Cas utilizes Nck2 to activate Cdc42 and regulate cell
polarization during cell migration in response to wound
healing
Kohei Funasaka
1
, Satoko Ito
2
, Hitoki Hasegawa
2
, Gary S.Goldberg
3
, Yoshiki Hirooka
1
,
Hidemi Goto
1
, Michinari Hamaguchi
2
and Takeshi Senga
2
1 Department of Gastroenterology, Nagoya University Graduate School of Medicine, Japan
2 Division of Cancer Biology, Nagoya University Graduate School of Medicine, Japan
3 Molecular Biology Department, University of Medicine and Dentistry of New Jersey, Stratford, NJ, USA
Introduction
The establishment of cell polarity is essential for a
variety of cellular functions, such as cell division, dif-
ferentiation and migration; however, the molecular
mechanisms underlying cell polarization have not been
elucidated thoroughly. Genetic and cell biological stud-
ies have identified several molecules that are important
for cell polarity. Among these proteins, Cdc42, a Rho

2010, accepted 28 June 2010)
doi:10.1111/j.1742-4658.2010.07752.x
Integrin-mediated activation of Cdc42 is essential for cell polarization,
whereas the integrin adaptor protein Cas is required for cell migration dur-
ing wound healing. After phosphorylation on tyrosine residues, Cas recruits
the adaptor proteins Crk and Nck to execute integrin-mediated signals.
However, the mechanisms leading to Cdc42 activation and its relationship
with Cas, Crk and Nck have not been elucidated clearly. In the present
study, we demonstrate that Cas utilizes Nck2 to activate Cdc42 and induce
cell polarization in response to wounding. By contrast, Cas recruits CrkII
to activate Rac1 and promote the extension of cell protrusions needed for
cell motility. These results indicate that Cas utilizes Nck2 and CrkII in a
coordinated set of distinct pathways leading to cell migration.
Structured digital abstract
l
MINT-7909509: Cas (uniprotkb:Q61140) and Nck2 (uniprotkb:Q8BQ28) colocalize (MI:0403)
by fluorescence microscopy (
MI:0416)
Abbreviations
CasKo, homozygous null Cas knockout; CasWt, CasKo transfected with wild-type Cas; DAPI, 4¢,6¢-diamino-2-phenylindole dihydrochloride;
GST, glutathione S-transferase; PAK, p21-activated kinase; PBD, p21 binding domain; PIX, PAK-interacting guanine nucleotide exchange
factor; PP2, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-
D]pyramidine; siRNA, small interfering siRNA.
3502 FEBS Journal 277 (2010) 3502–3513 ª 2010 The Authors Journal compilation ª 2010 FEBS
that associate with SH2 domains to direct protein
interactions mediating signaling events leading to cell
migration [12,13].
Cas is ubiquitously expressed and its deletion in
mice is embryonic lethal [14]. Fibroblasts derived from
Cas-deficient mice showed cytoskeletal abnormalities

Cdc42 activity during cell migration in response to
wound healing.
Results
Cas is required for the polarization of migrating
cells
To examine the role of Cas in the establishment of cell
polarity during cell migration, we performed a wound-
healing assay using Cas deficient CasKo cells (homo-
zygous null Cas knockout cells) and CasWt cells
(generated by transfecting CasKo cells with wild-type
Cas). Cas expression in CasWt cells was similar to that
in Balb3T3 cells, and Cas was absent in CasKo cells
(Fig. 1A). As shown in Fig. 1B, CasWt cells migrated
faster than CasKo cells in this assay. In addition to
the wound healing assay, CasWt cells also migrated
approximately 40% better than CasKo cells through a
modified Boyden chamber (Fig. 1C).
Because cell polarization is an important prelude to
migration [26], we examined the effects of Cas on cell
polarization in response to wound healing. As shown
in Fig. 2A, CasWt cells at the wound edge started to
extend protrusions toward the free space within 4 h,
and over 90% of the cells at the edge were polarized,
with one side pointed toward the wound within 6 h.
By contrast, < 10% of the CasKo cells at the wound
edge displayed a polarized morphology 6 h after the
wound was made.
Measurement of protrusion length also indicated
that Cas was required for the formation of cell protru-
sions. As shown in Fig. 2B, CasWt cells exhibited cell

