Integrin a3b1-dependent Activation of FAK/Src Regulates Rac1-Mediated Keratinocyte Polarization on Laminin-5

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1 ORIGINAL ARTICLE Integrin 31-Dependent Activtion of FAK/Src Regultes Rc1-Medited Kertinocyte Polriztion on Lminin-5 Dvid P. Chom 1, Vincenzo Milno 1, Kevin M. Pumigli 1 nd C. Michel DiPersio 1 Upon epiderml wounding, kertinocytes t the wound edge ecome ctivted, deposit newly synthesized lminin-5 into the extrcellulr mtrix, nd migrte into the wound ed. The interction etween integrin 31 nd lminin-5 is essentil for estlishment of stle, leding lmellipodium nd persistent kertinocyte migrtion. We previously showed tht integrin 31 ctivtes the Rho fmily GTPse Rc1 nd regultes Rc1- dependent formtion of polrized, leding lmellipodi in migrting kertinocytes. In the present study, we explored the role of focl dhesion kinse (FAK) nd src signling in this process. We show tht overexpression of the FAK inhiitor FAK-relted non-kinse or of the FAK(Y397F) uto-phosphoryltion mutnt, induced norml, non-polrized spreding of kertinocytes on lminin-5. Integrin 31 ws required for full FAK utophosphoryltion t Y397, nd susequent src kinse-dependent phosphoryltion of FAK t residues Y861 nd Y925, sites responsile for promoting signl trnsduction downstrem of FAK, indicting tht 31 regultes the coordintion of FAK/src signl trnsduction. Inhiiting either src kinse ctivity or FAK signling interfered with 31-medited Rc1 ctivtion nd polrized cell spreding. These findings revel novel pthwy in migrtory kertinocytes wherein 31 lminin-5 interctions regulte src kinse signling through FAK, promoting Rc1 ctivtion nd polrized lmellipodium extension. Journl of Investigtive Dermtology (2007) 127, doi: /sj.jid ; pulished online 17 August 2006 INTRODUCTION During cutneous wound heling, wound edge epiderml kertinocytes ecome highly motile, depositing newly synthesized lminin-5/lminin-322 (LN-5) onto the extrcellulr mtrix (ECM) s they migrte into the wound (Nguyen et l., 2000). Similrly, cultured kertinocytes t the edge of scrpe-wounded monolyer or epiderml outgrowth show chrcteristic phenotypes of ctivted wound edge cells, including new LN-5 synthesis, enhnced gp junction communiction, nd polrized protrusion of lmellipodi (Lmpe et l., 1998; Nguyen et l., 2000; Chom et l., 2004). Integrins re the mjor receptors for cell dhesion to ECM, nd they ply criticl roles in cytoskeletl dynmics, cell polriztion, nd cell migrtion (Hynes, 2002). Studies with integrin knockout mice hve reveled importnt roles 1 Center for Cell Biology nd Cncer Reserch, Alny Medicl College, Alny, New York, USA Correspondence: Dr C. Michel DiPersio, Center for Cell Biology nd Cncer Reserch, Alny Medicl College, Mil Code 165, Room MS-326, 47 New Scotlnd Avenue, Alny, NewYork , USA. E-mil: dipersm@mil.mc.edu Arevitions: ECM, extrcellulr mtrix; ERK, extrcellulr signl-regulted kinse; FAK, focl dhesion kinse; FRNK, FAK-relted non-kinse; GFP, green fluorescent protein; LN-5, lminin-5 ( 332); MK, mouse kertinocyte; PP2, 4-mino-5-(4-chlorophenyl)-7-(t-utyl)pyrzolo[3,4-d]pyrimidine Received 20 Ferury 2006; revised 15 My 2006; ccepted 21 June 2006; pulished online 17 August 2006 for 1 integrins in polriztion nd migrtion of epiderml kertinocytes in vivo nd in vitro (Grose et l., 2002; Rghvn et l., 2003; Chom et l., 2004). Interctions etween integrin 31 nd LN-5 ply prticulrly prominent role in estlishment of stle, leding lmellipodium nd initition of persistent cell migrtion tht is chrcteristic of ctivted kertinocytes (Chom et l., 2004; Frnk nd Crter, 2004). However, signling pthwys tht link integrin-medited dhesion to kertinocyte polriztion re uncler. Kertinocytes normlly migrte s contiguous sheet within which cell cell dhesions re mintined y cdherins. However, individully migrting kertinocytes disply gene expression profiles nd polrized morphology tht re chrcteristic of ctivted cells t wound edge, indicting tht cell ECM dhesions re sufficient for mny spects of the ctivted phenotype (Chom et l., 2004; Frnk nd Crter, 2004; Hrper et l., 2005). Thus, nlysis of individully migrting kertinocytes provides powerful experimentl system for dissecting integrin-signling pthwys tht regulte epithelil cell polriztion nd formtion of stle leding lmellipodi. The polrized morphology of migrting cell is estlished nd mintined y intrcellulr signling events tht control lmellipodium extension t the leding edge nd cell retrction t the triling edge (reviewed y Ridley et l., 2003). Directionl migrtion is mintined through loclized chnges in integrin-signling complexes tht effect differentil & 2006 The Society for Investigtive Dermtology 31

