A Crucial Role for DOK1 in PDGF-BB-Stimulated Glioma Cell Invasion Through P130cas and Rap1 Signalling

ß 2014. Published by The Company of Biologists Ltd | Journal of Cell Science (2014) 127, 3397 doi:10.1242/jcs.158576

CORRECTION A crucial role for DOK1 in PDGF-BB-stimulated glioma cell invasion through p130Cas and Rap1 signalling

Angela Barrett, Ian M. Evans, Antonina Frolov, Gary Britton, Caroline Pellet-Many, Maiko Yamaji, Vedanta Mehta, Rina Bandopadhyay, Ningning Li, Sebastian Brandner, Ian C. Zachary and Paul Frankel

There was an error published in J. Cell Sci. 127, 2647-2658.

In the Abstract and Introduction, TERF2IP was incorrectly given as an alternative name for Rap1. This should have referred to the small GTPase Rap1 (which has two isoforms, Rap1a and Rap1b).

We apologise to the readers for any confusion that this error might have caused.

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College 6JJ, University WC1E Neurology, London of Institute Disease, Neurodegenerative neaBarrett signalling Angela cell Rap1 glioma and PDGF-BB-stimulated p130Cas in through DOK1 invasion for role crucial A ARTICLE RESEARCH eevd3 a 03 cetd2 ac 2014 March 26 Accepted 2013; May 31 Received ` breast melanoma, 2004). glioblastoma, 1 a in including Cross, deregulated is cancers, and receptor of PDGF (Jones and variety PDGF cells of expression cancer The both in and motility factor cell growth regulating non-transformed ligand derived important Platelet an understood. is (PDGF)-BB well (3D) three-dimensional not and are motility in invasion cell involved tumour of pathways 2011). regulation as signalling Weinberg, the and described chemotactic (Hanahan is specific cancer’ and of However, ‘hallmarks tissue, the the surrounding cells of tumour their one gives invade motility to cell capacity increased of acquisition The INTRODUCTION p130Cas, DOK1, signalling, PDGF-BB- GTPase PDGF motility, of Cell WORDS: regulation KEY the signalling p130Cas–Rap1 in a DOK1 through pathway. motility for cell PDGF- data role tumour inhibited mediated These crucial DOK1FF invasion. a of spheroid show expression (3D) three-dimensional and of mediated Rap1 knockdown and and DOK1 migration, PDGF-BB- inhibited cell Rap1 inhibited chemotactic and DOK1 PDGF-BB-induced (p130Cas15F) of Knockdown p130Cas activation. Rap1 mediated of mutant domain non-tyrosine-phosphorylatable substrate a of Expression cells. PDGF-BB-treated of membrane cell with the colocalises at DOK1 p130Cas phosphorylated TERF2IP). tyrosine PDGF-BB-induced as known the (also Rap1 both the of activation and of BCAR1) as inhibition known (also p130Cas in of phosphorylation tyrosine resulted 398, and residues at Tyr 362 of place in Phe containing (DOK1FF) mutant DOK1 expression a of or DOK1 PDGF-BB of Knockdown high-grade cells. upon glioma from human phosphorylated of biopsies stimulation tyrosine tumour becomes in DOK1 and gliomas. lines cell glioma expressed the several is DOK1 in on (DOK1). dependent 1 kinase are of downstream migration protein adaptor and invasion cell PDGF-BB- glioma that are here mediated report motility We metastasis. cell and invasion tumour for regulating essential Mechanisms motility. cell glioma (PDGF)-BB-stimulated factor growth platelet-derived regulates DOK1 ABSTRACT nttt fNuooia Studies. Neurological of Institute eat Mehta Vedanta uhrfrcrepnec ([email protected]) correspondence for Author etefrCrivsua ilg n eiie iiino eiie Rayne Medicine, of Division Medicine, and Biology Cardiovascular for Centre 04 ulse yTeCmayo ilgssLd|Junlo elSine(04 2,24–68doi:10.1242/jcs.135988 2647–2658 127, (2014) Science Cell of Journal | Ltd Biologists of Company The by Published 2014. 1, 1 iaBandopadhyay Rina , ,InM Evans M. Ian *, 3 iiino erptooyadDprmn of Department and Neuropathology of Division 1, ,Atnn Frolov Antonina *, 2 igigLi Ningning , 2 eaLl Weston Lila Reta 1 3 ayBritton Gary , eata Brandner Sebastian , loacl oiiy ihipiain o h mechanisms the for glioblastoma. implications human of with high-grade pathogenesis role the PDGF-BB-mediated motility, the crucial underlying from in a cell pathway with plays biopsies p130Cas–Rap1 glioma DOK1 together a tumour that regulating Taken indicate in in results Rap1. these DOK1 gliomas, and are of invasion DOK1 radial expression 3D on pathway and migration a dependent cell PDGF-BB through small glioma and of TERF2IP), phosphorylation, that the stimulation tyrosine as p130Cas of known and on PDGF-BB- dependent (also activation 398, Rap1 for Furthermore, PDGF-BB-induced GTPase crucial p130Cas. and mediates of is 362 DOK1 phosphorylation residues tyrosine stimulates Tyr these stimulated PDGF-BB at on that in phosphorylation found phosphorylation signalling We PDGF-BB- p130Cas al., in DOK1 cells. chemotactic et DOK1 glioma the and of Pellet-Many malignant role motility in 2011; the cell al., role investigated mediated et we key vascular (Evans Here, a and 2011). cells cells plays muscle glioma BCAR1) U87 smooth of as migration known PDGF-BB-induced cellular (also coordinated p130Cas produce to 2009). al., et molecules (Mashima responses signalling of other formation regulating the temporally in and/or that protein spatially adaptor proposed complexes, or multi-molecular scaffold been a has as role It a 2009). has pleckstrin C- DOK1 al., the et in N-terminal (Mashima residues region tyrosine terminal an phosphotyrosine-binding multiple containing a and with by domain (PTB) followed structure, domain, (PH) domain homology modular has DOK1 1997). Baltimore, a and Yamanashi 1997; al., kinases et tyrosine (Carpino non-receptor and receptor both by phosphorylated signalling to these responses how chemotactic understood produce fully to PDGF. been yet coordinated have motility are multi- not complexes molecules cell these is large of Although it regulation 2008). into described, al., the kinases, GTPases et for (Takahashi protein small required proteins, complexes and protein adaptor phosphatases of proliferation migration, glioma cell protein recruitment and stimulates of the PDGF-BB survival variety 2006). through a motility, Holland, regulating and including in (Shih by PDGF- role functions, produced 2006). important al., cell factors diffuse an et growth (Farin plays a PDGF-BB by BB as in vessels such cells glioma cells, blood resulting that endothelial to proposed vessels, been attracted has blood It are tissue. and brain of tracts infiltration preferentially fibre proliferate and along migrate cells Glioblastoma tumour. leukaemia of 1999). forms Westermark, various and and (Heldin carcinoma prostate carcinoma, rvosy erpre httrsn hshrlto of phosphorylation tyrosine that reported we Previously, is that protein 62-kDa a is (DOK1) 1 kinase of Downstream brain malignant primary invasive highly a is Glioblastoma 1 aoiePellet-Many Caroline , 3 a .Zachary C. Ian , 1 ak Yamaji Maiko , 1 n alFrankel Paul and 1 , 2647 1, `

Journal of Cell Science sntdtce nnra ri (http://www.proteinatlas.org/ brain importantly normal and samples in is glioma detected DOK1 of that not majority found is We the (supplementary glioma. in in cancers expression expressed Protein DOK1 Human brain mRNA the for through explored and Atlas DOK1 we available Additionally, breast in S1B). Fig. are lung, material increases that significant in sets found expression data We to (TCGA) Oncomine. Cancer addition The Atlas using In expression Genome S1A). mRNA investigated Fig. also in we material reduced COPA, moderately (supplementary was cancer it whereas lung cancers, both kidney in overexpressed and moderately breast was normal mRNA versus DOK1 cancer that of showed Analysis (COPA) 2006). normal, Analysis Ghosh, updated Profile versus and Outlier (MacDonald continuously cancer Cancer and of the cancer analysis in versus cancer searching on expression based by mRNA database, Oncomine examined DOK1 1A). was (Fig. human cancer expression multiple stimulation DOK1 PDGF-BB examined to responsiveness and We their for lines tumours. protein 2001; cell DOK1 glioma about human al., known et is in little Hosooka very expression yet 2010; 2011), al., al., et et Mercier in (Berger roles negative and progression positive both tumour play to reported been has DOK1 RESULTS ARTICLE RESEARCH yae fsvrlbose fhmnGM(tgs2 n ,alpoie ihsprt oenmes eaae ygleetohrssadpoe using probed and electrophoresis gel by separated numbers) code separate with 2648 provided all 4, and 3 2, (stages and ( GBM DOK1 without biopsies. human against or glioma of (+) antibodies malignant with biopsies human phospho-ERK several and and of total lines and lysates cell phospho-PDGFRB glioma and total human of in Levels expression DOK1 1. Fig. b atn lt hw eeadi l usqetfgrsaerpeettv fa es he eaaeexperiments. separate three least at of representative are figures subsequent all in and here shown Blots -actin. lhuhPG-Bsiuaincue outices in increase robust a PDGF- caused to stimulation responses PDGF-BB investigated Although we PDGFR of 2009), activation receptor AA-mediated al., PDGF et to the response (Martinho Because in cells 2011). that U87MG al., (PDGFR of tyrosine protein et migration the (Evans adapter DOK1 in PDGF-BB levels an role of key PDGF-BB-induced p130Cas, basal a of timecourse plays of that to phosphorylation to The declined similar tyrosine 2A). very it (Fig. was and phosphorylation min 60 5 post-treatment, between peaking after cells min transient, 15 was at U87MG increase DOK1 This and of of 398. phosphorylation and the we 362 treatment in Tyr increase biopsies, PDGF-BB marked glioma a invasion. PDGF-BB-stimulated stimulated human in cell DOK1 in of role glioma and the investigate lines to decided cell which tumour tissue, glioma brain normal 1B). (Fig. with was expression (ranging little compared protein very biopsies showed 2–4) DOK1 (GBM) of grades multiforme expression from human glioblastoma the from in biopsies we that in elevated found levels protein results, We DOK1 gliomas. at these look to decided Considering ENSG00000115325/cancer). aigetbihdta O1i xrse nsvrlhuman several in expressed is DOK1 that established Having 2 -i ramn ih5 gm DFB r loson B Protein (B) shown. also are PDGF-BB ng/ml 50 with treatment 5-min a ) ora fCl cec 21)17 6725 doi:10.1242/jcs.135988 2647–2658 127, (2014) Science Cell of Journal a a ensont eurgltdi gliomas in upregulated be to shown been has ) A O1i xrse nmlil ua loacl lines. cell glioma human multiple in expressed is DOK1 (A) a inligi 8M cells. U87MG in signalling a

