RESEARCH ARTICLE

MEKK1-dependent phosphorylation of calponin-3 tunes cellcontractilityHiroaki Hirata1,*, Wei-Chi Ku2,‡, Ai Kia Yip3, Chaitanya Prashant Ursekar1, Keiko Kawauchi1,§, Amrita Roy1,Alvin Kunyao Guo1,¶, Sri Ram Krishna Vedula1,**, Ichiro Harada4,5, Keng-Hwee Chiam1,3, Yasushi Ishihama2,Chwee Teck Lim1,6, Yasuhiro Sawada1,4,7,‡‡,§§ and Masahiro Sokabe1,8,§§

ABSTRACTMEKK1 (also known as MAP3K1), which plays a major role in MAPKsignaling, has been implicated in mechanical processes in cells, suchas migration. Here, we identify the actin-binding protein calponin-3 asa new MEKK1 substrate in the signaling that regulates actomyosin-based cellular contractility. MEKK1 colocalizes with calponin-3 atthe actin cytoskeleton and phosphorylates it, leading to an increase inthe cell-generated traction stress. MEKK1-mediated calponin-3phosphorylation is attenuated by the inhibition of myosin II activity,the disruption of actin cytoskeletal integrity and adhesion to softextracellular substrates, whereas it is enhanced upon cell stretching.Our results reveal the importance of theMEKK1–calponin-3 signalingpathway to cell contractility.

KEYWORDS: Actomyosin, Mechanotransduction, Phosphorylation,Substrate rigidity

INTRODUCTIONMEKK1 (also known as MAP3K1), is a mitogen-activated proteinkinase (MAPK) kinase kinase that activates the c-Jun N-terminalkinase (JNK) and extracellular signal-regulated kinase (ERK)MAPK signaling pathways (Yujiri et al., 1998; Hagemann andBlank, 2001). Full-length MEKK1 contributes to pro-survivalsignaling, whereas its cleavage by caspase 3 generates a C-terminalkinase domain fragment that promotes apoptosis (Pham et al.,2013). In vivo analysis using MEKK1-deficient mice, which exhibita failure in eyelid closure caused by impaired epithelial migration

(Yujiri et al., 2000; Zhang et al., 2003), highlights regulation of cellmigration as a physiological role of MEKK1. This is furthersupported by in vitro studies showing that MEKK1 is cruciallyinvolved in directed migration of various types of cells (Yujiri et al.,2000; Zhang et al., 2003, 2005; Cuevas et al., 2003, 2006; Denget al., 2006).

In migrating cells, actomyosin-generated contractile forces pullthe cell body forward and promote retraction of the cell rear (Ridleyet al., 2003; Parsons et al., 2010). Thus, actomyosin contraction isessential for driving directed cell migration (Parsons et al., 2010).MEKK1 has been reported to contribute to the formation of actinstress fibers (Zhang et al., 2003, 2005), a major contractile apparatusin non-muscle cells, as well as the retraction of cell rear tails (Cuevaset al., 2003). This implies that MEKK1 might promote cellmigration by regulating actomyosin contractility. AlthoughMEKK1 localizes to the actin cytoskeleton (Christerson et al.,1999; Cuevas et al., 2003) and functions downstream of RhoA instress fiber formation (Zhang et al., 2005), little is known about theMEKK1-mediated signaling that regulates cell contractility.

Calponins, which comprise a family of actin-binding proteins, arereportedly involved in the regulation of a variety of cell motilebehaviors such as migration, contraction and morphogenesis(Rozenblum and Gimona, 2008; Wu and Jin, 2008). There arethree genetic isoforms of calponin: h1 or basic calponin (calponin-1; CNN1), h2 or neutral calponin (calponin-2; CNN2), and h3 oracidic calponin (calponin-3; CNN3). CNN1 is exclusivelyexpressed in smooth muscle cells, whereas CNN2 and CNN3 areexpressed more ubiquitously. The amino acid sequences arehighly conserved among these isoforms; only the C-terminal tailsdiffer to a large extent (Fig. S1). CNN1 bound to actin filamentsinhibits actin-activated myosin ATPase activity without affectingphosphorylation of the myosin regulatory light chain (MLC), andattenuates contraction of smooth muscle (Abe et al., 1990; Winderand Walsh, 1990; Itoh et al., 1994; Horowitz et al., 1996;Obara et al., 1996; Tang et al., 1996). The inhibitory effect ofCNN1 on actomyosin contraction is, however, alleviated uponphosphorylation at Ser175 by protein kinase C or Ca2+/calmodulin-dependent kinase II (Winder and Walsh, 1990; Itoh et al., 1994;Obara et al., 1996; Tang et al., 1996). Therefore, CNN1 has beenconsidered as a troponin-like molecular switch for actomyosincontraction in smooth muscle (Rozenblum and Gimona, 2008; Wuand Jin, 2008). In contrast to CNN1, the functions of the non-musclecalponins, CNN2 and CNN3, have been poorly documented.However, CNN3 reportedly plays a role in stress fiber formation andcell migration (Appel et al., 2010; Daimon et al., 2013). It has alsobeen reported that CNN3 can be phosphorylated. Among severalphosphorylation sites known in CNN3, Ser293 phosphorylation isinvolved in its binding to F-actin (Abouzaglou et al., 2004;Shibukawa et al., 2010, 2013). However, it remains unclear whetherReceived 15 March 2016; Accepted 10 August 2016

