rga,Uiest fWsigo,Sate A915 USA. 98195, WA Seattle, Washington, of University Program, 2dSre,Bleu,W 80,USA. 98005, WA Bellevue, Street, 32nd ariwAeu ,Sate A919 USA. 98109, WA Seattle, N, Avenue Fairview naiTeckchandani Anjali cell Src-dependent inhibit to transformation Cas destabilizes 5 Cullin ARTICLE RESEARCH ß eevd2 aur 03 cetd3 coe 2013 October 30 Accepted 2013; January 24 Received " Research Cancer USA. 98109, Hutchinson WA Fred Seattle, Division, Center, Research Clinical address: *Present often are or polypeptides 1 CRL mono- encoding or specificity. inhibit stimulates include substrate can their Consequently, substrates CRLs on depending CRL so CRL to transformation , stimulate degradation. substrate anti-oncogenic the for and a targets oncogenic cases, 26S most to the in adaptors. that, substrate-specific binding polyubiquitylation alternative of number and Substrate large Joazeiro, RING a cullin a and of a between bridge one contains (Deshaies a CRL forms Each that backbone 2005). protein largest Deshaies, and E3 the Petroski 2009; of constitute class complexes known (CRL) Cullin–RING– INTRODUCTION Cul5, 5, Cas Cullin Src, Ubiquitin, Transformation, Migration, that WORDS: KEY show by ruffling Src–Cas-induced results transformation of SOCS6. cell Our through inhibition including epithelial transformation. pathways, suppresses several of a Cul5 aspects of endogenous use other or but SOCS6 ruffling, not membrane of stimulates transformation mutant knockdown Cas the Either degradation-resistant for cells. necessary Cul5-deficient is of Cas degradation. proteasome- consequent to dependent and SOCS6 binding protein Src adaptor Cas Cul5-CRL and (Crk- stimulates the p130Cas Cul5 Phosphorylation not substrate that substrate). Src is is the associated found alone of Src We degradation activation stimulate Src. transformation. Src together cause kinase but to tyrosine cells, sufficient endogenous Cul5-deficient are in the which activated on dysmorphogenesis, dependent colony cells all and epithelial dynamics migration, and membrane growth mammary growth-factor-independent stimulates Removal pathway. Cul5 signaling that of Src–Cas a by transformation report prevent (Cul5-CRLs) to kinase complexes we protein cullin-5–RING–E3-ubiquitin-ligase terminate require Here, to mechanism signaling. irreversible an degradation provides and ubiquitylation protein Phosphorylation-dependent ABSTRACT rga,Uiest fClrd,Dne,Clrd 01,USA. 80217, Colorado Denver, Colorado, of University Program, *uhrfrcrepnec ([email protected]) correspondence for **Author lxne .Strait A. Alexander iiino ai cecs rdHthno acrRsac etr 1100 Center, Research Cancer Hutchinson Fred Sciences, Basic of Division hs uhr otiue qal oti work this to equally contributed authors These 04 ulse yTeCmayo ilgssLd|Junlo elSine(04 2,5950doi:10.1242/jcs.127829 509–520 127, (2014) Science Cell of Journal | Ltd Biologists of Company The by Published 2014. 1,§ 1, " { n oahnA Cooper A. Jonathan and rsn drs:Booto ytm,182SE 12822 Systems, Biocontrol address: Present ereS Laszlo S. George , § rsn drs:Boeia Sciences Biomedical address: Present 2 oeua n ellrBiology Cellular and Molecular 1, * , " eg Simo Sergi , 1,2, ** ´ 1 ih hrfr eslce eas hi osihbt unvrof signaling. oncogenic turnover stimulates inhibits and loss proteins their pY because selected be therefore might of silencing genetic heterozygosity and of loss Lai mutations, kinases cancers Loss-of-function 1994; human Tyrosine 2009). in 2006). al., activated (Hunter, frequently al., are et et and oncogenic, Wang Hampton be proteins 2010; to 2003; al., tend al., et oncogenic Sasi et 2010; al., Fay include et 2008; substrates al., suggesting et cancer, CRL (Elliott human of SOCS–Cul5 types some that in decreased is proteins nw hte u5tresocgncpoen rtumor or proteins infection. oncogenic virus not of targets absence is the it in Cul5 However, suppressors suppressor, viruses. whether tumor these Cul5 by 2010). known Zhou, oncogenesis the and mediates (Lee to thus p53 of bind herpesvirus degradation that inducing thereby overexpressed. and adaptors Kaposi’s-sarcoma-associated or Cul5 Cul2 encode amplified and Cul1, commonly several to Adenoviruses is ubiquitylates which suppressors, oncoproteins example, Cul4A, tumor For target whereas 2010). respectively, which Cul3 VHL Zhou, Fbw7, Keap1, in and mutations and loss-of-function (Lee have often cells cells cancer cancer in altered nldn nui,Selfco n G.Tefl opeiyof complexity factors full unknown. The growth is EGF. substrates peptide and CRL and SOCS–Cul5 factor Steel cytokines insulin, by including tissues signaling family many inhibit in remaining expressed and The widely They mechanisms. are chemokines. SOCS4–SOCS7, Cul5-dependent members, and as Cul5-independent well cytokines by leukocytes signaling as by kinase in tyrosine induced family studied JAK inhibit intensively are been they SOCS1– have family these, where CisH SOCS-SH2 Of eight and CisH. are al., and SOCS3 There (pY) SOCS1–SOCS7 et 2001). named (Okumura phosphotyrosine al., members, polyubiquitylation et containing Pawson for 2012; Cul5-CRL substrates to target degrons their thus understanding can and substrates hindered specific has significance. complexity of biological This substrates. discovery in of domains, the variety substrate-interaction a These bind WD40 2012). turn, domains, domains al., Tubby-like et their and SH2 (Okumura repeats to Ankyrin as according domains, SPRY classified such motifs, be domains, can which 40 substrate-interaction EloB approximately proteins, encodes box binds genome mammalian SOCS Kamura also The 2009; 2004). in EloC al., al., resulting et et 2002). (Rnf7), (Huang Rbx2 al., N- CRLs protein the RING adaptor–EloBC–Cul5–Rbx2 et binds and Cul5 Tceb1) (Kile and (EloC, (Tceb2), Cul5 C elongin of protein, box terminus SOCS linker a a called binds sequence al., a et that contain (Kile adaptors substrates Cul5 select Most to 2002). uses it proteins adaptor alternative haiShah Khyati , u5rsmlsohrcli rtisi h ag ubrof number large the in proteins cullin other resembles Cul5 xrsinof Expression and domains SH2 contains proteins box SOCS of family One 1, CUL5 { ais Pilling Carissa , n eea ee noigSOCS-SH2 encoding genes several and CUL5 n OSgnsi acrcells cancer in genes SOCS and 1,2 , 509

Journal of Cell Science htCl upesstetasomto feihla el by cells epithelial substrates degradation. of Src for unidentified transformation other and Cas the phosphorylated targeting suppresses suggest results Cul5 The not phenotype. thusthat Cul5-deficient but the of and ruffling of expression aspects membrane or other residues SOCS6 stimulates tyrosine of Cas Removal specific degradation-resistant Cas. of at turnover phosphorylated stimulates in is when required Cas is binds Cas SOCS6 It adaptor proliferation Cul5 2000). 2006; The migration. al., growth-factor-independent increased al., et and for Flier et der cells (Cabodi van Cul5-deficient cells 2011; the al., cancer for important et of is Tornillo GTPases Cas proliferation small 2012). and al., regulate et motility that Matsui 2001; adaptors al., to et (Bouton binds cytoskeletal to thus response and proteins in tension, adhesion phosphorylated focal tyrosine with becomes and interacts and Src Cas for substrate kinases. a tyrosine cancer is breast other Cas as BCAR1). known 1, (also resistance that p130Cas anti-estrogen found stabilizes We Cul5 involved. present, of are removal is substrates Cul5 Cul5 when other transformation that induce suggesting not is does Src Src endogenous However,ectopic but transformation. for Src, required of and activated activation enzymatically genetic require not Transformation does transformation. induces cells Cul5 epithelial Which mammary Two absent? (2) is activated? Cul5 identified. also when is transformation been Src drive Cul5-deficient if substrates Are not transformed (1) only unanswered. have cells crucial. remain also substrates questions are important substrates these Cul5 additional However, that suggesting Src, Cul5-deficient, of transformation SRC The 2009). Cooper, and (Laszlo by the of either is deletion Src activated, which genetically in Cul5 fibroblasts also Moreover, of 2011). transformation al., these that et induces Src, in Pan knockdown 2009; found turnover active Cooper, Src and have stabilizes for (Laszlo required cells fibroblasts others are mouse Cul5-CRLs and that in