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Journal of Cell Science K ciiycmae ihtevhcecnrl(i.3A). (Fig. reduced that control also DOK1 indicated vehicle and the microscopy 398, PDGF-stimulated with immunofluorescence Tyr compared and Furthermore, activity reduced 362 Tyr AKT inhibitor at PI3K strongly the phosphorylation with cells DOK1 U87MG Zhao of 2000; LY294002 for Treatment al., 2001). et required DOK1 Dijk al., (van et be for fibroblasts and in to cells localisation membrane reported haematopoietic 3-kinase in been phosphorylation Phosphatidylinositide tyrosine has 2B). activity of (Fig. phosphorylation (PI3K) p130Cas tyrosine and PDGF-BB-stimulated had DOK1 2007), on al., et effect (Slack-Davis kinase the PTK2B) no FAK as of known of (also activity PYK2 treatment the at and block PF573228, contrast, inhibitor, specifically family that By FAK concentrations potent 2C). the with (Fig. cells U87MG p130Cas both of and phosphorylation DOK1 significantly 1296 tyrosine AG PDGF-BB-stimulated selective or 1296. PP2 reduced AG additional with inhibitor cells an U87MG PDGFR signalling of selective with and Pre-treatment the and cells inhibitor PP2, SRC treated inhibitor, SFK the we of PDGFR, also 2B). specificity (Fig. through SU6656 the phosphorylation YXXP. DOK1 confirm tyrosine within To sequence, p130Cas residues PDGF-BB-stimulated consensus inhibited 2B), (Fig. strongly substrate of 398 SRC and inhibition 362 the to Tyr the on response phosphorylation in in SU6656 activated inhibitor SFK become resulted the to with Treatment known stimulation. PDGF-BB are which (SFKs), S2B). Fig. material (supplementary mohmsl el nrae yoiepopoyainof phosphorylation artery tyrosine coronary increased human cells of PDGFR muscle treatment cells. these PDGF-AA smooth in events cells, contrast, signalling p130Cas U87MG these By on in effect increased activation no had Fig. ERK PDGF-AA PDGF-BB and material phosphorylation although (supplementary tyrosine Furthermore, PDGF-BB or S2A). PDGF-AA either kinases. ( family cells SRC U87MG for (A) requirement tyrosine the p130Cas and and DOK1 phosphorylation PDGF-BB-stimulated of Timecourse 2. Fig. ARTICLE RESEARCH 2650 PDGFR PDGFR P28 t0.1 at (PF228) and prepared ( for were SFM cells in lysates U87MG incubated Cell (B) (min). indicated. times as indicated immunoblotted ( the control for vehicle (+) plus PDGF-BB SFM either with treatment O1(ideadlwrpnl) * panels). lower and (middle the DOK1 show are and experiments (RU), independent units mean three relative least phosphorylation at in as from shown presented Data as ImageJ, panel. tyrosine using right-hand of densitometry the Quantification by panel. performed with separate left-hand was probed three the phosphorylation and least in blotted at shown prepared, of are then blots experiments were Representative lysates antibodies. Cell indicated min. ( the 5 control for SFM with (+) treatment BB by followed ‘C’), DMSO, C 8M el ( cells U87MG (C) cmae ihPG-tmltdcnrl C) * control, ‘C’); control, PDGF-stimulated with (compared tmltdcnrl ‘C’); control, stimulated hshrlto a efre si rgthn panel). (right-hand B in tyrosine as of performed with separate Quantification was probed three panel). phosphorylation and least (left-hand blotted at shown prepared, of are then blots experiments were Representative lysates antibodies. Cell indicated min. ( the 5 control for SFM with (+) treatment BB PP2 by inhibitor followed kinase ‘C’), family DMSO, Src the either with (10 min 30 for pre-incubation enx okda h oeo R aiytrsn kinases tyrosine family SRC of role the at looked next We m ) h DF niio G19 (10 1296 AG inhibitor PDGFR the M), 6 ...Dt r omlsdt ihrttlp3Cs(o ae)or panel) (top p130Cas total either to normalised are Data s.e.m. 2 a a b ). sw eotdpeiul Ple-aye l,2011) al., et (Pellet-Many previously reported we as , yoiepopoyainwsdtcal nrsos to response in detectable was phosphorylation tyrosine yoiepopoyain(i.1) oices in increase no 1A), (Fig. phosphorylation tyrosine m Mor5 , , 0 ofun)wr nuae nSMfor SFM in incubated were confluent) 80% 0 ofun)wr nuae nSMfor SFM in incubated were confluent) 80% , 8hpirt r-nuainfr3 i ihPF573228 with min 30 for pre-incubation to prior h 18 # m P ,S65 t2 at SU6656 M, , .5(oprdwt F control, SFM with (compared 0.05 P , .1(oprdwt PDGF-BB- with (compared 0.01 m rtevhcecnrl(0.05% control vehicle the or M m )o h eil (0.05% vehicle the or M) P , 2 , 0 ofun)were confluent) 80% rSMpu 0ng/ml 50 plus SFM or ) .5(oprdwt SFM with (compared 0.05 2 2 r5 gm PDGF- ng/ml 50 or ) r5 gm PDGF- ng/ml 50 or ) $ P , , , 2 8hpirto prior h 18 0.01 8hpirto prior h 18 ). i.SB.W ute netgtdterl fDK nPDGF- generated in We assay. DOK1 spheroid of 3D role a the using motility investigated material BB-stimulated further supplementary We to 7A,B; S3B). cells (Fig. these Fig. either of PDGF-BB In of ability towards the knockdown assay. inhibited migrate cells, significantly migration Rap1 glioma or transwell U251MG DOK1 a and U87MG using levels both assessed Rap1–GTP was on migration effect no had PDGF- 6D). to Ad.DOK1 (Fig. of response in resulted whereas activation also Rap1 expression BB, in expression decrease Ad.DOK1FF significant whereas a effect. in no phosphorylation, had PDGF-BB-stimulated Ad.DOK1 tyrosine decreased significantly p130Cas expression 6C, Ad.DOK1FF mutated Fig. 398 in of and shown 362 As wild- Tyr (Ad.DOK1FF). with phenylalanine either to DOK1 encoding or (Ad.DOK1) we adenoviruses DOK1 this, with type do cells To Rap1. U87MG mediating and for infected p130Cas important through was signalling 398 and PDGF-BB with 362 Tyr transfected at were phosphorylation that cells shown). in not (data reduced siRNA not DOK1 Rac1–GTP but was Rac1, increased GTP-bound effect active modestly of this pulldown cells by determined U87MG we as PDGF- levels, activation. of p130Cas, Rac1 in of treatment DOK1 non- downstream BB for role activated the possible a become al., investigated also to overexpressed has et GTPase shown Rac1 (Evans that the been was Because 6B) controls. with or (Fig. effect compared with 2011) mutant transfected This 6A) p130Cas15F (Fig. either Rap1. phosphorylatable were siRNAs that GTP-bound cells DOK1 active in reduced of significantly pulldown determined of as levels, treatment cellular by Rap1–GTP PDGF-BB increased cells. strongly on cells glioma U87MG in effect activity Rap1 similar regulating a p130Cas. has and DOK1 S2C), proteins of PDGF-BB Fig. redistribution selective these material that the (supplementary with of 1296 indicating pre-treatment AG by colocalisation inhibitor blocked PDGFR was the a which by co- 5), in accompanied (Fig. membrane, performing cell increase the by to significant cells, p130Cas and U87MG DOK1 localisation of the in localisation increased the PDGF-BB p130Cas staining. on also immunofluorescent and treatment We DOK1 PDGF-BB 4B). were of (Fig. of migration, siRNA effect DOK1 the PDGF-BB-mediated cell molecules with examined regulating signalling treatment in two by because DOK1 unaffected roles AKT, well-established by and selective, with ERK phosphorylation of was tyrosine phosphorylation p130Cas knockdown S3A). in of Fig. siRNAs material of (supplementary DOK1 Inhibition U251MG multiple line the phosphorylation cell by glioma by inhibited the tyrosine supported similarly further was PDGF-BB-induced p130Cas were against that directed results siRNAs These finding multiple tyrosine 4A). with (Fig. p130Cas reduced treated DOK1 markedly with were of was that cells cells effect level when an min, the U87MG 5 p130Cas after increased phosphorylation of the PDGF-BB-stimulated strongly testing Treatment on by PDGF-BB phosphorylation. silencing examined the was DOK1 tyrosine in This of signalling motility. effect p130Cas cell of of regulation mediator endogenous important was that PDGF- following 3B). DOK1 cells (Fig. U87MG of treatment in BB membrane amount cell the the at localised reduced treatment LY294002 h oeo O1i DFB-eitdchemotactic PDGF-BB-mediated in DOK1 of role The tyrosine DOK1 whether examine to us led results These in p130Cas and DOK1 for role possible a investigated next We an be could DOK1 that suggested 2 Fig. in results The ora fCl cec 21)17 6725 doi:10.1242/jcs.135988 2647–2658 127, (2014) Science Cell of Journal