during wound healing
Cdc42 is a Rho GTPase that traffics to the leading
edge of cell protrusions and regulates cell polarity dur-
ing wound healing [1]. The effects of Cas on Cdc42
localization during wound healing were evaluated by
K. Funasaka et al. Cas ⁄ Nck2 regulates cell polarity
FEBS Journal 277 (2010) 3502–3513 ª 2010 The Authors Journal compilation ª 2010 FEBS 3503
immunofluorescence microscopy. As shown in Fig. 3A,
whereas more than 50% of the CasWt cells at the
wound edge contained Cdc42 localized on the leading
edge, < 10% of the CasKo cells at the wound edge
showed localization of Cdc42 on the leading edge.
Thus, Cas is required for trafficking of Cdc42 to the
leading edge of migrating cells.
In addition to intracellular location, the effects of
Cas on Cdc42 activation were also examined. A previ-
ous study demonstrated the activation of Cdc42 during
wound healing [1]. Cdc42 activity was assessed by
affinity precipitation of Cdc42-GTP with a glutathione
S-transferase–p21-activated kinase–p21 binding domain
(GST-PAK-PBD) fusion protein. As shown in Fig. 3B,
wound-induced activation of Cdc42 was reduced in
CasKo cells compared to CasWt cells. To further con-
firm the reduced activation of Cdc42 in CasKo cells,
we examined the activity of Cdc42 in both cell lines
3 h after wounding. Three independent experiments
demonstrated that the Cdc42 activity 3 h after wounding
in CasWt cells was almost twice that of CasKo cells
(Fig. 3C).
Silencing of Cas in Balb3T3 cells inhibits cell

(24 h)
*
200 µm
Migration assay (3 h)
CasKo CasWt
0
50
100
150
200
250
300
350
400
450
CasKo CasWt
No. of migrated cells per field
*
CasKo CasWt
Cas
Actin
Balb3T3
A
B
C
Fig. 1. Cas is essential for cell migration.
(A) Western blot analysis of Cas in CasWt,
CasKo and Balb3T3 cells. (B) Confluent
monolayers of CasWt and CasKo cells were
wounded with a pipette tip and incubated

The Src tyrosine kinase phosphorylates Cas to pro-
mote cell migration [18]. We employed a Src kinase
inhibitor [4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyra-
zolo[3,4-d]pyramidine; PP2] to determine whether Cas
phosphorylation was needed for the establishment of
polarity during wound healing. As shown in Fig. 5A,
tyrosine phosphorylation of Cas was induced by
wounding, which was effectively suppressed by PP2
treatment. This inhibition of Cas phosphorylation by
PP2 caused a decrease in cell elongation during wound
healing. As shown in Fig. 5B, cells treated with PP2 did
not extend protrusions into the wound area within 6 h
after wounding. In addition, PP2 treatment reduced
Golgi mobilization between the nucleus and wound
edge to levels seen in CasKo cells (Fig. 5C). These data
suggest that Src phosphorylates Cas to induce cell
polarization and migration during wound healing.
Nck2 is crucial for cell polarization and Cdc42
activation during wound healing
Crk and Nck are adaptor proteins that can associate
with phosphorylated tyrosine residues of Cas [8]. Two
024
6 (h)
CasKo
CasWt
Percentage of cells with
polarized golgi
0
10
20

A
B
C
D
Fig. 2. Cas promotes wound-induced cell
polarization. (A) Confluent monolayers of
CasKo and CasWt cells were wounded and
cells were incubated at 37 °C with 5% CO
2.
Photographs were taken at the indicated
time points (scale bar = 100 lm). (B) Three
hours after wounding, the cells were fixed,
immunostained with anti-a-tubulin serum
and DAPI, and the length of the protrusions
of wound edge cells was measured. Thirty
cells in randomly selected fields were mea-
sured in each of three independent experi-
ments. Data are the distance (mean ± SD)
between the leading edge and the nucleus
(*P < 0.01). (C) Three hours after wounding,
the cells were fixed and immunostained
with anti-a-tubulin serum and DAPI (scale
bar = 20 lm). (D) CasWt and CasKo cells
were wounded, fixed and immunostained
with anti-GM130 serum and DAPI at the
indicated time points to evaluate the per-
centage of cells with Golgi located in the
120° arc facing the wound. One hundred
cells were evaluated for Golgi localization in
each of two independent experiments. Data

CrkII knockdown cells but not in Nck2 knockdown
cells (Fig. 6D, F). CrkII siRNA reduced CrkII expres-
sion by approximately 50%, leading to a significant
reduction in cell protrusion distance of approximately
30% compared to control cells (t-test: P < 0.01).
These results indicate that Cas ⁄ CrkII association was
required for the formation of protrusions, whereas
Cas ⁄ Nck2 association was essential for the polariza-
tion of cells.
To further confirm the role of Nck2 for cell polari-
zation in cells expressing Cas, its localization and
effects on Cdc42 activity during wound healing were
examined. As shown in Fig. 7A, Nck2 co-localized
with Cas on the leading edge of cells. By contrast,
localization of Nck2 on the leading edge was not
observed in CasKo cells (Fig. 7B), indicating that Cas
was required for the polarized localization of Nck2.
CasKo
CasWt
0
10
20
30
40
50
60
70
CasKo CasWt
0
0.5

