ERK and Rho Are Separate Signaling Pathways Necessary
for LPA-induced Cell Chemotaxis--
It is known that LPA induces ERK
activation through a PTX-sensitive pathway (17), whereas Rho activation
is through a PTX-insensitive mechanism (18). This prompted the
hypothesis that ERK and Rho may be distinct signaling pathways
responsible for cell proliferation and migration, respectively.
However, we found that exposure of cells to PTX prevented LPA-induced
cell chemotaxis independent of changes in Rho activation (Fig.
1A). Furthermore, inhibition of Rho activity by expression of a dominant negative Rho (19) prevented
chemotaxis independent of ERK activation (Fig. 1C). These
findings implicate ERK and a PTX-sensitive G
i signaling pathway in the regulation of cell chemotaxis in response to LPA (20,
21). PTX and dominant negative Rho did not affect cell attachment to
the extracellular matrix (ECM), demonstrating that these signals
regulate chemotaxis and do not prevent ECM recognition and attachment
(Fig. 1, B and D). These findings indicate that ERK and Rho represent two distinct signaling pathways necessary for
LPA-mediated chemotaxis.

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Fig. 1.
ERK and Rho are separate signaling pathways
necessary for LPA-induced chemotaxis. A, serum-starved
COS-7 cells were treated overnight in the absence ( ) or presence (+)
of 50 µg/ml PTX. Cells were examined for chemotaxis for 3 h
using 5 µg/ml collagen-coated (on the top and bottom) 8.0-µm porous
Boyden chambers in the absence ( ) or presence (+) of 100 ng/ml LPA
placed in the bottom compartments. The number of migratory
cells/microscopic field (×200) on the underside of the membrane was
counted as described under "Experimental Procedures." Cells on a
dish were treated with PTX as described above and stimulated with 100 ng/ml LPA for 10 min. Lysates were then immunoblotted using either
phosphospecific ERK1/2 or anti-ERK2 antibodies as indicated. Activated
Rho (Rho GTP) was determined using the Rhotekin binding domain, which
selectively binds Rho GTP. Total Rho protein was determined by
immunoblotting using an anti-RhoA antibody. B, an aliquot of
cells treated as described in A was allowed to attach to
collagen-coated wells for 3 h and then fixed and stained with
crystal violet. The number of adherent cells was counted as described
above in the migration experiment. C, cells expressing a
dominant mutant of Rho (RhoN19) (+) or the mock empty vector ( ) were
allowed to migrate for 3 h as described in A in the
absence ( ) or presence (+) of 100 ng/ml LPA in the bottom
compartment. Transfected cells on a dish were treated with 100 ng/ml
LPA for 10 min. Lysates were harvested and immunoblotted for
phospho-ERK (ERK1/2) and total ERK (ERK2 loading control) as described
above. D, an aliquot of cells treated as described in
C was allowed to attach to collagen-coated wells for 3 h and then fixed and stained with crystal violet as described in
B. Results shown reflect the mean ± S.D. (error
bars) of three replicate experiments.
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ERK Activation Is Amplified in Response to a Gradient but Not a
Uniform Concentration of Chemoattractant--
Chemotactic cells move
toward shallow chemoattractant gradients, indicating that they can
compare and process extremely small differences in concentrations of
extracellular stimuli (2). This is attributable, in part, to the fact
that chemotactic cells localize and amplify G protein-coupled signaling
pathways in the leading pseudopodium (22). We showed previously that
cells exposed to a gradient of LPA readily chemotax, whereas cells
exposed to a uniform concentration do not (16). These findings suggest that cells may differentially regulate ERK activity during
chemoattractant sensing and cell translocation compared with cells that
encounter a uniform concentration of chemokine. Indeed, ERK activation
was more rapid and significantly elevated in cells exposed to a
chemoattractant gradient compared with cells exposed to a uniform
concentration of chemokine (Fig.
2A). On the other hand, cells
exposed to a gradient of insulin, which does not induce chemotaxis
(16), showed significantly delayed and reduced ERK activity compared with cells exposed to a uniform concentration of growth factor (Fig.