Journal of Cell Science EERHARTICLE RESEARCH ta. 00 ece ta. 01.B otat hr r reports are there tumour contrast, By a 2011). as al., (Berger et DOK1 progression Mercier tumour for 2010; of al., role regulator et a negative remains or suggest and suppressor emerging studies still is Some tumorigenesis unclear. in DOK1 of role The DISCUSSION 7E). all (Fig. spheroid I siRNAs U87MG collagen radial in Rap1-specific invasion of cell stimulation different the reduced in Three invasion significantly Rap1 using siRNAs. of expression radial Rap1 role silencing targeted The by 3D addressed PDGF-BB-stimulated 7D). was Fig. invasion and in radial S4; Fig. decrease phosphorylation material (supplementary significant tyrosine U87MG in a Y398F p130Cas DOK1 caused or Y398F. Y362F cells and DOK1 PDGF-BB- Y362F either mutations on of point Expression 398) adenoviruses DOK1 with Tyr single cells the and infecting expressing 362 by each signalling (Tyr of DOK1 contribution residues stimulated no the tyrosine investigated the had also of We Ad.LACZ Ad.DOK1 the 7D). with (Fig. whereas compared control invasion radial PDGF-BB, on effect by inhibited significant of significantly induced role cells the U87MG invasion. invasion examined in radial expression next PDGF-BB-mediated Ad.DOK1FF We in treated was 7C). phosphorylation cells (Fig. PDGF-BB DOK1 from siRNAs of generated DOK1 effect spheroids with the in control h and the reduced 48 SFM, with significantly for in compared treatment incubated invasion plugs radial spheroids PDGF-BB I enhanced PDGF-BB. in collagen resulted containing in (SFM) SFM medium them serum-free or either with embedded overlaid PDGF-BB and and supplemented upon membrane spheroids the to localisation U87MG DOK1 increased of areas indicate coverslips Arrows glass experiments. onto separate seeded three were cells least U87MG at treatment. (B) of antibodies. representative indicated are the Images with probed and blotted ( prepared, control then for were SFM in lysates incubated Cell (10 and min. LY294002 5 with membrane. for min the (+) to 30 PDGF-BB recruitment for and phosphorylation pre-incubation DOK1 to for prior required is PI3K 3. Fig. 2 r5 gm DFB + o i.Cnoa mgn a efre sdsrbdi aeil n ehd,wt O1sann hw ngreen. in shown staining DOK1 with Methods, and Materials in described as performed was imaging Confocal min. 5 for (+) PDGF-BB ng/ml 50 or ) , 8hpirt r-nuainfr3 i ihL240 (10 LY294002 with min 30 for pre-incubation to prior h 18 m )o h eil 00%DS,‘’,floe ytetetwt F eil oto ( control vehicle SFM with treatment by followed ‘C’), DMSO, (0.05% vehicle the or M) xrsin efudta O1poeni xrse nthe in expressed mRNA is DOK1 protein with DOK1 We agreement that function. In found and Atlas. we using Protein expression, expression cancer in Human protein expression the protein to DOK1 expression investigated this limitations therefore are mRNA there correlating cancers, specific increased GBM in in DOK1 including Although for role in cancers, a brain. suggests increases several normal significant in versus found expression (non- normal mRNA We cancer corresponding DOK1 individual samples. with for tissue comparisons presented as cancerous) are well several sets TCGA, as on Data search types cancers. information and to of genetic cancer Oncomine types high-resolution liver used contains and we kidney which addition, brain, In with particularly lymphoma. associated DOK1 cancers, of strongly overexpression several that is in revealed also DOK1 COPA indicate but we of leukaemia, COPA, is not levels Using reduced ERBB2. does analysis of found upregulation analysis this significant a and reveals Standard COPA strong of whereas cancers. upregulation, in mRNA value ERBB2 overexpressed breast significant is The of which oncogene, 25% cancers. ERBB2 the human by highlighted expression cancer various DOK1 a analyse within is to population in 2006), total expression Ghosh, the and in gene (MacDonald samples type high of subset where identify a to in profile be seen developed expression algorithm to an (Wang oncogene COPA, found population an used cells cell We was 2004). invasive al., carcinoma DOK1 the et in invasive tumours, and upregulated of mammary significantly progression a study primary in tumour Indeed, profiling 2011). versus in al., expression et role Mercier gene 2001; positive al., et a (Hosooka motility plays DOK1 that m )o h eil 00%DS) olwdb ramn ihSMvehicle SFM with treatment by followed DMSO), (0.05% vehicle the or M) A 8M el ( cells U87MG (A) ora fCl cec 21)17 6725 doi:10.1242/jcs.135988 2647–2658 127, (2014) Science Cell of Journal , 0 ofun)wr nuae nSMfor SFM in incubated were confluent) 80% 2 r5 ng/ml 50 or ) , 2651 8h 18

Journal of Cell Science EERHARTICLE RESEARCH Ease l,21;Ple-aye l,21) O1hsbeen has cells DOK1 muscle 2011). 2652 al., smooth et vascular Pellet-Many 2011; and al., cells et glioma (Evans PDGF-BB-dependent in of role is key migration a PI3K plays p130Cas that SFK. protein by adaptor such suggests phosphorylation and its This membrane, for cell 2001). needed the be al., Dijk membrane, to might (van recruitment recruitment et cell DOK1 types Zhao for cell the required 2000; other at in al., localisation findings et previous DOK1 with reduced consistent strongly also inhibitor phosphorylation PI3K and tyrosine a cells, DOK1 with PDGF-BB-stimulated glioma treatment decreased in Furthermore, that, SFK. dependent demonstrate is on data phosphorylation tyrosine Our DOK1 2009). SFK- PDGF-BB-induced al., 2002; both al., the et of et Senis (Liang reports by mechanisms with DOK1 SFK-independent phosphorylation, and regulated of unclear, dependent mechanism is tyrosine is exact phosphorylation DOK1 The tyrosine phosphorylation pathway. in signalling DOK1 PDGF-BB increase that transient indicating are resulted a stimulation motility PDGF-BB study, cell in this and tumour In DOK1 understood. in well regulating pathways not in signalling involved Ling downstream mechanisms 2004; its al., the et 2005), (Lee al., motility et cell protein muscle smooth DOK1 and epithelial which biopsies in glioma tissue, 4 undetectable. and brain was DOK1 3 expression normal higher 2, with grade showed in several compared detected results in not our expression is protein and Furthermore, samples brain. glioma normal available the of majority for SFM in incubated were cells post-transfection, h 48 At nM). 25 (siScr, siRNA scrambled control with or nM) p130Cas. of 25 phosphorylation (siDOK1, tyrosine DOK1 PDGF-BB-stimulated targeting regulates siRNAs specifically DOK1 4. Fig. niae niois uniiaino 10a hshrlto a efre ydnioer sn mgJ aafo he needn xeiet are experiments mean independent the three as from shown Data and ImageJ. (RU) using units densitometry relative by performed phosphorylation was p130Cas phosphorylation as p130Cas presented ( of control Quantification vehicle antibodies. containing indicated SFM with treatment to cmae ihlgn-tmltdsSr.()U7Gclswr rae saoeadpoe ihteidctdantibodies. indicated the with probed and above as treated were cells U87MG (B) siScr). ligand-stimulated with (compared ercnl eotdta yoiepopoyaino the of phosphorylation tyrosine that reported recently We of regulation the in implicated been has DOK1 Although 2 r5 gm DFB + o iue.Cl yae eete rprd lte n rbdwt the with probed and blotted prepared, then were lysates Cell minutes. 5 for (+) PDGF-BB ng/ml 50 or ) a1 n t ciaintruhecag fGPfrGP In GTP. are that for pathways GDP signalling of multiple drives GTPase exchange activation small through Rap1 the turn, activation of its as recruitment and the known the Rap1, (also in to C3G results This (GEF) binds the RAPGEF1). factor with associate domain, exchange particular, Crk nucleotide and In SH3 p130Cas guanine both 2011). and N-terminal Crk, al., protein Ras its et adaptor for Stefano factors through exchange (Di p130Cas, with GTPases complexes p130Cas small of of role family formation key A the 2006). are in motility al., that et cell is Defilippi complexes during 2013; cytoskeleton al., signalling et the of (Barrett of assembly remodelling the for for required scaffold a pathways. major signalling as pathways, AKT other and transduction ERK affect signal the of (RTK) significantly activation including kinase not PDGF-BB. because tyrosine PDGF- did p130Cas, receptor with regulating of knockdown in stimulation role phosphorylation DOK1 selective tyrosine following a colocalise BB-stimulated has results p130Cas membrane tyrosine DOK1 and the Furthermore, p130Cas the DOK1 PDGF-BB-induced both to that to that for herein contrast and show required we phosphorylation in Nevertheless, is al. association p130Cas, we et DOK1 any Abramson observe Indeed, and by to reported 2003). unable DOK1 were endogenous al., but between with DOK1, et p130Cas tagged and co-immunoprecipitate (Abramson phosphorylation tyrosine to determined p130Cas attempted on not effect the was and unclear, Fc is 1 type of (Fc stimulation upon receptors p130Cas with associate to reported 6 hog t utpedmis 10a stogtt function to thought is p130Cas domains, multiple its Through ...Dt r omlsdt oa 10a.* p130Cas. total to normalised are Data s.e.m. ora fCl cec 21)17 6725 doi:10.1242/jcs.135988 2647–2658 127, (2014) Science Cell of Journal I nms el,bttentr fti association this of nature the but cells, mast in eRI) A 8M el eetasetdwt he independent three with transfected were cells U87MG (A) P , .5 ** 0.05; P , , 0.01 8hprior h 18 e