suggesting which We Cul5 unclear involved. been of has and knockdown is It 1993). polyubiquitylated ligase Imamoto al., is 1999; et ubiquitin al., Nada Src et 1993; pathway: Harris Soriano, active is 1999; and but Martin, ubiquitin-proteasome Src and stable 2012). (Hakak the degraded is al., cancers Src by et human inactive Krishnan in regulated 2004; implicated negatively Parsons, strongly and is (Ishizawar that kinase tyrosine EERHARTICLE RESEARCH 510 Cul5- whether tested transformed. then were cells Cul5 We deficient 1A). that (shRNA) (Fig. selected confirmed inhibited We and was expression Cul2. RNA lines against cell shRNA a control, polyclonal short-hairpin express a puromycin-resistant, that as and or, retroviruses Cul5 with targeting infected were Muthuswamy Cells puromycin-resistance 2005; 2001). for Brugge, al., extensively and et used (Debnath been has studies human which transformation immortalized MCF10A, line spontaneously knockdown Cul5 cell non-tumorigenic, to endogenous mammary the interference whether al., in RNA test et used further Cul5 Johnson we To tumor 2003; transformation, 2010). al., al., inhibits a et et be (Fay Lai lines cell survival 2007; might cancer or some in proliferation Cul5 carcinomas (VACM1) inhibits several Cul5 that in of expression reduced second, suggest is and expression evidence Cul5 first, of suppressor: cells lines epithelial transforms Two expression Cul5 of Inhibition RESULTS enwso htihbto of inhibition that show now We r,tepoenecddb the by encoded protein the Src, uatclsi o u ipyt h nrae ciiyof activity increased the to simply due not is cells mutant CSK ee hc noe r-niioykinase Src-inhibitory a encodes which gene, CUL5 SRC SRC rt-noee sa is proto-oncogene, eemtto rby or mutation gene xrsini human in expression nvitro in htwr agrta h oto ooisadsoe increased showed and colonies control the than colonies larger dysmorphic were formed transformedthat cells other Like Cul5-deficient 2004). cells, al., MCF10A et Wrobel 2005; have 2002; al., al., proliferate, et et Reginato (Debnath to polarized poorly form continue are of receptors, and that kinase combination decreased cells cells tyrosine containing the activated colonies contrast, or and (vSrc), solid Bcl2, By Src cells and D1 viral epithelium. inner activated polarized dying by quiescent, and transformed form outer, dead cells Normal Cul5 comprising an 2003). colonies al., endogenous et hollow (Debnath Matrigel that in formation suggests This proliferation. cell EGF-independent S1B). inhibits specifically Fig. (supplementary stimulated material significantly was in growth siRNA sequences independent with Cul5 Cul5 different not the of of knockdown targeting effects did transient off-target performed possible Kamura we for Cul2 2009; shRNA, control al., To of et 2004). al., (Huang et knockdown S1A) EGF- Fig. allowed material whereas also (supplementary proteins, cullin growth, protein other RING not the independent of but Rbx2, absence Cul5 of the by knockdown in needed Stable cells 1B,C). control (Fig. than EGF (EGF), better factor significantly growth grew epidermal but contains which medium, normal needn irto,dsuto ffclahsos n the and lamellipodium. adhesions, ruffling extended, focal an of of formation disruption migration, including EGF- independent dysmorphogenesis, transformation, colony of proliferation, cells hallmarks EGF-independent several Cul5-deficient suppresses that Cul5 suggesting 2C). (Fig. nucleus, migration the during normally of polarize oriented also was front complex were Golgi fibers in the antibodies Stress prominent However, 2B). 2B,C). the with (Fig. (Fig. to cells decreased contrast detected cells normal in density of adhesions sites, pY-Cas, Cul5-deficient focal low and contact FAK at vinculin, S1C,D). tiny against medium many Fig. EGF-deficient contained material in plated Cul5-deficient (supplementary when were evident time- Enlarged also cells by shown). were not ruffling confirmed (data and cells was lamellipodia migrating membrane ruffling of micrography indicating Increased lapse 2A). regions, (Fig. leading phase-dark ruffling elongated and an Fig. exhibited lamellipodium material they supplementary Characteristically, 2; (Fig. S1C,D). sub-confluent or migrating using also effects. down off-target was of knocked chance also the transiently chamber of reducing was regions was epithelial– Migration different Boyden targeting 1I). Single-cell Cul5 the siRNAs (Fig. a when absent 2004). undergo was in but stimulated al., not Cul5 EGF when 1G,H) did et (Fig. increased towards and (Wrobel cells migration sheet transition control a than mesenchymal as cells rapidly Cul5-deficient remained more microscopy. and wounded, phase-contrast migrated medium, by EGF-deficient Cul5-deficient to monitored and transferred Boyden control were and of cells wounds monolayers scratch Confluent using chambers. assays migration performed red). 1De,f, oriented (Fig. colonies some not control and in was than red) diffuse GM130, more apoptosis Golgi 1Dc,d, basal The (Fig. reduced lacked interior cells green). and the to 3, red) consistently caspase Ki67, 1Dc,d, (Fig. 1Da,b, (Fig. proliferation rnfraino C1Aclscnb sae ycolony by assayed be can cells MCF10A of Transformation in cells control to rate similar a at grew cells Cul5-deficient ae oehr hs eut ugs htendogenous that suggest results these together, Taken when altered morphologically were cells Cul5-deficient we motility, cell regulates Cul5 whether investigate To ora fCl cec 21)17 0–2 doi:10.1242/jcs.127829 509–520 127, (2014) Science Cell of Journal a nern(i.1ef re) -ahrnwas E-cadherin green). 1De,f, (Fig. integrin 6 CUL5 CUL5 RA gi,EGF- Again, mRNA. RA(e below), (see mRNA

Journal of Cell Science EERHARTICLE RESEARCH C1Aclsi aiu obntosadpolyclonal and combinations various into introduced in were MCF10A Src cells and Cul5-deficient vectors Cul5 MCF10A Src against retrovirus of whether shRNAs puromycin-resistant transformation tested encoding and We Hygromycin- for 2004). cells. or al., required et expression is (Wrobel Src cells regulating MCF10A by as well as that a proteins stability. Src suggest regulating by using results activate partly perhaps The that activity, detected 3A). Tyr416, Src (Fig. represses site activity, Cul5 increased autophosphorylation consistently Src to was antibody but variable quite phosphoepitope levels, was experiments, protein supplementary Src mRNA in 3A; between increase (Fig. The though S2A). unaltered Fig. were material even RT-PCR, by cells, measured in increased MCF10A MCF10A levels protein in Src Cul5-deficient Src that showed regulates blotting We also Western 2011). cells. Cul5 al., et endogenous Pan inhibited whether 2009; not Cooper, tested but and activated (Laszlo fibroblasts of in degradation Src the cells for Cul5-deficient required of is transformation Cul5 for required * is EGF. ng/ml Src 20 and serum absenc the horse in 5% assay s.e.; to healing and Scratch-wound response Mean (G,H) ap distance. in type. and Migration filter each (H) proliferation of collagen-coated wounding. of colonies a after Quantification five hours through in (E,F) comp 24 cells migrated shown. monolayers stained Golgi are of of fi arrowheads), colonies images percentage were green the representative Phase-contrast or Colonies s.e.; determine 3, (G) of (B) days. to and caspase centers used 3 14 Mean the cleaved were EGF. or for through (c,d, sections of 2 EGF optical sections cells absence 1, ng/ml Optical apoptotic the days arrowhead). 