2B). In our studies, we have seen that ERK1 and ERK2
activities (i.e. ERK-P) are the same and are representative
of changes in ERK activity overall. These findings support the idea
that ERK activation is a downstream process of chemosensing
mechanisms necessary for directional cell migration.

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Fig. 2.
ERK is amplified in response to a gradient
but not a uniform concentration of chemoattractant. Serum-starved
COS-7 cells were allowed attach to collagen-coated 3.0-µm porous
Boyden chambers for 2 h, and 100 ng/ml LPA (A) or 25 µg/ml insulin (B) was added either to the upper and lower
chamber (Uniform) or to the lower chamber only
(Gradient) for the indicated times. Total cellular protein
was isolated, and immunoblotting was performed for phospho-ERK and
total ERK as described above. In our studies, ERK1 and ERK2
phosphorylations are similar, and thus a representative blot is shown
with ERK2 and is representative of at least three replicate
experiments.
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|
ERK Is Necessary for Pseudopodial Extension but Not
Retraction--
We next wanted to determine how ERK activity controls
the migration machinery of cells. Stationary cells are often observed to extend and retract pseudopodial-like processes from their cell body
as they interact with the surrounding extracellular environment (23-25). However, directional cell migration or chemotaxis is
characterized by protrusion of a dominant leading pseudopodium in the
direction of a chemoattractant gradient followed by cell body
translocation (1, 4). Therefore, it is likely that establishment of a leading pseudopodium involves the engagement of localized membrane protrusive activity and the uncoupling of retraction mechanisms. Although little is known about the molecular processes that coordinate this event, actin-myosin contractility is likely to be important. ERK
has been reported to regulate the actin-myosin contractile machinery of
cells through its ability to phosphorylate MLCK leading to increased
MLC phosphorylation and contractile activity (26). Furthermore, recent
reports indicate that MLCK activity and MLC phosphorylation are
elevated in the leading front of migrating cells (8, 11, 24). However,
it is not known how these events are regulated or whether they
contribute to pseudopodial growth or retraction, per se. To
investigate a possible role for ERK and MLCK in pseudopodial dynamics,
we utilized an in vitro model that allows for quantification
as well as biochemical analysis of pseudopodia undergoing growth or
retraction in response to a chemoattractant gradient (16). Cells were
plated on 3.0-µm porous membranes attached to a Boyden chamber and
then polarized toward an LPA gradient. Cells then extend leading
pseudopodia through the pores to the underside of the chamber in the
direction of the gradient. Importantly, removal of the LPA gradient
from the bottom chamber causes pseudopodia to retract back to the cell body on the upper surface (16). The cell body and extending or
retracting pseudopodia can be harvested differentially from the
different sides of the filter for analysis. Our previous work had shown
that pseudopodia extension through the micropore filters in response to
an LPA gradient is linear for 30-120 min, whereas retraction is linear
for up to 120 min (16). Interestingly, we found that ERK activity was
increased significantly in extending pseudopodia and decreased during
retraction (Fig. 3, A and
B). ERK activity in extending pseudopodia was persistent for
15-120 min, whereas initiation of pseudopodia retraction caused a
rapid decrease in ERK activity that reached basal levels by 10 min
(Fig. 3B). Therefore, ERK activation is associated with
extension and growth of pseudopodial processes and not retraction.
Inhibition of ERK activity in cells with PTX (27), expression of a
dominant blocking form of Raf-1, or treatment with the MEK inhibitor
PD98059 (28) prevented pseudopodia extension (Fig. 3, C,
D, and E, respectively). However, treatment with
an inhibitor of the EGF receptor, AG1478 (29), did not alter
pseudopodia growth (Fig. 3E), suggesting that LPA
stimulation of pseudopodial extension does not require EGF
receptor-mediated ERK activity in trans as suggested
previously (30-32). In addition, expression of a constitutively active
form of MEK (33) did not alter basal chemokine-induced pseudopodial extension, suggesting that the activation of the ERK pathway alone is
not sufficient to facilitate directional membrane protrusion (Fig.