1Mechanobiology Institute, National University of Singapore, 117411 Singapore.2Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto 606-8501,Japan. 3A*STAR Bioinformatics Institute, 138671 Singapore. 4LocomotiveSyndrome Research Institute, Nadogaya Hospital, Kashiwa 277-0032, Japan.5Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology,Yokohama 226-8501, Japan. 6Department of Biomedical Engineering, NationalUniversity of Singapore, 117583 Singapore. 7Department of Biological Sciences,National University of Singapore, 117543 Singapore. 8Mechanobiology Laboratory,Nagoya University Graduate School of Medicine, Nagoya 466-8550, Japan.*Present address: R-Pharm Japan, and Nagoya University Graduate School ofMedicine, Nagoya 466-8550, Japan. ‡Present address: School of Medicine,College of Medicine, Fu Jen Catholic University, New Taipei 24205, Taiwan.§Present address: Frontiers of Innovative Research in Science and Technology(FIRST), Konan University, Kobe 650-0047, Japan. ¶Present address: Cancer &Stem Cell Biology Program, Duke-NUS Graduate Medical School Singapore,169857 Singapore. **Present address: L’oreal Research and Innovation,138648 Singapore. ‡‡Present address: Department of Rehabilitation for theMovement functions, Research Institute, National Rehabilitation Center for Personswith Disabilities, Tokorozawa 359-8555, Japan.

§§Authors for correspondence (ys454-ind@umin.ac.jp;msokabe@med.nagoya-u.ac.jp)

H.H., 0000-0002-2604-9158; C.P.U., 0000-0001-9253-0559; M.S., 0000-0001-7791-0166

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http://jcs.biologists.org/lookup/doi/10.1242/jcs.189415.supplementalmailto:ys454-ind@umin.ac.jpmailto:msokabe@med.nagoya-u.ac.jphttp://orcid.org/0000-0002-2604-9158http://orcid.org/0000-0001-9253-0559http://orcid.org/0000-0001-7791-0166http://orcid.org/0000-0001-7791-0166

phosphorylation of CNN3 at the other sites is implicated in actin- oractomyosin-related cell functions.Here, we show that MEKK1 regulates cellular contractility.

Furthermore, we find that MEKK1 phosphorylates CNN3 atThr288. MEKK1-dependent phosphorylation of CNN3 increasesthe traction stress that cells generate. Importantly, the CNN3phosphorylation, which depends on myosin II activity and actincytoskeletal integrity, is enhanced upon adhesion to rigidextracellular substrates or application of external stretching force.This suggests that the MEKK1–CNN3 signaling is mechanicallyregulated. Collectively, we propose that MEKK1 and CNN3comprise a new pathway involved in a positive-feedbackregulatory mechanism of cellular contractility.

RESULTSMEKK1 mediates traction stress generation of cellsTo examine the role of MEKK1 in regulating cellular contractility,we used traction force microscopy to measure the traction stressexerted by cells on extracellular substrates (Pelham andWang, 1999;Dembo and Wang, 1999). RNA interference (RNAi)-mediateddepletion of MEKK1 expression [short hairpin RNA (shRNA) forMEKK1] in mouse myoblastic C2C12 cells significantly reducedthe magnitude of cell-generated traction stress (Fig. 1B,C). Notably,phosphorylation of MLC (myosin regulatory light chain 2, MYL2),a crucial step in the activation of non-muscle myosin II (Tan et al.,1992), was not affected by depleting MEKK1 expression (Fig. 1A),suggesting that MEKK1 regulation of cellular contractility does notinvolve alteration of MLC phosphorylation.

Phosphorylation of CNN3 at Thr288, a new phosphorylationsite, is blocked by inhibition of myosin IIWe then sought molecular details behind the regulation of cellularcontractility by MEKK1. Contractility of non-muscle cells isreportedly modulated by the mechanical properties of thesurrounding environments (Geiger et al., 2009; Zaidel-Bar et al.,

