abig B23T UK. 3AT, CB22 Cambridge ohnr Fukui Yoshinori ereDamoulakis George in Rac polarity GPCR-driven regulate and to signalling RhoG activates directly P-Rex1 ARTICLE RESEARCH eevd1 ac 04 cetd1 ac 2014 March 12 Accepted 2014; March 10 Received § ` 1 between crosstalk al., revealed have et studies spatio- (Rossman recent Crucially, (GEF) classes inactivation 2005). rapid factor several by and guanine-nucleotide-exchange pathways undergo signalling activation of upstream to of linked They are cycles that adhesion changes. co-ordinated migration, temporally morphological motility, cell cytoskeletal and the for Rho of the aspects necessary key within reorganisation regulate subgroup , a small ), of and family Rac2 (Rac1, Rac INTRODUCTION GTPase oxidase, Small GEF, NADPH GPCR Rho migration, , Cell of signalling, Cell understanding WORDS: . KEY actin our the and for GTPases Rho implications to findings signalling broad These have DOCK2. indirectly of functions thus Rac-dependent recruitment regulates RhoG-dependent RhoG, through for acting P-Rex1, GEF which a in together, hierarchy Taken as signalling neutrophils. new a migrating reveal of also results edge our F- RhoG leading and of DOCK2, the Loss GEF to they Rac system. actin, the this of suggesting in recruitment GPCR-driven Rac activity, impaired of upstream oxidase function NADPH both Rac-dependent activation or Rac P-Rex1 GPCR-driven and in either the reductions of for equivalent caused loss GEF RhoG Furthermore, a neutrophils. as mouse acts primary both also RhoG, P-Rex1 GTPase that Rac-related show we activity Rac1, GEF its towards to Although attributed been respectively. have , functions and and P-Rex1-dependent signalling cancer tumorigenic breast promotes in and metastasis types, 3- cell phosphoinositide small many and in of GPCRs (PI3K) of subfamily downstream P-Rex1 Rac engaged (GEF) is the factor activating guanine-nucleotide-exchange The by GTPases. cytoskeleton of organisation actin the the regulate (GPCRs) receptors G--coupled ABSTRACT eateto hraooy nvriyo ot aoiaa hplHl,450 Hill, Chapel USA. Carolina, at North Carolina Hill, North Chapel Drive, of West University Pharmacology, of Department acrRsac,CetrBat aoaois 3 uhmRa,Lno SW3 London Road, Fulham of 237 Institute UK. Laboratories, Biology, 6JB, Beatty Cancer Chester of Research, Division Cancer Team, Oncogene address: Fukuoka, *Present University, Kyushu Bioregulation, Japan. of Institute Medical Neuroscience, hs uhr otiue qal oti work this to equally contributed authors These uhrfrcrepnec ([email protected]) correspondence for Author Campus, Research Babraham Institute, Babraham laboratory, Inositide 04 ulse yTeCmayo ilgssLd|Junlo elSine(04 2,28–60doi:10.1242/jcs.153049 2589–2600 127, (2014) Science Cell of Journal | Ltd Biologists of Company The by Published 2014. 3 ed .Welch C. Heidi , 2 ieegrCmrhnieCne etrand Center Cancer Comprehensive Lineberger 1, ,LueGambardella Laure *, nvitro in 3 eateto muoilg and Immunobiology of Department 1 n nGPCR-stimulated in and hnigJ Der J. Channing , 1 etL Rossman L. Kent , 2 e .Stephens R. Len , iae(IK inltasuto aha Sehn tal., et (Stephens PtdIns(3,4,5)P a pathway as identified transduction was P-Rex1 signal 2008). (PI3K) kinase lhuhsvrllnso vdnepitt ioa oefor role pivotal understood, a poorly to point still evidence are of proteins PtdIns(3,4,5) lines Rac several GTP/ on although regulate they exchange which by GDP mechanisms the Nishihara but movement, 2011; cell al., are 2008). et al., Lindsay GEFs (Berger et 2012; Sanz-Moreno Rac progression al., 2002; et al., and and/or et Hodis 2012; Rac1 initiation is al., there in disease et and with metastasis, that associated and evidence invasion migration strong cell these cancer of to drives deregulation addition immunity, processes In and development 2008). normal their al., during al., to et et key (Hung Sanz-Moreno contexts as 2013; subcellular and isoforms cellular different and in subfamilies function GTPase Rho different otasueGC-rvnsgasdrcl hog Rac, through directly signals P-Rex1 GPCR-driven placed from ideally and neutrophils catalytic being transduce 2002), major P-Rex1 al., its despite et is to However, Welch this cells. 2005; that in al., function assumed et have Hill studies 2007; subsequent al., et (Barber the and ta. 01.PRx a lobe hw oihbttetumour the inhibit to of shown been PtdIns(3,4,5) P drivers also and Lindsay has 2012; suppressor as P-Rex2 al., metastasis 2011). et reduced al., identified (Berger mice et P-Rex1 in been of melanoma N-Ras-driven deletion of recently genetic and have the dominant related melanoma, the direct than in Mutations the Rac activation. context-dependent P-Rex2 as Rac and of P-Rex1 GPCRs or between of regulation link complex idea P-Rex1-mediated satisfying more conceptually the is 2005). that al., signalling et suggests Welch 2005; al., This et (Dong functions Rac-dependent ietyitrc ihcascl RBdmi-otiigRc,2 Rac1, CRIB-domain-containing classical, with interact directly GPCR of cells. primary variety in function 2003). GTPase a and GPCR-driven to GEF (Dinauer, of studies biochemical rapidly for ideal (ROS) them and of making ligands, robustly generation species respond the oxygen