for the localization of Cdc42. The percent-
age of these cells with Cdc42 localized on
the leading edge is presented as the
mean ± SEM (*P < 0.01). (B) Forty
scratches were made on the confluent
monolayers of cells, and cells were lysed at
the indicated time points to detect total
Cdc42 and active, GTP bound, Cdc42. (C)
Forty scratches were made and, 3 h later,
cells were lysed to detect total and active
Cdc42. Three independent experiments
were performed and relative ratios of Cdc42
activity are shown as the mean ± SD.
A representative result from the western
blotting is shown.
Cas ⁄ Nck2 regulates cell polarity K. Funasaka et al.
3506 FEBS Journal 277 (2010) 3502–3513 ª 2010 The Authors Journal compilation ª 2010 FEBS
In addition to cell polarization, activation of Cdc42
during wound healing was dependent on Nck2. As
shown in Fig. 7B, cells transfected with Nck2 siRNA
displayed approximately 50% of the Cdc42 activity
seen in control transfectants during wound healing,
whereas depletion of CrkII did not affect Cdc42 acti-
vation.
Discussion
Polarization of cells in the direction of migration is
required for the organized movement of cells during
embryonic development and wound healing [5].
Because integrin-mediated signaling pathways are cru-
cial for cell polarization [1], we studied the role of Cas,

Ctrl Cas
Ctrl
siRNA
Ctrl
siRNA
Relative ratio of
active Cdc42
*
0
0.5
1
1.5
Ctrl Cas
Ctrl Cas
Percentage of cells with
polarized golgi
20 µm
20 µm
siRNA
siRNA
siRNA
siRNA
siRNA
A
B
CD
Fig. 4. Silencing of Cas in Balb3T3 cells
inhibits wound-induced cell polarization and
activation of Cdc42. (A) Balb3T3 cells were
transfected with either control or Cas siRNA

vious studies demonstrating that PP2 treatment dis-
rupted polarization of astrocytes during migration
[1]. Crk and Nck proteins are adaptor proteins that
associate with tyrosine-phosphorylated Cas through
SH2 domains [20–22]. Interestingly, silencing of CrkII
reduced the elongation of protrusions but did not
disrupt the reorientation of the Golgi. Cas ⁄ CrkII asso-
ciation regulates the activation of Rac via a functional
cooperation with GTPase-activating protein DOCK180
[30–32]. Activation of Rac is essential for the for-
mation of protrusions [1]; therefore, the Cas ⁄ CrkII
pathway appears to regulate protrusion formation by
0
10
20
30
40
50
60
70
80
Ctrl PP2
*
Percentage of cells with polarized golgi
–+ +
PP2
pTyr
Cas
IP: Cas
Scratch (3 h)

Golgi that lied within the 120° arc facing the
wound. One hundred cells in each of three
independent experiments were evaluated
for Golgi localization (*P < 0.01).
Cas ⁄ Nck2 regulates cell polarity K. Funasaka et al.
3508 FEBS Journal 277 (2010) 3502–3513 ª 2010 The Authors Journal compilation ª 2010 FEBS
activating Rac during the wound-healing assay. In
addition, knockdown of Nck2 expression resulted in
randomly oriented protrusions and Golgi reorienta-
tion, which indicates that the Cas ⁄ Nck2 pathway is
essential for the establishment of cell polarity.
As shown in Fig. 8, these data suggest that Cas uti-
lizes CrkII and Nck2 in parallel pathways to promote
cell migration. Cas associates with Nck2 to activate
Cdc42 and induce cell polarization. At the same time,
Cas also associates with CrkII to induce Rac1 activa-
tion, leading to cell protrusion and elongation.
Transfection of Nck1 siRNA into CasWt cells par-
tially disrupted the reorientation of the Golgi. Nck1
and Nck2 have 68% identity at the amino acid
sequence level and are considered to have redundant
functions [24], although some proteins have been
reported to specifically associate with Nck2. For exam-
ple, Pinch1, which is an essential adaptor protein for
integrin-mediated signaling, specifically interacts with
the SH3 domain of Nck2 [33]. Signaling pathways spe-
cifically regulated by Nck2 may mediate polarization;
however, we cannot rule out the possibility that Nck2
is more abundantly expressed in CasWt cells and, thus,
Nck2-knockdown resulted in a more significant disrup-