2 regultion of Rho fmily GTPses nd cytoskeletl remodeling long the nterior posterior xis of the cell. Restriction of Rc ctivtion to the leding edge of migrting cell promotes stle formtion of leding lmellipodium (Noes nd Hll, 1999; Ridley et l., 2003). For exmple, the locliztion of n 4 integrin pxillin Arf-GAP complex restricts Rc ctivtion to the leding edge in some migrting cells (Nishiy et l., 2005). However, this mechnism involves direct pxillin integrin interction tht is specific to 4 integrins (Liu et l., 1999), which re restricted to certin cell types. Therefore, there re likely to e distinct mechnisms in kertinocytes wherey integrins ctivte Rc1. Focl dhesion kinse (FAK) regultes cytoskeletl reorgniztion in response to mny integrins (Cry nd Gun, 1999). Studies of FAK-deficient cells hve estlished role for FAK in lmellipodium formtion, focl dhesion turnover, nd cell migrtion (Ilic et l., 1995; Tilghmn et l., 2005). Furthermore, FAK is overexpressed nd highly ctivted in invsive tumor cells (Agochiy et l., 1999), nd epidermis-specific knockout of FAK suppresses tumor formtion nd progression in vivo (McLen et l., 2004). FAK functions s n ctivtle scffolding pltform for the inding of signling nd dptor proteins (Schlepfer nd Mitr, 2004). For exmple, upon integrin-medited dhesion FAK is uto-phosphorylted t Y397, which serves s inding site for the SH2 domin of src fmily kinses. Once recruited to FAK, src kinses phosphorylte other tyrosine residues on FAK, some of which serve s inding sites for signling or dptor molecules tht ctivte downstrem signling pthwys (Schlepfer nd Mitr, 2004). Among these residues, Y861 is thought to enhnce uto-phosphoryltion of FAK (Leu nd M, 2002), nd Y925 leds to extrcellulr signlregulted kinse 1/2 (ERK1/2) ctivtion through inding of the Gr2 dptor protein (Schlepfer et l., 1994). FAK lso contins severl conserved motifs tht ind other dptor proteins, such s p130 cs (Cry nd Gun, 1999). Using kertinocytes derived from integrin 3-null mice, we recently demonstrted tht 31 is required for stiliztion of initil protrusions into polrized lmellipodi, nd tht this polriztion is pre-requisite for persistent cell migrtion (Chom et l., 2004). We lso identified requirement for Rc1 in 31-dependent kertinocyte polriztion (Chom et l., 2004). In the current study, we identify novel role for 31 in the ctivtion of FAK/src signling tht promotes Rc1-medited polriztion of kertinocytes on LN-5. Although dhesion-dependent src kinse ctivtion occurred in the sence of 31, src-dependent phosphoryltion of FAK residues Y861 nd Y925 required 31, indicting tht 31 enles ctivted src kinses to signl through FAK. Furthermore, FAK/src signling ws required for full Rc1 ctivtion nd polrized kertinocyte spreding, linking 31-medited ctivtion of FAK/src to Rc1-dependent formtion of leding lmellipodium. 31-dependent ctivtion of ERK1/2, which we previously linked to kertinocyte survivl (Mnohr et l., 2004), lso required src signling; however, ERK signling ws not required for cell polriztion. These findings support model wherein interction of 31 with LN-5 promotes FAK/src signling, leding to ctivtion of divergent pthwys tht regulte distinct functions in epiderml kertinocytes. RESULTS Inhiition of FAK leds to norml kertinocyte spreding Three kertinocyte cell lines were used in this work: mouse kertinocyte (MK) þ / þ cells were derived from wild-type mouse, MK / cells were derived from n 3 integrin-null mouse, nd MK3 cells re MK / cells trnsfected stly with humn 3 (DiPersio et l., 2000). To test the effects of inhiiting FAK on kertinocyte spreding, we infected MK3 cells with denovirus encoding green fluorescent protein (GFP)-tgged FAK-relted non-kinse (FRNK), which interferes with endogenous FAK function (Heidkmp et l., 2002). In ddition, we tested GFP-tgged FAK mutnt, FA- K(Y397F), which is mutted t the Y397 uto-phosphoryltion site nd inhiits lmellipodium extension nd migrtion in other cells (Zho et l., 1998; Owen et l., 1999). Expression of GFP-tgged wild-type FAK (GFP-FAK(wt)) served s control. GFP-tgged proteins were expressed t 5- to 10-fold higher levels thn endogenous FAK protein, s determined y quntittive immunolot with n ntiody ginst the C-terminus of FAK tht recognizes endogenous FAK nd ech fusion protein (Figure 1). These were the lowest expression levels t which we were still le to detect reduced phosphoryltion of endogenous FAK Y397. As expected, expression of GFP-FAK(wt) incresed phosphoryltion of endogenous FAK Y397 nd ws itself phosphorylted t Y397, wheres neither GFP-FRNK nor GFP-FAK(Y397F) ws detected y nti-py397, s they ech lck this residue (Figure 1). Prllel cultures of uninfected or infected MK3 cells were plted onto lminin-5 ( 332) (LN-5) ECM nd cell spreding ws compred (Figure 1 f). Adenovirl infection efficiency ws greter thn 90% for ll constructs, nd t lest B80% of cells were well spred for ech condition, llowing us to ssess the individul effects of GFP-fusion proteins on spreding morphology. Among spred cells, pproximtely 27% of uninfected cells ssumed well-polrized phenotype with lrge, leding-edge lmellipodium tht is typicl of migrting kertinocytes (Figure 1, left