2015), including the substrate rigidity, as we have observed(Fig. 1C), and as we and others have reported previously (Loet al., 2000; Paszek et al., 2005; Saez et al., 2005; Yip et al., 2013).We postulated that MEKK1 might be involved in the positive-feedback mechanism that mediates substrate-rigidity-dependentregulation of cell contractility (Giannone and Sheetz, 2006;Buxboim et al., 2010; Trichet et al., 2012), and examined whetherthe activity of MEKK1 was modulated by actomyosin contraction,the major source of cellular contractility (Beningo et al., 2006).MEKK1 activity was assessed by evaluating the level of MEKK1phosphorylation at Thr1381 in its activation loop, theautophosphorylation that reportedly represents MEKK1 activation(Matsuzawa et al., 2008; Enzler et al., 2009; Saha et al., 2014). In animmunoblot analysis of C2C12 cell lysate using an antibody thatwas raised against Thr1381-phosphorylated MEKK1 (Fig. S2), wedid not detect a distinct band at the molecular mass of full-lengthMEKK1 (196 kDa) (asterisk in Fig. S3A). Instead, the anti-Thr1381-phospho-MEKK1 antibody blot demonstrated a band atthe apparent molecular mass of 39 kDa, which disappeared in thelysate from myosin-II-inhibited cells (arrow in Fig. S3A). Toidentify the protein represented by the 39-kDa band,immunoprecipitates with the anti-Thr1381-phosphorylatedMEKK1 antibody were resolved by SDS-PAGE. A massspectrometric analysis of the excised gel piece containing the 39-kDa band (indicated by a square parenthesis in Fig. S3B) revealedthat the sample included peptides that share the sequences withCNN3 (Fig. S3C). Given that CNN3 contains the sequence(S285QGTG289), which is similar to the region containingthe phosphorylation site (Thr1381) in MEKK1 (S1378KGTG1382),we hypothesized that the 39-kDa band detected by the anti-Thr1381-phosphorylated MEKK1 blot might represent CNN3phosphorylated at Thr288.

To test this hypothesis, we conducted anti-Thr1381-phosphorylated MEKK1 immunoblot analysis of anti-CNN3immunoprecipitates and observed a distinct 39-kDa band

Fig. 1. MEKK1 increases cellular contractility.(A) C2C12 cells infected with retrovirus forexpression of control shRNA (shControl) orshRNA against MEKK1 (shMEKK1) were lysedand immunoblotted for MEKK1, phosphorylatedMLC (pMLC), MLC and β-actin. Theposition of molecular mass markers is indicated.(B) Traction stress exerted by C2C12 cellsinfected with shControl or shMEKK1. Differentialinterference contrast images of cells on 6-kPapolyacrylamide gel substrates are imposed withtraction stress vectors (red arrows). Scale bars:10 µm (black, for size); 1 kPa (red, stress vector).(C) C2C12 cells infected with shControl orshMEKK1 were grown on polyacrylamide gelsubstrates with different rigidities (6, 14 and23 kPa), and their traction stresses weremeasured. Magnitudes of traction stressaveraged over individual cell areas are shown.Each plot represents themean±s.e.m. for 9 cells.*P

(Fig. S3D), suggesting that the anti-Thr1381-phosphorylatedMEKK1 antibody could cross-react to phosphorylated CNN3. Tofurther examine whether Thr288 of CNN3 was phosphorylated incells, we raised a polyclonal antibody against Thr288-phosphorylated CNN3. When we analyzed C2C12 cell lysate byimmunoblotting using this antibody, we detected a distinct 39-kDaband (Fig. 2A). Furthermore, the intensity of the 39-kDa band wasmarkedly decreased in the lysate from CNN3-depleted cells(Fig. 2B). In addition, exogenously expressed FLAG-taggedCNN3 wild-type (FLAG–CNN3 WT), but not its threonine-replaced mutant (FLAG–CNN3 T288A), could be detected byanti-Thr288-phosphorylated CNN3 immunoblotting (Fig. 2C).These results indicate that Thr288 of CNN3 is phosphorylated incells.

CNN3 is phosphorylated by MEKK1Thr1381 is the autophosphorylation site of MEKK1 (Deak et al.,1997; Siow et al., 1997; Chadee et al., 2002). Considering thesimilarity in the sequences surrounding Thr1381 of MEKK1 andThr288 of CNN3, we postulated that Thr288 of CNN3 might bephosphorylated by MEKK1. To test this, we first examined theassociation between MEKK1 and CNN3. FLAG-tagged CNN3(FLAG–CNN3) localized along stress fibers in a punctate fashion(Fig. 3A), where α-actinin1 also localized (Fig. 3B). Consistentwith a previous report showing the colocalization of MEKK1 withα-actinin (Christerson et al., 1999), HA-tagged MEKK1 (HA–MEKK1) colocalized with FLAG–CNN3 at these puncta (Fig. 3C).Interaction betweenMEKK1 and CNN3was further assessed by co-immunoprecipitation experiments, which showed that HA–MEKK1was co-precipitated with CNN3 (Fig. 3D). These results reveal thatMEKK1 forms a complex with CNN3 in cells.We next examined whether modulation of MEKK1 expression

altered CNN3 phosphorylation. When expression of MEKK1 wasdepleted using shRNA, CNN3 phosphorylation was greatlyattenuated (Fig. 3E). By contrast, overexpression of wild-typeMEKK1, but not its kinase-dead mutant (MEKK1 D1369A) (Xuet al., 1996), increased CNN3 phosphorylation (Fig. 3F).Furthermore, when immunopurified HA–MEKK1 was incubated

with a GST-fused recombinant protein of the C-terminal region ofhuman CNN3 (amino acids 243–329; GST–CNN3243-329; Fig. S4)in vitro, GST–CNN3243-329 was phosphorylated in an ATP-dependent manner (Fig. 3G). These results strongly suggest thatThr288 of CNN3 is phosphorylated by MEKK1.

