Báo cáo khoa học: Cas utilizes Nck2 to activate Cdc42 and regulate cell polarization during cell migration in response to wound healing docx

Cas utilizes Nck2 to activate Cdc42 and regulate cell
polarization during cell migration in response to wound
healing
Kohei Funasaka1, Satoko Ito2, Hitoki Hasegawa2, Gary S.Goldberg3, Yoshiki Hirooka1,
Hidemi Goto1, Michinari Hamaguchi2 and Takeshi Senga2
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

Keywords
Cas; Cdc42; Crk; Nck; polarity
Correspondence
T. Senga, Division of Cancer Biology,
Nagoya University Graduate School of
Medicine, 65 Tsurumai-cho, Showa-ku,
Nagoya 466-8550, Japan
Fax: +81 52 744 2464
Tel: +81 52 744 2463
E-mail:
(Received 14 April 2010, revised 1 June
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 during 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)

Introduction
The establishment of cell polarity is essential for a
variety of cellular functions, such as cell division, differentiation and migration; however, the molecular
mechanisms underlying cell polarization have not been
elucidated thoroughly. Genetic and cell biological studies have identified several molecules that are important
for cell polarity. Among these proteins, Cdc42, a Rho
family GTPase conserved in a wide range of organisms, has been found to play a pivotal role for the
establishment of cell polarity [1–3]. In yeast, Cdc42 is
required for polarized bud formation during cell division and morphological changes in response to pheromone signaling [4]. In multicellular organisms, cell

polarity is determined by extracellular stimuli, such as
chemoattractant gradients and cell–cell contact. Localization and activation of Cdc42 in response to these
environmental changes are key events leading to cell
polarization [5,6].
Cas is a multiadaptor protein that regulates various
signaling pathways in response to extracellular stimuli,
including growth factors and integrin-mediated cell
adhesion [7–9]. Cas was originally identified as a
highly phosphorylated protein in cells transformed by
v-Src and v-Crk [10,11]. Cas contains an N-terminal
SH3 domain, proline-rich regions and a substrate
domain with multiple tyrosine phosphorylation sites

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

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Cas ⁄ Nck2 regulates cell polarity

K. Funasaka et al.

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
and defects in cell migration and spreading, indicating
an essential role of Cas for integrin-mediated signals
[15]. Tyrosine phosphorylation of Cas is mostly mediated by the Src family kinases, and its phosphorylation
is required for Cas-mediated cell migration and transformation [16–19]. Phosphorylated Cas recruits adaptor proteins such as Crk and Nck [20–22]. Association
of Crk with Cas enhances cell migration and spreading
by activating Rac1 [23]. Nck is important for regulating signals from cell surface receptors to the actin
cystoskeleton, as well as for cell movement. A number
of signaling molecules have been found to associate
with Nck; however, the physiological importance of
these interactions remains uncertain [24].

A wound-healing assay comprises a simple in vitro
experiment used to examine cell migration that is
enabled as a result of the release of physical constraints. A scratch in the confluent monolayer initiates
cell migration in the direction perpendicular to the
scratch until the gap is filled with cells [3]. Several
hours after the wound is made, cells on the edge of the
wound develop a polarized morphology [1]. Polarized
cells on the wound edge extend membrane protrusions
and reorient the Golgi in the direction of migration
[25]. Integrin-mediated activation of Cdc42 has been
shown to be critical for this polarization during cell
migration [1]; however, the signaling molecules
involved in the integrin-mediated activation of Cdc42
remain unknown. In the present study, we show that
Cas utilizes Nck2 to regulate cell polarization and
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 woundhealing assay using Cas deficient CasKo cells (homozygous 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 protrusions. As shown in Fig. 2B, CasWt cells exhibited cell
protrusions with a length of 61 ± 23 lm (mean ± SD)
by 3 h after wounding. This was almost twice the average protrusion length exhibited by CasKo cells, which
measured 35 ± 15 lm.
Microtubule elongation forms toward the leading
edge of cells during wound healing [1]. Tubulin staining indicates that Cas promoted this directional formation of microtubules within 3 h after wounding. As
shown in Fig. 2C, elongation of microtubules between
the nucleus and wound was observed in over 80%
of the CasWt cells on the wound edge. By contrast,
< 10% of the CasKo cells displayed this directional
organization of microtubules.
When cells are polarized for migration, the Golgi
becomes oriented between the nucleus and the direction of migration [3]. To examine the effects of Cas on
Golgi orientation, the localization of the Golgi matrix
protein, GM130 [27], was examined in CasKo and
CasWt cells on the wound edge after wounding. As
shown in Fig. 2D, polarized localization of the Golgi