grph; exmple shown in Figure 1c, g). Compred with uninfected cells, MK3 cells infected with GFP-FRNK or GFP-FAK(Y397F) showed sustntil increse in the proportion of cells with n errnt spreding phenotype chrcterized y multiple, rndomly oriented protrusions (Figure 1, right grph; exmples shown in Figures 1e, i nd f, j). This increse in normlly spred cells ws ccompnied y reduced numer of polrized cells. Anormlly spred cells often displyed prominent ctin fiers or undles (i.e. Figure 1e), possily reflecting enhnced cellulr contrctility s reported for FAK-null firolsts (Chen et l., 2002). Importntly, norml spreding ws not non-specific effect of denovirl infection or GFP-fusion protein expression, s it occurred in only 5 7% of uninfected cells or cells infected with GFP-FAK(wt) (Figure 1, right grph). GFP-tgged FAK, FRNK, nd FAK(Y397F) ech coloclized with integrin 31 in lmellipodil extensions (Figure 1k m), consistent with 32 Journl of Investigtive Dermtology (2007), Volume 127

3 their known ilities to loclize to focl dhesions (Owen et l., 1999; Heidkmp et l., 2002; Tilghmn et l., 2005). These results indicte tht inhiiting FAK promotes norml kertinocyte spreding on LN-5. Src kinse-dependent phosphoryltion of FAK is regulted y 31 Auto-phosphoryltion of FAK Y397 is regulted y integrinmedited dhesion (Cry nd Gun, 1999). Accordingly, phosphoryltion of FAK Y397 in 31-expressing MK þ / þ or MK3 cells ws completely dhesion-dependent (Figure 2). Phosphoryltion of FAK Y397 ws reduced considerly in 31-deficient MK / cells on either LN-5 ECM or purified LN-5 (Figure 2), consistent with role for 31 in FAK ctivtion. It is possile tht the sl levels of dhesiondependent FAK ctivtion in MK / cells ws medited y integrin 64, s dhesion of these cells to LN-5 ECM is completely dependent on 64 (Mnohr et l., 2004), nd t lest one study hs implicted 64 in FAK ctivtion (Adel- Ghny et l., 2002). Alterntively, low levels of FAK ctivtion my occur in response to rpidly deposited fironectin or other endogenous ECM protein, s MK cells lso express severl other integrins (DiPersio et l., 2000). The SH2 domin of certin src fmily kinses inds to phosphorylted Y397 of FAK, llowing src to phosphorylte Y861 nd Y925 on FAK nd crete inding sites for other signling nd dptor proteins (Cry nd Gun, 1999; Schlepfer nd Mitr, 2004). Phosphoryltion of FAK t oth Y861 nd Y925 ws dhesion dependent in 31-expressing MK cells (Figure 2 nd c). In contrst, phosphoryltion of Y861 nd Y925 ws reduced in MK / cells dhered to LN-5 ECM or purified LN-5 (Figure 2 nd c), demonstrting tht phosphoryltion of these src kinse-dependent sites is regulted y 31. A previous study reported tht phosphoryltion of FAK Y861 occurs independently of integrin-medited cell dhesion in highly metsttic prostte crcinom cells, wheres poorly tumorigenic cells showed dhesiondependent Y861 phosphoryltion (Slck et l., 2001). We hve determined tht the MK cell lines used in the current study re not tumorigenic (J. Lmr nd C.M. DiPersio, unpulished), consistent with the dhesion-dependent Y861 phosphoryltion tht we oserved (Figure 2). MK þ / þ or MK3 cells treted with 4-mino-5-(4-chlorophenyl)-7-(t-utyl)pyrzolo[3,4-d]pyrimidine (PP2) showed complete inhiition of phosphoryltion t oth Y861 nd Y925 of FAK (Figure 3 nd, nd dt not shown), indicting tht src kinse ctivity ws required for phosphoryltion t these sites. PP2 potently inhiited phosphoryltion of the stimultory uto-phosphoryltion site of src, Y418, confirming inhiition of src ctivtion (Figure 3d). In contrst, PP2 hd little effect on Y529 phosphoryltion (Figure 3d), s this src residue is phosphorylted y cellulr src kinse. Only slight inhiition of FAK Y397 phosphoryltion ws evident in PP2 treted cells when normlized to totl FAK (Figure 3c), perhps reflecting role for src kinses in enhncing sl levels of FAK uto-phosphor- Totl FAK FAK 397 K14 Uninfected FAK(wt) FRNK FAK(Y397F) GFP-FAK/GFP-FAK(Y397F) Endogenous FAK GFP-FRNK GFP-FAK Endogenous FAK % cell with polrized morphology Uninfected FAK(wt) FRNK FAK(Y397F) % cell with norml morphology Uninfected FAK(wt) * * FRNK FAK(Y397F) Figure 1. Inhiition of FAK results in norml kertinocyte spreding. MK3 cells were infected for 48 hours with denovirus expressing GFP-tgged wild-type FAK (FAK(wt)), FRNK, or FAK(Y397F), then plted on LN-5 ECM under serum-free conditions for 90 minutes. Identicl prllel cultures were () lysed to ssess expression of GFP-fusion proteins, or (, c f) fixed nd stined for F-ctin to ssess cell morphology. () Uninfected or infected MK3 cells were immunolotted with n ntiody tht recognizes endogenous FAK nd ech GFP-tgged fusion protein, s indicted (totl FAK); identities of GFP-tgged proteins were confirmed y immunolot with nti-gfp (dt not shown). GFP-FAK(wt) levels were B10-fold higher, nd GFP-FRNK or GFP-FAK(Y397F) levels were B5-fold higher, thn endogenous FAK levels. Phosphoryltion of FAK Y397 ws ssessed y immunolot with