Journal of Cell Science EERHARTICLE RESEARCH 10a yoiepopoyainadRp ciainsuggests activation The Rap1 and PDGF-BB-stimulated cells. for phosphorylation crucial glioma tyrosine are 398 PDGF- p130Cas U87MG and of 362 in regulation Tyr that the finding signalling in role Rap1 important BB-stimulated an play invasion. p130Cas and and motility PDGF-BB-mediated cell regulating for in essential is DOK1 establish which for spheroid further signalling, role results inhibited These important DOK1 silencing. Ad.DOK1FF an core. Rap1 did spheroid of radial as the expression directional outgrowth, of produce or rim the does knockdown from a it towards outwards movement source, movement supplied of model exogenously chemotactic defined a with not single by a is model problem be this Although invasion establishing this to PDGF-BB. overcome spheroid in proven to the has difficulties sought using environment We the gradient. 3D to chemotactic a owing in difficult, for invasion chemotactic required cell of is Examination tumour DOK1 motility. that signalling cell showed PDGF-BB-stimulated invasion a 3D mediating and chemotaxis in role Rap1. and key p130Cas involving a tyrosine pathway DOK1 plays PDGF-BB-induced Ad.DOK1FF activation that phosphorylation Rap1 of conclusion that and the overexpression finding phosphorylation supports tyrosine Van Our or p130Cas 2010). and knockdown inhibits Zwartkruis, (Boettner DOK1 and motility either Frische cell 2009; RTK-mediated Aelst, for required ae oehr hs eut hwfrtefrttm htDOK1 that time first the for show results these together, Taken of assays activation, Rap1 in role its with Consistent uat O1Y6Fad oalse xet O1Y398F DOK1 extent, single lesser the a of to expression and, in warrants Y362F that Abl this DOK1 found for and mutants role We cells, a investigation. glioma preclude further not in described do signalling pathway This DOK1-mediated results DOK1-dependent p130Cas for these shown). important the However, be here. through not not signalling might (data PDGF-BB Abl phosphorylation that and suggests DOK1 of tyrosine of effect stimulation significant p130Cas PDGF-BB-mediated a on demonstrate Abl therefore to knockdown. silencing unable siRNA-mediated We were using in 2004). we Abl implicated al., However, of been role et has the (Woodring investigated pathway motility Abl cell and an regulating (Yamanashi and kinase binding tyrosine 1997), Abl a Baltimore, the as for described substrate originally and protein was DOK1 partner(s). shown). binding PDGF-BB-stimulated not However, that (data on downstream suggesting further 1999). DOK1, effect act or might al., p130Cas they significant of et phosphorylation no tyrosine Noguchi had p130Cas to 2006; siRNAs and ( DOK1 Crk al., to knockdown proteins bind et to adaptor reported (Defilippi The been have complexes. NCK partners and non-functional binding manner, form DOK1 dominant-negative endogenous the to with a that competing show in by data possibly behaves these Furthermore, mutant one residues. either Ad.DOK1FF these bind that of protein(s) both signalling or the for role important an ti ieyta O1ascae ihohras-yet-unidentified other with associates DOK1 that likely is It ora fCl cec 21)17 6725 doi:10.1242/jcs.135988 2647–2658 127, (2014) Science Cell of Journal 0)o ihrCko C ihtargeted with NCK or Crk either of ,80%) aaso h mean the show Data colocalisation. of coefficient Pearson’s as expressed three experiments from independent data represents (lower and shown panel) is Methods) and in Materials described (as colocalisation phospho- p130Cas and DOK1 of Quantification treatment. PDGF-BB upon increased colocalisation of areas and regions membrane indicate three Arrows least experiments. at separate of representative (yellow) are p130Cas and phospho and DOK1 co-staining of show images (upper Merged red panel). in shown phospho- (Y410) and p130Cas green in shown DOK1 staining with Methods, in and described Materials as Confocal performed min. was 5 imaging for PDGF-BB or ng/ml free) 50 (serum control SFM with treatment for SFM and in coverslips incubated glass onto seeded were p130Cas. phosphorylated and DOK1 of in colocalisation increase PDGF-BB-mediated 5. Fig. oprdwt F oto (two-tailed Student’s control SFM with compared t-test). 6 , ...* s.e.m. 8hpirto prior h 18 8M cells U87MG P , 0.05, 2653

Journal of Cell Science EERHARTICLE RESEARCH 2654 for SFM in incubated were cells post-transfection, h 48 At nM). 25 (siScr, siRNA activation. scrambled Rap1 control for with or required nM) are 25 p130Cas (siDOK1, and DOK1 6. Fig. ofun)wr netdwt dLc,A.O1o dDKF tMI f20 t4 fe neto,clswr nuae nSMfor SFM in incubated were cells infection, after h 48 At 200. of MOIs ( at control Ad.DOK1FF SFM or Ad.DOK1 with Ad.LacZ, with infected were confluent) a efre ydnioer sn mgJ aafo he needn xeiet r rsne sRp–T eaieuis(U n hwthe show and (RU) units relative levels Rap1–GTP Rap1–GTP as of presented Quantification are antibodies. experiments indicated independent the three with from probed Data and ImageJ. blotted using were mean densitometry samples by extract performed whole-cell and was Rap1–GTP Methods. and Materials ( control vehicle containing nlsduigaRp ciainasya ecie nMtrasadMtos uniiaino a1GPlvl a efre sfrA * A; for as performed was levels Rap1–GTP of Quantification Methods. and Materials PDGF-BB. in plus described Ad.LacZ as assay with activation Rap1 a using analysed ** quantified. was phosphorylation dLc,A.10a rA.10a1Fa Oso 5.A 8hatrifcin el eetetda ecie bv n uniiaino a1GPlevels Rap1–GTP of quantification and above described as treated were cells infection, after * h A. 48 At for 250. as of performed MOIs was at Ad.p130Cas15F or Ad.p130Cas Ad.LacZ, 6 ...Dt r omlsdt oa a1 ** Rap1. total to normalised are Data s.e.m. 2 r5 gm DFB +.Cl yae eete rprd lte n rbdwt h niae niois n p130Cas and antibodies, indicated the with probed and blotted prepared, then were lysates Cell (+). PDGF-BB ng/ml 50 or ) P 2 , r5 gm DFB + o i.Cl yae eepeae n sae sn a1atvto sa,a ecie in described as assay, activation Rap1 a using assayed and prepared were lysates Cell min. 5 for (+) PDGF-BB ng/ml 50 or ) .5(oprdwt iadsiuae Ad.LacZ); ligand-stimulated with (compared 0.05 P , .1(oprdwt dLc lsPG-B.()U7Gclswr rae si ,lsdi a1atvto ufrand buffer activation Rap1 in lysed C, in as treated were cells U87MG (D) PDGF-BB). plus Ad.LacZ with (compared 0.01 P , .1(oprdwt iadsiuae ic) B 8M el ( cells U87MG (B) siScr). ligand-stimulated with (compared 0.01 A 8M el eetasetdwt he needn iNstreigDOK1 targeting siRNAs independent three with transfected were cells U87MG (A) § P , .5(oprdwt dLc F oto) C 8M el ( cells U87MG (C) control). SFM Ad.LacZ with (compared 0.05 ora fCl cec 21)17 6725 doi:10.1242/jcs.135988 2647–2658 127, (2014) Science Cell of Journal , 0 ofun)wr netdwith infected were confluent) 80% , 8hpirt ramn ihSFM with treatment to prior h 18 , 8hpirt treatment to prior h 18 P , .5compared 0.05 , 80%