Neutrophils for responsible reactive complex, of microbicidal oxidase components essential NADPH be neutrophil migration to the the al., known regulating et are to of proteins Sanz-Moreno addition Rac 2008; In cytoskeleton, al., 2008). form al., et et stem (Kay Stephens amoeboid leukocytes, cells 2008; tumour other rapid, some including and types, cells a cell many of exhibit Rac- characteristic of they understanding control of because their pressing. task and more the the GEFs, all family becomes findings, functions, P-Rex these dependent of of biology light the In 2009). E ciiyta ssnritclyrgltdb PtdIns(3,4,5) by regulated synergistically is that activity GEF -rti-ope eetr GCs r ct euaosof regulators acute are (GPCRs) receptors G-protein-coupled hGi orhmme fteRcsbaiy u osnot does but subfamily, Rac the of member fourth a is RhoG dynamics actin into studies for model attractive an are Neutrophils 2 apelD Lawson D. Campbell , bc uuiso eetrculdhtrtiei proteins G heterotrimeric receptor-coupled of subunits P 3 1, eeae ytecasIpoponstd 3- phosphoinositide I class the by generated ` n hli .Hawkins T. Phillip and 2 / 2 iedslyol ata eet in defects partial only display mice 3 hshts,PE Fn tal., et (Fine PTEN phosphatase, 1 ae .Anderson E. Karen , 1, ` 3 ,§ dpnetRac -dependent 1 2589 , P 3

Journal of Cell Science PR,uigacmiaino iceia n genetic prevailing downstream RhoG the for and GEF to a as biochemical contrary acts P-Rex1 that, of function, its suggest of combination view data of Our downstream a activation approaches. using Rac and GPCRs, ROS RhoG between GPCR-stimulated 2006). relationship al., in et (Kunisaki defects migration cell in severe and protein Furthermore, production DOCK 2007). have major al., the et neutrophils, DOCK2, Buul neutrophils lacking van neutrophils mouse 2010; al., et mouse al., in Hiramoto-Yamaki et 2006; al., ligands Samson et 2010; Condliffe GPCR 2011; al., by cell et (Bass oxidase EGF-dependent of activation NADPH trans- efficient in for the leukocyte required roles is and RhoG migration, to endothelial outgrowth, addition In migration, 2003). Negishi, r mean are fRci oessesb eriigabprieRcGEF Rac bipartite (Co a and families 1–5) recruiting 1–3) (isoforms by DOCK (isoforms the systems ELMO of members some comprising upstream function in complex to reported Rac been has of it Instead, effectors. 3 and ARTICLE RESEARCH 2590 wortmannin nM 100 of absence or presence the in incubated were mice WT PI3K from and Neutrophils WT (A–C) from levels. Neutrophils 10 Rac (D) with or RhoG stimulated total and to (WM), PI3K normalised a were through levels receptor fMLP Rac G-protein-coupled the of downstream activated neutrophils. is RhoG 1. Fig. mean EF etohl rmW n P-Rex1 and WT from Neutrophils (E,F) 0s aaaemean are Data s. 10 nlgto h bv ok estott xlr the explore to out set we work, above the of light In 6 ..o he needn xeiet.* experiments. independent three of s.d. 6 ...o he E n ag fto()idpneteprmns G etohl rmW n Vav123 and WT from Neutrophils (G) experiments. independent (F) two of range and (E) three of s.e.m. hGadRcatvto eemaue yafnt ulon(sn S–LO n S–A-RB epciey n T-hGadGTP- and GTP-RhoG and respectively) GST–PAK-CRIB, and GST–ELMO1 (using pulldown affinity by measured were activation Rac and RhoG 6 ...o oridpneteprmns H TadP-Rex12 and WT (H) experiments. independent four of s.e.m. m MPfrteidctdtms aaaemean are Data times. indicated the for fMLP M c 2 / 2 2 iewr tmltdwt 10 with stimulated were mice / 2 ie()o ienl o ohPRx n -e- (P-Rex12 P-Rex-2 and P-Rex1 both for null mice or (E) mice ˆ te ´ ta. 02 ao and Katoh 2002; al., et P # .5 ** 0.05, P # .1 ya nardStudent’s unpaired an by 0.01, m MPfr1 .Dt r mean are Data s. 10 for fMLP M 6 ...o or()adrneo w BC needn experiments. independent (B,C) two of range and (A) four of s.e.m. rvnpoessidrcl nti ytmb rmtn RhoG- DOCK2. promoting GEF Rac by the system Rac- of this recruitment regulates in dependent and indirectly neutrophils, processes mouse driven in engagement GPCR of os etohl ihfL eutdi ai rnin iein rise transient rapid a of in resulted Stimulation fMLP Methods). with and neutrophils Materials mouse (see autocrine peptide factors by paracrine ligands, pre-activation formylated or minimal GPCR with to that the RhoG (fMLP), rapidly state phenylalanine and of as quiescent robustly for a regulation respond such procedure in to the refined them neutrophils a allows assess mouse developed primary to we GPCR- purifying any GPCRs, order in GPCR of measured In downstream of be system. to downstream its yet however, RhoG driven has 2006); of regulation al., and et role activation (Condliffe a neutrophils for in engagement evidence is There receptor fMLP PI3K G-protein-coupled a the through by activated is RhoG RESULTS 2 / 2 iewr tmltdwt 10 with stimulated were mice ora fCl cec 21)17 5920 doi:10.1242/jcs.153049 2589–2600 127, (2014) Science Cell of Journal t-test. c n -e1dpnetmcaimi rmr mouse primary in mechanism P-Rex1-dependent and - c dpnetmechanism -dependent 2 / 6 2 F eesiuae ih10 with stimulated were (F) ) ...o oridpnetexperiments. independent four of s.e.m. 2 / 2 iewr tmltdwt 10 with stimulated were mice m MPfrteidctdtms aaare Data times. indicated the for fMLP M N -formyl-methionyl-leucyl- m MPfr1 .Data s. 10 for fMLP M m MPfor fMLP M