Phosphorylation of CNN3 is involved in traction forcegeneration by cellsWe next examined whether Thr288 phosphorylation of CNN3 wasinvolved in cellular contractility regulated by MEKK1. To testwhether CNN3 phosphorylation modulated cellular contractility, wedepleted endogenous CNN3 expression from C2C12 cells usingshRNA, and introduced either a wild-type (WT) or phospho-defective (T288A) mutant FLAG-tagged shRNA-resistant form ofCNN3 (Fig. 4A), and measured the cell-generated traction stresseson substrates with different rigidities. We found that CNN3-T288A-expressing cells exerted smaller traction stress compared withCNN3-WT-expressing cells on both the 6 kPa and the 24 kPasubstrates (Fig. 4B,C). We speculate that a lack of statisticalsignificance in the difference on the 14 kPa substrate might be dueto the large variability in traction stresses that cells generate on∼15 kPa substrates (Yip et al., 2013). Taken together, these resultsfrom our traction force microscopy analysis indicate that CNN3phosphorylation at Thr288 positively regulates cellular contractility.

The phosphorylation level of MLC was not affected by thephosphorylation status of CNN3 Thr288 (Fig. 4A) in the samemanner as it was not by the expression of MEKK1 (Fig. 1A).Therefore, CNN3 phosphorylation-mediated regulation of cellularcontractility appears to be distinct from modulation of MLCphosphorylation.

CNN3phosphorylation dependsoncytoskeletal integrityandtensionWe then asked whether the MEKK1–CNN3 pathway participates inthe positive-feedback mechanism that regulates cellular contractility(Giannone and Sheetz, 2006; Buxboim et al., 2010; Trichet et al.,2012), and tested whether actomyosin contraction modulatedMEKK1 activity and CNN3 phosphorylation. Although inhibition

Fig. 2. Thr288 of CNN3 isphosphorylated. (A) C2C12 cell lysatewas immunoblotted with anti-Thr288-phosphorylated CNN3 antibody(α-pCNN3) and anti-CNN3 antibody(α-CNN3). (B) C2C12 cells infected withretrovirus for expression of controlshRNA (shControl) or shRNA againstCNN3 (shCNN3) were lysed andimmunoblotted with anti-Thr288-phosphorylated CNN3 (α-pCNN3), anti-CNN3 and anti-β-actin antibodies.(C) C2C12 cells transfected with eitherthe empty vector (vector), FLAG–CNN3WT or FLAG–CNN3 T288A were lysedand immunoblotted with anti-Thr288-phosphorylated CNN3 (α-pCNN3), anti-CNN3 (α-CNN3), anti-FLAG and anti-β-actin antibodies. In anti-Thr288-phosphorylated CNN3 and anti-CNN3blots, bands representing endogenousCNN3 (CNN3) and FLAG-tagged CNN3(FLAG–CNN3) are indicated. Theposition of molecular mass markers isindicated for each blot.

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of myosin II did not apparently affect the activity of MEKK1(Fig. 5A), it did significantly decrease CNN3 phosphorylation(Fig. 5B). Disruption of the actin cytoskeleton (Fig. 5C) andadhesion to softer substrates (Fig. 5D) also attenuated the CNN3phosphorylation. Furthermore, higher cell density, which gives riseto attenuated actin stress fiber formation (Bereiter-Hahn andKajstura, 1988), resulted in lower CNN3 phosphorylation levels(Fig. 5E). These results suggest that CNN3 phosphorylation atThr288 is attenuated under the conditions where development ofactomyosin-based cytoskeletal tension is hampered. By contrast,

sustained equibiaxial stretching (3%, 5 min) of substrates to whichcells adhered (Ursekar et al., 2014) caused an increase in CNN3phosphorylation (Fig. 5F). Collectively, we suggest that Thr288phosphorylation of CNN3 depends upon cytoskeletal tension.Notably, even though the distributions of CNN3 andMEKK1 alongthe stress fibers became less punctate upon myosin II inhibition,their colocalization appeared to be preserved (Fig. 5G). Thissuggests that the formation of the complex between CNN3 andMEKK1 is neither based on actomyosin contractility nor dependenton CNN3 phosphorylation.

Fig. 3. Thr288 of CNN3 is phosphorylated by MEKK1. (A) C2C12 cells co-transfected with F-Tractin–EGFP and FLAG–CNN3 were immunostained for FLAG.Magnified images of the boxed region are shown in the lower panels. (B) C2C12 cells co-transfected with α-actinin–mCherry and FLAG–CNN3 wereimmunostained for FLAG. (C) C2C12 cells co-transfected with HA–MEKK1 and FLAG–CNN3 were immunostained for HA and FLAG. Scale bars: 20 µm(A, upper panels); 10 µm (A, lower panels, B,C). (D) Lysate fromC2C12 cells transfected with HA–MEKK1was subjected to immunoprecipitation (IP) with the anti-CNN3 antibody (α-CNN3) or control rabbit IgG (IgG). The immunoprecipitates were analyzed by immunoblotting for HA and CNN3. (E) C2C12 cells infected withretrovirus for expression of control shRNA (shControl) or shRNA against MEKK1 (shMEKK1) were lysed and immunoblotted for MEKK1, Thr288-phosphorylatedCNN3 (pCNN3), CNN3 and β-actin. (F) C2C12 cells transfected with either the empty vector (vector), HA-tagged wild-type MEKK1 (HA–MEKK1 WT) orHA-tagged kinase-dead MEKK1 (HA–MEKK1 KD) were lysed and immunoblotted for Thr288-phosphorylated CNN3 (pCNN3), CNN3, HA and β-actin.(G) Recombinant GST–CNN3243-329 and immunopurified HA–MEKK1 were mixed in the indicated combinations in the presence or absence of 10 µM ATP. Afterkinase reactions for 30 min at 30°C, the products were analyzed by immunoblotting for Thr288-phosphorylated CNN3 (pCNN3), HA and GST. The position ofmolecular mass markers is indicated for each blot.