3G). Importantly, treatment of retracting pseudopodia with PD98059 or the expression of constitutively active MEK1 did not alter
membrane retraction, per se. These findings demonstrate that
ERK activity was not necessary for this response and that an
independent pathway controls this process (Fig. 3, F and
H, respectively). It is noteworthy that total ERK protein
was the same in the cell body and pseudopodium under growth and
retraction conditions, indicating that ERK protein levels remain
constant under these conditions (Fig. 3, A and
B). Similarly, MEK, the upstream activator of ERK, showed
increased activity in growing pseudopodia and decreased during
retraction (data not shown). The level of MEK protein was also the same
in both conditions (data not shown). Together these findings
demonstrate that ERK activity is necessary for pseudopodium extension
but not retraction. These findings also indicate that ERK activity is
spatially localized to the leading pseudopodium of cells polarized
toward a chemoattractant gradient.

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Fig. 3.
ERK is necessary for pseudopodial extension
but not retraction. A, serum-starved COS-7 cells were
allowed to extend pseudopodia on 5 µg/ml collagen-coated 3.0-µm
porous Boyden chambers toward a 100 ng/ml LPA gradient for the
indicated times. The cell body on the upper membrane surface or
pseudopodia (Pseudo) on the lower membrane surface were
isolated and analyzed by one-dimensional SDS-PAGE as described under
"Experimental Procedures." Total cellular proteins were also
isolated from whole cells attached to the porous filters which were not
treated with LPA (NT). Immunoblotting was performed for
phospho-ERK and total ERK as described above. B, cells were
allowed to extend pseudopodia for 60 min (time 0) as
described in A, and then the LPA gradient was removed from
the bottom compartment and pseudopodia allowed to retract for the
indicated times. The cell body and pseudopodium were purified and
immunoblotted for phospho-ERK and total ERK as described above. In our
studies we have seen that ERK1 and ERK2 activities correlate well with
one another and that in B, ERK2 phosphorylation is
representative of changes in ERK activity in general. C,
cells were pretreated overnight in the absence ( ) or presence (+) of
50 µg/ml PTX. Cells were then examined for pseudopodia extension in
response to a 100 ng/ml LPA gradient (+) or not treated ( ) for 60 min. Pseudopodia protein on the underside of the membrane was
determined as described under "Experimental Procedures."
D, COS-7 cells were transfected with the empty vector
(Mock), the vector encoding HA-tagged kinase dead Raf-1
mutant (HA-Raf-KD), or HA-tagged wild type Raf-1 (HA-Raf-WT). Cells
were examined for pseudopodia formation in response to LPA as described
above. Expression of HA-Raf-KD and HA-Raf-WT was verified using total
protein from cells on a dish and immunoblotted for Raf using anti-HA
antibody. E, cells were allowed to attach to collagen-coated
membranes for 2 h. Cells were then pretreated for 60 min in the
absence ( ) or presence (+) of 50 µM PD98059, to inhibit
MEK activity, or 1 µM AG1478, to inhibit EGF receptor
activity. Pseudopodia formation was initiated using LPA as a gradient
and pseudopodium protein determined as described above. F,
cells were allowed to extend pseudopodia toward an LPA gradient for 60 min as described above. The gradient of LPA was then removed, and
either buffer (NT) or PD98059 was added to the upper and
lower chambers, and the pseudopodia were allowed to retract for the
indicated times. The pseudopodia retraction was quantitated as
described above. G, COS-7 cells were transfected with either
the empty vector or the vector encoding a constitutively active MEK1
mutant (MEK+). Cells were examined for pseudopodia formation
in response to LPA as described above. Overexpression of MEK1 was
verified using total protein from cells on a dish and immunoblotted for
MEK1 using anti-MEK1 antibody. H, COS-7 cells were
transfected with either the empty vector or the vector encoding a
constitutively active MEK1 mutant. Cells were examined for pseudopodia
retraction as described above. Results shown are representative of
three replicate experiments for A and B and
reflect the mean ± S.D. (error bars) of three
replicate experiments for C-H.