nti-py397. Equl loding ws confirmed y immunolot with nti-kertin 14 (K14). () Grphs show the percentge of spred cells tht were well polrized (left grph) or normlly spred (right grph). Results represent mens þ SEM, n ¼ 4; *Po0.05 compred with uninfected control (one-wy nlysis of vrince with Dunnett post-hoc test). (c j) Corresponding imges of (c f) tetrmethyl rhodmine isothiocynte-phlloidin/4-6-dimidino-2-phenylindole, (g) phse contrst, or (h j) GFP/4-6-dimidino-2-phenylindole re shown for (c, g) uninfected cells or for cells expressing (d, h) GFP-FAK(wt), (e, i) GFP-FRNK, or (f, j) GFP-FAK(Y397F). Arrows in (c, g) nd (d, h) indicte leding lmellipodi of polrized cells; rrowheds in (e, i) nd (f, j) indicte rndomly oriented protrusions in normlly spred cells. Br ¼ 10 mm. (k m) Deconvolved imges of 3 immunofluorescence (3 column), GFP fluorescence (GFP column), merged fluorescence (3/GFP column), or phse contrst (phse column) re shown for MK3 cells infected with (k) GFP-FAK(wt), (l) GFP-FRNK, or (m) GFP-FAK(Y397F). Arrows point to exmples of regions where 31 coloclized with GFP-fusion protein. Br ¼ 10 mm. (Fig 1 continued on following pge) 33

4 Uninfected GFP-FAK(wt) GFP-FRNK GFP-FAK(Y397F) c d e f g h i j k α3 GFP α3/gfp Phse FAK(wt) l FRNK m FAK(Y397F) Figure 1. Continued yltion (Cry et l., 2002; Leu nd M, 2002). However, this difference ws not sttisticlly significnt in three independent experiments. Src kinse ctivity is required for kertinocyte spreding on LN-5 To test the effect of inhiiting src kinses on kertinocyte spreding, cells were incuted with PP2 efore plting onto LN-5 ECM-coted coverslips. PP2 potently inhiited postttchment spreding of oth MK þ / þ nd MK3 cells (Figure 4). This inhiition my not e ccounted for solely y crippling of FAK/src signling, s src kinse ctivity is likely required for spects of cell spreding tht do not involve FAK (Frme, 2004). Importntly, however, FAK Y397 phosphoryltion ws lrgely preserved in PP2-treted MK þ / þ or MK3 cells (Figure 3c, nd dt not shown); thus, the results in Figure 4 indicte tht 31-medited FAK ctivtion is not sufficient to cuse kertinocyte spreding nd tht src kinse ctivity is lso necessry. These findings re consistent with studies of src/yes/fyn-null firolsts, in which restoring expression of wild-type src, ut not ctlyticlly inctive src, restored cell spreding (Cry et l., 2002). Results in Figures 3c nd 4 further show tht src-dependent cell spreding is not required for FAK Y397 phosphoryltion, indicting tht 31-medited ctivtion of FAK (Figure 2) precedes cell spreding. The ltter finding is consistent with previous report tht 31 inding to solule LN-5 cn enhnce FAK Y397 phosphoryltion in the sence of cell spreding (Shng et l., 2001). Tken together, results in Figures 1 4 suggest tht 31-LN-5 interctions induce coordinted FAK/src signling tht permits polrized kertinocyte spreding. Adhesion-medited stimultion of src kinse ctivity occurs independently of 31 in kertinocytes on LN-5 The crucil role of src kinses in regulting the consequences of FAK uto-phosphoryltion rises the question of whether dhesion regultes src kinse ctivity in MK cells. Tyrosine 34 Journl of Investigtive Dermtology (2007), Volume 127

5 Suspension LN-5 ECM Purified LN-5 MK +/+ MK / MK +/+ MK / MK / Phse Phlloidin FAK 397 Totl FAK DMSO FAK 861 Suspension LN-5 ECM Purified LN-5 MK +/+ MK / MK +/+ MK / MK / MK +/+ Totl FAK c FAK 925 Totl FAK Suspension LN-5 ECM Purified LN-5 MK +/+ MK / MK +/+ MK / MK / Figure 2. FAK phosphoryltion t Y397, Y861, nd Y925 is 31-dependent in kertinocytes dhered to LN-5. MK þ / þ,mk /,ormk3 cells were either kept in suspension or plted on LN-5 ECM or purified LN-5 (20 mg/ml) for 30 minutes. Immunolot ws performed with ntiodies specific for FAK phosphorylted t () Y397, () Y861, or (c) Y925, or with ntiody ginst totl FAK. Experiments with purified LN-5 were performed in duplicte; other results re representtive of t lest four experiments. PP2 DMSO PP2 DMSO PP2 FAK 861 FAK 925 Totl FAK c FAK 397 Totl FAK DMSO PP2 Totl FAK src 529 Totl src DMSO PP2 DMSO PP2 src 418 Figure Dependent phosphoryltion of Y861 nd Y925 of FAK requires src kinse ctivity. MK3 cells were plted on LN-5 ECM in the presence of DMSO or PP2 (5 mm) for 30minutes. ( c) FAK phosphoryltion t () Y861, () Y925, or (c) Y397 ws ssessed s in Figure 2. (d) The ility of PP2 to interfere with src kinse ctivity ws confirmed y immunolot for phospho-y418, phospho-y529, nd totl src protein. Results re representtive of t lest three experiments performed in MK3 nd MK þ / þ cells. phosphoryltion t Y418 correltes with incresed src kinse ctivity, wheres tyrosine phosphoryltion t Y529 cn negtively regulte src kinse ctivity (Schwrtzerg, 1998). MK cells showed enhnced Y418 phosphoryltion when dhered to LN-5 ECM or purified LN-5, compred with cells in suspension (Figure 5). This dhesion-dependent increse in Y418 phosphoryltion ws independent