in CasKo cells was clearly delayed compared to that of
CasWt cells. Approximately one-third of the Golgi was
localized within a 120° arc between the nucleus and
the wound edge upon the wounding, which was the
result of chance because cells were sectioned into three
120° arcs. Three hours after wounding, approximately
two-thirds of CasWt showed polarized localization of
the Golgi, whereas < 40% of CasKo cells showed
polarized localization of the Golgi.
Cas promotes Cdc42 activation and trafficking
during wound healing
Cdc42 is a
edge of cell
ing wound
localization

FEBS Journal 277 (2010) 3502–3513 ª 2010 The Authors Journal compilation ª 2010 FEBS

Rho GTPase that traffics to the leading
protrusions and regulates cell polarity durhealing [1]. The effects of Cas on Cdc42
during wound healing were evaluated by
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Cas ⁄ Nck2 regulates cell polarity

A

CasKo

CasWt

K. Funasaka et al.

Balb3T3
Cas

Actin

B

Wound healing assay
CasKo
CasWt

Distance of migration
(24 h)

(µm)
600
500

0

400

*

300
200

24
100
(h)

0

200 µm

Migration assay (3 h)
CasKo
CasWt

No. of migrated cells per field

C

CasKo
450
400
350
300
250

*

200
150
100
50
0

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 previous 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 confirm 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
3504

CasWt

CasKo

CasWt

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
for 24 h. Data are the mean ± SD of the
distance that leading edge of the monolayer
traveled into the wound area in five
randomly selected fields from three
independent experiments (*P < 0.01); scale
bar = 200 lm. (C) 5 · 104 CasKo and CasWt
cells were loaded onto the upper surface
of Boyden chambers, incubated for 3 h,
fixed, and examined by microscopy. Cells
that migrated to the lower surface of the
chamber are shown as the mean ± SD from
five randomly selected fields in three
independent experiments (*P < 0.01).

in CasWt cells was almost twice that of CasKo cells
(Fig. 3C).
Silencing of Cas in Balb3T3 cells inhibits cell
polarization
To further evaluate the requirement of Cas for cell
polarization, we used small interfering RNA (siRNA) to
knockdown Cas expression in Balb3T3 cells. As shown
in Fig. 4A, transfection with Cas siRNA effectively suppressed Cas expression. Three days after the transfection
of either control or Cas siRNA, orientation of the Golgi
during wound healing was examined by immunostaining. As shown in Fig. 4B, an average of 29 ± 3.8% of
the cells transfected with Cas siRNA contained polarized Golgi by 3 h after wounding compared to an average of 66 ± 3.2% seen in control transfectants.
In addition to reducing cell polarization, Cas siRNA
transfection also reduced Cdc42 activation and trafficking during wound healing. As shown in Fig. 4C,

FEBS Journal 277 (2010) 3502–3513 ª 2010 The Authors Journal compilation ª 2010 FEBS


Cas ⁄ Nck2 regulates cell polarity

K. Funasaka et al.

0

A

2

6

4

(h)

CasKo

CasWt

100 µm
Distance of cell protrusion
(µm)

B
C

90
80
70
60
50
40
30
20
10
0

CasKo

CasWt

*

CasKo

20 µm

CasWt

CasKo

D
Percentage of cells with
polarized golgi

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% CO2.
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 measured in each of three independent experiments. 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 percentage 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
are the mean ± SEM (*P < 0.01). Images
on the right panel are representative images
of immunostained cells 3 h after wounding.
White lines indicate wound direction (green,
GM130; blue, DAPI; scale bar = 20 lm).