5 arrowheads), in red on with (e,f, white cadherin, grown Violet membrane Matrigel Ki67, cells basal Crystal factor-depleted (a,b, arrowheads), control with growth cells red to stained in dividing EGF, GM130, normalized cultured for of were cells control stained absence counts MCF10A internal and and Cell Cul5-deficient an permeabilized presence measured. and and the was Control Cul5 in intensity culture. against grown and Matrigel antibodies were (C) with cells only probed MCF10A 3 lysates Cul5-deficient day cell and MCF10A acini. Control transformed (pMXpuroII-shCul5) (B,C) of Cul5-deficient (actin). formation and and migration vector) proliferation, (pMXpuroII EGF-independent control suppresses 5 Cullin 1. Fig. oeocgnsrqieedgnu r otransform to Src endogenous require oncogenes Some a nern re rohasidct rsne re tr niaeasne n aea ebae(,,E- (e,f, membrane lateral and absence) indicate stars green presence; indicate arrowheads green integrin, 6 ta. 05.Teeclsaenttasomdi omlmedium, normal in transformed not are (4HT)- (Reginato cells These 4-hydroxytamoxifen protein 2005). an fusion al., a of receptor) et use (estrogen expressing vSrc–ER made line regulated we migration, cell and MCF10A proliferation cell MCF10A kinase Src require cells and Cul5-deficient activity. migration of increased the colonies that material dysmorphic indicate results acini (supplementary These normal S2E). cells Fig. Cul5-deficient restored of The to SU6656, migration S2C,D). morphogenesis increased inhibitor, Fig. the Src material for pharmacological (supplementary migration required and cells not was Src, Cul5-deficient transiently (data Src and were assayed. Cul5 versa cells targeting was effects, vice siRNAs off-target with or for transfected control vector To hygromycin-resistant shown). shRNA shRNA Src Cul5 a and vector whether from puromycin-resistant obtained a material from were supplementary expressed results was 3B; Similar (Fig. S2B). knockdown Fig. Cul5 migration expression by and Src proliferation induced EGF-independent inhibited the shRNA suppressed Src and selected. were populations ots hte r ciaini ufcett stimulate to sufficient is activation Src whether test To ora fCl cec 21)17 0–2 doi:10.1242/jcs.127829 509–520 127, (2014) Science Cell of Journal P , .5 ** 0.05; P n , 5 .1ad*** and 0.01 .()Mgaini odncabrasy Cells assay. chamber Boyden a in Migration (I) 3. A fiinyo u5dpein etr ltof blot Western depletion. Cul5 of Efficiency (A) P , .0 yStudent’s by 0.001 n 5 .()Got in Growth (D) 3. poi.Serial optosis. t -test. e (c,d, lex protein fEGF. of e xed, 511

Journal of Cell Science EERHARTICLE RESEARCH rmcnrl u5 n u2dfcetMF0 el and cells MCF10A Cul2-deficient 512 and Cul5- recovered control, were peptides pY from Briefly, CRLs. by This SH2-SOCS–Cul5 for migration 4A). (Fig. and siRNA adaptors. proliferation SH2-SOCS Cul5 through suppresses proteins as pY inhibiting Cul5 extent that similar suggests a to SOCS4, and migration proliferation SOCS2, EGF-independent stimulated against SOCS6 siRNA and of SOCS5 effects combined the Indeed, recapitulate siRNA. would Cul5 siRNA with SOCS4, SOCS6 SOCS2, and of SOCS5 knockdown combined SH2- whether S3A). other tested Fig. than then material We (supplementary level cells RNA MCF10A adaptor the in at genes SOCS abundant SOCS Src more domain-containing are SOCS5 SOCS6 be SOCS4, SH2 and SOCS2, to that Cul5 indicated the could likely analysis are RT-PCR other proteins. by substrates transformation Cul5 recognized that pY to Such be speculated pY. contain contribute We and substrates bound. that is substrates which adaptor on depending substrates, has substrate many Cul5 ubiquitylate cells. and to Cul5-deficient substrates potential of Cul5 the transformation be the might that to proteins contribute are cells other Cul5-deficient for of searched transformation We substrates for required is Cas different inhibiting Src. by 3D). to it transformation that (Fig. addition Src, in suppresses proteins endogenous cells Cul5 other possible activated that control likely and seems is not vSrc–ER than by did faster phosphorylated it cells vSrc–ER migrate Src Although the or greater 3C; cells, (Fig. having proliferate Cul5-deficient pY-Cas Despite or than S2F). proteins activity as total Fig. 4HT, in of material pY the dose supplementary low for in a blotting even by by active further judged stimulated partially and al., was 4HT, et vSrc–ER of (Reginato absence that 4HT found of presence We the 2005). in transformed become but esree o Ycnann rtista ih etargets be might that proteins pY-containing for screened We eiin el,sgetn htScadCswr ntesame migration the in normal work Cas and restored Src Cul5- pathway. that of signaling also migration suggesting Src, and cells, proliferation like increased deficient siRNA Cas, the for Therefore, required S3D,E). is with Fig. cells knockdown material Cul5-deficient transient (supplementary Cas effects, to off-target for area of control normal To ruffle the 4E). and restored (Fig. knockdown length Cas addition, lamellipodium Cul5-deficient In of 4D). (Fig. migration cells and the cells inhibited proliferation Cul5-deficient knockdown and Cas EGF-independent control S3C). in Fig. Cas material down (supplementary knock to Retroviruses required cells. used is MCF10A were Cas van Cul5-deficient whether 2011; of tested 2001; al., transformation therefore et We for al., Tornillo Src- 2000). 2012; al., et al., et resist by Flier et (Bouton der Matsui invasion 2006; cells al., fibroblasts cancer et stimulates Cabodi and in and fibroblasts knockdown mouse ruffling transformed Cas membrane overexpression and Cas-knockout Cas induces transformation. Src, suppresses cells mutant cancer 2012). breast active by al., transformation (supplementary et Cas detectably inhibits Cas Cul5 increase that 4C). post-transcriptionally. suggest (Fig. expression results not protein These down S2A). did were knocked Fig. material 6 SOCS5 transiently levels SOCS4, and SOCS2, were mRNA 5 or Cul5 SOCS6 4, Cul5- when pY- and increased SOCS2, of in also Levels or 4B,C). were increased (Fig. Src Cul5 siRNA Western were with when down knocked levels S3B). transiently and Cas Cul2- cells Fig. or that deficient 2005). material control of confirmed from in (supplementary sample al., detected blotting pY249 the cells not in et times were and deficient 18 but of (Zhang cells pY128 total Cul5-deficient a identified to spectrometry were p130Cas corresponding mass Phosphopeptides by identified a saScsbtaeipiae ntasomto (Matsui transformation in implicated substrate Src a is Cas sdt ti ci ies h ht oe niaeteregions the indicate boxes insets. white the The in fibers. enlarged actin stain to 3 used (C, marker Golgi a adhesions focal 0.2 visualize single anti- (B, to with anti-pY-Cas stained and and anti-FAK permeabilized vinculin, fixed, were (arrowheads). Cells ruffling (B,C) leading membrane elongated increased an and of have lamellipodium direction cells indicate Arrows Cul5-deficient later. (A) at hours migration. 6 cells imaged and edge scratched leading medium, the cells EGF-deficient MCF10A to Cul5-deficient transferred and were control adhesions. of smaller monolayers and Confluent lamella membrane leading increased larger show ruffling, cells Cullin-5-deficient 2. Fig. ora fCl cec 21)17 0–2 doi:10.1242/jcs.127829 509–520 127, (2014) Science Cell of Journal m lcsa h otmo h el)adanti-GM130, and cells) the of bottom the at slices m m flattened m Z poetos.Palii was Phalloidin -projections).

Journal of Cell Science nciae u5mtn Cl 79)i Cul5-deficient T7- in with co-precipitated K799R) 5C). (Fig. pY-Cas Cul5 (Cul5 HA-tagged, tagged T7-tagged, cells. mutant with CskR222 Cas Cul5 Cul5 HA-tagged and co-expressing Cas inactivated by between proteasome-dependent assayed interaction Physical was and Cas. Cul5- Cul5- the of show promotes with degradation consistent to Src are feasible that results technically hypothesis the ubiquitylation, not Cas was dependent it Although S4A). 94.Cspopoyain sae ihanti-pTyr165 Csk than with cells CskR222 assayed Cooper, in and greater phosphorylation, Howell was 1999; antibodies, Cas Martin, and 1994). (Hakak respectively EERHARTICLE RESEARCH cie eue ace os irbatlnsdrvdfo a is from (Csk Src derived Csk when lines type Cul5 fibroblast by mouse regulated matched Csk is used stability SOCS6 we through Cas active, Cas whether binds test and stability To protein Cas regulates increased was Cul5 cell Phosphorylation of cells. phosphorylation Cul5-deficient tyrosine or increased normal and with EGF protein compared of vSrc–ER (4HT) presence the 4-hydroxy-tamoxifen and of of absence phosphorylation absence the the the in 0.25 Note in numbers with protein. cells cell fusion vSrc–ER of vSrc–ER in Ratios a proteins days. expressing 5–7 cells after and counted cells and EGF wes s.e.; of of and absence Quantification Mean and constant. remained presence across levels the averaged protein cells. in were Actin Cul5-deficient cultured phospho-Src. s.e.; of were and and migration cells Src mean of and MCF10A levels right; proliferation the the EGF-independent increases on for deficiency shown sufficient, 5 Cullin not lysates. but cell necessary, MCF10A is Src 3. Fig. pxmcn rtaoeihbtr(i.5,lns5 ) Cas 6). 