Journal of Cell Science sddfraino T-hG(i.1,) ugsigthese suggesting 1B,C), (Fig. linked. GTP-RhoG be of might responses formation wortmannin-sensitivity Under and 2000). did kinetics al., neutrophils as similar et exhibited GTP-Rac1 Sasaki mouse -Rac2 of 2000; and formation al., in fMLP-stimulated et conditions, GPCRs Li equivalent 2000; of al., et downstream (Hirsch activity PI3Kc PI3K identifying studies previous PI3Kc with on dependent significantly was RhoG mean ftefL eetr etetdmuenurpiswt the with neutrophils mouse treated downstream we specifically receptor, or fMLP feature neutrophils, the general the in more of whether a signalling assess is GPCR To P-Rex stimuli. on of activity of RhoG variety of a dependence to rapidly respond to ciaino hGrqie PtdIns(3,4,5)P requires GPCR-driven RhoG suggesting basal of wortmannin, sensitive to activation inhibitor acutely returning was pan-PI3K response and This the s 1A). to 10 (Fig. min at 1 peaking within levels GTP-RhoG, of levels the ARTICLE RESEARCH i.2 oiiefebc hog a2 PtdIns(3,4,5) Rac2, through feedback Positive 2. Fig. (P-Rex1 we P-Rex1 of therefore loss regulation and that the found in 2005), families we al., GEF Strikingly, two RhoG. et these of of Welch role 2011; the neutrophils investigated al., in activation et major as Rac1/2 identified (Lawson been fMLP-stimulated also of have Vav1 regulators in and (Samson family RhoG P-Rex1 Vav 2010). for the al., GEFs of et as members including identified systems, been cell have other RhoG Rac of for activation GEFs fMLP-driven Certain for required is P-Rex1 IK.Ti a upre ymaueet nnurpisfrom neutrophils in measurements by PI3K supported was This PI3Ks. o -e2(oade l,20) ossetwt this, with a from Consistent displayed neutrophils in P-Rex2 seen 2004). and that to P-Rex1 P-Rex1 similar al., activation both RhoG in of et defect loss cells (Donald with these neutrophils in P-Rex2 identified P- previously is effect role neutrophils for mouse functional significant 1E), in no no (Fig. P-Rex with had of Rex1, isoform isoforms major activation Vav The 1G). three RhoG (Fig. all of fMLP-driven loss whereas impaired severely TadRac2 and WT mean arnui Lt)adsiuae o h niae ie ih10 with times indicated the for stimulated and (LatB) B Latrunculin Student’s etohl xrs ihlvl fmn PR loigthem allowing GPCRs many of levels high express Neutrophils 6 6 c 2 ...o he needn xeiet.()Nurpisfo TadRhoG and WT from Neutrophils (C) experiments. independent three of s.e.m. ag ftoidpneteprmns B PtdIns(3,4,5) (B) experiments. independent two of range 2 / 2 t / -test. 2 ie hc eeldta MPdie ciainof activation fMLP-driven that revealed which mice, ie(i.1F). (Fig. mice 2 / 2 iewr tmltdwt 10 with stimulated were mice m stedmnn ls I class dominant the as MPfr1 .Ro ciainwsmaue yafnt ulonuigGTEM1 aaare Data GST–ELMO1. using pulldown affinity by measured was activation RhoG s. 10 for fMLP M 3 Fg D,consistent 1D), (Fig. ytei ycasI class by synthesis P 3 n -ci r o epnil o euaino hGatvt yP-Rex1. by activity RhoG of regulation for responsible not are F-actin and P 3 m a esrdb CEIM n omlsdt h eeso PtdIns(4,5) of levels the to normalised and LC-ESI/MS by measured was MP aaaemean are Data fMLP. M 2 / 2 ) a o eue nnurpisfo Rac2 activity from RhoG three neutrophils fMLP-driven at in that reduced feedback found the not required such We was activation Rac2. of RhoG signalling of role whether of presence assessed potential interpretation we (Stephens the First, the GTPases levels. tested obfuscate Rho-family can We to and data. signalling 2008) al., GPCR et in play in to role engagement known GPCR a are loops of positive-feedback downstream However, pathway neutrophils. rapid the in induced activation 1H). (Fig. C5a P-Rex-dependent and strongly was fMLP, mediator that RhoG inflammatory Like of activation potent chemoattractant. a neutrophil C5a, factor complement easse MPdie ls IKatvt.W on no found PtdIns(3,4,5)P We activity. fMLP-stimulated PI3K in I Second, class an Rac2. difference not fMLP-driven towards is assessed activity P-Rex1 P-Rex1 we by of activity consequence RhoG indirect of regulation that indicating h rti C0(i.3)ad motnl,Ro Fg 3D). (Fig. Cdc42-like cells) RhoG the Sf9 importantly, baculovirus-infected 3C), from and, (Fig. (purified 3F) Rac3 P-Rex1 (Fig. activity Full-length as shown TC10 well previously exchange protein as as Rho 2002), substantial 3A,B,E), al., (Fig. et Cdc42 exhibited (Welch and P-Rex1 Rac1/2 In towards domain. assay, DH-PH P-Rex1 purified this the for substrates as GTPases ewe idtp W)adRhoG and (WT) wild-type between rvoswr a niae htPRx a c saGFfrRac for GEF a as act can vitro P-Rex1 proteins in that indicated RhoG has for work GEF Previous efficient an is P-Rex1 hs odtossgetn htatnplmrsto snot is 2C). (Fig. polymerisation RhoG actin or under actin P-Rex1 activation block through signalling that RhoG initiate to to in suggesting required B reduction conditions no latrunculin RhoG these PI3Kc observed fMLP-induced used on We of effect activity. its we measured downstream and Third, polymerisation, functions system. RhoG this that confirming noprto sa sn ae fHis of panel a using assay incorporation P- of specificity substrate Rex1 the investigated therefore We reported. 2 hs eut ugs htPRx susra fRhoG of upstream is P-Rex1 that suggest results These / 2 iewr nuae o 0mni h rsneo bec f25 of absence or presence the in min 10 for incubated were mice nvitro in ora fCl cec 21)17 5920 doi:10.1242/jcs.153049 2589–2600 127, (2014) Science Cell of Journal 6 nvitro in ...o oridpneteprmns * experiments. independent four of s.e.m. nmr eal efrtepoe mant-GTP a employed first We detail. more in u t ciiytwrsRo a o been not has RhoG towards activity its but 2 6 / 2 2 tge h family Rho -tagged P / # 2 A etohl from Neutrophils (A) .5b nunpaired an by 0.05 ie(i.2B), (Fig. mice 3 ie(i.2A), (Fig. mice P accumulation 2 aaare Data . 2591 m in M