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DISCUSSIONActomyosin contraction primarily depends on MLCphosphorylation, which is regulated by Rho to Rho kinase and/ormyosin light chain kinase signaling (Fukata et al., 2001). In additionto this well-documented mechanism, we have revealed in this studythat MEKK1 and CNN3 comprise a new pathway for regulation ofcellular contractility; MEKK1 mediates CNN3 phosphorylation atThr288, which results in an increase in cellular contractility.At present, it is unclear how Thr288 phosphorylation of CNN3

instigates an increase in cellular contractility. However, studies onsmooth muscle CNN1 might provide an insight into theunderlying mechanism. CNN1 bound to actin filamentsdecreases the ATPase activity of MLC-phosphorylated myosin(Abe et al., 1990; Winder and Walsh, 1990). Given that both theactin-binding and the myosin-ATPase-inhibitory regions arehighly conserved between CNN1 and CNN3 (Fig. S1) (Winderet al., 1998), CNN3 might also have a similar inhibitory effect onmyosin ATPase activity. Because Ser175 phosphorylation ofCNN1 by protein kinase C or Ca2+/calmodulin-dependent kinaseII alleviates the inhibitory effect of CNN1 (Winder and Walsh,1990; Itoh et al., 1994; Obara et al., 1996; Tang et al., 1996),Thr288 phosphorylation of CNN3 might also attenuate itsinhibitory effect on the myosin ATPase activity, therebyincreasing the actomyosin contractility.Apart from these expected similarities among calponins, it is

noteworthy that Thr288 resides in the C-terminal tail region ofCNN3, where the sequence is not conserved in other calponinisoforms (Fig. S1). Therefore, the regulatory mechanism ofcellular contractility through Thr288 phosphorylation by MEKK1appears to be specific to CNN3. At present, however, it is unclearhow cytoskeletal contractility promotes MEKK1-dependentphosphorylation of Thr288 in CNN3. Because MEKK1phosphorylation was not decreased by myosin II inhibition(Fig. 5A), it is unlikely that actomyosin contraction enhancesCNN3 phosphorylation by increasing the kinase activity ofMEKK1.Given that the interaction of CNN3 and MEKK1 appears to beindependent of actomyosin contraction (Fig. 5G), the susceptibility

of CNN3 to phosphorylation by MEKK1 might be enhanced bymechanical extension as we previously reported in the case ofphosphorylation of p130Cas (also known as BCAR1) by Src(Sawada et al., 2006). Alternatively, increased cell contractilitymight lead to inactivation of phosphatase(s) responsible fordephosphorylation of CNN3. Although further studies are neededto uncover the mechanism behind the cytoskeletal-tension-dependent regulation of CNN3 phosphorylation, localization of theMEKK1–CNN3 complex on stress fibers designates stress fibers perse as a distinct signaling platform for mechanotransduction (Hirataet al., 2015).

Although CNN3 phosphorylation by MEKK1 increases cellularcontractility, the resulting development of cytoskeletal tension inturn promotes CNN3 phosphorylation, which leads to formation ofa positive-feedback loop concerning the mechanical regulation ofcell functions. In addition to CNN3, CNN2 is also expressed in non-muscle cells (Wu and Jin, 2008). Interestingly, expression of CNN2is reportedly upregulated under conditions in which cytoskeletaltension is higher (Hossain et al., 2005, 2006; Jiang et al., 2014).Given that CNN2 stabilizes actin stress fibers (Hossain et al., 2005),the cytoskeletal-tension-dependent increase in CNN2 expressionmight also participate in a positive feedback type of cellularcontractility regulation. In contrast, CNN3 expression was notaffected by the status of cytoskeletal tension (Fig. 5). Theseobservations suggest that there is a difference between CNN2 andCNN3 in the mechanisms by which they contribute to thecytoskeletal-tension-associated regulation of cellular contractility,and indicate the importance of threonine phosphorylation in the tailregion, which is unique for CNN3.

Cellular contractility is implicated in various fundamentalcell functions including migration, morphogenesis, survival,proliferation and differentiation (Clark et al., 2007). The in vivocontribution of MEKK1 and CNN3 to wound closure (Deng et al.,2006; Daimon et al., 2013), in which actomyosin contraction playsan important role (Shaw and Martin, 2009), might involve theMEKK1-dependent CNN3 phosphorylation that we havedemonstrated in this study.