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Pseudopodial Extension toward a Gradient of the ECM Requires ERK
Activation--
ECM gradients also provide spatial cues that cause
directional pseudopodial extension and haptotactic cell migration (34, 35). ERK activity was elevated in haptotactic pseudopodia extending toward a collagen gradient, and PD98059 inhibited this response (Fig.
4, A and B). Again,
ERK protein levels were the same in the pseudopodium and cell body. In
addition, PTX did not block pseudopodium extension under these
conditions, indicating that PTX-sensitive heterotrimeric G proteins are
not involved in this response (data not shown). Haptotactic pseudopodia
do not readily retract and therefore were not investigated further.
These findings provide additional evidence that ERK is necessary for
directional pseudopodial extension and establishment of cell
polarity.

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Fig. 4.
Pseudopodial extension toward a gradient of
ECM requires ERK activation. A, COS-7 cells were
allowed to extend pseudopodia through 3.0-µm porous membranes coated
only on the underside with 10 µg/ml collagen I for 60 and 120 min.
Cell body and pseudopodia (Pseudo) lysates were harvested at
the indicated times as described above. Lysates from cells in
suspension for 15 min (Suspension) or attached to 10 µg/ml
collagen I for 15 min (Attached) were also harvested for
comparison. Immunoblotting was performed for phospho-ERK and total ERK
as described above. B, cells pretreated in the absence
(NT) or presence of 50 µM PD98059 for 1 h
were allowed to extend pseudopodia toward collagen I for the indicated
times, and pseudopodia protein was quantitated as described above.
Results shown are representative of three replicate experiments for
A and reflect the mean ± S.D. (error bars)
of three replicate experiments for B.
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MLCK Is Necessary for Pseudopodial Extension but Not
Retraction--
ERK phosphorylates MLCK leading to increased kinase
activity and MLC phosphorylation. MLC phosphorylation at serine 19 (MLC-19) leads to increased actin-myosin association and contractility. Expression of a dominant blocking form of MLCK in cells or treatment with the MLCK kinase inhibitor ML-7 (36) prevented pseudopodia formation and MLC-19 phosphorylation in cells (Fig. 5,
A and B). Importantly, as with ERK, inhibition of MLCK activity in retracting pseudopodia with ML-7 did not alter membrane retraction or MLC-19 phosphorylation (Fig. 5, C and D). MLCK (data not
shown) and MLC protein levels were the same in the cell body and
pseudopodium (Fig. 5D). Thus, ERK and MLCK activity are
specifically required for pseudopodial extension but not
retraction.

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Fig. 5.
MLCK is necessary for pseudopodial extension
but not retraction. A, COS-7 cells were transfected
with either the empty vector (Mock) or the vector encoding
kinase dead MLCK (MLCK-KD), which serves as a dominant
negative protein. Cells were examined for pseudopodia in the absence
( ) or presence (+) of 100 ng/ml LPA in the bottom compartment.
Extending pseudopodia were quantitated as described above. Transfected
cells on a dish were treated uniformly on top in the absence ( ) or
presence (+) of 100 ng/ml LPA for 10 min, and MLC phosphorylation on
serine 19 (MLC-19-P) and total MLC phosphorylation was
determined by immunoblotting using either phosphospecific MLC-19-P or
anti-MLC antibodies as indicated. Densitometry and quantification were
performed as in A, and the -fold increase is relative to
MLC-19-P in control mock-transfected cells. B, cells were
pretreated in the absence ( ) or presence (+) of 5 µM
MLCK inhibitor, ML-7, or dimethyl sulfoxide (DMSO; control)
for 30 min and allowed to extend pseudopodia toward 100 ng/ml LPA for
60 min as described above. Total cellular protein from treated cells
was harvested and immunoblotted for MLC-19-P and MLC as described
above. Densitometry and MLC-19-P quantification were performed as in
A, and the -fold increase is relative to MLC-19-P in control
dimethyl sulfoxide-treated cells. C, cells were allowed to
extend pseudopodia toward an LPA gradient for 60 min as described
above. The gradient of LPA was then removed, and either buffer
(NT) or 5 µM ML-7 was added to the upper and
lower chambers and the pseudopodia allowed to retract for the indicated
times. Retracting pseudopodia were quantitated as described above.