Journal of Cell Science EERHARTICLE RESEARCH ora fCl cec 21)17 6725 doi:10.1242/jcs.135988 2647–2658 127, (2014) Science Cell of Journal irtn e il.* $ field. per migrating mean and the experiments show separate Methods. three and of representative Materials are transwell in Data a detailed in as used assay At were nM). migration cells 25 (siScr, post-transfection, siRNA h scrambled 48 control with (siDOK1, or DOK1 nM) targeting 25 siRNAs independent three with spheroids. U87MG PDGF-BB- invasion radial of for 3D required and are motility chemotactic Rap1 stimulated and DOK1 7. Fig. oto cabe iN 2 M.** nM). with (25 or siRNA nM) scrambled (25 control DOK1 targeting siRNAs independent ( control). SFM ehd.Dt r rsne sfrA * A. for and as Materials presented in are detailed Data used as Methods. were assay cells migration post-transfection, transwell h a scrambled in 48 control At with nM). or (25 nM) siRNA 25 siRNAs (siRap1, independent Rap1 three targeting with transfected were cells fRp n AD xrsinaeson(left-hand shown are ** expression panel). GAPDH levels and Representative Rap1 nM). of with (25 or siRNA nM) scrambled (25 control Rap1 targeting siRNAs independent ( PDGF-BB. ** 200. at of Y398F MOIs Ad.DOK1 or Y362F Ad.DOK1, Ad.DOK1 Ad.LacZ, Ad.DOK1FF, with infected were confluent) ihsSrpu DFB) ( PDGF-BB). plus siScr with cmae ihsiScr); with (compared nt R)adso h mean the show area and relative (RU) three as units four least presented least at are at from experiments for Data independent core condition. the per of spheroids area different the (red rim minus invasion line) the dashed to corresponding area 4% by the in determined measuring fixed was were invasion Spheroids and h. paraformaldehyde 48 additional SFM an in for incubated ( and control gel vehicle collagen containing spheroids a production, in spheroid imbedded after were h and 24 Materials At to in Methods. used described were as cells spheroids of generate amounts equal and suspended ( C–E P , .5(oprdwt ic F oto) ( control). SFM siScr with (compared 0.05 t2 otifcino rnfcin el eere- were cells transfection, or infection post h 24 At ) P , E .1(oprdwt ic lsPDGF-BB). plus siScr with (compared 0.01 8M el eetasetdwt three with transfected were cells U87MG ) C 6 P 8M el eetasetdwt three with transfected were cells U87MG ) , ... xrse stenme fcells of number the as expressed s.e.m., .1cmae ihA.aZplus Ad.LacZ with compared 0.01 P , $ ( .1(oprdwt siScr); with (compared 0.01 A P 8M el eetransfected were cells U87MG ) , 2 .5(oprdwt siScr with (compared 0.05 D r5 gm DFB (+) PDGF-BB ng/ml 50 or ) 8M el ( cells U87MG ) 6 s.e.m. P , .1(compared 0.01 P , 0.01 , B 80% U87MG ) 2655

Journal of Cell Science CAACAGTGTA-3 AAAGTCAAAGATCAATGTTAA-3 eeprhsdfo ign(rwe,U) lSasngtv control; negative AllStars 5 UK): (Crawley, siDOK1-3, Qiagen from purchased were infcnl dac u nesadn fDK inligand signalling DOK1 of Rap1 function. understanding and motility our p130Cas involving advance findings cell pathway significantly Our signalling glioma novel motility. PDGF-BB-mediated a cell through regulates tumour DOK1 of regulation important that positive these the investigations. future in of of basis role the be precise will cell residues the and tyrosine activation Understanding Rap1 multi- for for a motility. that required required form the complex to is to signalling proteins indicating due protein 398 DOK1-binding likely specific is Tyr invasion, This of or signalling. requirement PDGF-BB 362 in radial Tyr function DOK1 either 3D of phosphorylation and tyrosine p130Cas phosphorylation PDGF-BB-stimulated decreased significantly ARTICLE RESEARCH 2656 of concentration siRNA final using a by nM. transfected with 25 were (Invitrogen), confluence 60% 2000 at Lipofectamine cells glioma U251 and U87 transfection siRNA 5 siDOK1-2, N- DOK1 (Y398), phospho-DOK1 against phospho-PDGFR- (E-16), andterminus Antibodies (Y416) MA). phospho-Src (Danvers, phospho-p130Cas (T202/Y204), (Y402), phospho-ERK phospho-PDGFR- phospho-PYK2 ERK, PYK2, (Y410), against Antibodies siRNA and reagents Antibodies, phosphatase and protease at spun with then were homogenates buffer The 10,000 of sonication. RIPA using Department (Roche) in inhibitors the homogenised Samples into processing. tissue were transferred for dissected were immediately they was where Neuropathology, and cells or and post-mortem theatre operating tissue the in and at of obtained 08/0077 were (LREC use biopsies Committee Neurosurgical Ethics 02/N093). The Hospital National HTA 12198). the (UK; by Act and approved Tissue 12054 Human the The numbers by intervention. governed Licence surgical is tissue the human before of consent storage informed gave patients All biopsies surgical of Derivation 10% containing DMEM in (1:100; Sigma). penicillin-streptomycin cultured with P4333, supplemented were (FCS) cells serum calf glioma fetal U251 and U87 culture Cell METHODS AND MATERIALS Wrigo,U) iO11 5 siDOK1-1, UK): (Warrington, (Bristol, UK). Bioscience (London, Peprotech Tocris Fak/Pyk2 from purchased from the was purchased PDGF-BB and UK). all PP1 were 1-naphthyl PF573228 inhibitor inhibitor Src/Abl the Src SU6656, The inhibitor UK). (Paisley, and Invitrogen from anti-rabbit-IgG were Alexa-Fluor-555–phalloidin donkey donkey Alexa-Fluor-486-conjugated Alexa-Fluor-546-conjugated UK). anti-goat-IgG, (Paisley, (Y397) Invitrogen phospho-FAK from against ECM antibody was from and was KY) (Y362) (Versailles, phospho-DOK1 Biosciences against Antibody UK). and (Oxford, Taiwan) Laboratories Transduction City, BD (Taipei from Abnova was antibody monoclonal from Biotechnology.anti-p130Cas was Cruz UK), DOK1 (Cambridge, Santa total Abcam against from from antibody was also C-terminus DOK1 were the IgGs against Antibody rabbit and goat against Santamouse, antibodies from Secondary were Germany). (TU-02) (Heidelberg, Biotechnology tubulin and Cruz (V-18) GAPDH Rap1, (A-17), FAK kit. assay protein Bio-Rad the using measured was supernatant h olwn iNswr ucae rmApidBiosystems Applied from purchased were siRNAs following The hsi h is td lal salsigarl o DOK1 for role a establishing clearly study first the is This g o 0mn n h rti ocnrto fteresulting the of concentration protein the and min, 10 for 9 -GGATGCATGGTGGTGCCAA-3 9 -CCGCCTGGACTGCAAAGTGAT-3 b 9 ;siRap1A-3,5 Y5)wr rmCl inln Technology Signaling Cell from were (Y751) 9 -CCCAACGATAGAAGATTCCTA-3 a Y5) PDGFR- (Y754), 9 9 -GGGCCTTTATGATCTGCCT-3 ;siRap1A-2,5 9 h olwn siRNAs following The . 9 a 9 -AAGTCGATTGC- ;siRap1A-1,5 C2) PDGFR- (C-20), 9 b 9 9 . - ; , A.O1 rDKF A.O1F tamlilct finfection of DOK1 multiplicity a (Ad.LacZ), 200. at LacZ of (Ad.DOK1FF) either (MOI) DOK1FF expressing or adenovirus (Ad.DOK1) with infected eesdfo h ed pnteadto f2 of addition the upon beads the from (GE was Rap1–GTP released Rap1. GTP-bound beads precipitate to glutathione–Sepharose probe incubated bead-bound the were to with Lysates UK). coupled Buckinghamshire, binding Sciences, was Life GST–Rap1 Healthcare complete RalGDS The inhibitors]. of EDTA-free phosphatase domain and (TCEP), inhibitor tris-(2-carboxyethyl)phosphine protease mM 1 eesa eemndb etr ltaayi ftewoecl lysate. whole-cell the of analysis blot Rap1 western total by to determined normalised as and samples levels pulldown on out carried was blotting h eutn ero’ ofiin fclclsto a determined. and was analysed, colocalisation were of channels Cordelie coefficient p130Cas and Pearson’s Y410 resultant (Bolte and the JACoP DOK1 plugin the (Y410) ImageJ Both imaging 2006). p130Cas the using phosphorylated sequential upright quantified and SPE2 using was DOK1 LEICA of software a Co-localisation LEICA-LAS capture. using performed running was microscope imaging Confocal PBS. aie(B-)fr1ha omtmeaue eoebigpoe ihthe with 4 probed at being incubation before overnight temperature, by room antibody at primary h 1 phosphate-buffered in for Tween-20 (PBS-T) (v/v) saline 0.1% and milk with dry non-fat blocked (w/v) were 5% Membranes (Invitrogen). membranes (PVDF) polyvinylidene fluoride onto Bis-Tris electrotransfer 4–12% by complete on followed SDS-PAGE Invitrogen), EDTA, (Nupage, by gels analysed mM were 5 and lysates (Sigma), NaCl, cell II resulting and mM the I 150 inhibitors phosphatase X-100, and (Roche) Triton inhibitor protease Tris- 1% mM 7.5, 50 pH containing solution HCl a in lysed were cells immunoblotting, For Immunoblotting instructions: manufacturer’s the to according CTG-3 mutagenesis, multi-site-directed used 5 for Y362F, and used UK). Multi- were Cheshire, QuikChange designed primers Technologies, the indicated following (Agilent using the The Kit vector, generated Mutagenesis pENTR3C were DNA Site-Directed the DOK1FF by into in verified cloning mutations to were Prior and sequencing. Invitrogen) GATEWAY (pAd/CMV/V5-DEST; the using Y398F), generated vector were (Y362F, Y398F DOK1 DOK1FF and Y362F DOK1, DOK1 wild-type expressing Adenoviruses infection and construction Adenoviral K eeisre noa2-elpae eu-remedium Serum-free plate. 24-well a into inserted Oxford, Biosciences, 2011). BD were al., (Falcon; et inserts (Evans UK) culture previously cell described transwell as Briefly, performed was assay This assay migration chemotactic Transwell MgCl assay. mM buffer activation activation 5 NP-40, in 1% glycerol, specific harvested 10% Tris-HCl, and mM a indicated [50 as using treated were determined cells U87MG were levels Rap1–GTP assay activation Rap1 4% in fixed performed were incubations were in Antibody permeabilisation min. by 30 4 cells at followed for overnight min X-100 60 Triton staining, for 0.2% PBS in immunofluorescent paraformaldehyde imaging For confocal and staining Immunofluorescent im ihaclbainsrpadaayi ydnioer sn ImageJ using the densitometry by following Health). analysis of of and scanning Institutes UK), strip (National by calibration Chalfont, a quantified using with Little were films detection Immunoblots Healthcare, protocol. and (GE manufacturer’s Biotechnolgy) (HRP)-linked reagents peroxidase horseradish (Santa-Cruz ECL a with antibody temperature room secondary at h 1 for Qikie dnvrsTtrImnasykt elBoas San at Biolabs, chloride immunoassay stored Cell caesium by was kit; determined Adenovirus by Immunoassay was CA). and Titer cells purified titre Diego, Adenovirus HEK293A virus recombination, were host the (QuickTiter by into particles and transfection centrifugation, Viral generated by produced (Invitrogen). were was adenovirus Invitrogen) V5-DEST; TACAACCCT-3 9 38,5 Y398F, ; ora fCl cec 21)17 6725 doi:10.1242/jcs.135988 2647–2658 127, (2014) Science Cell of Journal 9 -CCCAAAGAGGATCCCATCTTTGATGAACCTGAGGGC- ˚ n1 oiesrmabmn(S) .%Ten2 in 20 Tween 0.1% (BSA), albumin serum bovine 1% in C 9 O1aeoia xrsinvcos(pAd/CMV/ vectors expression adenoviral DOK1 . 9 -CGGGTGAAGGAGGAGGGCTTTGAGCTCCCC- 2 20 ˚ ,floe yincubation by followed C, ˚ 6 .U7Gclswere cells U87MG C. D ufr Western buffer. SDS 2 0 MNaCl, mM 100 , ` res, TM