Journal of Cell Science ienl o ohPRx n -e2(i.3K). from (Fig. P-Rex2 neutrophils and in P-Rex1 unaffected both Cdc42 for were null fMLP-driven GTP-Cdc42 activated, mice detect of were Rac levels not and RhoG and could where a conditions (our we have under cells activation neutrophils, to these mouse known in in is of role Cdc42 expressed analysis Although not observations). Transcriptome is unpublished TC10 3G–J). (Fig. suggests and RhoB neutrophils TCL RhoA, RhoG, not for but or GEF TC10, and efficient RhoC Cdc42 an Rac3, as and act Rac2 to Rac1, potential the and specificity 3L). (Fig. EERHARTICLE RESEARCH usin hrfr,i h xett hc ciaino hGis RhoG of 2592 activation which to coupling extent GEF, key the A dominant is RhoG. therefore, on a exchange question, as nucleotide to P-Rex1 directly activation identify GPCR data above The and activity Rac oxidase fMLP-driven NADPH regulate RhoG and P-Rex1 as well as RhoG, immunoprecipitation for on GEF [ based efficient assay, an of alternative as an act using to Rac1, able also was (P-Rex12 P-Rex-2 and P-Rex1 both for null Rho mice indicated and the mice WT on from exchange neutrophils nucleotide BM Guanine Isolated (K) GTPases. experiments. family independent Rho two expressed of representative bacterially are into shown mant-GTP of (2 incorporation GTPase RhoG. the family on monitor exchange that nucleotide assays guanine catalyses GEF directly P-Rex1 3. Fig. needn xeiet.()Rcmiat f-eie ERo n a1wr nuae o 0mnwt rwtotE–-e1 ntepeec fanionic of presence the in EE–P-Rex1, without or with min 10 for incubated were Rac1 PtdIns(3,4,5) and and EE-RhoG liposomes Sf9-derived Recombinant, (L) experiments. independent 10 with stimulated were onigo on aincetd.A oiiecnrlfrncetd-idn aaiiy Tae eeicbtdfr1 i ntepeec fET.Dt are Data EDTA. of presence the in min 10 for incubated were GTPases capability, nucleotide-binding for control positive a mean As radionucleotide. bound of counting hs eut ugs htPRx a eaieybodsubstrate broad relatively has P-Rex1 that suggest results These 35 6 ...o he needn xeiet efre ndpiae* duplicate in performed experiments independent three of s.e.m. S]GTP c -oddadE-agdlpdmdfe RhoG lipid-modified EE-tagged and S-loaded m )wsasse ntepeec rdcrls rasne(lesurs fPRx 20n) rosidct ieo -e1adto.Data addition. P-Rex1 of time indicate Arrows nM). (200 P-Rex1 of squares) (blue absence or circles) (red presence the in assessed was M) m MPfr1 .Cc2atvto a esrdb fiiypldw sn S–A-RB aaaemean are Data GST–PAK-CRIB. using pulldown affinity by measured was activation Cdc42 s. 10 for fMLP M P 3 n h noprto f[ of incorporation the and , 35 S]GTP c a sesdb muorcptto fE–hGo Rc n iudscintillation liquid and –Rac1 or EE–RhoG of immunoprecipitation by assessed was S P # .5b nupie Student’s unpaired an by 0.05 AJ h eobnn HP oano -e1wsue nfluorescent-based in used was P-Rex1 of domain DH-PH recombinant The (A–J) paetars ag ffL ocnrtos lhuhthey although doses. lower concentrations, were at fMLP formation greater of ROS proportionately range were in a defects NADPH across These fMLP-driven apparent 4). in (Fig. activity defects oxidase severe comparable displayed rvsPRxdpnetRo ciain etohl from neutrophils activation, stimulation in fMLP RhoG directly RhoG where deletion P-Rex-dependent conditions RhoG and drives Under P-Rex assays. sought of and our effects equivalent studies, the these with differences this compare for by used in to explained conflict conditions RhoG be experimental of might the thus this regulator in that major and reasoned the We defects is system. deletion, severe P-Rex1 more that RhoG the observation with upon deletion al., contrast P-Rex1 et studies reported of Welch these effects 2005; in small al., observed relatively et the Dong have However, 2006; 2005). al., NADPH studies et the Rac2- of (Condliffe activation some Separate oxidase as regulate such alternative P-Rex1 Rac2. functions, neutrophil and its dependent RhoG and of the that activation reported Rac1 additionally, previously the and, substrates to P-Rex1 function potential of this of function relationship important most the 2 / ora fCl cec 21)17 5920 doi:10.1242/jcs.153049 2589–2600 127, (2014) Science Cell of Journal 2 ieadmc ulfrbt -e1adP-Rex2 and P-Rex1 both for null mice and mice t -test. 6 ...o three of s.e.m. 2 / 2 )