D, cells were allowed to extend pseudopodia for 60 min
(time 0) toward an LPA gradient and then allowed to retract
as described above for 60 min in the presence of dimethyl sulfoxide
( ) or ML-7 (+). Cell body and retracting pseudopodia were purified
and immunoblotted for MLC-19-P, and total MLC and MLC-19-P were
quantified as described above. The -fold increase represents the change
in MLC-19-P (ratio of MLC-19-P to total MLC) relative to basal MLC-19-P
in the corresponding cell body sample for each condition. Results shown
reflect the mean ± S.D. (error bars) of three
replicate experiments for A-C and are representative of
three replicate experiments for D.
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Rho Activity Is Necessary for Pseudopodia Retraction but Not
Extension--
It is noteworthy that MLC-19 phosphorylation was
increased in the pseudopodium compared with the cell body proper, and
this does not change during extension or retraction (Fig.
5D). This suggests that MLC phosphorylation and
contractility are important for both the growth and retraction
processes. If ERK and MLCK activity are not necessary for pseudopodial
retraction, what signal regulates this process? Rho may control
pseudopodial dynamics through inactivation of myosin phosphatase,
leading to increased MLC-19 phosphorylation and contraction (37).
Indeed, inhibition of Rho activity in cells with a dominant blocking
form of Rho (Fig. 6B),
treatment with the Rho inhibitor C3 transferase (38) (Fig.
6A), or treatment with the Rho kinase inhibitor Y-27632 (data not shown) specifically prevented pseudopodial retraction but not
extension. In fact, pseudopodial extension was increased compared with
control cells (Fig. 6A). Associated with inhibition of Rho
activity was decreased MLC-19 phosphorylation in retracting but not
extending pseudopodia (Fig. 6, D and C). The
increased expression of Rho by itself did not perturb pseudopodial
dynamics because expression of wild type Rho in cells did not alter
this process (Fig. 6A). Importantly, our previous findings
indicated that Rho activity is highest in the extending pseudopodia and decreased during retraction (16). However, Rho activity does not
completely return to basal levels in retracting pseudopodia (16) and
therefore may be sufficient to facilitate the myosin-mediated contractile process under these conditions. The increased Rho activity
seen in the extending pseudopodium may be important as a negative
feedback mechanism to regulate Rac activity and protrusive processes as
suggested previously (16, 39). In support of this, suppression of Rho
activity in cells did increase pseudopodial extension (Fig.
6A). Alternatively, Rho activity in the extending pseudopodium may serve a function(s) independent of pseudopodial dynamics. Together these results indicate that ERK and MLCK are important for pseudopodial extension, whereas Rho is necessary for
pseudopodial retraction during cell polarization and chemotaxis. These
distinct signaling pathways converge on myosin, a key player in
pseudopodia dynamics, to regulate chemotaxis differentially. A model
depicting the role of ERK and Rho is shown in Fig.
7 and discussed below.

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|
Fig. 6.
Rho activity is necessary for pseudopodia
retraction but not extension. A, COS-7 cells were
transfected with the empty vector (Mock), the vector
encoding HA-tagged dominant negative RhoA mutant (Rho N19),
or wild type Rho (wt Rho). Serum-starved cells were examined
for pseudopodia extension for the indicated times and quantitated as
described above. B, cells pretreated overnight in the
absence (NT) or presence of 50 µg/ml C3 toxin
(C3) were allowed to extend pseudopodia toward an LPA
gradient and quantified as described above. C, cells were
transfected with either the empty vector or a HA-tagged dominant
negative RhoA mutant. Cells were then examined for pseudopodia
retraction for the indicated times as described above. D,
cells were pretreated with C3 toxin and were also examined for
pseudopodia retraction for the indicated times and quantified as
described above. E, purified cell bodies and pseudopodia
proteins from cells treated as in A were examined for
MLC-19-P and MLC as described above. Quantification for densitometric
analysis in E was made relative to control mock-transfected
cells. F, cells were transfected and treated as described in
C, and cell body and retracting pseudopodia
(Pseudo) lysates were harvested and examined for MLC-19-P
and MLC as described above. Quantification for densitometric analysis
in F, consistent with other legends, was made relative to
control mock-transfected cells. The -fold increase represents the
change in MLC-19-P (ratio of MLC-19-P to total MLC) relative to
MLC-19-P in the corresponding cell body control for each condition.