Fig. 4. CNN3 phosphorylation at Thr288increases cellular contractility. (A) C2C12 cellswere co-infected with retrovirus for the expressionof control shRNA (shControl) or shRNA againstCNN3 (shCNN3), together retrovirus theexpression of either the FLAG–CNN3 WT- orFLAG–CNN3 T288A. The cells were lysed andimmunoblotted for FLAG, CNN3, phosphorylatedMLC (pMLC), MLC and β-actin. The position ofmolecular mass markers is indicated. (B) Tractionstress exerted by C2C12 cells co-infected with theshRNA against CNN3, together with either FLAG–CNN3WT (WT)- or FLAG–CNN3 T288A (T288A).Differential interference contrast images of cellson 6-kPa polyacrylamide gel substrates areimposed with traction stress vectors (red arrows).Scale bars: 30 µm (black, for size); 3 kPa (red,stress vector). (C) C2C12 cells co-infected withshRNA against CNN3, together with either FLAG–CNN3 WT (WT) or FLAG–CNN3 T288A (T288A)were grown on polyacrylamide gel substrates withdifferent rigidities (6, 14 and 23 kPa), and theirtraction stresses were measured. Magnitudes oftraction stress averaged over individual cell areasare shown. Each plot represents the mean±s.e.m.for ≥9 cells. *P

MATERIALS AND METHODSCell culture, transfection and retroviral infectionC2C12, NIH3T3 and 293T cells were maintained in Dulbecco’smodified Eagle’s medium (Life Technologies, Carlsbad, CA)supplemented with 10% fetal bovine serum (Life Technologies) at37°C in 5% CO2. For transient transfection of cells with plasmids, theLipofectamine 2000 transfection reagent (Life Technologies) was usedaccording to the manufacturer’s instruction. Retroviral infection wasconducted as described previously (Kawauchi et al., 2008). Infectedcells were selected with 4 µg/ml puromycin and/or 1000 µg/mlhygromycin.

PlasmidspcDNA3-HA-mouse MEKK1 was a gift from Isao Naguro (University ofTokyo, Tokyo, Japan). Human CNN3 cDNA was a gift from YoshinaoWada and Yukinao Shibukawa (Osaka Medical Center and ResearchInstitute for Maternal and Child Health, Osaka, Japan). pcDNA3-α-actinin-mCherry was a gift from Hiroaki Machiyama (National University ofSingapore, Singapore). The F-Tractin–EGFP construct (Johnson and Schell,2009) was a gift from Michael J. Schell (Uniformed Services University,Bethesda, MD). The FLAG and human CNN3 sequences were subclonedinto the pBabe.hygro vector. Site-directed mutants of MEKK1 (D1369A)and CNN3 (T288A and shRNA-resistant mutants) were generated by the

Fig. 5. CNN3 phosphorylation at Thr288 depends on cytoskeletal tension. (A) NIH3T3 cells transfected with HA–MEKK1were treated with DMSO (control) or100 µM blebbistatin (Blebb) for 30 min. The cells were lysed and immunoblotted for Thr1381-phosphorylated MEKK1 (pMEKK1), MEKK1, HA and β-actin.(B,C) Top panels, NIH3T3 cells treated with DMSO (control), 100 µM blebbistatin (Blebb) or 1 µM cytochalasin D (CytoD) for 30 min were lysed andimmunoblotted for Thr288-phosphorylatedCNN3 (pCNN3) andCNN3. Bottom panels, quantification of the densitometric ratio of pCNN3 against CNN3. Each barrepresents the mean±s.d. (n=3). *P

QuikChange mutagenesis method (Agilent Technologies, Santa Clara, CA).For shRNA-mediated depletion of protein expression, the target sequencewas inserted into the pSUPER.retro.puro vector (Oligoengine, Seattle, WA).The target sequences used were: 5′-GGAACCGGTGCAGGAGAGT-3′ formouse MEKK1 (Map3k1), and 5′-GTATGCAGAAAAACAAACA-3′ formouse CNN3 (Cnn3). For bacterial expression of GST–CNN3243-329, theamino acid 243–329 region of CNN3 was amplified by PCR and subclonedinto the pGEX-5X-2 vector.

Antibodies and inhibitorsRabbit polyclonal antibody (pAb) against Thr288-phosphorylated CNN3was raised against the phospho-peptide NGSQG(pT)GTNGS (Abmart,Shanghai, China). The rabbit pAb against CNN3 (cat. no. sc-28546) andcontrol rabbit IgG (cat. no. sc-2027) were purchased from Santa CruzBiotechnology (Dallas, TX). The rabbit pAb against Thr1400-phosphorylated human MEKK1 (cat. no. PAB0513) was from Abnova(Taipei, Taiwan). The rabbit pAb against MEKK1 (cat. no. A302-396A) wasfrom Bethyl Laboratories (Montgomery, TX). The mouse monoclonalantibodies (mAbs) against β-actin (cat. no. A5441) and FLAG (cat. no.F1804) were from Sigma-Aldrich (St. Louis, MO). The rabbit pAbs againstSer19-phosphorylated myosin light chain 2 (cat. no. 3671) and total myosinlight chain 2 (cat. no. 3672) were from Cell Signaling Technology (Danvers,MA). The rat mAb against HA (cat. no. 11867423001) was from Roche(Basel, Switzerland). The rabbit pAb against GST (cat. no. PM013) was fromMedical & Biological Laboratories (Nagoya, Japan). Horseradish peroxidase(HRP)-conjugated anti-mouse and -rabbit-IgG antibodies were from GEHealthcare (Little Chalfont, UK). Alexa-Fluor-488-conjugated goat anti-mouse-IgG, Alexa-Fluor-488-conjugated goat anti-rat-IgG and Alexa-Fluor-546-conjugated goat anti-mouse-IgG antibodies were from LifeTechnologies. Blebbistatin and cytochalasin D were from TorontoResearch Chemicals (North York, Canada) and Sigma-Aldrich, respectively.