Journal of Cell Science iclrdsac rmteeg ftecr oteeg fcontiguous of edge the to core the 2007). al., of the et was as (Stein edge the determined cells was the and invading invasion invasion core from of spheroid Spheroid rim distance The the circular ImageJ. formaldehyde. of using area invasion 4% of circular rim the h, in measuring 48 for by fixation invade determined to allowed by were Spheroids followed (+). PDGF-BB ng/ml 50 plus f1%FSi MMadmtycluoe peod eepoue by produced were Spheroids methylcellulose. 100 and pipetting DMEM in FCS 10% of otmdnntsu-utr lt n nuaigfr2 (37 h 24 for incubating and plate non-tissue-culture bottomed neeic nee graticule. indexed eyepiece an eecutdi orrno ilsprwl t20 at membrane well the West per of Immucor, side fields lower random (IBG the four kit to in migrated counted Quik-Diff had were Reastain that Cells a were UK). with membranes Sussex, before stained removed were and membrane the fixed of side upper the to oa DF eesadeullaigwsasse ywsenblotting. a western by using nm. assessed 595 was immediately, of loading correction equal determined wavelength and a levels was with PDGFR nm Total well 450 to each set reader microplate of absorbance The dH either with supplemented was The solution (Nutacon, I protocol collagen manufacturer’s The the Netherlands). dilution with by mg/ml) accordance PureCol) (2.1 in mg/ml; DMEM, plugs (3.1 in I I collagen collagen bovine fibrillar in from embedded prepared and collected were Spheroids el/eli eu-reDE)wr de otetpcabrand chamber top the to added (1.5 the were suspension 37 to in at DMEM) incubated cells added serum-free glioma was U251 in vehicle or cells/well or U87 and PDGF-BB chamber, without bottom or with supplemented ARTICLE RESEARCH aaadwoeteppr .. .. .M n ..promdteexperiments. the performed M.Y. and C.PM. G.B., A.F., the paper. analysed the experiments, wrote the and performed data study, the designed P.F. and I.M.E. A.B., contributions Author interests. competing no declare authors The interests Competing or (ANOVA) variance of performed P was analysis analysis representing Statistical bars two-way (s.e.m.). error mean by with the means, of are error graphs standard the the on displayed data The analysis Statistical Na 10% activated NaCl, mM mM 1 137 and 8.0, EDTA pH Tris-HCl mM mM 2 20 glycerol, P40, 100 Nonidet 1% adding diluent: before blocked times and 7.2) three 300 pH with PBS, h in four 2 20 washed for Tween was (0.05% well buffer each washing day, with next times The temperature. room at incubation 4 100 of a to concentration tyrosine- diluted was the working antibody only capture detect The HRP. to using used receptor, After phosphorylated antibody was tyrosine detection PDGFR. phosphorylated HRP-conjugated for nonphosphorylated an specific material, unbound and away phosphorylated recognises washing PDGFR tyrosine human for specific both antibody capture immobilised activity An PDGFR for assay ELISA previously described as cells cells siRNA period, transfection 3 U87 and as and and infection trypsinised the on were Following infection technique 2011). out al., Virus et metho-cellulose (Evans carried 2004). the were (Augustin, transfection using previously generated described were Spheroids assay invasion spheroid 3D H72 3 MNC,00%Ten2 n .%BAwsadded 50 was with incubation BSA by three followed after 0.1% Finally, min, h. and 20 Tris-HCl 2 20 further 100 a mM washes, Tween for 20 incubation further before 0.05% well, in the NaCl, to recommendations directly mM 137 manufacturer’s 7.2, h the pH 2 for to incubate to 100 left Then, diluted again. was washed plate and The temperature blank. room a at as used was protein without ( 2 , r5 gm DFB +.Puswr vradwt F ( SFM with overlaid were Plugs (+). 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V.M., reagents. equally. contributed G.B. and A.F. basn . oebu,G n eh,I. Pecht, and G. Rozenblum, J., Abramson, References at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.135988/-/DC1 online available material Supplementary material (S.B.). Supplementary for scheme Institute funding National Centre’s Health’s of Research Department Biomedical UK (NIHR) the received Research who from Health UCLH/UCL Tumour funding at Brain of undertaken the proportion was work and a This C.P-M.), 8-128). grant and Grant project G.B. (NL; M.Y., and Charity (I.E., RG/06/003 I.C.Z. grant to programme PG/12/65/29840 Foundation and Heart Biological (A.B. P.F. British and to a Biotechnology BB/K013068/1) A.F.), and the (BB/G017921/1 from Council grants Research by Sciences supported was work This Funding art,A,Ple-ay . ahr,I . vn,I .adFakl P. Frankel, and M. I. Evans, C., I. Zachary, C., Pellet-Many, A., Barrett, G. H. Augustin, egr .H,Nk,M,Mrti . alr .S,Sci .D,Vae A., Viale, D., N. Socci, S., B. Taylor, A., Morotti, M., Niki, H., A. Berger, vn,I . aai . rto,G,Ple-ay . oke . ahr,I C. I. Zachary, C., Lockie, C., Pellet-Many, G., Britton, M., Yamaji, A., M., Pincini, I. Evans, D., Repetto, B., Bisaro, G., Tornillo, P., M. Leal, P., Stefano, Di S. Cabodi, and P. Stefano, Di P., Defilippi, apn,N,Wsiwk,D,Srf,A,Mrhk . oaah,R,Stillman, R., Kobayashi, D., Marshak, A., Strife, D., Wisniewski, N., Carpino, L. Aelst, Van and B. Boettner, ai,A,Szk,S . ekr . oda,J . rc,J .adCnl,P. Canoll, and N. J. Bruce, E., J. Goldman, M., Weiker, O., S. Suzuki, A., Farin, ot,S n Cordelie and S. Bolte, edn .H n etrak B. Westermark, and H. C. Heldin, J. F. Zwartkruis, and W. E. Frische, aaa,D n eneg .A. R. Weinberg, and D. Hanahan, oe,A .adCos .C. N. Cross, and V. A. Jones, M. Ichihashi, T., Matozaki, T., Horikawa, H., Nagai, T., Noguchi, T., Hosooka, ece,P . ahaoa . lne . rgie . ead .C,Ghani, C., M. Renaud, J., Gregoire, M., Plante, M., Bachvarova, L., P. Mercier, Y. Yamanashi, and T. Tezuka, Y., Hishida, R., Mashima, C., Pinheiro, A., Martins, B., M. Lambros, A., Longatto-Filho, O., Martinho, R. D. Clemmons, and J. Badley-Clarke, A., L. Maile, Y., Ling, aDnl,J .adGoh D. Ghosh, and W. J. MacDonald, D. M. Resh, and B. V., Clarkson, Shivakrupa, A., Pouchkine, Strife, D., J., Wisniewski, X., Auclair, Liang, O., Destaing, F., Saltel, C., Andrieu, S., Lee, 10a:akysgaln oei elhaddisease. and health in node signalling key a p130Cas: receptor. Fcepsilon 1 type Springer. the by mediated signaling Immunol. J. in involved are nsgaignetworks. signaling in suppressors. al. tumor et lung B. P. as Rothman, genes N., DOK Motoi, of J., Szoke, C., Brennan, hshrlto sesnilfrgot atrdpnetmgaino glioma of migration factor-dependent growth cells. endothelial for and and essential migration is P. phosphorylation cell Frankel, in and hubs molecular as cells. cancer al. p130CAS of et invasion and S. Cabodi, p140CAP E., Turco, proteins N., Sharma, E., Santopietro, A-soitdpoeni hoi ylgnu ekmapoeio cells. progenitor leukemia myelogenous 88 chronic in protein GAP-associated B. Clarkson, and B. signaling. 20) rnpatdgim el irt n rlfrt nhs brain host on proliferate and migrate analysis. cells dynamic a glioma vasculature: Transplanted (2006). ooaiainaayi nlgtmicroscopy. light in analysis colocalization ltltdrvdgot factor. growth platelet-derived generation. GTPases. like rwhfco receptors. factor growth Dok-1. of mutants negative dominant 5437-5446. by cells melanoma M. Kasuga, and . et,B,Biai .adBcvrv D. Bachvarov, and I. Bairati, Teˆtu, B., K., signaling. immunoreceptor in adapters gliomas. growth al. family in platelet-derived et PDGFA ligand F. of its Milanezi, analysis and A., (PDGFRA) number 101 Mackay, A copy receptor J., and factor Amorim, mutation F., Expression, Pardal, A., Silva, nui-iegot atrIsgaigi utrdvsua mohmsl cells. muscle regulates Bioinformatics smooth thereby vascular and cultured in integrin Chem. signaling Biol. alphaVbeta3 I J. the factor to growth insulin-like binding SHP-2 mediates for signaling. c-Kit required in are Dok-1 kinases of Chem. family recruitment membrane Src and and phosphorylation 3-kinase Phosphatidylinositol gamma or (2002). IL-1, al. TNF, to et response P. in Jurdic, serines R., radiation. Dok1 Kobayashi, phosphorylates beta B., kinase Salaun, J., Michelon, 197-204. , 973-982. , ora fCl cec 21)17 6725 doi:10.1242/jcs.135988 2647–2658 127, (2014) Science Cell of Journal 277 rc al cd c.USA Sci. Acad. Natl. Proc. ur pn elBiol. Cell Opin. Curr. 13732-13738. , Cell 33 e.Biol. Dev. 22 3151-3158. , 280 85-91. , 20) ehd nedteilcl ilg.Bri;NwYork: New Berlin; biology. cell endothelial in Methods (2004). 144 20) niiino h oiiyadgot fB61 mouse B16F10 of growth and motility the of Inhibition (2001). 2950-2951. , 21) erpln1sgaigtruhp3Cstyrosine p130Cas through signaling Neuropilin-1 (2011). 646-674. , 19) 6(o) osiuieytyrosine-phosphorylated, constitutively a p62(dok): (1997). o.Cl.Biol. Cell. Mol. rnsCl Biol. Cell Trends `res,F.P. subcellular into tour guided A (2006). el o.Lf Sci. Life Mol. Cell. m .Cn Res. Can. J. Am. 340 20) oto fcl deindnmc yRap1 by dynamics adhesion cell of Control (2009). 1-9. , 20) noei eiaie fplatelet-derived of derivatives Oncogenic (2004). hso.Rev. Physiol. 21 20) OA–cne ule rfl analysis. profile outlier cancer – COPA (2006). 19) ehns fato n nvv oeof role vivo in and action of Mechanism (1999). Glia 684-693. , 21) a1 ecnr mn h Ras- the among mercenary a Rap1, (2010). 31 101 21) alak fcne:tenext the cancer: of Hallmarks (2011). 53 1174-1185. , 16 799-808. , 17416-17421. , 20) 10a:avraiescaffold versatile a p130Cas: (2006). 257-263. , .Microsc. J. 21) hrceiaino O1 a DOK1, of Characterization (2011). 1 20) o rti aiymembers family protein Dok (2003). a.Genet. Nat. 61 79 663-673. , muo.Rev. Immunol. 2912-2923. , 1283-1316. , el Signal. Cell. 224 20) h oe fDok of roles The (2009). 42 216-223. , 21) Identification (2010). 213-232. , 21) h adaptor The (2011). o.Cl.Biol. Cell. Mol. 232 20) IkappaB (2004). 20) DOK1 (2005). 273-285. , r .Cancer J. Br. 25 766-777. , (2009). (2013). .Biol. J. 2657 Eur. Cell 21 ,