Journal of Cell Science rmbt RhoG both from losgetsm hneln faRc n a2po othe RhoG alone. to and/or Rac2 P-Rex1 pool or of Rac2 Rac1 effects through and some not Rac1 that are or a occur, of must channelling oxidase RhoG they Rac2, some of and suggest Rac1 effects of also bigger activation than much formation ROS the on effect deletion given an this through Furthermore, system in RhoG. this P-Rex in on P-Rex indirectly for Rac2 can P-Rex1 and role or that Rac1 implies major regulate and GTP- RhoG RhoG the regulate to or that is of network ROS suggests signalling of -Rac2, effects 10 formation and fMLP-stimulated Rac1 in at either equivalence on activation deletion The Rac also isoforms 6E,F). reduced Vav (Fig. al., three et partially all Lawson for 2003; displayed null al., mice from et neutrophils (Kim 2011), consistent data again published and be regard, previously can this with In activation receptor. fMLP Rac P- the other, to by that engaged routes were and RhoG-independent, system deletion and/or this Rex- in P-Rex stimulus-strength- present and is suggesting redundancy RhoG 6A–D), dependant (Fig. of smaller effects significantly the data, (10 fMLP published of 5G,H). dose higher (Fig. conditions a Vav these At under reduced three activation partially Rac2) all displayed not (but also for Rac1 Vav3) null and mice Vav2 (Vav1, from isoforms Neutrophils 5C–F). (Fig. Rex2 EERHARTICLE RESEARCH eih,20) LOpoen xs osiuieybudto bound constitutively and exist Katoh proteins 2009; the al., ELMO of et 2003). (Ho those mediate Negishi, cells to only in demonstrated proteins, processes convincingly RhoG-dependent been effector have RhoG Of family neutrophils. ELMO identified in and activation few P-Rex1 Rac which the control by might mechanism the signalling identify RhoG to sought DOCK2 next of We translocation fMLP-driven regulates RhoG Fg AB htwssbtnily( substantially was that 5A,B) (Fig. (1 tmlto fnurpiswt u-aia oeo fMLP of dose sub-maximal a with neutrophils of Stimulation m )eiie ai omto fGPRc n GTP-Rac2 and GTP-Rac1 of formation rapid elicited M) 2 / 2 ieadmc ulfrbt -e1adP- and P-Rex1 both for null mice and mice m ) n ossetwt previously with consistent and M), § 0)rdcdi neutrophils in reduced 50%) m MfMLP ebae rsmbytruha neato ihEM.We ELMO. plasma RhoG and with the DOCK2–GFP crossing interaction by to hypothesis an this recruitment tested through DOCK2 presumably promoting membrane, by neutrophils in in observed as for. translocation, unaccounted remains DOCK2 studies, those into input independent lse fDC2GPtasoaineet,wihw termed RhoG and we WT which Both ‘large’. events, and translocation ‘small’ DOCK2–GFP of classes membrane ruffling. in membrane increases with any associated for control density Cell to us dye allowed membrane Orange the Mask with Counterstaining disc microscopy. undergoing spinning using confocal neutrophils coverslips of glass in on translocation membrane chemokinesis fMLP-induced plasma the the examined to and DOCK2–GFP controls, WT GFP-expressing n npr eitdb h OK H- oankonto known domain DHR-1 DOCK2 the PtdIns(3,4,5)P PI3K-dependent by mediated bind plasma is part the neutrophils in to and fMLP-stimulated translocation in DOCK2 those membrane that of demonstrated authors a the studies expressing transgene, mice 2006; from DOCK2–GFP al., neutrophils germline-encoded Using et 2009). (Kunisaki al., DOCK of neutrophils et regulator major Nishikimi crucial in a a activity DOCK2, be to Rac 2005). shown GPCR-driven al., been et has leukocytes, Lu al., in 2004; et isoform al., Kulkarni 2002; et al., Lu et 2011; through (Brugnera Rac domain on DHR-2 exchange DOCK bipartite the nucleotide a form catalyses they that together, complex and, GEF family DOCK the of members oto ffL-rvnRcatvt nneutrophils. in activity RhoG Rac RhoG-mediated and fMLP-driven that and P-Rex1- of for membrane control indicate basis plasma mechanistic the data a provide to thus These only DOCK2 of neutrophils. were translocation promotes WT events 7A,B). (Fig. in translocation protrusions observed DOCK2–GFP DOCK2–GFP-positive large small However, form to ognrt OK–F-xrsigRhoG DOCK2–GFP-expressing generate to eraoe htRo ih euaefL-rvnRcactivity Rac fMLP-driven regulate might RhoG that reasoned We vrtecus fteeeprmns eosre w discrete two observed we experiments, these of course the Over ora fCl cec 21)17 5920 doi:10.1242/jcs.153049 2589–2600 127, (2014) Science Cell of Journal 3 experiments. and (B) mean three are of Data (B,D). levels ROS integrated xeiet sn 10 from second] using per experiments ROS (RLU) units of light rate [relative the synthesis of traces as kinetic presented representative are Data HRP. of presence the in by chemiluminescence measured of luminol-dependent (ROS) production species and fMLP, oxygen of reactive doses indicated the with AB rRhoG or (A,B) ulfrbt -e1adPRx2(P-Rex12 P-Rex-2 and P-Rex1 both for null mouse primary neutrophils. in fMLP-driven activity regulate oxidase P-Rex NADPH and RhoG 4. Fig. (Co ˆ te ´ ta. 05.Hwvr DHR-1- a However, 2005). al., et etohl rmW ieadmice and mice WT from Neutrophils 2 / 2 6 ie(,)mc eestimulated were mice (C,D) mice ag fto()independent (D) two of range m 2 MP(,) n total and (A,C), fMLP M / 2 2 / etohl eeable were neutrophils 2 ieadDOCK2– and mice 2 / 6 2 2 / s.e.m. 2 mice, 2593 )