5, lanes Csk 5A, in 5B; (Fig. stable (Fig. inhibitor was by repressed proteasome inhibited was was a Src turnover epoxomycin, Cas or S4A). absent Fig. 8). was material supplementary 7, Cul5 4, when of 3, hours was lanes 10 lines Cas 5A, was (Fig. cycloheximide, generated half-life Cul5 Cas on with We dependent When inhibited degraded, 3). Cul5. rapidly was against and shRNA synthesis or 1 protein vector expressing lanes cells CskR222 5A, (Fig. expected eln sa nteasneo G.Dsac esrd2 or fe onig enads.e.; and Mean wounding. after hours 24 measured Distance EGF. of absence the in assay healing uateby.Ete iaedfcieCk(22 rwild- or (R222) Csk kinase-defective Either embryo. mutant m H.()Poieain elcut eenraie ocnrlclsgoni h bec fEF enads.e.; and mean EGF; of absence the in grown cells control to normalized were counts cell Proliferation: (D) 4HT. M n + 5 a eepesd od-ers n ers Src, repress and de-repress to re-expressed, was ) .(,)Atv r sntsfiin oidc rlfrto rmgain C etr ltaayi flstso oto n Cul5-deficient and control of lysates of analysis blot Western (C) migration. or proliferation induce to sufficient not is Src Active (C,D) 4. , + n – or nCk22clsbtgetrthan greater but cells CskR222 in hours 2–3 el Fg A upeetr aeilFig. material supplementary 5A; (Fig. cells 5 xeiet.Mgain cac-on eln sa nteasneo G.Dsac esrd2 or fe wounding. after hours 24 measured Distance EGF. of absence the in assay healing scratch-wound Migration: experiments. 3 n 5 .()Tetasomdpeoye fCl-eiin el eur r.Poieain igeaddul knockdown double and single Proliferation: Src. require cells Cul5-deficient of phenotypes transformed The (B) 3. + el,as cells, xPbnigst o h r H oanadtesequence the and domain SH3 Src the for site binding PxxP YD YD nldn r n c,wihln omlil downstream proteins, multiple 15 signaling to the link of whether which tested variety pYxVP Nck, We a the and pathways. to of Crk binding phosphorylation including increased induces binding, repeats Src After otis1 eetdtrsn sequences region central tyrosine The 2012). repeated al., targeting 15 et adhesion (Matsui contains SH3 focal 5E) N-terminal (Fig. C-terminal an domain with and (FAT) of protein region binding scaffold central the a is domain, stimulating Cas was S4C). Src SOCS6. Fig. with to phosphorylation material Cas consistent when (supplementary are or decreased PP2 results with These binding activated inhibited whereas was was Src pervanadate, with Src proteins increased SOCS MEFs when other was binding not CskR222 Cas–SOC6 T7-SOCS detected but More S4B). SOCS6 Cul5-deficient, Fig. of to material (supplementary binds into co-transfection Cas that HA-Cas Similarly, revealed were with 5D). co-immunoprecipitated (Fig. pY proteins that proteins Cas. protein Cas inhibit endogenous SOCS SOCS with only for to the blotted pY-Cas, was pervanadate SOCS6 western increase with and immunoprecipitated treatment and brief overexpressed phosphatases transiently after were and, proteins SH2-SOCS T7-tagged etse hc H-OSpoen ol idt pY-Cas. to bind could proteins SH2-SOCS which tested We Vtrsnswr eurdfrbnigt OS.HA-tagged SOCS6. to binding for required were tyrosines YV domain. SH2 Src the binds phosphorylated, when which YV, ora fCl cec 21)17 0–2 doi:10.1242/jcs.127829 509–520 127, (2014) Science Cell of Journal n 5 .* 3. P , .5 ** 0.05, P , .1ad*** and 0.01 A etr ltaayi of analysis blot Western (A) n 5 xPtrsnso the or tyrosines YxVP P .Mgain scratch-wound Migration: 3. , xP olwdb a by followed YxVP, .0 yStudent’s by 0.001 enbosis blots tern further t -test. 513

Journal of Cell Science ( idn oSC6adCl-eedn a degradation. Cas Cul5-dependent and SOCS6 to binding EERHARTICLE RESEARCH RAbtntohrSC RA splmnaymaterial (supplementary individual 514 mRNAs five SOCS of other each not because of but expression to inhibited effects mRNA and unlikely other Cas off-target is increased for siRNAs Cas target to SOCS6 in a is increase due Src The be that 6A). suggesting (Fig. Src, CRLs state not steady SOCS–Cul5 but the Cas increased and the SOCS6. of siRNA Cas down SOCS6 level for knocking that stabilized confirmed by necessary we first substrates We migration, is CRL in SOCS6–Cul5 Cas increase other is the that Cas whether for test suggested To sufficient cells. far Cul5-deficient of migration so increased degradation for results Cas targeting Our by ruffling membrane regulates SOCS6 membr phase-dark of area (all and 15F edge) leading type, w the edge to wild leading nucleus the Cas the at from cells for (distance s.e. of length averaged and images lamellipodia were mean Phase-contrast the EGF the Mea scratched. show cell, of wounding. then Graphs each presence after measured. and For and were hours medium scratching. absence ruffles 24 EGF-deficient after the measured to hours in Distance transferred 6 numbers EGF. were captured cell of monolayers of absence Confluent Ratios the blots. area. days. in western ruffle 5–7 assay of after healing Quantification counted scratch-wound s.e., constant. and Migration: and remained EGF experiments. levels of s.e.; independent protein presence and three Actin and Mean across pY-Src. absence EGF blots. and the of western Cas in absence of of cultured the Quantification levels in constant. the later. s.e.; assay remained increases days and healing levels 4 SOCS6 Mean scratch-wound protein analyzed and Migration: actin and SOCS5 EGF. control, proteins, SOCS4, of s.e.; a SOCS SOCS2, absence and As or the Mean Cas. Cul5 in wounding. of control, grown Cul5. after levels against by cells hours siRNA regulated control 24 with is to measured and incubated normalized Distance cells were were Cul5-deficient cells counts of MCF10A Cell abnormalities Cul5. Proliferation: edge of leading knockdown for phenocopies necessary SOCS6 is Cas 4. Fig. hshrlto fCsa the at Cas of phosphorylation when a Cas15F at or reduced 5G). with expressed type consistent (Fig. Src, wild also degradation with the cells mutation was into than transfected CasFF transiently level Cas FF 5F). higher inhibited (Fig. partly significantly SOCS6 The mutation to 15F was the binding co-immunoprecipitation. and binding inhibited and completely by MEFs Cul5-deficient measured CskR222, into SOCS6 YD ae oehr h eut ugs htSrc-catalyzed that suggest results the together, Taken Vmttdt DV uat eec-rnfce ihT7- with co-transfected were mutants FDFV) to mutated YV n 5 .Tecnrldt r h aea hs hw nFg B E a eoa rmCl-eiin C1Aclsrsudlmlioimlnt and length lamellipodium rescued cells MCF10A Cul5-deficient from removal Cas (E) 3B. Fig. in shown those as same the are data control The 4. n 5 .()Tetasomdpeoye fCl-eiin el eur a.Poieain ige n obekokonMF0 el were cells MCF10A double-knockdown and single- Proliferation: Cas. require cells Cul5-deficient of phenotypes transformed The (D) 8. xPstsmttdt xP n FF and FxVP) to mutated sites YxVP YD Vsqec stimulates sequence YV , 0clscniinfrom cells/condition 50 n 5 .(,)Wsenbo nlsso C1Acl yae.()Cli eiinyicessthe increases deficiency 5 Cullin (B) lysates. cell MCF10A of analysis blot Western (B,C) 3. SOCS6 te hntpso u5dfcetclsivleohrSOCS other 6D). involve (Fig. cells substrates stimulating other by Cul5-deficient and and migration proteins cells of increased migrating phenotypes that and of other pY-Cas, edge of downregulation leading the the at ruffling unclear. by or is increased decreased is least is expression turnover its and its because ruffling CasWT stimulates CasWT, than strongly CasFF more by Whether by Cas15F. most less signaling-defective stimulated did CasFF, was but Ruffling degradation-resistant S4E) 6B,C). not (Fig. Fig. did ruffling level, material (re)-expression stimulate (supplementary higher Cas migration a stability. stimulate at increased with expressed consistent was CasFF S4F). to Fig. in Degradation-resistant fused material Cas endogenous (supplementary 15F, to cells Cas-deficient level MCF10A or similar Cul5-deficient at FF established was expressed CasWT, was Cas we CasWT re-expressing YFP. of lines ruffling, stabilization cell stimulate MCF10A whether for S4E), to test sufficient is To sufficient substrate Fig. 6B,C). SOCS6 (Fig. that another ruffling not suggesting or material Cas increased, of did significantly stabilization (supplementary the was knockdown ruffling whether to membrane SOCS6 migration tested sufficient was then Although SOCS6 stimulate We of migration. shown). knockdown by stimulate not Cas data of stabilization and S4D Fig. n h eut ugs htSC6Cl niismembrane inhibits SOCS6–Cul5 that suggest results The 5 xeiet.* experiments. 3 ora fCl cec 21)17 0–2 doi:10.1242/jcs.127829 509–520 127, (2014) Science Cell of Journal P , .5 ** 0.05, A eoa fSC2 OS,SC5and SOCS5 SOCS4, SOCS2, of Removal (A) P n , 5 .1ad*** and 0.01 .()Tasetkokono u5or Cul5 of knockdown Transient (C) 5. P , .0 yStudent’s by 0.001 ere t . ane -test. n