of 31 (Figure 5). Phosphoryltion of src Y529 ppered constitutive, showing neither dhesion-dependence nor 31-dependence when normlized to totl src (Figure 5). Thus, lthough 31 ws not required for src kinse ctivtion, it ws required for src-dependent phosphoryltion of specific residues of FAK (Figure 3), indicting tht 31 regultes the ility of ctivted src kinses to signl through FAK. These dt lso indicte tht src ctivtion precedes 31-dependent MK cell d Figure 4. Inhiition of src kinse prevents kertinocyte spreding on LN-5. () MK þ / þ cells or () MK3 cells were plted on LN-5 ECM-coted coverslips in the presence of DMSO or PP2 (5 mm) for 30 minutes. Cells were fixed nd F-ctin ws stined with tetrmethyl rhodmine isothiocynteleled phlloidin; corresponding phse imges re shown. Results re representtive of four seprte experiments in MK þ / þ or MK3 cells. Br ¼ 10 mm. scr 418 Totl scr Suspension LN-5 ECM Purified LN-5 MK +/+ MK / MK +/+ MK / MK +/+ MK / Suspension LN-5 ECM MK +/+ MK / MK +/+ MK / scr 529 Totl scr Figure 5. Adhesion to LN-5 increses levels of ctive src kinse independently of 31. MK þ / þ,mk /,ormk3 cells were either kept in suspension, or plted on LN-5 ECM or purified LN-5 (20 mg/ml) for 30 minutes, s indicted. Immunolot ws performed with ntiodies specific for () phospho-src Y418, () phospho-src Y529, or totl src. Experiments with purified LN-5 were performed in duplicte; other results re representtive of t lest four experiments. spreding, s MK / cells showed src ctivtion upon dhesion to LN-5 (Figure 5), ut they spred poorly on this sustrte within the time course of these ssys (Chom et l., 2004). Inhiition of FAK/src signling reduces ctivtion of Rc1 in kertinocytes on LN-5 31-dependent ctivtion of Rc1 is required for formtion of polrized lmellipodi y MK cells (Chom et l., 2004). 35

6 DMSO PP2 Uninf. GFP FRNK Phse Phlloidin Rc1 in pull down DMSO Rc1 in lyste % ctive Rc1 remining 100% 19% 100% 73% 26% MK +/+ Figure 6. Rc1 ctivity requires FAK/src signling. Following dhesion of MK3 cells to LN-5 ECM for 30 minutes, pull-down ssys for ctive Rc1 were performed using GST fusion-protein corresponding to the PBD of PAK-1. () Serum-deprived cells were cultured in the presence of either DMSO or PP2 (5 mm). Results re representtive of three seprte experiments. () Cells were either uninfected (uninf.), or infected with denovirus expressing GFP or GFP- FRNK, s indicted. Results re representtive of two seprte experiments. Levels of ctive Rc1 ound to GST-PAK p21-inding domin (top pnels) were quntified nd normlized to levels of totl Rc1 in the strting lystes (ottom pnels). The percent of ctive Rc1 reltive to the corresponding control (100%) is shown elow ech lne. U0126 DMSO Suspension MK +/+ MK / perk1/2 Totl ERK1/2 MK +/+ DMSO PP2 DMSO PP2 perk1/2 Totl ERK1/2 LN-5 MK +/+ MK / Figure dependent ERK1/2 ctivtion requires src kinse ctivity. () MK þ / þ,mk /,ormk3 cells were either kept in suspension or plted on LN-5 ECM for 30 minutes. Levels of ctivted ERK1/2 were ssessed y immunolot with n ntiody specific for phosphorylted ERK1/2 (p-erk1/2) or totl ERK1/2 (totl ERK1/2). () MK þ / þ or MK3 cells were plted on LN-5 ECM-coted coverslips in the presence of DMSO or PP2 (5 mm) for 30 minutes. ERK1/2 ctivtion ws ssessed s in (). Results re representtive of t lest three seprte experiments. Therefore, we tested whether inhiiting src or FAK leds to reduced levels of ctive Rc1 in 31-expressing cells. MK3 cells treted with PP2 showed B5-fold reduction in levels of ctive Rc1 compred to DMSO-treted cells (Figure 6), indicting requirement for src kinses. Similrly, MK3 cells expressing GFP-FRNK showed B4-fold reduction in levels of ctive Rc1 compred with uninfected cells, nd B3-fold reduction compred with cells expressing GFP lone (Figure 6), indicting role for FAK. These results support model wherein 31 promotes lmellipodi formtion through ctivtion of pthwy tht involves FAK, src, nd Rc1. 31-Dependent ERK1/2 ctivtion is dependent on src kinse ctivity ut is not required for estlishment of polrized lmellipodi ERK1/2 cn e ctivted y integrins nd hs een shown to regulte cell migrtion (Aplin nd Julino, 1999; We et l., 2004). We therefore tested whether src kinse ctivity U0126 c MK +/+ DMSO U0126 DMSO U0126 perk1/2 Totl ERK1/2 Figure 8. MEK/ERK ctivity is not required for polrized lmellipodi formtion y 31-expressing MK cells. () MK þ / þ or () MK3 cells were plted onto LN-5 ECM-coted coverslips in the presence of DMSO or U0126 (10 mm) for 30 minutes. Cells were fixed nd F-ctin ws stined with tetrmethyl rhodmine isothiocynte-leled phlloidin; corresponding phse imges re shown. Br ¼ 10 mm. (c) MK þ / þ or MK3 cells were treted s in ( nd ), nd ERK1/2 ctivtion ws ssessed s in Figure 7. regultes 31-dependent ERK1/2 ctivtion. MK cells were plted onto LN-5 ECM where ERK1/2 phosphoryltion is 31-dependent (Figure 7) (Mnohr et l., 2004). Incution with PP2 completely