Journal of Cell Science CD,adRhoG and (C,D), EERHARTICLE RESEARCH 2594 DOCK2 F-actin from of Neutrophils polarisation fMLP-driven regulates RhoG neutrophils. mouse primary in agonist of doses low by driven 1 activity with Rac stimulated regulate P-Rex were and RhoG 5. Fig. u aasgetta hGcnata nusra regulator upstream an of distribution as the act visualised we can activity, actin RhoG that Rac in Given DOCK2-driven that spike DOCK2. of suggest of global loss data rapid by our a unaffected is follows that this and independent to polymerisation be gradients, localised to be agonist upon appears normally of polarisation would edge to spontaneous that due leading This activity presumably region. Rac the 2006), of al., at loss et the F-actin (Kunisaki fMLP concentrate with treatment to ability their n a2wr esrdb fiiypldw sn S–A-RB eeso T-on a1o A2wr omlsdt h oa a1adRc level. Rac2 and Rac1 total the to normalised were RAc2 mean or are Rac1 Data GTP-bound presented. of also Levels are GST–PAK-CRIB. * blots using western pulldown representative affinity of by Images measured were Rac2 and P # .5 ** 0.05, P # .1b nupie Student’s unpaired an by 0.01 2 / 2 ie(,)o ienl o l he a sfrs(Vav123 isoforms Vav three all for null mice or (E,F) mice m MPfrteidctdtms CH sltdB etohl rmW ie ienl o ohPRx n -e- (P-Rex12 P-Rex-2 and P-Rex1 both for null mice mice, WT from neutrophils BM Isolated (C–H) times. indicated the for fMLP M 2 / 2 ieehbtapoon eetin defect profound a exhibit mice t-test. 6 ...o or(–) he D,fv E n he FH needn experiments. independent (F–H) three and (E) five (D), three (A–C), four of s.e.m. 2 / 2 aclt h itiuino -ci ywide-field by F-actin ( polymerisation actin of studies rapid previous process, in this As distribution 7C,G). of (Fig. fixed microscopy the then and epifluorescence visualise to polarisation, us stimulated allowing spontaneous calculate TRITC–phalloidin, were allow with Cells stained to and process. suspension, polarisation of in loss this whether affects assess to RhoG neutrophils fMLP-stimulated in F-actin ihapeiul eotdlc fefc fDC2 hsevent this DOCK2, Consistent of phalloidin perimeter. effect of of cell lack ring entire reported bright the previously a out a with as marked that visualised staining be could treatment) GH eesiuae ih1 with stimulated were (G,H) ) ora fCl cec 21)17 5920 doi:10.1242/jcs.153049 2589–2600 127, (2014) Science Cell of Journal m AB sltdB etohl rmW mice WT from neutrophils BM Isolated (A,B) MPfr1 .Atvto fRac1 of Activation s. 10 for fMLP M , 5spost-fMLP s 15 2 / 2 )

Journal of Cell Science EERHARTICLE RESEARCH el xiiigFatnplrsto,adteetn fF-actin of of number RhoG extent the in the both reduced and min, significantly 2 were polarisation, at and polarisation, F-actin 1 began At exhibiting min. F-actin cells 2 total by polymerisation of complete of actin largely Polarisation not fMLP-driven analysis does 7D). RhoG global (Fig. FACS of loss curtail that deletion. confirmed significantly also RhoG levels F-actin by cellular unaffected was n ciiyo OK uigfL-eitdneutrophil fMLP-mediated during localisation are the DOCK2 results controlling of in These polarisation. RhoG activity 7E,F). for (Fig. role and a neutrophils with WT consistent to compared , 2 0sadwas and s 30 / 2 neutrophils hGi hssse.Ti stefrtsuyt successfully to study first the on is exchange This nucleotide system. guanine this for in the as responsible RhoG P-Rex1 redox GEF identify and major function, and neutrophil GPCR-driven secretion in remodelling, cellular actin dynamic of as understanding signalling. GTPase GTPases, our such rapid of to small processes, study central The multiple family. is between, Rho regulation the the interplay of modulating those and by particularly behaviour of, cell in activity changes rapid elicit GPCRs DISCUSSION u aarva hGatvto sarpdadcuilevent crucial and rapid a as activation RhoG reveal data Our ora fCl cec 21)17 5920 doi:10.1242/jcs.153049 2589–2600 127, (2014) Science Cell of Journal (Vav123 n ienl o ohPRx n -e- (P-Rex12 P-Rex-2 and P-Rex1 both for null mice at and predominates agonist. activity of Rac on doses deletion high Vav of Effect 6. Fig. ersnaiewsenbosaeas rsne.Dt are of Data Images presented. level. also mean Rac2 are and blots Rac1 western total representative Rac2 the or to Rac1 normalised GTP-bound were pulldown of affinity Levels by measured GST–PAK-CRIB. were using Rac2 and Rac1 of Activation AB,RhoG (A,B), 6 ag fto(,,,)idpnetexperiments. independent (A,B,E,F) two of range 2 / 2 EF eesiuae ih10 with stimulated were (E,F) ) 2 / 2 ie(,)o ienl o l he a isoforms Vav three all for null mice or (C,D) mice sltdB etohl rmW mice WT from neutrophils BM Isolated m MPfr1 s. 10 for fMLP M 2 / 2 ) 2595