Journal of Cell Science eltMn,C,Fakl . vn,I . ezg . Ju¨ B., nemann-Ramı Herzog, M., I. Evans, P., Frankel, C., Pellet-Many, ti,A . euh . oly . ees .adSne,L M. L. Sander, and M. Berens, D., Mobley, T., Demuth, J., M., E. A. Ung, Stein, M., Iwanicki, W., R. Tilghman, H., K. Martin, K., J. Slack-Davis, Y., Kitamura, K., Fukunaga, M., Tsuda, K., Inagaki, T., Matozaki, T., Noguchi, ei,Y . Antrobus, A., Y. Senis, hh .H n oln,E C. E. Holland, and H. A. Shih, EERHARTICLE RESEARCH 2658 ucecl irto n inligvap130Cas. via signalling and migration cell muscle C. I. Zachary, ellrcaatrzto fanvlfclahso iaeinhibitor. kinase adhesion al. focal et G. novel W. Roberts, a C., Chem. J. of Kath, B., characterization Cooper, Cellular J., M. Luzzio, C., Autry, M. role Kasuga, possible insulin: and and migration. adhesion Y. cell cell Yamanashi, by in induced p62(Dok) K., of phosphorylation Shii, T., Kitamura, 438-453. ahmtclmdlo lolsoatmrshri naini three- a in invasion spheroid tumor experiment. glioblastoma vitro in of dimensional model mathematical la tumorigenesis. glial and Dok-1 interactions. of SHIP-1 to phosphorylation leading Dok-3 Src-kinase-independent reveals signaling outside-in Watson,S.P.andGarcı cancer. ovarian epithelial in gene, suppressor tumor candidate 282 14845-14852. , 21) erpln1mdae DFsiuaino aclrsmooth vascular of stimulation PDGF mediates Neuropilin-1 (2011). MOJ. EMBO acrLett. Cancer . eei,S,Parguin S., Severin, R., a A. ´a, 18 20) ltltdrvdgot atr(DF and (PDGF) factor growth Platelet-derived (2006). 20) rtoi nlsso nernalphaIIbbeta3 integrin of analysis Proteomic (2009). 1748-1760. , ipy.J. Biophys. 232 .Trmoi n Haemostasis and Thrombosis J. 139-147. , 92 ,A . oa . izan N., Zitzmann, I., Rosa, F., A. a, ˜ 356-365. , ice.J. Biochem. 435 19) Tyrosine (1999). o.Oncol. Mol. 609-618. , 7 1718-1726. , rz .and M. ´rez, 20) A (2007). .Biol. J. (2007). 5 , ho . cmt,A . i,Y,D rsoao . adli .P n Van and P. P. Pandolfi, A., Cristofano, Di Y., Qin, A., A. Schmitz, M., Zhao, Lo¨ B. H., wenberg, Mano, P., M. Amelsvoort, E., Akker, Den van B., T. Dijk, and van K. Hirata, W., Ikeda, T., Hara, Y., Nagamatsu, Y., Rikitake, M., Takahashi, aaah,Y n atmr,D. Baltimore, and Y. Yamanashi, Shah, J., Field, L., G. Zhou, A., S. Johnson, J., Meisenhelder, J., E., P. Woodring, Sahai, B., J. Wyckoff, L., A. Wells, K., Lapidus, S., Goswami, W., Wang, fp2dk sesnilfrisngtv feto ioe-ciae protein mitogen-activated on effect negative its for activation. essential kinase (MAP) is p62(dok) of L. Dok. Aelst, protein, docking a as protein 211. kDa 62 rasGAP-associated cells. hematopoietic in formation Blood complex Lyn/Tec/Dok-1 kinase-dependent M. Lindern, von cells. and NIH3T3 of edges leading at locally PDGF Y. Takai, . ld,F,Pwo,T,Nk,M,Pnof,P .e al. et P. P. spreading. Pandolfi, cell M., during Niki, filopodia promote T., 165 to Pawson, Dok1 F., phosphorylates Bladt, K., S. primary J. within cells Condeelis, carcinoma invasive and tumors. of signature mammary E. expression gene J. a of Segall, testing H., R. Singer, 493-503. , 96 ora fCl cec 21)17 6725 doi:10.1242/jcs.135988 2647–2658 127, (2014) Science Cell of Journal 3406-3413. , 20) hshioiie3kns-eedn ebaerecruitment membrane 3-kinase-dependent Phosphoinositide (2001). 20) eunilatvto fRp n a1salGpoen by proteins G small Rac1 and Rap1 of activation Sequential (2008). acrRes. Cancer 20) tmcl atridcspopaiyioio 39- phosphatidylinositol induces factor cell Stem (2000). .Ep Med. Exp. J. 64 8585-8594. , 19) dniiaino h b-and Abl- the of Identification (1997). 194 265-274. , ee Cells Genes 20) dniiainand Identification (2004). 13 549-569. , 20) c-Abl (2004). Cell .Cl Biol. Cell J. 88 205- ,