rogted 31-medited ctivtion of ERK1/2 (Figure 7), demonstrting requirement for src kinse ctivity. However, src-dependent ctivtion of ERK1/2 ws not required for extension of polrized lmellipodium, s polrized spreding of MK þ / þ or MK3 cells ws not inhiited y tretment with the MEK inhiitor U0126 (Figure 8 nd ). Inhiition of ERK1/2 ctivtion y U0126 t the concentrtion used ws confirmed y immunolot for phosphorylted ERK1/2 (Figure 8c). DISCUSSION Cell polriztion is n erly phenotypic chnge visile in ctivted epiderml kertinocytes t the wound edge (Singer nd Clrk, 1999). Previously, we estlished role for integrin 31 in promoting Rc1-dependent polriztion of kertinocytes on LN-5 (Chom et l., 2004), finding tht ws recently confirmed in study y Hmelers et l. (2005). In the current study, we identified novel role for 31 s n ctivtor of FAK/src signling tht occurs upstrem of 36 Journl of Investigtive Dermtology (2007), Volume 127

7 LN-5 α3β1 418 Y scr Y Y FAK 397 Y tlin Rc1 ERK1/2 Cell polriztion Bck Front 1. Cell survivl (Mnohr et l., 2004) 2. Adhesion complex turnover (We et l., 2004) Figure 9. Model for the role of 31-medited FAK/src signling in kertinocyte function. Adhesion to LN-5 increses src kinse ctivity in MK cells. In the presence of 31 LN-5 interction, FAK is recruited to dhesion complexes nd uto-phosphorylted t Y397. Once uto-phosphorylted, FAK recruits ctive src kinses to dhesion complexes, resulting in srcmedited phosphoryltion of dditionl sites on FAK nd leding to ctivtion of ERK1/2 nd Rc1 y divergent downstrem pthwys. Although ERK1/2 ws not required for kertinocyte polriztion (Figure 8), it cn regulte dhesion complex turnover in migrting epithelil cells (We et l., 2004) nd cell survivl (Mnohr et l., 2004). Rc1-dependent kertinocyte polriztion. In norml epidermis, sl kertinocytes re nchored to the sement memrne primrily through interctions of integrin 64 in hemidesmosomes with LN-5, rther thn y 31 (Dowling et l., 1996; Georges-Louesse et l., 1996; vn der Neut et l., 1996; DiPersio et l., 2000). Upon wounding, ctivted kertinocytes rpidly redistriute 31 to leding edge lmellipodi. They lso egin to synthesize nd secrete LN-5 efore initition of cell migrtion, nd they continue to deposit LN-5 s they migrte over exposed derml collgen (Nguyen et l., 2000). As previous work hs shown tht kertinocytes deficient in LN-5 expression show defective migrtion on collgen (Ryn et l., 1999), we suggest tht the interction of 31 nd newly deposited LN- 5 is criticl pltform for enhncing FAK/src signling in ctivted kertinocytes. Importntly, other integrins tht ind to lignds in the provisionl wound mtrix, such s collgen nd fironectin, cn lso ctivte FAK nd regulte kertinocyte migrtion (Gincotti nd Ruoslhti, 1999; Pilcher et l., 1999; Nguyen et l., 2000; Wtt, 2002), suggesting tht kertinocyte ehvior/migrtion during wound heling is likely to involve coordinted responses to oth newly deposited LN-5 nd existing dhesion lignds in the provisionl ECM. FAK ws previously reported to orgnize into dhesion complexes in the leding lmellipodium of migrting kertinocyte (Frnk nd Crter, 2004). However, to our knowledge, role for FAK in kertinocyte polriztion hs not een descried previously. Here we showed tht inhiition of endogenous FAK y either GFP-FRNK or GFP- FAK(Y397F) promoted non-polrized, norml cell spreding tht included multiple, rndomly oriented protrusions, similr to norml spreding phenotypes descried for FAKdeficient firolsts (Tilghmn et l., 2005). Recently, the FAK/src signling complex hs ecome recognized s distinct functionl unit tht regultes integrin-dependent cell functions (Plyford nd Schller, 2004; Westhoff et l., 2004), including cell motility nd dhesion complex turnover in migrting cells (We et l., 2004; Brunton et l., 2005). We oserved tht in kertinocytes cultured on LN-5, FAK phosphoryltion t Y861 nd Y925 ws dependent on oth 31 nd src kinse ctivity, nd tht Rc1 ctivtion ws regulted y oth FAK nd src kinse. Tken together, these dt suggest tht 31 regultes the ility of ctive src kinses to interct with nd signl through FAK (Figure 9). This model ssumes tht the interction of src kinses nd FAK is medited y inding of the src SH2 domin with phosphorylted Y397 of FAK (Schller et l., 1994). It hs een suggested tht FAK regultes src signling in firolsts y regulting the sucellulr locliztion of src kinses (Schller et l., 1999), consistent with our oservtion tht muttion of the src-inding site on FAK (Y397F) leds to errnt cell spreding. We oserved tht uto-phosphoryltion of src t Y418 ws incresed y kertinocyte dhesion to LN-5, ut tht this occurred independently of 31. We hve not determined whether this event is required for 31-dependent phosphoryltion of FAK t Y861 or Y925, s phosphoryltion t src Y418 enhnces, ut is not required for, sl src kinse ctivity (Schwrtzerg, 1998). Nonetheless, s src ctivtion ws independent of 31, yet FAK uto-phosphoryltion/ ctivtion nd