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 Department of Gastroenterology, Nagoya University Graduate School of Medicine, Japan Division of Cancer Biology, Nagoya University Graduate School of Medicine, Japan 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: tsenga@med.nagoya-u.ac.jp (Received 14 April 2010, revised 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 FEBS Journal 277 (2010) 3502–3513 ª 2010 The Authors Journal compilation ª 2010 FEBS 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 h, and over 90% of the cells at the edge were polarized, with one side pointed toward the wound within h By contrast, < 10% of the CasKo cells at the wound edge displayed a polarized morphology 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 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 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 3503 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) 600 500 400 * 300 200 24 100 (h) 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 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 h after wounding Three independent experiments demonstrated that the Cdc42 activity h after wounding 3504 CasWt CasKo CasWt Fig 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) · 104 CasKo and CasWt cells were loaded onto the upper surface of Boyden chambers, incubated for 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 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 A (h) CasKo CasWt 100 µm Distance of cell protrusion (µm) B C 90 80 70 60 50 40 30 20 10 CasKo CasWt * CasKo 20 µm CasWt CasKo D Percentage of cells with polarized golgi Fig 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 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 * * 0h cells transfected with Cas siRNA exhibited approximately half of the Cdc42 activity found in control transfectants 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 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 3505 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 CasKo CasWt 20 µm B Time (h) CasWt CasKo Active Cdc42 Total Cdc42 Active Cdc42 Total Cdc42 CasKo CasWt Relative ratio of active Cdc42 1.5 C 0.5 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 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, 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 20 µm Ctrl Cas siRNA siRNA Ctrl Cas C D Active Cdc42 Ctrl siRNA Total Cdc42 Relative ratio of active Cdc42 Fig 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, 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, days later, cells were fixed 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 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, 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 0.5 Ctrl siRNA 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 3507 Cas ⁄ Nck2 regulates cell polarity A K Funasaka et al IP: Cas – – Scratch (3 h) PP2 + – + + pTyr Cas B (h) PP2 (10 µM) Ctrl Percentage of cells with polarized golgi 100 µm C 80 70 60 50 40 * 30 20 10 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 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 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 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 Ctrl Nck1 Nck2 CrkII CrkL siRNA F 100 90 80 70 60 50 40 30 20 10 * 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 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, days later, cells were lysed and expression of indicated proteins was evaluated by immunoblotting (C) CasWt cells were transfected with the indicated siRNAs and, 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, 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 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 3509 Cas ⁄ Nck2 regulates cell polarity 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 0.5 siRNA Ctrl CrkII Ctrl Nck2 siRNA Active Cdc42 Total Cdc42 Cas Nck2 Cdc42 Polarization CrkII Rac1 Protrusion Cell migration Fig 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 Nck2 is localized to the leading edge and is required for the activation of Cdc42 (A) Confluent monolayers of CasWt cells were wounded and, h later, cells were fixed and immunostained for Cas and Nck2 (scale bars = 20 lm) (B) CasWt and CasKo cells were wounded and, h later, cells were fixed and immunostained for Nck2 (C) CasWt cells were transfected with the indicated siRNAs and, 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, · 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 h Cells were fixed in 4% paraformaldehyde for 20 min, permeabilized with 0.5% Triton X-100 in NaCl ⁄ Pi for and incubated in 7% calf serum in NaCl ⁄ Pi for 30 Cells were incubated with primary antibody in NaCl ⁄ Pi for h, washed with NaCl ⁄ Pi for 15 min, incubated with fluorescein isothiocyanate- or Alexa Fluor 594-labeled secondary antibody in NaCl ⁄ Pi for h, incubated with 4¢,6¢-diamino-2phenylindole dihydrochloride (DAPI) for 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 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 to remove cell debris Cell lysates were incubated with GST-PAK-PBD (residues 67–150) fusion protein bound to glutathione-agarose beads for h at °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 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, days later when cells reached confluency, a scratch was made and the cells were fixed h later The cells were then stained with FEBS Journal 277 (2010) 3502–3513 ª 2010 The Authors Journal compilation ª 2010 FEBS 3511 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 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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... durhealing [1] The effects of Cas on Cdc42 during wound healing were evaluated by 3503 Cas ⁄ Nck2 regulates cell polarity A CasKo CasWt K Funasaka et al Balb3T3 Cas Actin B Wound healing assay CasKo... 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