Results shown reflect the mean ± S.D. (error bars) of
three replicate experiments for A-D and are representative
of three replicate experiments for E and F.
|
|

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|
Fig. 7.
Model depicting the distinction between ERK
and Rho regulation of pseudopodia function through the LPA
receptor. Based on our findings and the work of others, a model
can be proposed as to the role of ERK and Rho in mediating MLC function
in polarized migrating cells. As a cell encounters a gradient of LPA,
the LPA receptor (LPAR) becomes ligated and activated
leading to ERK activation, which is sustained and amplified on the side
of the cell facing the gradient through a PTX-sensitive
G i pathway leading to increased MLCK activity and MLC
phosphorylation. This modulates the generation of traction force
necessary for proper pseudopodial formation. When the LPA gradient is
removed or when the cell is exposed to a uniform chemokine
concentration, there is a loss of directional ERK signaling and
decreased traction force. Pseudopodium retraction is facilitated by
Rho-mediated contraction through down-regulation of myosin phosphatase,
producing actin-myosin force necessary to retract the pseudopodium.
Although it is evident that ERK and Rho are important players in the
regulation of MLC phosphorylation, most likely there are additional
pathways from the initial signal which help to mediate the extension or
retraction of pseudopodia. These signaling molecules include PAK1,
protein kinase A, p38, protein kinase C (PKC), and protein
kinase G (PKG). Positive regulators of MLC phosphorylation
include PAK, p38, and protein kinase C. Negative regulators of MLC
phosphorylation include PAK1, protein kinase A, and protein kinase
G.
|
|
 |
DISCUSSION |
It is intriguing that cells extend and retract pseudopodial-like
structures from their membrane surface in an apparent random fashion.
These exploratory pseudopodia appear to probe the extracellular environment and thus may serve as antennae sensing spatial adhesive cues, soluble gradients of chemoattractant, and the proximity of
neighboring cells. This process may be important for transmitting information to the interior cell body allowing for proper decisions to
be made, such as whether to migrate or remain stationary. However, it
is clear that once a directional signal is detected, random pseudopodia
extension/retraction ceases, and a single pseudopodium is protruded
from the cell body only in the direction of the stimulus. This suggests
that molecular components that mediate membrane protrusion are rapidly
localized to the side facing the directional cue, whereas retraction
mechanisms are suppressed or turned off in this region. In contrast,
protrusive mechanisms would be expected to be suppressed, and
retractive mechanisms would remain operational on the side opposed to
the directional cue. Together these signals are likely to regulate
directional migration including pseudopodial extension/retraction as
well as the physical turning of the pseudopodium in response to
gradient changes. Therefore, it is important to identify specific
signals and their spatio-temporal organization within the
chemotactic cell to understand how morphological polarity and cell
movement are achieved.
