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

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

polarization during cell migration in response to wound healing

Kohei Funasaka1, Satoko Ito2, Hitoki Hasegawa2, Gary S.Goldberg3, Yoshiki Hirooka1,

Hidemi Goto1, Michinari Hamaguchi2and 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

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

family GTPase conserved in a wide range of

organ-isms, 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

divi-sion and morphological changes in response to

phero-mone signaling [4] In multicellular organisms, cell

polarity is determined by extracellular stimuli, such as chemoattractant gradients and cell–cell contact Locali-zation 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

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: tsenga@med.nagoya-u.ac.jp

(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 dur-ing wound healdur-ing 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

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.

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

medi-ated by the Src family kinases, and its phosphorylation

is required for Cas-mediated cell migration and

trans-formation [16–19] Phosphorylated Cas recruits

adap-tor 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

regulat-ing 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

con-straints 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

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 protrusions with a length of 61 ± 23 lm (mean ± SD)

by 3 h after wounding This was almost twice the aver-age 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 stain-ing indicates that Cas promoted this directional forma-tion 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 direc-tion of migradirec-tion [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 Rho GTPase that traffics to the leading edge of cell protrusions and regulates cell polarity dur-ing wound healdur-ing [1] The effects of Cas on Cdc42 localization during wound healing were evaluated by

immunofluorescence microscopy As shown inFig 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 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

inFig 4A, transfection with Cas siRNA effectively sup-pressed Cas expression Three days after the transfection

of either control or Cas siRNA, orientation of the Golgi during wound healing was examined by immunostain-ing As shown in Fig 4B, an average of 29 ± 3.8% of the cells transfected with Cas siRNA contained polar-ized Golgi by 3 h after wounding compared to an aver-age of 66 ± 3.2% seen in control transfectants

In addition to reducing cell polarization, Cas siRNA transfection also reduced Cdc42 activation and traf-ficking during wound healing As shown in Fig 4C,

0 100 200 300 400 500 600

CasKo CasWt

Distance of migration CasKo CasWt

0

24

Wound healing assay

(h)

(µm)

(24 h)

*

200 µm

Migration assay (3 h)

0 50 100 150 200 250 300 350 400 450

CasKo CasWt

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 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 · 10 4

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).

cells transfected with Cas siRNA exhibited

approxi-mately half of the Cdc42 activity found in control

transfectants 3 h after wounding Cdc42 was also

evi-dent 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

com-ponent of the signaling cascade that directs cell

polari-zation, 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

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

CasKo

CasWt

0 10 20 30 40 50 60 70 80 90

CasKo CasWt

*

100 µm

20 µm

CasKo CasWt

20 µm

0 10 20 30 40 50 60 70 80

0 h 1 h 3 h 6 h

CasKo CasWt

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

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).

Crk family members, CrkII and CrkL, can associate

with phosphorylated Cas to regulate the actin

cytoskel-eton, 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

polar-ization of CasWt cells

In addition to inhibiting orientation of the Golgi,

cell protrusions were more randomly oriented in

Nck2 knockdown cells compared to either control or CrkII siRNA-transfected cells (Fig 6D, E) Interest-ingly, the elongation of protrusions was reduced in 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 localipolari-zation 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 1 1.5

CasKo CasWt

Relative ratio of active Cdc42

*

20 µm

Active Cdc42

Total Cdc42

CasWt CasKo Time (h) 0 1 3 0 1 3

CasKo CasWt

Total Cdc42

Active Cdc42

A

B

C

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 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.

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,

which is an adaptor protein that mediates integrin sig-naling 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 vari-ous extracellular stimuli, including integrin-mediated

0 10 20 30 40 50 60 70 80

Ctrl Cas

Cas

Actin

Total Cdc42 Active Cdc42

Ctrl Cas

Ctrl siRNA

Ctrl siRNA

Relative ratio of active Cdc42

*

0 0.5 1 1.5

Ctrl Cas

Ctrl Cas

20 µm

20 µm siRNA

siRNA

siRNA

siRNA

siRNA

A

B

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

immuno-stained 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

per-centage of cells (mean ± SEM, n = 300)

dis-playing 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

expres-sion Arrows indicate Cdc42 localized on the

leading edge.

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

dis-rupted reorientation of the Golgi in the direction of

migration, which is consistent with the findings of

pre-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

*

PP2

pTyr

Cas

IP: Cas Scratch (3 h)

PP2

(10 µ M )

Ctrl

100 µm

A

B

C

Fig 5 Src-mediated tyrosine phosphoryla-tion is required for wound-induced cell polar-ization (A) CasWt cells were wounded and treated with 20 l M Src kinase inhibitor, PP2, for 3 h Cell were lysed and immunoprecipi-tated 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).

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-tion of polarizadisrup-tion 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 stimula-tion 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

Nck2 Ctrl Nck2

Actin

CrkII Ctrl

Actin

CrkL Ctrl CrkL

Actin

CrkII

A

0 10 20 30 40 50 60 70 80 90

Ctrl Nck1 Nck2 CrkII CrkL

C

E

D

F

*

0 10 20 30 40 50 60 70 80 90 100

Ctrl CrkII Nck2

Ctrl siRNA Nck2

siRNA

CrkII siRNA

Nck1 Ctrl Nck1

Actin

B

CasKo CasWt

CrkII CrkL Nck1 Nck2 Actin

0 10 20 30 40 50 60

Ctrl CrkII Nck2

20 µm

siRNA

*

*

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

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

distance (mean ± SD) between the leading

edge and the nucleus (*P < 0.01 compared

to control and Nck2 siRNA-transfected

cells).

important roles for Nck proteins in the regulation of

Cdc42; however, the mechanism by which Nck

pro-teins regulate Cdc42 activation has not been

eluci-dated PAK proteins are serine⁄ threonine kinases that

associate with Nck and are involved in a wide range of

biological activities, including actin cytoskeleton reor-ganization [35] Recently, it was reported that PAK1 functions as a scaffold protein to regulate Cdc42 acti-vation [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 proline-rich 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 activa-tion 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 inter-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

C

Nck2

Cas Merge

Total Cdc42

Active Cdc42

Ctrl Nck2

Relative ratio of active Cdc42

20 µm

20 µm

siRNA

siRNA

Total Cdc42

Active Cdc42

Ctrl CrkII siRNA

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 indi-cates the relative activity of Cdc42 (mean ± SD).

Protrusion Polarization

CrkII Nck2

Rac1 Cdc42

Cas

Cell migration

Fig 8 Schematic presentation of regulation of cell migration by

Cas Cas utilizes Nck2 to activate Cdc42 and induce cell

polariza-tion Cas also utilizes CrkII to augment Rac1 activity, leading to cell

elongation Acting together, these pathways result in cell migration.

Further studies will be required to elucidate more fully

the roles of these focal adhesion proteins in cell

polari-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

37C 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%

metha-nol 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

inde-pendent 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

accor-dance with the manufacturer’s instructions

Immunofluorescence analysis

Cells were cultured on glass coverslips coated with fibronec-tin Confluent monolayers of cells were scratched with a pipette tip to achieve a wound of approximately 800 lm in width and incubated at 37C with 5% CO2for 3 h Cells were fixed in 4% paraformaldehyde for 20 min, permeabi-lized 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⁄ Pifor 1 h, washed with NaCl⁄ Pifor 15 min, incubated with fluorescein isothiocyanate- or Alexa Fluor 594-labeled secondary anti-body in NaCl⁄ Pi 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, MgCl210 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 4C 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 deter-mined to be in the polarized position One hundred cells in ten randomly selected fields were evaluated for Golgi locali-zation 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