src-medited phosphoryltion of FAK were dependent on the integrin, we conclude tht 31 is required for src to signl through FAK in kertinocytes dhered to LN-5. The dhesion-medited increse in src Y418 phosphoryltion, nd presumly kinse ctivity, ppered independent of cell spreding, s 31-deficient MK / cells spred poorly within the time frme of src kinse ctivtion. We hve not yet determined whether nother integrin, such s 64, is responsile for the dhesion-medited increse in src kinse ctivity. Our previous studies showed tht 31-medited dhesion to LN-5 promotes oth kertinocyte survivl vi FAK nd ERK1/2 (Mnohr et l., 2004) nd formtion of stle lmellipodium y Rc1-dependent pthwy (Chom et l., 2004). Results from the current study egin to elucidte how these distinct signling functions of 31 my e integrted through FAK. Migrting kertinocytes nd invsive crcinom cells must not only undergo drmtic cytoskeletl rerrngements to promote motility, ut they must lso engge mechnisms tht protect them from noikis induced y leving n orgnized sement memrne for provisionl wound mtrix or tumor microenvironment tht contins unfmilir lignds. Severl groups hve reported tht ERK1/2 ctivtion protects cells from noikis (Schwrtz nd Assoin, 2001; Reginto et l., 2003), wheres the cytoskeletl regultory effects of smll GTPses re well documented (Noes nd Hll, 1999). As multidomin signling protein, FAK is cple of stimulting oth ERK1/2 nd Rc1 ctivity (Schlepfer nd Mitr, 2004), nd the ility of FAK to stimulte divergent pthwys in migrting cells hs een proposed previously (Cho nd Klemke, 2000). We found tht, wheres ERK1/2 nd Rc1 re ech regulted y 31 in kertinocytes (Chom et l., 2004; Mnohr et l., 2004), the 37

8 phenotypes tht they regulte, cell survivl nd cell polriztion, do not pper to e interdependent. Indeed, lthough oth FAK nd ERK1/2 signling were involved in kertinocyte survivl (Mnohr et l., 2004), in the current study we found tht ERK1/2 ctivity ws not required for formtion of polrized lmellipodi. It remins possile tht ERK1/2 regultes kertinocyte migrtion susequent to polriztion, s ERK1/2 hs een shown to regulte dhesion complex turnover (We et l., 2004). FAK my promote Rc1 ctivtion vi its interctions with p130 cs, which cn led to ctivtion of the Rc1 GEF, Dock180 (Kiyokw et l., 1998; Klemke et l., 1998). Indeed, 31 inding to LN-10/11 ws reported to ctivte p130 cs / CrkII/DOCK180/Rc1 pthwy in lung denocrcinom cells (Gu et l., 2001), nd we previously reported tht phosphoryltion of p130 Cs is regulted y 31 in kertinocytes (Mnohr et l., 2004). Alterntively, recent study identified requirement for the Rc1 GEF, Tim1, in kertinocyte spreding nd migrtion on LN-5 (Hmelers et l., 2005). In summry, our results suggest model in which dhesion of 31 to LN-5 in migrtory kertinocytes enhnces the ility of src kinses to signl through FAK (Figure 9). This model plces 31 in role s conduit for FAK/src signling in kertinocytes, where integrin-medited recruitment nd uto-phosphoryltion of FAK is single essentil event in process tht is suject to significnt upstrem regultion, nd src kinses re importnt in regulting the consequences of 31-dependent FAK ctivtion. Once ctivted, FAK/src signling promotes distinct phenotypic chnges required for successful kertinocyte migrtion, polrized lmellipodi formtion nd enhnced survivl, tht re dependent on Rc1 nd ERK1/2, respectively. MATERIALS AND METHODS Cell culture Derivtion of the MK þ / þ (MK-1.16) nd MK / (MK-5.4.6) kertinocyte cell lines from wild-type nd 3-null mice, respectively, ws pulished previously (DiPersio et l., 2000). MK / cells stly trnsfected with full-length humn 3 cdna (i.e. MK3 cells) express high levels of 31 on the cell surfce (DiPersio et l., 2000). MK growth medium consisted of Egle s minimum essentil medium supplemented with 4% fetl ovine serum (BioWhittker, Wlkersville, MD) from which C 2 þ ws chelted, 0.05 mm CCl 2, 0.4 mg/ml hydrocortisone, 5 mg/ml insulin, M T3, 10 units/ml INFg (Sigm, St Louis, MO), 10 ng/ml epiderml growth fctor, 100 units/ml penicillin, 100 mg/ml streptomycin, nd L-glutmine (Invitrogen, Crlsd, CA). Stocks of MK cell lines were mintined t 331C, 8% CO 2, on dishes coted with 30 mg/ml dentured rt-til collgen (Cohesion, Plo Alto, CA). For experiments, MK cells were sucultured on LN-5 ECM prepred from the SCC-25 squmous cell crcinom line, s descried previously (DiPersio et l., 2000). All experiments nd regents descried in this study were pproved y the Institutionl Biosfety Committee of Alny Medicl College. Adenovirl infection of MK cells Adenoviruses were constructed s descried (Medows et l., 2001) using the AdEsy system (He et l., 1998). Cells were seeded onto collgen-coted 10 cm dishes nd infected for 24 or 48 hours. Infection conditions were optimized to chieve GFP-FAK(wt), GFP- FRNK, or GFP-FAK(Y397F) expression levels tht were 5- to 10-fold higher thn levels of endogenous FAK. Adenovirl infection efficiency ws greter