LPA is an abundant lipid component present in the serum and is a potent
physiological mediator of cell migration, angiogenesis, and of
pathological conditions associated with cancer (40-43). Indeed, high
levels of LPA have been detected in the plasma and ascitic fluid of
ovarian carcinomas (44, 45) where it has been shown to stimulate
ovarian tumor cell growth by increasing angiogenesis (46). In addition,
increased levels of LPA have been shown to stimulate prostate and
breast cancer proliferation (47-50). Thus, it is important to
understand how LPA mediates signals and controls cellular functions
including cell migration. Our findings demonstrate that ERK and RhoA
are two necessary signaling pathways downstream from the LPA receptor
which control the migration machinery of chemotactic cells through
regulation of pseudopodia growth and retraction, respectively. Previous
findings support the ability for cross-talk between ERK and Rho in
motility (38, 51). However, in our system, ERK and Rho regulate
migration distinctly. Cell migration was inhibited by PTX, suggesting
the involvement of the ERK pathway but not Rho. The use of an in
vitro model system that allows for biochemical isolation of
growing or retracting pseudopodia revealed that ERK and RhoA
differentially modulate pseudopodial dynamics in response to an LPA
gradient. An interesting finding in this study was that these signals
differentially regulate MLC phosphorylation at serine 19, which is
necessary for actin-myosin-mediated tension and force. It is likely
that ERK phosphorylation of MLC modulates adhesive processes and
traction forces as activated ERK localizes to focal adhesions of
migratory cells (52). On the other hand, activated ERK is not necessary for formation of actin-mediated membrane ruffles and thus may not be
directly involved in regulation of actin polymerization and membrane
protrusion during pseudopodium extension (7).
Based on our findings and the work of others, a model can be proposed
as to the role of ERK and Rho in mediating MLC function in polarized
migrating cells (Fig. 7). As a cell encounters a gradient of LPA, Rac
and Cdc42 are activated on the side facing the gradient which
facilitates actin-mediated pseudopodia extension, which occurs
independently of the ECM as shown previously (16, 53). ERK is also
spatially activated on the side facing the gradient through a
PTX-sensitive G
i pathway, leading to increased MLCK
activity and MLC phosphorylation. Interaction of the protruding membrane with the ECM stabilizes the pseudopodium and promotes sustained ERK/MLC activation necessary for generation of traction force
during pseudopodial expansion (11, 54). Activated ERK has been shown to
localize to newly forming focal adhesions at the edge of spreading
cells, and this localization event requires myosin-mediated
contractility (19, 52). Thus, it is possible that persistent ERK
activity observed in the pseudopodium spatially regulates focal
adhesion dynamics and tension at the cell-substratum interface. The
continued formation of new focal adhesions at the advancing front may
then stimulate additional ERK activity and MLC-mediated contractility
in a positive feedback loop. Small adhesions near the leading edge of
motile cells have been shown to transmit strong propulsive tractions,
which allow the cell to migrate while maintaining its spread morphology
(55). ERK may also directly target other cytoskeletal-associated
components such as caldesmon, microtubules, Scar/WAVE, or modulate
integrin affinity, which are likely to be involved in this process
(56-60).
It is also intriguing that LPA stimulates Rho activity in cells and
that Rho is strongly activated in the pseudopodium, yet this signaling
event is not necessary for pseudopodia extension per se. In
fact, blocking Rho activity increased pseudopodia extension. Localized
Rho activity in the pseudopodium may serve as a negative feedback
mechanism to control protrusive signals such as Rac and Cdc42, which
mediate pseudopodia extension as proposed previously (14, 61). RhoA has
been shown to down-regulate Cdc42 and Rac1 activity (61). Importantly,
in our previous work we showed that Rho activity was elevated
significantly in extending pseudopodia, but we also noted that Rho
activity did not return to basal levels during pseudopodial retraction
(16, 19). Thus, a pool of Rho activity is maintained in the retracting
pseudopodium and available to facilitate MLC-mediated retraction.
We have shown previously that pseudopodial growth requires the assembly
of a CAS/CrkII scaffold, which facilitates translocation and activation
of Rac1 at the leading edge (16). We have found that Rac1 mediates
pseudopodial extension via CAS/CrkII coupling and that this occurs
independently of the ERK pathway (62). It is also likely that there are
alternative means by which MLC becomes phosphorylated in migrating
cells. The serine/threonine p21-activated kinase, PAK (63, 64), is an
effector for both Rac and Cdc42 (63-65) and has been shown to increase
MLC phosphorylation, leading to increased contraction and focal
adhesions (66). In addition, PAK can directly phosphorylate and
activate Raf-1 leading to activation of ERK (67, 68). Previous reports
in other systems showed that an active PAK mutant decreased the
phosphorylation of MLC in baby hamster kidney and in HeLa cells (69)
but increased phosphorylation in 3T3 cells (70) (Fig. 7). However,
recent evidence indicates that PAK is not required for extension of
lamellipodia per se but may be involved in regulation of
focal contact turnover (66).