Journal of Cell Science EERHARTICLE RESEARCH e cuuaino ahsbtaei ml eas nythe only because small increases is the but substrate downregulated, the is each The subpopulation phosphorylated rapidly. of by less accumulation degraded expression are downregulation net Cul5 phosphoproteins these When for reduced, 6D). is (Fig. substrates system tyrosine target ubiquitin/proteasome adaptors Src vital restraining SOCS transformation. different which by have in phosphorylated Src-dependent model a inhibiting cells suggest CRLs data and Our epithelial SOCS–Cul5 signaling normal that kinase in suggests functions evidence The DISCUSSION Csk and Src) (active SOCS6. CskR222 through (shCul5) Cas Cul5-deficient binds and and (vector) stability control protein of Cas lysates regulates Cul5 5. Fig. a T Fo 5 eetasetytasetdit eaclswt cieScadGPa oto.Qatfcto fwsenbosi hw nrgt me right; on shown is blots western of Quantification control. a as GFP and co- Src active of with Quantification cells antibodies. HeLa FF into T7 transfected and with transiently co-expre 15F s.e.; immunoprecipitated were s.e.; were WT, 15F and SOCS6 and Cas or T7-tagged lysed mean FF of and WT, pervanadate, right; Cas Scheme Cas with HA-tagged on (E) mutant treated shown anti-Cas. or were is with Wild-type Cells binding. immunoprecipitation immunoblotted SOCS6 MEFs. and for CskR222 antibody required Cul5-deficient are anti-T7 sites in with phosphorylation tyrosine immunoprecipitated Cas were (F) lysates mutants. Cell lysed. and pervanadate lt rbdfrH-a,p-a n oa Ypoen.Nt htmn Ypoen eepeeti h ellst,btCswsteol n immunoprecipi one only the was Cas but lysate, cell the in T7 present were against proteins antibodies pY with many immunoprecipitated that Note prepared, proteins. were pY T7-Cul5 lysates total with and phosphorylation, pY-Cas tyrosine HA-Cas, stimulate for to probed blots pervanadate with treated were Cells aiu ie ihccoeiiet nii e rti ytei.GPHwsue sacnrl aflf uniiaini hw nbto ae.(C panel. bottom in fo shown treated is MEFs, quantification CskR222 Half-life Cul5-deficient control. and a vector as of used lysates was T7-Cul5 in GAPDH mutant Cas synthesis. neddylation of protein and blots HA-Cas new Cul5. Western inhibit with degradation. to associates Cas cycloheximide (B) with hours. times 10 various for epoxomycin and cycloheximide n 5 .* 3. P K799R , .5 ** 0.05, D a soitswt OS.HL el eetasetdwt oto etr 7tge OS–OS rT-agdCs,tetdwith treated CisH, T7-tagged or SOCS1–SOCS7 T7-tagged vector, control with transfected were cells HeLa SOCS6. with associates Cas (D) . P , .1ad*** and 0.01 P , .0 yStudent’s by 0.001 n 5 .()CsFwsepesda infcnl ihrlvlta h idtp rCs5.HA-tagged Cas15F. or type wild the than level higher significantly at expressed was CasFF (G) 4. t -test. K799R eectasetdit u5dfcet sR2 Es nwihedgnu r sactive. is Src endogenous which in MEFs, CskR222 Cul5-deficient, into cotransfected were A u5dpnetCstroe eurstepoesm.Wsenbosof blots Western proteasome. the requires turnover Cas Cul5-dependent (A) o epsil fCswsiatvtdsll yp phosphatases. pY would becomes by that solely inactivated activity too was Cas Cas may if of it possible control be system spatial not of until and negative-feedback pool temporal delayed the unaffected provide and This is Cas signaling, phosphorylated. that after Cas means and degraded site unphosphorylated activation only phosphorylation of are same dependence molecules the The on activation. degradation Cas in involved u5sbtaei a,wihbnst OS otneto Src- p on a contingent SOCS6 at to phosphorylation binds key which One dependent Cas, transformation. is cause substrate to combine Cul5 proteins pY several in + rpesdSc Es nuae ntepeec rasneof absence or presence the in incubated MEFs, Src) (repressed ora fCl cec 21)17 0–2 doi:10.1242/jcs.127829 509–520 127, (2014) Science Cell of Journal DVScS2bnigsite binding Src-SH2 YDYV n western and Cas ) nand an tated ssed 515 r

Journal of Cell Science EERHARTICLE RESEARCH c2gnrtsfle ut-cnrsrcue iia othose to similar 516 structures multi-acinar and filled D1 without cyclin generates apoptosis protection of from not Bcl2 expression Combined protects but proliferation. of Bcl2 proliferation increasing overexpression whereas cell apoptosis, example, excess from For induces D1 degrees. cyclin different cell to and of apoptosis polarity genetic those invasion, Single proliferation, with mechanisms. regulate and compared other manipulations by be adhesions highly transformed can cells focal phenotypes MCF10A lamellipodium, reduced These extended fibers. and stress ruffling greatly membrane was a morphology dynamic their and including EGF, of altered, absence when proliferation the and in proliferation, migration increased Their were increased decreased medium. semi-solid and with in cultured apoptosis acini, reduced dysmorphic polarity, formed transformation. cell cells epithelial suppresses Cul5-deficient Cul5 Indeed, that signaling. here kinase found tyrosine we counteract also possibility to the families raising evolved 2008), phosphatase Cul5 anenome (Liongue al., that and et Bilateria sea (King kinase metazoans in tyrosine the in burgeoned The members in 2012). eight metazoans, al., present to in et genes four arose SOCS and also Nematostella genes ancestral SOCS four multicellular 1994). to with al., restricted et is Cul5 (Hampton of Cullins, CRLs SOCS–Cul5 other by actions Unlike transformation Src-dependent transforming of Suppression the inhibit kinases. substrates are to tyrosine By CRLs deregulated their SH2-SOCS–Cul5 transformation. adapted pY, of contain activity and exquisitely that aspects Cas proteins proteins of other to increased binding edge to SOCS leading Cul5, contribute Other the presumably or at cells. SOCS6 ruffling migrating membrane of dynamic absence stimulates the In xrse nseii acrtps xrsinof expression under- types: also cancer are (http://www.oncomine.org, specific genes large 2012) SOCS a in al., Other in et expressed 2012). Sriram underexpressed October, (Lai stomach 2010; accessed is and colorectum al., gene lung, et of the carcinomas of of allele and an percentage (Hampton addition, lost In neuroblastoma 2006). al., frequently and et Wang 1994; cancer al., et breast al., et in Lai 2010). heterozygosity 2007; and al., al., gastric et et The (Johnson Sasi of 2010). culture 2010; proliferation in al., cells inhibits et cancer (Elliott Cul5 Lai mammary 2003; of cancer al., expression gastric et Ectopic Fay and 2008; breast al., et poor-prognosis abnormal in of decreased the to Expression cancer humans. suppress of CRLs attributed in SOCS–Cul5 that suggestions be all previous with might not remain cells activity. but cells Src Cul5-deficient increased modest Cul5-deficient some of the whereas phenotypes Therefore, Src by mutant rounding, Cul5- activated simply spread. highly cell of explained Moreover, activity. colonies causes be Src dysmorphic in cannot direct increase and However, cells and a 2011). migration Src al., deficient is et inhibits acinar increased (Lin Src that cells in the phosphatase MCF10A and by pY changes invasion 2005) a inhibits similar PTPN23, al., for causes et substrate Src (Reginato mutant Indeed, morphogenesis suggests transformation. active for This sufficient highly be Src. could transformation endogenous activation Src CSF1R, requires that or cells of HER2 Cul5-deficient knockdown by here, of show transformation we Like CSF1R as or, Cul5. autocrine-activated 2005) Brugge, HER2, and mutant (Debnath activated by created upeso ftasomto yCul5 by transformation of Suppression ora fCl cec 21)17 0–2 doi:10.1242/jcs.127829 509–520 127, (2014) Science Cell of Journal CUL5 eemp oargo ffeun osof loss frequent of region a to maps gene t rCl sasn ssfiin osiuaemembrane stimulate to sufficient is absent is SOCS6 Cul5 when or upregulation whose pY-Cas, is SOCS6–Cul5 CRL the for substrate One transformedphenotype. the to contributing pathways molecular different suppress complexes Various CRL proteasome. SOCS–Cul5 the pY- pY-B, by (pY-A, downregulation substrates for C) Src of subset a Cul5 targets that propose We EGF-independence. and migration increased ruffling, membrane Cul5 including cells. phenotypes, epithelial transformed in Src-dependent Src represses and Cul5 of roles the for s.e. from and cells/condition Mean scratching. after hours measured 6 was ruffles area membrane The phase-dark (C) of migration. of direction The indicates (arrowheads). arrow Cas Cul5, overexpressing lacking or cells SOCS6 in ruffling membrane wounded show the area into migrating cells of Phase- images (B) contrast scratched. then and medium EGF- deficient to transferred were edge. monolayers leading Confluent the at Cas ruffling of membrane overexpression stimulates or SOCS6 of knockdown s.e.; and mean right; on shown levels is protein blots western Actin of Src. Quantification constant. not remained but protein Cas the of increases level SOCS6 of knockdown Transient (A) ruffling. membrane for regulates Cas and active degradation targets SOCS6 6. Fig. * other cells. for Cul5-deficient required of also phenotypes transformed are Src and Cas ruffling. -test. P , .5 ** 0.05, CUL5 P , n OSgnsi frequently is genes SOCS and .1ad*** and 0.01 n 5 xeiet.