C
E
D
F
*
0
10
20
30
40
50
60
70
80
90
100
Ctrl CrkII Nck2
Percentage of cells
with directional protrusions
Ctrl
siRNA
Nck2
siRNA
CrkII
siRNA
Nck1
Ctrl Nck1
Actin
B
CasKo CasWt

cell polarization. (A) Expression of indicated
proteins in CasKo and CasWt cells was
examined by western blotting. (B) CasWt
cells were transfected with the indicated
siRNAs and, 3 days later, cells were lysed
and expression of indicated proteins was
evaluated by immunoblotting. (C) CasWt
cells were transfected with the indicated
siRNAs and, 3 days later, cells were fixed
and immunostained with GM130 to visualize
Golgi, and nuclei were stained with DAPI.
The graph indicates the percentage of cells
(mean ± SEM, n = 100) that have the Golgi
in the 120° arc facing the wound. (D) CasWt
cells transfected with either Nck2 or CrkII
siRNA were wounded and, 3 h later, cells
were fixed and immunostained for a-tubulin
and the nucleus. White lines indicate the
wound direction (scale bar = 20 lm). (E)
Data are presented as the percentage of
cells (mean ± SEM, n = 150) displaying pro-
trusions within the 60° arc in the direction
of migration. Fifty cells were counted in
each of three independent experiments
(*P < 0.01 compared to control and CrkII
siRNA-transfected cells). (F) The length of
the protrusions from cells on the wound
edge was measured. Thirty cells were mea-
sured in each of three independent
experiments. Data are presented as

acting with bPIX [38,41].
In conclusion, in the present study, we have shown
that Cas utilizes Nck2 to activate Cdc42 and induce
cell polarization, whereas Cas also recruits CrkII to
activate Rac1 to form cell protrusions and elongation
for promotion of cell migration during wound healing.
0
0.5
1
1.5
Ctrl Nck2
AB
C
Nck2
Cas Merge
Total Cdc42
Active Cdc42
Ctrl Nck2
Relative ratio of
active Cdc42
CasWt CasKo
20 µm
20 µm
siRNA
siRNA
Total Cdc42
Active Cdc42
Ctrl CrkII
siRNA
Fig. 7. Nck2 is localized to the leading edge

zation and migration.
Materials and methods
Cells, antibodies and reagents
Cells from homozygous null Cas knockout mouse embryos
were transfected with wild-type Cas (CasWt cells) or the
parental transfection vector pBabeHygro (CasKo cells),
selected for resistance to hygromycin, and maintained as
described previously [14,18]. Clones were not taken for sub-
sequent experiments to minimize potential effects of clonal
variation. The antibodies used in the experiments were:
anti-Cas, anti-GM130, anti-Nck1 and anti-Cdc42 sera (BD
Transduction Laboratories, San Jose, CA, USA); anti-Crk
serum (Cell Signaling, Danvers, MA, USA); anti-Nck2
serum (Millipore, Billerica, MA, USA); anti-CrkL serum
(Santa Cruz Biotechnology, Santa Cruz, CA, USA);
fluorescein isothiocyanate-conjugated anti-a-tubulin serum
(Sigma, St Louis, MO, USA). PP2 was purchased from
Funakoshi (Tokyo, Japan).
Cell migration assays
Wound healing assays were performed by scratching con-
fluent cell monolayers with a pipette tip and incubating at
37 °C with 5% CO
2
. Twenty-four hours later, the distance
that leading edge of the monolayer traveled into the
wound area was measured in five randomly selected fields
from three independent experiments. To measure cell
migration using Boyden chambers, 5 · 10
4
cells were

incubated in 7% calf serum in NaCl ⁄ P
i
for 30 min. Cells
were incubated with primary antibody in NaCl ⁄ P
i
for 1 h,
washed with NaCl ⁄ P
i
for 15 min, incubated with fluorescein
isothiocyanate- or Alexa Fluor 594-labeled secondary anti-
body in NaCl ⁄ P
i
for 1 h, incubated with 4¢,6¢-diamino-2-
phenylindole dihydrochloride (DAPI) for 5 min and then
analyzed under a fluorescence microscope (BX60; Olympus,
Tokyo, Japan).
Cdc42-activity assay
Forty scratches approximately 800 lm in width and the
length of the dish were made on confluent monolayers of
cells in 10 cm dishes. Cells were then incubated for 3 h,
lysed with lysis buffer (Tris–HCl 25 mm, pH 7.4, NaCl
150 mm, MgCl
2
10 mm, NP40 1%) with protease inhibitor
cocktail (Roche Diagnostics, Basel, Switzerland) and centri-
fuged at 21 880 g. for 20 min to remove cell debris. Cell
lysates were incubated with GST-PAK-PBD (residues
67–150) fusion protein bound to glutathione-agarose beads
for 1 h at 4 °C. Beads were washed with lysis buffer
four times and then subjected to western blotting with

ments, 30 cells in randomly selected fields were evaluated to
calculate the average length of these protrusions.
Acknowledgements
We thank the members of the Division of Cancer Biol-
ogy for helpful discussions and technical assistance.
This research was funded in part by a grant from the
Ministry of Education, Culture, Sports, Science and
Technology of Japan.
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