thn 90% for ll constructs, s ssessed y visulizing GFP-positive cells. Infected MK cells were sucultured for fluorescence or signl trnsduction nlysis s descried elow. Fluorescence nlysis nd microscopy MK cells were serum-deprived for 4 8 hours then trypsinized from stock pltes, resuspended in either serum-contining Egle s minimum essentil medium (4% chelted fetl ovine serum) or serumfree Egle s minimum essentil medium (0.5% BSA), supplemented with 0.05 mm CCl 2, nd pre-incuted t 331C for 30 minutes with rottion efore plting onto LN-5 ECM-coted coverslips t density of pproximtely cells/coverslip. After 30 minutes of dhesion, cells were fixed in 4% prformldehyde, permeilized with 0.2% Triton X-100, nd locked in 1% BSA. Stining for F-ctin ws performed with tetrmethyl rhodmine isothiocynte-phlloidin (Sigm, St Louis, MO). Immunofluorescence ws performed with rit polyclonl ntiody ginst 3 integrin suunit (DiPersio et l., 1995) nd Alex Fluor 594 got nti-rit secondry ntiody (Invitrogen, Crlsd, CA). Cells were viewed on n Olympus BX60 upright microscope. Imges were generted using SlideBook (Intelligent Imging Innovtion, Denver, CO) nd processed using Adoe Photoshop (version 7.0). For Figure 1k m, deconvolution ws performed on single-plne imges using the noneighors function. To ssess cell morphology (Figure 1), uninfected or infected cells were plted onto LN-5 ECM-coted coverslips for 90 minutes in serum-free medium then stined with tetrmethyl rhodmine isothiocynte-phlloidin, s descried ove. For ech smple, t lest 70 spred cells from rndomly chosen fields were scored s either spred/non-polrized, polrized, or normlly spred, nd the proportions of cells with polrized or norml phenotypes were clculted s percentge of the totl numer of spred cells (see the text for detiled descriptions of phenotypes). Scoring ws performed in lind fshion; n ¼ 4 for ech condition. Sttisticl nlysis ws performed using Prism (GrphPd Softwre, Inc., Sn Diego, CA). Immunolot nlysis of dhesion-dependent signling Cells were either kept in suspension or dhered for 30 minutes to LN-5 ECM or purified humn LN-5 (Chemicon, Temecul, CA; 20 mg/ml in phosphte-uffered sline). For inhiitor experiments, cells were treted during the pre-incution period nd fter plting with DMSO, PP2, or U0126 (Cliochem, Sn Diego, CA) t concentrtions indicted in the figure legends. Cells were lysed in Cell Signling Lysis uffer (Cell Signling Technology, Beverly, MA), nd 30 mg of totl cellulr protein ws run on 10% SDS-PAGE gel, trnsferred to nitrocellulose, nd proed with the following ntiodies: nti-fak py397, py861, or py925 (BioSource Interntionl, Cmrillo, CA); totl FAK (Upstte Biotechnology, Lke Plcid, NY); nti-src py418, py529, or nti-pn src (BioSource Interntionl, Cmrillo, CA); nti-perk1/2 (Cell Signling Technology, Beverly, MA) or nti-erk1/2 (Promeg, Mdison, WI); ntikertin 14 (Covnce, Richmond, CA). Chemiluminescence ws performed using the SuperSignl kit (Pierce, Rockford, IL) nd quntified on Fluor-S MultiImger (Bio-Rd, Hercules, CA) using Quntity-One softwre. 38 Journl of Investigtive Dermtology (2007), Volume 127

9 Rc1 ctivity ssy Levels of ctive Rc1 were determined using the Rc1 Assy Regent (Upstte Biotechnology, Lke Plcid, NY). Serum-deprived MK cells were plted for 30 minutes onto LN-5-rich ECM t density of pproximtely cells/60 mm dish. For inhiitor experiments, cells were treted with PP2 s descried ove. Cell lystes were prepred in 250 ml ice-cold Mg 2 þ Lysis/Wsh uffer, nd ctive Rc1 ws precipitted with PAK-1 p21-inding domin-grose conjugte ccording to the mnufcturer s instructions, soluilized in reducing smple uffer, nd suject to 12% SDS-PAGE. Strting lyste (5 ml) ws included s loding control. Rc1 ws detected y immunolot with nti- Rc1 (clone 102; BD Trnsduction Lortories, Sn Jose, CA) nd HRP-linked got ntimouse secondry ntiody (Pierce, Rockford, IL). CONFLICT OF INTEREST The uthors stte no conflict of interest. ACKNOWLEDGMENTS We thnk Dr Gng Liu for ssistnce with microscopy, Dr Vndn Iyer, John Lmr, Ptrick Brynt, nd Dr Peter Vincent for helpful comments nd technicl ssistnce, nd Dr Andrew Aplin for criticl reding of the mnuscript. This reserch ws supported y grnt from the Ntionl Institutes of Helth to CMD (R01CA84238) nd y grnt from the Ntionl Institutes of Helth to KP (R01CA081419). D.P.C. ws supported y predoctorl fellowship from the New York Stte Affilite of the Americn Hert Assocition ( T). REFERENCES Adel-Ghny M, Cheng HC, Elle RC, Puli BU (2002) Focl dhesion kinse ctivted y et(4) integrin ligtion to mclca1 medites erly metsttic growth. J Biol Chem 277: Agochiy M, Brunton VG, Owens DW, Prkinson EK, Prskev C, Keith WN et l. (1999) Incresed dosge nd mplifiction of the focl dhesion kinse gene in humn cncer cells. Oncogene 18: Aplin AE, Julino RL (1999) Integrin nd cytoskeletl regultion of growth fctor signling to the MAP kinse pthwy. 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