CasWt
CasKo

80
70
60
50
40
30
20
10
0

*

*

0h

cells transfected with Cas siRNA exhibited approximately half of the Cdc42 activity found in control
transfectants 3 h after wounding. Cdc42 was also evident at the ends of cell protrusions on the wound edge
in control transfectants, although it was not detected
in cells transfected with Cas siRNA (Fig. 4D). Taken
together with the results obtained from Cas knockout
cells, these data indicate that Cas is an important component of the signaling cascade that directs cell polarization, Cdc42 activity and cell migration in response
to wound healing.
Src kinase inhibition disrupts polarization of
migrating cells
The Src tyrosine kinase phosphorylates Cas to promote cell migration [18]. We employed a Src kinase
inhibitor [4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyramidine; PP2] to determine whether Cas

CasWt

1h

3h

6h

20 µm

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

FEBS Journal 277 (2010) 3502–3513 ª 2010 The Authors Journal compilation ª 2010 FEBS

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Cas ⁄ Nck2 regulates cell polarity

K. Funasaka et al.

Percentage of cells with
Cdc42 localized on the leading edge

A

CasKo

CasWt

70
60
50
40
30
20

*

10
0
CasKo

CasWt

20 µm

B
Time (h)

0

CasWt
1
3

CasKo
0

1

3

Active Cdc42
Total Cdc42

Active Cdc42
Total Cdc42

CasKo

CasWt

Relative ratio of
active Cdc42

1.5

C

1
0.5
0
CasKo

Crk family members, CrkII and CrkL, can associate
with phosphorylated Cas to regulate the actin cytoskeleton, cell migration, invasion and survival [28,29]. The
Nck family has two known members, Nck1 and Nck2,
and both proteins can associate with phosphorylated
Cas [20,22,24].
As shown in Fig. 6A, Crk and Nck proteins were
expressed to similar levels in CasKo and CasWt cells.
We performed siRNA knockdown experiments to
determine whether these proteins were involved in the
Cas-mediated polarization of cells. As shown in
Fig. 6B, transfection of specific siRNA to CasWt cells
effectively suppressed the expression of target Crk or
Nck proteins, but not other proteins. Cells transfected
with Nck2 siRNA displayed significantly less polarized
Golgi than other transfectants during wound healing,
indicating that Nck2 played a critical role in the polarization of CasWt cells.
In addition to inhibiting orientation of the Golgi,
cell protrusions were more randomly oriented in
3506

CasWt

Fig. 3. Cas promotes Cdc42 activation and
intracellular trafficking during wound healing.
(A) Three hours after wounding, cells were
fixed and immunostained for Cdc42. DAPI
was used to stain nuclei. Arrows indicate
Cdc42 localized on the leading edge. Fifty
cells on the wound edge in each of three
independent experiments were evaluated
for the localization of Cdc42. The percentage 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.

Nck2 knockdown cells compared to either control or
CrkII siRNA-transfected cells (Fig. 6D, E). Interestingly, the elongation of protrusions was reduced in
CrkII knockdown cells but not in Nck2 knockdown
cells (Fig. 6D, F). CrkII siRNA reduced CrkII expression 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 polarization of cells.
To further confirm the role of Nck2 for cell polarization 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.

FEBS Journal 277 (2010) 3502–3513 ª 2010 The Authors Journal compilation ª 2010 FEBS


Cas ⁄ Nck2 regulates cell polarity

K. Funasaka et al.

A

siRNA
Ctrl

Cas
Cas
Actin

80

siRNA

B

70
60
50
40

*

30
20
10

0

20 µm

Ctrl

Cas
siRNA

siRNA
Ctrl
Cas

C

D

Active Cdc42
Ctrl
siRNA

Total Cdc42

Relative ratio of
active Cdc42

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
and, 3 days later, cells were lysed and
immunoblotted with anti-Cas serum. b-actin
was used as a loading control. (B) Balb3T3
cells were transfected with either control or
Cas siRNA and, 3 days later, cells were
fixed 3 h after wounding and immunostained for GM130 to visualize the Golgi.
One hundred cells were evaluated for the
localization of the Golgi in each of three
independent experiments. Data are the percentage of cells (mean ± SEM, n = 300) displaying Golgi within the 120° arc facing the
wound (*P < 0.01). (C) Three days after
siRNA transfection, Balb3T3 cells were
scratched and then were examined 3 h later
for Cdc42 activation. The relative activity of
Cdc42 is indicated as a graph. (D) Three
days after siRNA transfection, Balb3T3 cells
were scratched and, 3 h later, cells were

fixed and immunostained for Cdc42 expression. Arrows indicate Cdc42 localized on the
leading edge.