()Mdlfor Model (D) experiments. 3 nvitro in P , n .0 yStudent’s by 0.001 5 .(,)Transient (B,C) 3. sconsistent is SOCS6 SOCS2 , 30 is

Journal of Cell Science upestransformation suppress sas ossetwt aafo ua uo el.Cas cells. tumor also human and invasion from cell data cancer stimulates with Src by consistent phosphorylation also is is and 2004). 2005; al., cancer et al., et breast ectopic (Farabegoli Furthermore, of 2004). cancers al., et grade Sutherland ovarian in worsening hypermethylated with decreases ARTICLE RESEARCH n rtoyi rvdsardclydfeetatraieto alternative different radically multiple requires turnover a Cas dephosphorylation. by provides inactivation involved. be to likely and fibers. are stress targets predictable of Cul5 formation with and and Src cytoskeleton, growth other Rho- However, adhesion actin inhibits focal Rac the focal in active Cul5-deficient defects in tiny that of tension front is the the mechanism dependent at possible regulate fibers One stress to cells. of be sufficient, lack also and not might adhesions it although ruffling, with involved, associated Src–Cas–Crk–Rac specifically the is Although pathway movement. membrane Rac. drive forward that and activates networks ruffling actin phosphorylation that dendritic stimulates complex then Rac Src-dependent Crk–DOCK180–ELMO by a stretched recruits phosphorylation. be Src-family-kinase- could for sites Cas dependent revealing leading-edge 2000). cytoskeleton, the al., other and in al., Sharma et for tension et 2007; Stupack al., by Klemke et Cas 2008; possibly occur Schrecengost 1999; Mayer, 2006; might al., al., and et et and and Sawada leading (Cheresh established 1998; Src mechanism well the following Cas is of the at ruffling importance and dynamics CRL of extension actin The SOCS6–Cul5 in turnover the edge. involved ruffling, that the leading-edge phosphoproteins the hypothesis induced increasing the Cas stimulates or with of SOCS6 ruffling. stability consistent inhibiting leading-edge in or either role expression that special observed a We has adhesion SOCS6 migration, and behavior. membrane migratory normal ensure that cytoskeletal, and proteins dynamics pY on regulate different effect target and collectively and significant SOCS5 non-redundant, SOCS4, a migration. are SOCS2, had of that SOCS6 suggests protein decreased speed This speed. SOCS or the migration single increases levels no substrates increased However, pY these absent, be of is could cells turnover Cul5 the normal of When in all of migration or proteins. limit many migration that that all increased infer substrates proteins we Cul5 the Src, SOCS because requires these cells and and Cul5-deficient Because domains, SOCS5 effect. SH2 a SOCS4, same contain as the SOCS2, migrated had of cells SOCS6 knockdown the Combined and fibers. Cul5 maintained sheet. stress leading were decreased of dynamic contacts and Cell–cell adhesions Knockdown oversized, focal an motility. tiny lamellipodium, creating cell migration, affects stimulated profoundly Cas and Cul5 SOCS6–Cul5 by regulation Cytoskeletal Cas. by as progression such cancer proteins of of phosphorylation aspects tyrosine some activating of to Summy expression contribute 2008; might decreased al., genes et 2003), (Kline tumors mice human Gallick, many in and oncogenic in weakly active only highly is Flier is der but Src van Because 2011; al., 2000). et Schrecengost al., Tornillo 2006; anti- 2000; factor- et al., al., et to et Stupack 2007; growth resistance Cabodi al., 2001; et induces al., pathway, cancer, et (Bouton breast anti-apoptotic estrogens in and, the independence Akt, stimulates h motneo a o rnfraino u5dfcetcells Cul5-deficient of transformation for Cas of importance The nciaino a ypopoyaindpnetubiquitylation phosphorylation-dependent by Cas of Inactivation normal for limiting is protein SOCS one no Although nvitro in Hraze l,21;Sutherland 2012; al., et (Herranz SOCS2 CUL5 and n SOCS and SOCS5 o a.TePRpout eectwith cut were products PCR The Cas. for LS-hr n LS-ha hN lsiswr aeby 2009). made Cooper, and (Laszlo were into plasmids plasmids shRNA of pLXSH-shCas ligation and pLXSH-shSrc Xho GCAAGGGGAGTT-3 euain nipratts o h uuewl eidentifying be will future the for cytoskeletal task in important role An unique regulation. a the reasons, plays these potentially For molecules. CRL Cas free SOCS6–Cul5 does of and pool delay local the time alter inherent not an adhesions. have focal in not still does is Dephosphorylation Cas while rapid occur signals typically could it is and reversible, dephosphorylation and and contrast, Cas By active time. more longer the is a In there for delay. SOCS6, or time Cul5 a and of with absence ubiquitylation feedback negative by for inactivation allows new proteolysis focal Thus for decrease. available incorporated Cas will in of be concentration assembly will local phosphorylation the Cas and rapidly, new more Cas turnover, stimulate increased of will rate adhesions the Locally, pY- match by turnover must synthesis. Cas mechanisms of -independent rate with and the dependent associate state, steady and at Globally, downstream SOCS6. signal enter, phosphorylated, might phospho-Cas molecules Cas become new individual adhesions, focal 2– Src, leaves pY-Cas As active of for with life active estimated be fibroblasts might half molecules an in Cas for hours overall proteasome. allow 3 and prior we process the stoichiometry If this phosphorylation of degradation. with stage of complete some association at to assembly activity loses and Cul5–Rbx2, Cas Presumably with chains interaction polyubiquitin EloB/C), (and SOCS6 with possibly association pY-Cas including presumably steps, te OSaatr n hi usrtsta euaecell individual regulate of activity move. that cells or as substrates regulated localization are the CRLs their SOCS–Cul5 how and and adaptors movements, SOCS other admsqec.Tetre eune sdwr:5 were: used sequences target loop The a sequence, an sequence. target sequence, sense target random the antisense by the followed sequence, pMXpuroII pMXpuroII-shCul5 in with promoter PCR 5 by previously primer 3 common made a were template, were as pMXpuroII- pMXpuroII, plasmid plasmids and pMXpuroII-shSrc plasmid shRNA The 2004). control shCas al., et vector (Kamura (shRbx2-1) empty described GGGTCCAGGTGATGGATGCCT the sequence and the (shCul2), target the GAGCTAGCATTGGATATGTGG (shCul5), Rbx2 sequence GCTGCAGACTGAATTAGTAG target Cul2 sequence anti-integrin target mouse Cul5 Center). P5G10, Research Cancer and Hutchinson Fred MA); Wayner, (Elizabeth Medical Boston, Harvard Brugge, (Joan School, MA); antibody monoclonal Billerica, mouse Millipore, anti-pTyr anti-Src (EMD 327 anti-T7 mouse mouse mouse Laboratories, TX); (Bethyl CA); Montgomery, anti-RhoGAP anti-HA NY); Inc., Carlsbad, rabbit IN); Technologies, Indianapolis, mouse Placid, (Life (Roche, anti-GFP anti-GFP Lake MA); rabbit and Inc., Beverly, (4G10) Biotechnology, Technology, (Upstate Signaling (Sigma- (Asp175) Caspase-3 anti-E-cadherin rabbit anti-cleaved (Cell (pTyr165); rabbit rat Cas and anti-phospho-p130 and (pTyr416) rabbit anti-phospho-Src anti-vinculin MO); Louis, Transduction, mouse St Aldrich, CA); BD anti-Ki67 mouse Jose, (ERK2, rabbit and San anti-GM130 kinase-2 CA); mouse signal-regulated Cruz, IL); anti-extracellular Grove, Santa Buffalo Inc., Biosystems, anti-actin, (Leica Biotechnology, goat anti-FAK Cruz rabbit sources: and anti-GAPDH (Santa following rabbit anti-Cas, the rabbit anti-Cul5, from rabbit obtained were chemicals Antibodies and constructs DNA Antibodies, METHODS AND MATERIALS 9 MprI otiigteuiutnpooe n hNsaantthe against shRNAs and promoter ubiquitin the containing pMXpuroII n nqepie otiigsqec oooost h U6 the to homologous sequence containing primer unique a and -u MprI-hu5 lnswr eune.pLXSH-shCul5, sequenced. were Clones pMXpuroII-shCul5. I-cut ora fCl cec 21)17 0–2 doi:10.1242/jcs.127829 509–520 127, (2014) Science Cell of Journal Not Not I- I- Xho Xho 9 rget rmcrepnigpMXpuroII corresponding from fragments I -u LS shCul5 pLXSH I-cut 5 and Src for , 21 iue eoedegradation. before minutes 12–18 9 -GGTCGACAGTGGTGTGTA-3 9 -GTCGACCACTGTGCTGGC- Not I- Xho 9 Xho speiul described previously as ie n i ae of bases six and site, I n lndinto cloned and I 9 -ACATGA- a IgG1 6 , Not 10% 517 I- 9