Journal of Cell Science Supplementary Figure S1A Disease summary for DOK1

Thresholds P-Value: 0.0004 Fold change: 2 Gene rank: Top 10%

Barrett, et al.

Journal of Cell Science | Supplementary Material Supplementary Figure S1B

DOK1 mRNA expression in TCGA data sets

Over-expression Gene Rank: 243/20,423 (in top 2%)

Over-expression Gene Rank: 1302/20,423 (in top 7%)

Over-expression Gene Rank: 2014/20,423 (in top 10%)

Over-expression Gene Rank: 2084/12,624 (in top 17%)

Barrett, et al.

Journal of Cell Science | Supplementary Material Supplementary Figure S2A

p-p130Cas (Y410)

PDGFRa PDGFRb Total p130Cas

b-actin b-actin p-ERK (T202/Y204)

ERK

Barrett, et al.

Journal of Cell Science | Supplementary Material Supplementary Figure S2B

PDGFRa PDGFRb

b-actin b-actin

Barrett, et al.

Journal of Cell Science | Supplementary Material Supplementary Figure S2C

DOK1 p130Cas (Y410) Merge

SFM & AG1296

PDGF-BB 5’ (50 ng/ml) & AG1296

Barrett, et al.

Journal of Cell Science | Supplementary Material upeetr iueS2C Figure Supplementary

Pearson's Coefficient 0.000 0.100 0.200 0.300 0.400 0.500 0.600 0.700 Journal ofCellScience AG1296 F & SFM | Supplementary Material DFB & PDGF-BB AG1296 art,e al. et Barrett, Supplementary Figure S3A

Total p130Cas

p-p130Cas (Y410)

Total DOK1

GAPDH

PDGF BB - + - + - + 5’ (50 ng/ml) siScr siDOK1 #1 siDOK1 #3

Barrett, et al.

Journal of Cell Science | Supplementary Material Supplementary Figure S3B

25 $

10 Number of cells migrated * * * * 5

0 - - - + - + PDGF BB + + - + 5’ (50 ng/ml) siScr DOK# 1 DOK1#3 RAP1#2 RAP1#3

Barrett, et al.

Journal of Cell Science | Supplementary Material Supplementary Figure S4

p-p130Cas (Y410)

Total p130Cas 1.4 $ $ 1.2

1.0 *

0.8

0.6 **

p130Cas Y410 (RU) **

0.4

Phospho 0.2

0.0 Total DOK1

GAPDH

PDGF BB - + - + - + - + - + 5’(50ng/ml) Ad.LacZ Ad.DOK1 Ad.DOK1FF Ad.DOK1 Ad.DOK1 (Y362F) (Y398F) Barrett, et al.

Journal of Cell Science | Supplementary Material Supplementary Figure Legends

Supplementary Figure S1: Oncomine Analysis: Disease Summary and TCGA analysis for DOK1. (A) The Oncomine database (OncomineTM, Compendia Bioscience, Ann Arbor,

MI). was queried for DOK1 expression in the available datasets based on the following analysis: cancer vs normal, cancer vs. cancer, and Cancer Outlier Profile Analysis (COPA).

Threshold values used were; Fold change=2 and p-value of 1E-4. The ‘red cells’ represents

DOK1 overexpression and the ‘blue cells’ represent DOK1 underexpression. The levels of expression are based on the gene rank percentile. (B) The Oncomine database was queried for

DOK1 mRNA expression within The Cancer Genome Atlas (TCGA) data sets indicated.

Individual cancers or comparisons, significance, fold change and gene overexpression ranking are listed for each cancer.

Supplementary Figure S2: PDGF-BB but not PDGF-AA mediates p130Cas tyrosine phosphorylation and PDGF receptor inhibition blocks p130Cas and DOK1 colocalisation. (A) U87MG cells were incubated overnight in serum-free medium and then treated for 10 min with serum-free medium (control), 30 ng/ml PDGF-AA or 30 ng/ml

PDGF-BB. Cells were then lysed, and the activity of either PDGFRα (left) or PDGFRβ

(right) was measured using a specific ELISA. Results are means ± S.E.M. obtained from three independent experiments each performed in triplicate. *P < 0.05 Serum Free versus plus

PDGF-BB. Similarly, total PDGFRs protein levels were assessed by Western blotting and equal loading was checked by assessing β-actin levels. (B) HCASMCs were incubated overnight in serum-free medium and then treated for 10 min with serum-free medium

(control), 30 ng/ml PDGF-AA or 30 ng/ml PDGF-BB. Cells were then lysed, and the activity of either PDGFRα (left) or PDGFRβ (right) was measured using a specific ELISA. Results are means± S.E.M. obtained from three independent experiments each performed in

Journal of Cell Science | Supplementary Material triplicate. *P < 0.05 Serum Free versus plus PDGF- AA or PDGF-BB. Total PDGFRs protein levels were assessed by Western blotting (sc-338 and sc-339 for PDGFRα and PDGFRβ respectively, Santa Cruz biotechnology, Inc.) and equal loading was checked by assessing β- actin levels (A5316, Sigma-Aldrich). (C) U87MG cells were seeded on glass cover slips and incubated in SFM for ~18h prior to treatment. Cells were then pre-treated with AG1296 at

10M or the vehicle (DMSO) for 30 minutes prior to treatment with SFM control (SFM) or treated with 50 ng/ml PDGF-BB for 5 minutes. Confocal imaging was performed as described in Materials and Methods, with DOK1 staining in green and Y410 phospho p130Cas in red. Merged images show co-staining of DOK1 and Y410 phospho p130Cas in yellow, and are representative of at least three separate experiments. Quantification of DOK1 and Y410 phosphop130Cas co-localisation (as described in Materials and Methods) is shown in the graphs and represents data from three independent experiments expressed as Pearson's

Coefficient of co-localisation (means +/- s.e.m). *p<0.05 compared to SFM Control by two- tailed student’s T-test.

Supplementary Figure 3: DOK1 is required in U251 cells for p130Cas tyrosine phosphorylation and DOK1 and Rap1 are required for PDGF-stimulated chemotactic motility. (A) U251MG cells were transfected with two independent siRNA targeting DOK1

(siDOK1) at a concentration of 25 nM, or 25 nM of a control scrambled siRNA (siScr). 48hr post transfection, cells were incubated in serum-free medium (SFM) for ~18hr prior to treatment with SFM vehicle control (-) or treated with 50 ng/ml PDGF-BB (+) for 5 minutes.

Cell lysates were then prepared, blotted, and probed with the indicated antibodies. The blots shown are representative of three independent experiments. (B) U251MG cells were transfected with two independent siRNA targeting DOK1 (siDOK1) or Rap1 (siRap1) at a concentration of 25 nM, or 25 nM of a control scrambled siRNA (siScr). 48hr post

Journal of Cell Science | Supplementary Material transfection, cells were used in a Transwell migration assay as detailed in Materials and

Methods. Values (n ≥ 3) are means ± s.e.m, expressed as the number of cells migrating per field; *p<0.01 compared to siScr, $ p<0.01 compared to untreated control (-).

Supplementary Figure 4: Tyrosine 362 and 398 on DOK1 are required for PDGF stimulated tyrosine phosphorylation of p130Cas and 3D radial invasion of U87MG spheroids. U87MG cells (~80% confluent) were infected with Ad.LacZ, Ad.DOK1,

Ad.DOK1FF, Ad.DOK1 (Y362F) or Ad.DOK1 (Y398F) at MOIs of 200 48 h after infection, cells were incubated in SFM for ~18hr prior to treatment with SFM control (-) or treated with

50 ng/ml PDGF-BB (+). Cell lysates were then prepared, blotted, and probed with the indicated antibodies, and p130Cas phosphorylation was quantified. **p<0.01 compared to

Ad.DOK1 plus PDGF. $p<0.05 compared to SFM control (-).

Journal of Cell Science | Supplementary Material