Cas

Percentage of cells with
polarized golgi

Ctrl

1.5
1
0.5

Ctrl
siRNA

0
Ctrl

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 activation.

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 crucial for cell polarization [1], we studied the role of Cas,

Cas
siRNA
20 µm

which is an adaptor protein that mediates integrin signaling leading to cell migration, in cell polarization. In
the present study, we found that Cas was essential for
the polarization of migrating cells. Scratch-induced
elongation of protrusions and reorientation of the
Golgi were more prominent in cells that expressed Cas
than in CasKo cells or cells treated with Cas siRNA.
In addition, we found that activation and localization
of Cdc42 on the leading edge of cells was disrupted in
CasKo and Cas siRNA-transfected cells, indicating
that Cas is crucial for the regulation of Cdc42 activity
during cell polarization.
Multiple tyrosine residues in the substrate-binding
domain of Cas are phosphorylated in response to various extracellular stimuli, including integrin-mediated

FEBS Journal 277 (2010) 3502–3513 ª 2010 The Authors Journal compilation ª 2010 FEBS

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Cas ⁄ Nck2 regulates cell polarity

A

K. Funasaka et al.

IP: Cas
–
–

Scratch (3 h)
PP2

+
–

+
+

pTyr
Cas

0

B

2

6

4

(h)

PP2
(10 µM)

Ctrl

Percentage of cells with polarized golgi

100 µm

C

80
70
60
50
40

*

30
20
10
0

Ctrl

PP2

adhesion. Among the tyrosine kinases required for
integrin-mediated signal transduction, Src is critical for

the phosphorylation of Cas [8]. PP2 treatment delayed
the protrusion of cells toward the wound and disrupted reorientation of the Golgi in the direction of
migration, which is consistent with the findings of previous studies demonstrating that PP2 treatment disrupted polarization of astrocytes during migration
[1]. Crk and Nck proteins are adaptor proteins that
3508

Fig. 5. Src-mediated tyrosine phosphorylation is required for wound-induced cell polarization. (A) CasWt cells were wounded and
treated with 20 lM Src kinase inhibitor, PP2,
for 3 h. Cell were lysed and immunoprecipitated with anti-Cas serum. Cells were
immunoblotted with anti-phosphotyrosine
and anti-Cas sera. (B) Confluent monolayers
of CasWt cells were wounded and then
incubated with dimethyl sulfoxide or PP2.
Photographs were taken at the indicated
time points (scale bar = 200 lm). (C)
Wounded CasWt cells were incubated for
3 h with dimethyl sulfoxide or PP2 and
immunostained with GM130 to visualize
Golgi, and nuclei were stained with DAPI.
Data are presented as the percentage of
cells (mean ± SEM, n = 300) displaying
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).

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 association regulates the activation of Rac via a functional

cooperation with GTPase-activating protein DOCK180
[30–32]. Activation of Rac is essential for the formation of protrusions [1]; therefore, the Cas ⁄ CrkII
pathway appears to regulate protrusion formation by

FEBS Journal 277 (2010) 3502–3513 ª 2010 The Authors Journal compilation ª 2010 FEBS


Cas ⁄ Nck2 regulates cell polarity

K. Funasaka et al.

A

CasKo

B

CasWt

Ctrl

Nck1

CrkII

Nck1

CrkL

Nck2

Actin

CrkII

Actin

CrkL
Nck1
Nck2

siRNA
Ctrl
CrkL

siRNA
Ctrl
Nck2
CrkL

Nck2

CrkII

Nck1

Actin

Actin

Actin

C
90
Percentage of cells with
polarized golgi

80

D

70

Ctrl
siRNA

60

*

50

CrkII
siRNA

Nck2
siRNA

40
30

20
10
0

Ctrl

Nck1 Nck2 CrkII CrkL
siRNA

F

100
90
80
70
60
50
40
30
20
10
0

*

Ctrl CrkII Nck2
siRNA

activating Rac during the wound-healing assay. In
addition, knockdown of Nck2 expression resulted in

randomly oriented protrusions and Golgi reorientation, 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 utilizes 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 activation, leading to cell protrusion and elongation.
Transfection of Nck1 siRNA into CasWt cells partially 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