Journal of Cell Science nii edlto sn h lgncetd ATCATACAAAT- to oligonucleotide Arg to the N- mutated the was using at Lys799 tag AATG epitope neddylation Residue T7 GFP. a inhibit inserting deleting by and modified pCAG- terminus been into inserted had were which They sequenced. GFP, and mRNA brain mouse from hc ak oooyt n nw amla ee saswere Assays gene. mammalian transfection. known AATTCTCCGAACGTGTCACGT, after any hours 96 to sequence performed homology the lacks which MD), targeted Germantown, plate (Qiagen, (Qiagen) 12-well Cas Waltham, or 1.25 a Scientific, Src and Fisher of CisH, for Thermo SOCS1–SOCS7, wells specific siGENOME, oligonucleotides MA), siRNA to pool four of (SMART directly pool Cul5 a of added pmol 50 and containing above) (described EERHARTICLE RESEARCH 518 was number 8. cell S4D, day in Fig. on increase and counted fold S3B was the Fig. number the S1A, and cell Fig. Violet 3 material Fig. day Crystal supplementary on with S1B, For and stained measured. Fig. 0) were (day material wells day triplicate supplementary next S4A, 1B,C; or Fig. 0 Fig. and and For serum S3D horse EGF. 2% containing ng/ml medium 20 growth in plated were Cells and Proliferation confluence Src blotting. near active western and by hours to GFP 24 analyzed lysed and 15F), plates were later or Cells Technologies). six-well FF (Life (WT, Lipofectamine2000 in using Cas HA-tagged grown with were transfected cells HeLa transfection DNA 2 transfection siRNA Technologies), (Life EGF ng/ml 20 0.5 Technologies), with (Life Technologies) serum (Life horse DMEM/F12 5% in maintained were cells infection MCF10A retroviral and culture Cell nNtria YPtgadmdfe oyikr .Antoku, 4- and S. by CA) Francisco, Sigma- Sigma-Aldrich. (San confirmed polylinker; from Sugen from from were hydroxy-tamoxifen were SU6656 modified C MO), Louis, mitomycin Clones (St and Aldrich and production. epoxomycin MG132, tag retrovirus sequencing. The for tag. EYFP HA unpublished) by with N-terminal N-terminal (pMSCV made an pMSCV_puro_EYFPC1 into an with was cloned expression were Cas) fragments transient rat same for in vector sequence Y764 the nHA in and Y669 Antoku. Y762 and Susumu Y667 to of corresponding mutation TN) (double YDYV, Nashville, mutant University, FF (Vanderbilt the Hanks and Steve from obtained were ilpr) 10 Millipore), Uiest fTrno oot,Cnd)(eiaoe l,20) 4- 2005). protein. fusion vSrc-ER al., the activate Muthuswamy et to Senthil assays (Reginato scratch-wound from Canada) gift Toronto, (0.25 hydroxy-tamoxifen kind Toronto, a of were MCF10A (University puromycin. vSrc-ER with expressing selection cells and infection pMSCV_puro_EYFPCas retrovirus by prepared with were promoter expressing MSCV stably the cells from the MCF10A EYFP-Cas in Cas-deficient maintained agent. were selective clones) not of pools, absence as (selected lines days, 10–15 for by 0.25–0.5 out in carried lines cell was stable of selection cells initial Following MCF10A 1989). Rosman, of packaged and (Miller infection protocols were standard and shCas cells or 293T shSrc HEK using shCul5, vector, empty empty pMXpuroII pLXSH vector, containing retroviruses Recombinant Technologies). (Life utrdi M upeetdwt 0 B n penicillin/ and FBS 10% with supplemented U/ml. before were 100 cells at described DME HeLa both 2009). streptomycin were Cooper, in and shCul5, Laszlo cultured 1994; or Cooper, empty and vector pLXSH (Howell containing empty retroviruses recombinant vector/pMXpuroII with infected lines, 6 os OSand SOCS Mouse os idtp n 5 uatCscN lns(hne l,2004) al., et (Shin clones cDNA Cas mutant 15F and wild-type Mouse V0lreTatgnimmortalized antigen large-T SV40 10 m /lhdootsn SgaAdih,0.1 (Sigma-Aldrich), hydrocortisone g/ml 4 CGAATGAGAAAGAAAATT. C1Aclswr eupne n800 in resuspended were cells MCF10A m ioetmn20 Lf ehoois.CnrlsiRNA Control Technologies). (Life Lipofectamine2000 l m /lisln n eiilnsrpoyi,bt t10U/ml 100 at penicillin-streptomycin, both and insulin, g/ml Bam Cul5 HI- m Not eecN lnswr bandb RT-PCR by obtained were clones cDNA gene ,SgaAdih a de 4husbefore hours 24 added was Sigma-Aldrich) M, m /lprmcno 50–100 or puromycin g/ml rget eecoe notepCAG2- the into cloned were fragments I Csk 2 / 2 m s n s 22cell R222 Csk and Csk , /lcoeatxn(EMD toxin cholera g/ml m fgot medium growth of l m /lhygromycin g/ml h diino lx-lo-aee eodr niois(Life antibodies 25 secondary at hour Alexa-Fluor-labeled 1 for 1:1000, of diluted Technologies), addition the otmsd eesandwt .%CytlVoe n2%ehnl Each the ethanol. on 20% triplicate. cells in in migrated Violet analyzed Crystal the was 0.1% and condition with removed, stained were were side membrane bottom the of side top MET n B o 0mntsa 37 trypsin, at 0.25% minutes of 20 for solution PBS a and using EDTA mM detached 2 were cells MCF10A 2 wells. with ng/ coated 20 and PA) 8 serum were An horse chamber 5% EGF. with the ml supplemented of DMEM/F12 wells with lower loaded The chamber chemotaxis MD). micro Gaithersburg, 48-well a (Neuroprobe, in performed were assays Migration assay microns. chamber start Boyden to the converted at was edges experiment intervals wound the at between of and end distance wounding and in after difference P200 shortly micrographs The taken lacking Phase-contrast a thereafter. were C. medium wounds with Mitomycin fresh triplicate ng/ml with surface of 1 replaced containing the medium and the scratching EGF EGF- and by were tip, monolayers wounded micropipette MCF10A confluent overnight, Briefly, starved 2007). described al., previously et as (Dow performed were assays migration Scratch-wound confocal laser-scanning wound LSM510 Scratch optical 20 Zeiss Sequential Apochromat Plan 2003). a Zeiss a al., using with et fitted microscope taken (Debnath described were been staining sections Immunofluorescence has 2003). al., acini et (Debnath of method, ng/ml 5 overlay at basement the EGF reconstituted with using on Matrigel) (growth-factor-depleted cells membrane MCF10A of culture imaging Three-dimensional and growth 3D Matrigel IAbfe otiig1 rtnX10 .%SS %sodium 1% SDS, 0.1% X-100, Triton 1% in lysed containing and (PBS) buffer saline phosphate-buffered RIPA in twice washed were Cells analysis Protein 40 using visualized were Cells Imaging X-100 25 Triton at 0.1% minutes with 5 permeabilized migration for and and fixed PBS wounding were in scratch cells After hours, 6 coverslips. for on grown were Cells cells migrating of Immunofluorescence S,5 omlga eu n B o 0mntsa 25 at minutes 30 for PBS and serum goat normal 5% BSA, ih1%FS el eersseddi MMF2a 5,0 cells/ 250,000 at DMEM/F12 in resuspended were cells FBS, 10% with ladaddt h o el.Tecabrwsicbtdi a in incubated was chamber The wells. top the CO 5% to of atmosphere added humidified and ml B iss oesiswr one nPoogGl ouin(Life solution Gold ProLong in mounted several were After Technologies). actin. coverslips visualize rinses, to used PBS was Technologies) (Life phalloidin nioiswr iue nbokn ouinadaddete for either added and solution blocking 25 at in hours diluted 3–4 were antibodies ahcl ln h on de vrg ufeae e elwas using cell assembled software. per were (Deneba) area Canvas Figures and replicates. ruffle (Adobe) biological Photoshop Average obtain edge. for to area repeated wound ruffle analyzed the measure for was areas to calculated along movie dense Health) for cell the of phase of minute Institutes each that frame (National every confirm one ImageJ to recorded then using used ruffling, were were high- dynamically movies Images were a 40 The used. A with minutes. camera. was 60 equipped CCD objective scientific system, HQ2 phase Coolsnap Field) Photometrics Swept resolution Technologies (Applied within (Prairie equal SoftWorx were scaling using image and deconvolved experiment. times an exposure and all and acquired Precision), Olympus). recorded camera; were were CCD HQ2 Images Images (Photometrics Olympus). settings camera (IX71, fixed microscope using DeltaVision a on oiso ebaerflswr atrdo io LiveScan Nikon a on captured were ruffles membrane of Movies ora fCl cec 21)17 0–2 doi:10.1242/jcs.127829 509–520 127, (2014) Science Cell of Journal , m 0clsaogtrewud.Teetr xeietwas experiment entire The wounds. three along cells 50 ˚ oesz oyabnt itr(W,Ws Chester, West (VWR, filter polycarbonate size pore m .Cvrlp eerne he ie nPSbefore PBS in times three rinsed were Coverslips C. m /lclae Vsprtdteupradlower and upper the separated IV collagen g/ml ˚ .Clswr lce naslto f2% of solution a in blocked were Cells C. 6 . 