ImmunoblottingCells were lysed with 2× lithium dodecyl sulfate sample buffer (LifeTechnologies) containing 2.5% β-mercaptoethanol. The lysate samples wereresolved by SDS-PAGE (4-12% Bis-Tris gel; Life Technologies),transferred onto nitrocellulose membranes (Merck Millipore, Billerica,MA), and probed with antibodies. Immunoreactive bands were detectedwith SuperSignalWest Pico or Femto Chemiluminescent Substrate (ThermoFisher Scientific, Rockford, IL). Antibodies were diluted to 1:10,000 inTris-buffered saline containing 0.1% Tween 20 and 1% skimmed milk.

ImmunoprecipitationPrimary antibodies and control rabbit IgG (50 μg/ml) were covalentlycoupled to protein-G-conjugated magnetic beads (Life Technologies) with20 mM dimethyl pimelimidate•2 HCl (DMP; Thermo Fisher Scientific)according to the manufacturer’s instructions. Cells were lysed with lysisbuffer (1% NP-40, 150 mM NaCl, 20 mM Tris, 1 mM EDTA, pH 8.0)supplemented with protease inhibitors (Roche) and phosphatase inhibitors(Sigma-Aldrich). The cell lysates were incubated for 30 min on ice and thencentrifuged for 40 min at 20,000 g. The protein concentration in thesupernatants was measured with the bicinchoninic acid (BCA) method(Thermo Fisher Scientific), and the concentration was equalized amongsamples by adding lysis buffer. The concentration-adjusted supernatants wereincubatedwith antibody-coupledmagnetic beads overnight at 4°C. The beadswere washed three times with lysis buffer, and, then, the precipitated proteinswere eluted with 2× lithium dodecyl sulfate sample buffer containing 2.5% β-mercaptoethanol. Immunoprecipitation samples were resolved by SDS-PAGE and then subjected to immunoblot or mass spectrometric analysis.

Mass spectrometric analysisSDS-PAGE-resolved immunoprecipitates were visualized by negativestaining (Wako Pure Chemical Industries, Osaka, Japan), and the gelpiece containing the 39-kDa protein band was excised. The gel piece waswashed with 50% acetonitrile, reduced with 10 mM dithiothreitol for45 min at 56°C, alkylated with 50 mM iodoacetamide for 30 min at roomtemperature, and digested with 5 ng/µl trypsin (Promega, Madison, WI)

overnight at 37°C. Digested peptides were extracted by 50% acetonitrileand 0.1% formic acid, vacuum dried, and analyzed by nano-liquidchromatography tandem mass spectrometry (nanoLC-MS/MS) using aTripleTOF 5600 mass spectrometry system (AB SCIEX, Foster City, CA)on-line coupled with an in-house packed ReproSil-Pur C18-AQ columndriven by an Ultimate 3000 RSLCnano system (Thermo Fisher Scientific) aspreviously described (Iwasaki et al., 2012).

Peptide identification was performed by Mascot version 2.3.01 (MatrixScience, Boston, MA) against the SwissProt database (version 2011_06)containing 16,376 mouse protein sequences. Peptides were considered to beidentified using the criteria as previously described (Iwasaki et al., 2012).

Preparation of polyacrylamide gel substratesPolyacrylamide gel substrates, to which fibronectin (Sigma-Aldrich) wasconjugated using sulfo-SANPAH (Thermo Fisher Scientific), were preparedas described previously (Dembo andWang, 1999). The Young’s modulus ofthe gels was measured by an AFM indentation assay (Vedula et al., 2014).For traction force microscopy, we used polyacrylamide gels co-polymerizedwith N-acryloyl-6-aminocaproic acid (ACA; Tokyo Chemical Industry,Tokyo, Japan), in which fluorescent beads (0.2 μm; Polysciences,Warrington, PA) were embedded. The ACA-copolymerized gels withYoung’s moduli of 6, 14 and 23 kPa were prepared on glass-bottom dishes,as described previously (Yip et al., 2013). Collagen type I (Koken, Tokyo,Japan) was covalently conjugated to the gel surface through ACA (Yip et al.,2013).