Distance of cell protrusion (µm)

20 µm

E
Percentage of cells
with directional protrusions

Fig. 6. Nck2 is required for wound-induced
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 protrusions 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 measured in each of three independent
experiments. Data are presented as
distance (mean ± SD) between the leading
edge and the nucleus (*P < 0.01 compared
to control and Nck2 siRNA-transfected
cells).

siRNA

siRNA
Ctrl
CrkII

60

50

*

40
30
20
10
0

Ctrl

CrkII Nck2
siRNA

reported to specifically associate with Nck2. For example, Pinch1, which is an essential adaptor protein for
integrin-mediated signaling, specifically interacts with
the SH3 domain of Nck2 [33]. Signaling pathways specifically 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 disruption of polarization than Nck1-knockdown did.
We found that Nck2 was required for the activation
of Cdc42 during wound healing. A previous study by
Miyamoto et al. [34] reported that Nck1 was essential
for the activation of Cdc42 by endothelin-1 stimulation. The same study also showed that the expression
of a membrane-bound form of Nck1 was sufficient to
activate Cdc42. These results suggest that there are

FEBS Journal 277 (2010) 3502–3513 ª 2010 The Authors Journal compilation ª 2010 FEBS

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K. Funasaka et al.

A

B

Merge

CasWt

Cas

CasKo

Nck2

20 µm

20 µm

C

1.5
Relative ratio of
active Cdc42

siRNA
Ctrl
Nck2
Active Cdc42
Total Cdc42

1
0.5
0

siRNA
Ctrl
CrkII

Ctrl

Nck2
siRNA

Active Cdc42
Total Cdc42

Cas

Nck2

Cdc42

Polarization

CrkII

Rac1

Protrusion

Cell migration
Fig. 8. Schematic presentation of regulation of cell migration by
Cas. Cas utilizes Nck2 to activate Cdc42 and induce cell polarization. Cas also utilizes CrkII to augment Rac1 activity, leading to cell
elongation. Acting together, these pathways result in cell migration.

important roles for Nck proteins in the regulation of
Cdc42; however, the mechanism by which Nck proteins regulate Cdc42 activation has not been elucidated. PAK proteins are serine ⁄ threonine kinases that
associate with Nck and are involved in a wide range of

3510

Fig. 7. Nck2 is localized to the leading edge
and is required for the activation of Cdc42.
(A) Confluent monolayers of CasWt cells
were wounded and, 3 h later, cells were
fixed and immunostained for Cas and Nck2
(scale bars = 20 lm). (B) CasWt and CasKo
cells were wounded and, 3 h later, cells
were fixed and immunostained for Nck2.
(C) CasWt cells were transfected with the
indicated siRNAs and, 3 days later, cells
were scratched and examined for Cdc42
activation. Three independent experiments

were performed to measure Cdc42 activity
in the absence of Nck2 and the graph indicates the relative activity of Cdc42
(mean ± SD).

biological activities, including actin cytoskeleton reorganization [35]. Recently, it was reported that PAK1
functions as a scaffold protein to regulate Cdc42 activation [36]. In that case, PAK1 associates with both
Gbc and PAK-interacting guanine nucleotide exchange
factor a (aPIX) to activate Cdc42 in response to
chemoattractants. bPIX, which has structural features
similar to aPIX, has been reported to regulate Cdc42
activity during wound-healing assays [37,38]. The SH3
domain of bPIX associates with a nontypical prolinerich region of PAK1 [39], whereas the SH3 domain of
Nck associates with the most N-terminal proline-rich
region of PAK1 [40]. Because Cas associates with the
SH2 domain of Nck, the protein complex of Cas–
Nck2–PAK1–bPIX may play a role in Cdc42 activation. Recent studies have also shown that Scrib, which
is a multidomain scaffold protein, is localized to the
leading edge of cells and regulates localization and
activation of Cdc42 during cell polarization by interacting 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.