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Journal of Cell Science excoae 0m rsHl H74 5 MNC,5m EDTA, mM 5 20 fluoride, NaCl, phenylmethylsulfonyl mM mM 150 1 EGTA, 7.4, mM pH 5 Tris-HCl, mM 20 deoxycholate, ARTICLE RESEARCH H74 ih10m al .%N-0adimmunoprecipitated and NP-40 digestion. 0.1% for NaCl, used Tris-HCl, mM mM was 4 100 100 at of overnight ratio with ml 0.5 substrate:trypsin 7.4, 2005), in pH resuspended al., 20:1 were protein et peptides and a (Zhang Lyophilized lysis described Cell that previously plates. as cm except 15 performed three was confluency on 90% digestion medium to growth grown were normal cells in shCul5 and control vector MCF10A Phosphoproteomics previously Src described Cas, encoding as mRNA of synthesized abundance The and cDNA 2009). Cooper, and and (Laszlo extracted was RNA RT-PCR 10 with some hour In 1 hours. 25 for 10 for with pre-treated vehicle various were treated for received cells then concentration) cells cases, final untreated confluence, ethanol Control near (1% times. vehicle to or grown cycloheximide were Cells chase Cycloheximide 4 at lysed then minutes 30 with incubated for were pervanadate cells sodium hours, to mM 48 proteins according 1 After Lipofectamine2000 SOCS protocols. using manufacturer’s T7-tagged cells the HeLa cells, into HeLa transfected were from immunoprecipitation For Immunoprecipitation eupne nLemibfe,te eovdb SDS-PAGE. by resolved then Csk buffer, or CskR222 Cul5-deficient Laemmli buffer, from lysis Immunoprecipitation in in times three resuspended washed was complex protein–antibody–bead evndt eeaddbfr yi n yae eepeae using prepared were 1994). lysates Cooper, and and (Howell lysis buffer before RIPA added were 10 pervanadate that except protocol, similar a 0m a,1 rtnX10adpoes niiosfloe by EDTA, followed mM 4 2 inhibitors at hours protease 7.4, 3 for pH and antibody 14,000 anti-T7 at HEPES, Triton-X-100 minutes 15 mM 1% for centrifugation 10 NaF, NaCl, mM 50 mM 150 containing 9 at rz.Baswr hnrne he ie ih05m of ml 0.5 with Phosphotyrosine-containing times NaCl. three mM 50 using 100 rinsed eluted 7.4, were then peptides pH were Tris-HCl, mM Beads 100 Cruz). Santa 99, yaeadatbd i o ora 4 at hour 1 for mix antibody and lysate 20 r itdi upeetr aeilTbeS1. Table material supplementary Primers Biosystems). in (Applied listed software 7900HT SDS are the and (Qiagen), was System kit PCR qPCR Time PCR genes, Real green bromide SOCS SYBR For ethidium QuantiTect range. and using cycles linear performed electrophoresis the PCR in gel 24 was and agarose Amplification 21 stain. 2% after by removed analyzed were and Samples 2009). Cooper, and 25 with followed concentration) times. final various for DMSO cycloheximide (0.1% vehicle osac h P os v.4 rti database. protein tyrosine (v3.44) on performed mouse Da were IPI 79.9663 the identifications of most search modification Peptide five to variable scan. the a using of MS 2003), X!Tandem scans al., the were with MS/MS et in five (Yi by ions samples described followed abundant CA) as scan MS configuration Dried one a Billerca, collecting in spectrometer instructions. (Millipore, mass FT Ziptips 5 manufacturer’s in microC18 resuspended the using following desalted were n iulzduigAesa C lsWsenBotn Detection Blotting Western NJ). Piscataway, Plus Biosciences, Healthcare ECL (GE Amersham System antibody using visualized secondary and peroxidase-conjugated anti-rabbit-horseradish or anti-mouse- goat either goat first by followed probed antibody, Tween, primary 0.1% indicated dry with the saline non-fat with Tris-buffered in 5% pY) for in BSA (or blocked milk MN), Minnetonka, (Osmonics, onto transferred nitrocellulose were Proteins ratio. acrylamide:bisacrylamide 75:1 or 30:1 adjusted were Samples 1 assay. to Bradford using assessed was concentration m 6 b /lartnn n Msdu rhvndt.Protein orthovanadate. sodium mM 1 and aprotinin, g/ml atnwsmaue yR-C speiul ecie (Laszlo described previously as RT-PCR by measured was -actin D apebfe,bie n eovdb 0 D-AEusing SDS-PAGE 10% by resolved and boiled buffer, sample SDS ˚ ih2.5 with C m f01 omcai n nlzdwt nLTQ- an with analyzed and acid formic 0.1% of l m fat-Yatbd-ojgtdbas(PY- beads antibody-conjugated anti-pY of g m f10m lcn H25 n samples and 2.5, pH glycine mM 100 of l ˚ .PoenAGbaswr de othe to added were beads A+G Protein C. m r niio P r1m sodium mM 1 or PP2 inhibitor Src M g yaewsrttdwt mouse with rotated was Lysate . ˚ .Atrcnrfgto,the centrifugation, After C. m pxmcnor epoxomycin M m /lleupeptin, g/ml + ˚ nbuffer in C Esused MEFs m m g/ml g/ml ern,H,Hn,X,Hn,N T. N. Hung, X., Hong, H., Herranz, ars .F,Soi . opr .M,Kmr . d,H n oly .M. P. Howley, and H. Oda, S., Kumar, M., E. Cooper, I., Shoji, F., G., K. Blanco, Harris, M., Alavaikko, R., Winqvist, A., Mannermaa, M., G. Hampton, S. G. Martin, and Y. Hakak, J., C. Mandernach, M., D. Shope, A., G. Karathanasis, A., K. Longo, J., M. Fay, M. Taffurelli, and D. Santini, C., Ceccarelli, F., A. Farabegoli, J. Johnston, S., and B. A. M. Peterson, Hookham, J., K., Elliott, Zarbalis, J., Caddy, S., J. Kauffman, E., L. Dow, un,D . yal,O,Hn,H . aebo,A . ua .M,Scott, M., D. Duda, M., A. Taherbhoy, W., H. Hunt, O., A. Ayrault, T., J. D. Huang, Cooper, and W. B. Howell, ehis .J n ozio .A. C. Joazeiro, and J. R. Deshaies, S. J. Brugge, and K. S. Muthuswamy, K. J., S. Debnath, Muthuswamy, J., M. Reginato, L., N. Collins, R., K. Mills, J., Debnath, S. J. Brugge, and J. Debnath, otn .H,Rgis .B n rc-tsa,P J. P. Bruce-Staskal, and B. R. Riggins, H., A. Bouton, References at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.127829/-/DC1 online available material Supplementary material Supplementary release for PMC in months. Science Deposited 12 C.P. National after to a Fellowship and Research G.L.]; Graduate Cancer to Foundation and American PF-07-287-01-MGO FHCRC; the number at from [grant Fund fellowship Society Project postdoctoral Pilot Foundation, Synergy Program Safeway Center Research the R01- Hutchinson Cancer numbers the Breast [grant from the Health grant Fund; of pilot Institutes a National R01-NS080194]; the CA41072, supported was work This Funding and G.S.L. A.T., paper. experiments. the interpreted wrote and J.A.C. performed designed, authors All contributions Author interests. financial competing no declare authors The interests Muthuswamy. Competing Senthil and Carter Markus Bill Clurman, Wayner, Bruce Elizabeth suggestions Kamura, Walter, and Takumi Roland reagents Hanks, Welcker, The valuable Steve research. without Antoku, the possible Susumu of been from course have and the not Ansari during would Moham assistance Toida, work technical Natalie Smirnov, for Denis Rivera to Melanie grateful very are We Acknowledgements hrs,D . eg .adKek,R L. R. Klemke, and J. Leng, A., D. Cheresh, Bisaro P., Stefano, Di A., Tinnirello, S., Cabodi, 19) bqii-eitddgaaino cieSctrsn kinase. USA tyrosine Sci. Src Acad. active of degradation Ubiquitin-mediated (1999). common a 11q23.3. carcinoma: and breast A. 11q22 human G. between sporadic Evans, and in region K. heterozygosity W. of Cavenee, Loss I., (1994). Newsham, H., Kiviniemi, J., P. Taskinen, Src. and tissues normal lines, cell cancer tissues. breast A. tumor cells, Husain, epithelial and breast K. in Pherson, expression A., Hicks, R., J. 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