Traction force microscopyCells were grown overnight on collagen-conjugated gel substrates inHEPES-buffered Dulbecco’s modified Eagle’s medium (Life Technologies)supplemented with 10% fetal bovine serum. Cells and fluorescent beadsembedded in gels were observed with a spinning-disk confocal microscopysystem (UltraVIEW VoX; PerkinElmer, Waltham, MA) equipped with aninverted microscope (IX81; Olympus, Tokyo, Japan), a 60× waterimmersion objective (NA 1.2, UPlanSApo; Olympus) and an electronmultiplying charge-coupled device camera (C9100-13; HamamatsuPhotonics, Hamamatsu, Japan) at 37°C. Differential interference contrastimages of cells as well as fluorescence images of embedded beads wereacquired before and after detaching the cells from the gel substrate bytreatment with trypsin and EDTA.

From the bead images before and after cell detachment, the entiredisplacement field in the gel substrate was calculated using MATLABsoftware (MathWorks, Natick, MA). The traction stress field was thenobtained by solving the inverse Boussinesq problem as described previously(Yip et al., 2013).

In vitro kinase assayThe GST–CNN3243-329 protein was expressed in Escherichia coli BL21cells. The cells were lysed with 1% Triton X-100, 1.9 mg/ml lysozyme,9 mM dithiothreitol, 20 mM Tris-HCl, 1 mM EDTA, 150 mM NaCl andprotease inhibitors (pH 8.0). The soluble fraction of the bacterial lysate wasapplied to GST SpinTrap columns (GE Healthcare). After repeated washingwith PBS, the purified recombinant protein was eluted with 20 mM reducedL-glutathione (Sigma-Aldrich) and 50 mM Tris-HCl (pH 8.0).

HA–MEKK1 expressed in NIH3T3 cells was immunoprecipitated withanti-HA-antibody-coupled magnetic beads. As a control, the lysate ofNIH3T3 cells transfected with the empty pcDNA3 vector was incubatedwith the anti-HA-antibody-coupled magnetic beads. After washing withwashing buffer (20 mMTris-HCl pH 8.0, 1 mMEDTA, 150 mMNaCl), theHA–MEKK1–magnetic-bead complexes were incubated with purifiedGST–CNN3243-329 in 25 mM HEPES, 50 mM NaCl, 5 mM MgCl2,0.5 mM dithiothreitol, protease inhibitors and phosphatase inhibitors (pH7.4) either in the presence or absence of 10 µMATP for 30 min at 30°C. Theproducts were analyzed by immunoblotting.

Stretching cellsCells were equibiaxially stretched using the device reported elsewhere(Ursekar et al., 2014). In brief, cells were grown overnight on

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polydimethylsiloxane (PDMS) stretch chambers coated with 10 µg/mlcollagen type I. After treatment of the cells with 100 µM blebbistatin for30 min at 37°C, the chambers were then equibiaxially stretched by 3% for5 min in the presence of blebbistatin. The cells were analyzed byimmunoblotting.

ImmunofluorescenceCells were fixed and permeabilized for 30 min with 4% formaldehyde and0.2% Triton X-100 in cytoskeleton-stabilizing buffer (137 mMNaCl, 5 mMKCl, 1.1 mM Na2HPO4, 0.4 mM KH2PO4, 4 mM NaHCO3, 2 mM MgCl2,5.5 mM glucose, 2 mM EGTA, and 5 mM PIPES, pH 6.1) (Hirata et al.,2008). This was followed by blockingwith 1%bovine serum albumin (BSA)in cytoskeleton-stabilizing buffer for 30 min. The cells were then incubatedwith primary antibodies for 40 min, washed and further incubated withsecondary antibodies for 40 min. Both primary and secondary antibodieswere diluted to 1:100 in the cytoskeleton-stabilizing buffer containing 1%BSA. The stained cells were observed with an epi-fluorescence invertedmicroscope (IX81, OLYMPUS, Tokyo, Japan) equipped with an oilimmersion objective (NA 1.45, 100×; PlanApo, OLYMPUS) and acharge-coupled device camera (CoolSNAP EZ, Photometrics, Tucson,AZ). Metamorph software (Molecular Devices, Sunnyvale, CA) was usedfor image acquisition.

Statistical analysisStatistical analyses were performed using Student’s two-tailed t-test (eitherpaired or unpaired).

AcknowledgementsWe thank Isao Naguro, Yoshinao Wada, Yukinao Shibukawa, Hiroaki Machiyamaand Michael J. Schell for kind gifts of plasmids.

Competing interestsThe authors declare no competing or financial interests.

Author contributionsH.H. and Y.S. designed the research. H.H., W.-C.K., C.P.U., A.K.G., A.R. andS.R.K.V. performed the experiments. K.K., I.H., Y.I. and C.T.L. provided technicaladvice on the experiments. H.H., A.K.Y. and K.-H.C. analyzed the data. H.H., Y.S.and M.S. wrote the manuscript. C.T.L., Y.S. and M.S. supervised the project.

FundingThis work was supported by the National Research Foundation Singapore, under itsResearch Centre of Excellence, the Mechanobiology Institute.

Supplementary informationSupplementary information available online athttp://jcs.biologists.org/lookup/doi/10.1242/jcs.189415.supplemental

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