FEBS Journal 277 (2010) 3502–3513 ª 2010 The Authors Journal compilation ª 2010 FEBS


Cas ⁄ Nck2 regulates cell polarity

K. Funasaka et al.

Further studies will be required to elucidate more fully
the roles of these focal adhesion proteins in cell polarization 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 subsequent 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 confluent cell monolayers with a pipette tip and incubating at
37 °C with 5% CO2. 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 · 104 cells were
seeded onto the upper surface of the chamber. The lower

surface of the filter was coated with fibronectin. Three
hours after seeding, cells were fixed with 70% methanol and stained with 0.5% of crystal violet. Cells that
migrated to the lower surface of the chambers were
counted in five randomly selected fields from three independent experiments.

siRNA transfection
siRNAs were designed and purchased from Sigma-Aldrich
(St. Louis, MO, USA). The sequences of siRNAs were: Cas
5¢-UCAUUUGACUAAUAGUCUATT-3¢; Nck1 5¢-GGA
UGAUUCCUGUCCCUUATT-3¢; Nck2 5¢-GGUCGCGA
GGCUGUAUGUAGU-3¢; CrkL 5¢-CUUACUAGAUCCG
UGAGUUAA-3¢; CrkII 5¢-GGAUCAACAGAAUCCCGA
UTT-3¢; Control (designed to target luciferase) 5¢- CUUA
CGCUGAGUACUUCGATT-3¢. Twenty nanomoles of
siRNA was transfected into cells using Lipofectamine
RNAiMAX (Invitrogen, Carlsbad, CA, USA) in accordance with the manufacturer’s instructions.

Immunofluorescence analysis
Cells were cultured on glass coverslips coated with fibronectin. Confluent monolayers of cells were scratched with a
pipette tip to achieve a wound of approximately 800 lm in
width and incubated at 37 °C with 5% CO2 for 3 h. Cells
were fixed in 4% paraformaldehyde for 20 min, permeabilized with 0.5% Triton X-100 in NaCl ⁄ Pi for 5 min and
incubated in 7% calf serum in NaCl ⁄ Pi for 30 min. Cells
were incubated with primary antibody in NaCl ⁄ Pi for 1 h,
washed with NaCl ⁄ Pi for 15 min, incubated with fluorescein
isothiocyanate- or Alexa Fluor 594-labeled secondary antibody in NaCl ⁄ Pi for 1 h, incubated with 4¢,6¢-diamino-2phenylindole 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, MgCl2 10 mm, NP40 1%) with protease inhibitor
cocktail (Roche Diagnostics, Basel, Switzerland) and centrifuged 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
anti-Cdc42 serum to detect active Cdc42 protein bound to
GST-PAK-PBD. Total Cdc42 protein was detected by
immunoblotting total cell lysates.

Golgi reorientation measurements
Measurement of Golgi reorientation was performed as
described previously [3]. In brief, confluent cells that had
been cultured on fibronectin-coated glass slides were
scratched with a pipette tip and incubated for 3 h. Cells
were fixed and stained for GM130 to visualize the Golgi.
Cells on the wound edge were divided equally into three
sectors, including the front sector between the nucleus and
the leading edge. The Golgi in the front sector was determined to be in the polarized position. One hundred cells in
ten randomly selected fields were evaluated for Golgi localization to determine the percentage of reoriented Golgi.

Measurement of protrusion orientation and
length
Cells were transfected with each siRNA and, 3 days later
when cells reached confluency, a scratch was made and the
cells were fixed 3 h later. The cells were then stained with

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Cas ⁄ Nck2 regulates cell polarity

K. Funasaka et al.

anti-tubulin serum and DAPI nuclear stain. Protrusions of
wound edge cells that had oriented within the 60° arc in the
direction of migration were regarded as directional protrusions. Fifty cells in ten randomly selected fields were evaluated for directional protrusions in each of three independent
experiments. To quantify the protrusions’ length, the distance of the leading edge from the nuclei of wound edge
cells was measured. In each of three independent experiments, 30 cells in randomly selected fields were evaluated to
calculate the average length of these protrusions.

12

13

Acknowledgements

14

We thank the members of the Division of Cancer Biology 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.

15

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