Although early work suggested that MLC contractility was important for
tail retraction in migrating cells, a growing body of evidence now
points to a prominent role for myosin-mediated contraction in
pseudopodia formation. MLCK activity and MLC-19 phosphorylation have
recently been shown to be elevated in the leading front of migrating
cells (8, 9, 71). These findings support a role for Rho-mediated myosin
regulation of pseudopodial protrusions. More directly, myosin
phosphorylation has been linked to cell migration and protrusive
events. For example, myosin IIB knockout studies show disordered cell
migration of neuroepithelial and differentiated cells in the
ventricular walls (71). In addition, neurons cultured from the superior
cervical ganglia of
B
/B
embryonic mice
showed decreased rates of neurite outgrowth and lamellipodia formation
in growth cones (71, 72). However, inhibition of myosin function by
microinjection of antibody led to increased migration and membrane
protrusion along the entire periphery of cells (73), indicating that
myosin activity plays a fundamental role in maintaining the
organization of the cytoskeleton of the cell.
Although it is evident that ERK and Rho are important players in the
regulation of MLC phosphorylation, most likely there are additional
pathways that mediate the extension or retraction of pseudopodia (Fig.
7). For example, the mitogen-activated protein kinase p38 has been
correlated with increased MLC phosphorylation (74), and activation of
protein kinase C induces the phosphorylation of MLC by inhibiting
myosin phosphatase (75). Protein kinase A phosphorylates MLCK on an
inhibitory site in vitro (76, 77) and may thus help to
decrease MLC phosphorylation. However, agents that increase cAMP
in vivo do not affect phosphorylation of this site or MLCK
activity (78, 79), suggesting an alternative means of regulation by
protein kinase A. Myosin phosphatase can be activated by protein kinase
G either directly (80) or indirectly through the inhibition of Rho
(81). These signaling molecules most likely work in concert with ERK
and Rho to control and fine tune pseudopodial dynamics.
Our findings show that ERK and Rho are distinct signals that
regulate pseudopodium formation and retraction, respectively, through
modulation of MLC phosphorylation. Understanding the temporal and
spatial regulation of pseudopodia dynamics is important as the
ability of cells to form a dominant leading pseudopodium is necessary
for cell migration. Our findings help provide an understanding at the
molecular level of how pseudopodia are regulated in chemotaxing cells.
We thank Dr. P. Gallagher (Indiana University
School of Medicine), Dr. C. J. Marshall (Chester Beatty
Laboratories, Institute of Cancer Research), Dr. M. A. Schwartz
(Scripps Research Institute), Dr. P. de Lanerolle (University of
Illinois at Chicago), Dr. M. Karin (University of California, San
Diego), and Dr. Matsumura (Rutgers University) for providing the reagents.
Published, JBC Papers in Press, February 5, 2003, DOI 10.1074/jbc.M211873200
The abbreviations used are:
ERK, extracellular
signal-regulated kinase;
CMV, cytomegalovirus;
dn, dominant negative;
ECM, extracellular matrix;
EGF, epidermal growth factor;
EGFP, enhanced
green fluorescent protein;
HA, hemagglutinin;
KD, kinase dead;
LPA, lysophosphatidic acid (1-oleoyl,
2-hydroxyl-sn-glycerol-3-phosphate);
MEK, mitogen-activated
protein kinase/extracellular signal-regulated kinase kinase;
MLC, myosin light chain;
MLC-19, MLC phosphorylation at serine 19;
MLCK, myosin light chain kinase;
PAK, p21-activated kinase;
PTX, pertussis
toxin;
WT, wild type.
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