Journal of Cell Science deo oaie etohl pnls fRo niae that indicates RhoG of loss upon The leading neutrophils 2003). the polarised to Negishi, recruitment of and DOCK2 edge in Katoh observed we 2006; adaptor defect their al., striking through et proteins (Hiramoto DOCK recruiting ELMO by Rac to signal 2002). rmG)adPtdIns(3,4,5)P and synergistic by demonstrated Gi) P-Rex has from work of previous regulation Thus activation. PI3K EERHARTICLE RESEARCH 2596 on dependence strong its and both response and this GPCRs, of of PI3K downstream speed activation the RhoG measure thsbe ieyrpre nohrssesta hGcan RhoG that systems other in reported widely been has It c c Bre ta. 07 ile l,20;Wlhe al., et Welch 2005; al., et Hill 2007; al., et (Barber ) n -e1idct rxmlrltosi oreceptor to relationship proximal a indicate P-Rex1 and bc 3 uuiso rtis(derived proteins G of subunits drvdfo G from (derived bc -stimulated ontemo PR nnurpis ti locnitn with consistent Rac also P-Rex and is indicating RhoG It data linking neutrophils. in previous for GPCRs responsible of also downstream is mechanism this ta. 05,RhoG 2005), al., et oe oe faoitadi motn o GPCR-stimulated for important This polarisation. is actin cooperative and 8). and (Fig. at formation agonist activation ROS simply activation of Rac cellular doses Rac total than in of lower into role important an input number rather plays pathway GEF by a hierarchical, of signalling, family unifies organisation Rho a of thus field indicating the Rac in for observations of pathway confusing new GPCR- control This in production. defects ROS GPCR-mediated display and all activity Rac neutrophils induced 2006) al., et (Kunisaki ora fCl cec 21)17 5920 doi:10.1242/jcs.153049 2589–2600 127, (2014) Science Cell of Journal 2 / 2 lutaino oaiainefcec (E efficiency polarisation of illustration (B). mean data are pooled Data as were presented mouse) and per counted cells 11 (average mice RhoG and (WT) control DOCK2–GFP-expressing primary of neutrophils. edge mouse leading the to polarisation F-actin and translocation fMLP-induced DOCK2 regulates RhoG 7. Fig. etohl rmRhoG from in neutrophils observed were events translocation ‘large’ no and WT RhoG in events ‘small’ translocation and DOCK2–GFP ‘large’ of microscopy. images confocal Representative by imaged 10 and with fMLP stimulated CMO, dye membrane aclto.E calculation. xeiet.* experiments. mean are Data ( the cells with of along number (G) presented of are stage analysis, each from cells, of representative Images (F). quantified efficiency the is polarisation with circumference) along cell of (E), 50% presented than F- less of to confinement actin as (defined polarised to deemed have cells of percentage The epifluorescence microscopy. wide-field by imaged and TRITC–phalloidin with stained fixed, permeabilised, being before times indicated 10 with stimulated were EG etohl rmW n RhoG and WT from Neutrophils (E–G) duplicate. in performed experiments are Data MFI. mean as presented are by cytometry, flow measured levels, F-actin Total TRITC– phalloidin. with stained and fixed, permeabilised being before times indicated 10 with stimulated were etohl rmfv TadtreRhoG three and WT five from neutrophils esstesdso ahcl (A cell each of front sides the the at versus (MFI) intensity fluorescence mean D etohl rmW n RhoG and WT from Neutrophils (D) nardStudent’s unpaired Cnlfee l,20)adDOCK2 and 2006) al., et (Condliffe 6 2 2 / / ...o he independent three of s.e.m. 2 2 2 etohl r hw A.Nt that Note (A). shown are neutrophils the with pre-labelled were mice / 2 P P Dn ta. 05 Welch 2005; al., et (Dong 6 6 a eemnda h ai of ratio the as determined was # n ...()Diagrammatic (C) s.e.m. ...o he independent three of s.e.m. .5 ** 0.05, nlsda ahstage. each at analysed ) t ts.Saebars: Scale -test. AB etohl from Neutrophils (A,B) 2 / m m 2 P MPfrthe for fMLP M MPfrthe for fMLP M ie vnsin Events mice. # .1b an by 0.01 y /A x 2 ) 2 / 2 P , 2 1. ) mice / 5 2 2 m m mice M / m. 2 2 / 2

Journal of Cell Science EERHARTICLE RESEARCH ob ciae ietyb G by directly shown activated been an be recently regard to has this complex [in activation –ELMO and, DOCK2 analogous 2002) parallel of Billadeau, pathways and other here, both (Turner possibly, GEFs involving family described Vav complex, of pathway activation Rac is of P-Rex1–RhoG–DOCK2 organisation GPCRs the the of that this clear downstream from within is activation Even it surfaces. interference context, factors plate simple paracrine culture minimal relatively or tissue to autocrine with adhesion by and/or isolation, generated signals in GPCR-driven background some in with and formation) least ROS neutrophils and precision at -Rac, quiescent and measure (GTP-RhoG of events to signalling stimulation us allowed Second, suspension (Stephens 2008). loops our al., cell feedback that et well-documented to probability as responses the such homeostatic rather stimulation, reducing convoluted seconds, days, reflect few or measurements a hours provides within minutes, First, approach happen fronts. than several this events their on data activation addition, through of GTPase interpretation In the regulators in mice. clarity and upstream further in GTPases of deletion endogenous involvement genetic of made activation the which we measure assess neutrophils, study, to primary present reflect of us the GPCR-sensitivity allowed always acute In the level. not of endogenous use might the or at active which events constitutively mutants, using systems dominant-negative overexpression on relied activation. families Rac GEF to context two pathway this these common in linking establish a for to in responsible important is be 2008; RhoG will al., it whether DOCK 2009; et 2010), al., Sanz-Moreno and al., 2011; et P-Rex et al., Fine et Sosa both 2012; Lindsay 2012; al., of al., et et role (Berger Hodis the cancer in in particular, members interest In family here. recent revealed the hierarchy 2000; potentially signalling given al., could the by et activity all) driven not Rac (Blangy be (if P-Rex-dependent many systems 2003), of of Negishi, and observations variety Katoh 2006; a al., et in Hiramoto potential functions Rac RhoG be that of given might However, upstream cells. Cdc42) in substrate and P-Rex for broad Rac substrates held The (including widely Rac. GTPases the family on challenge P-Rex exclusively of and acts selectivity cells, P-Rex in that proteins view P-Rex of role raiain(novn utpeipt notesm E and, GEF same of the type into this inputs by multiple conveyed properties (involving additional organisation The 2013)]. al., et aysmnlsuiso E n Taefnto have function GTPase and GEF of studies seminal Many general more the about questions important raise also data Our nvitro in culysget htsvrlRho- several that suggests actually bc uuisi irbat (Fritsch fibroblasts in subunits n Tcnrl eebe nprle.Rac2 parallel. in bred 2005) al., were et controls Welch 2008; WT al., et and (Donald previously described as obtained akrudwr rdwt RhoG with C57BL/6J bred a on were pathogen- 2006) al., opportunistic background et specific (Kunisaki mice under DOCK2–GFP conditions. isolators free within but facility same 6 akrudwr eeae ybedn Vav1 breeding by generated were background osdi niiulyvniae ae nabrirdaia facility. animal barriered a in cages ventilated Vav123 individually in housed prvdb h nmlWlaeEhc omte tteBabraham the at 80/2335. Committee PPL License Ethics Project Office Welfare Home Animal under Institute the by approved iea ecie rvosy(uiaae l,20)adW 1B mice B10Br WT P-Rex1 and controls. 2003) as al., used et were (Fujikawa previously described as mice RhoG Animals METHODS AND MATERIALS eeoyosprns Tltemtswr sda otoswhere controls as to born used litters were from littermates PI3K genotyping, WT possible. PCR parents. by heterozygous selected, were background DOCK2–GFP aall ienl o l he a sfrs(Vav123 isoforms in Vav bred three were controls all WT for and null 2000) Mice al., parallel. et (Hirsch previously described -e2(P-Rex12 P-Rex2 ewe –0weso g n g-ace hr osbe RhoG possible. Institute where Babraham at age-matched used and the were age Mice of from controls. weeks as obtained 8–20 use between for were Unit Breeding mice 1999) Services al., Biological C57BL/6 et (Roberts WT previously described and as obtained were background PI3K anrta prcae hi pta n eprlspecificity. observe temporal a directly and in spatial to level, their is endogenous appreciates field the that manner at this clear interactions a in and GEF–GTPase studies deduction, distinct future on based for This largely of challenge oxidase. still pools NADPH is of the however, as ‘streaming’ picture, such the effectors, for specific mechanism to GTP-Rac simple specificity a substrate allows broad also a exhibit provides GTPases, acting vitro naturally GEFs family other also Rho many different indeed on stimulation, and (conveying into P-Rex, the cell whereby on recruited involved mechanism of depends are which context GEF of GEFs precise composition Rac the that complexes, particular idea molecular The are each that (conveying specificity). processes to molecular to Rac to the likely Rac of distinct to of properties are coupling molecular pathways the they specific constituents, the of but through unclear, and, amplification sensitivity) still both are include GEFs) multiple also, RhoG c u a edrce odfeetseii usrtsi el.It cells. in substrates specific different to directed be can but , 2 2 / 2 2 / 2 ora fCl cec 21)17 5920 doi:10.1242/jcs.153049 2589–2600 127, (2014) Science Cell of Journal +/+ / 2 P-Rex1 , ie(ioioe l,20)o ie C57BL/6J mixed a on 2004) al., et (Vigorito mice Rac2 , itrae scnrl.Almuewr a umte oand to submitted was work mouse All controls. as littermates 6 c 2 RhoG 2 2 / 2 2 / / 2 2 / 2 ieo 2/l akrudwr bandas obtained were background 129/Ola a on mice namxdC57BL/6J mixed a on ) n soitdW oto iewr osdi the in housed were mice control WT associated and P-Rex12 , 2 / 2 sdfreprmnsaogwt DOCK2–GFP with along experiments for used 2 / 2 2 / ieadmc ulfrbt -e1and P-Rex1 both for null mice and mice 2 a E OK.PtdIns(3,4,5) DOCK2. GEF the Rac of activation GTP-RhoG RhoG-dependent of and formation the in resulting MPrlae G for releases GPCR fMLP Gi-coupled the of Activation fMLP neutrophils. the mouse by in Rac receptor of Activation 8. Fig. eti euaoyinput. less regulatory or certain indirect an represent broken arrows the input, a regulatory represent direct arrows bold Rac The of activity. localisation the into multiple inputs has of thus, edge and, leading neutrophils the to ELMO– complex the DOCK2 localise to shown been also PtdIns(3,4,5) PI3K with synergise 2 n soitdW oto iewere mice control WT associated and / 2 o tlattognrtosand generations two least at for 6 2 P 2 / 2 3 2/vbcgon were background 129/Sv / 2 Vav2 , oatvt P-Rex1, activate to bc ieo C57BL/6J a on mice uuis hc then which subunits, c -generated 2 2 / / 2 2 naB10/Br a on ) n Vav3 and 6 P 129/Sv 3 2597 has 2 2 / 2 in / 2 ,

Journal of Cell Science on a1adRc) n nuae ihrtto o 5mna 4 at min 15 for GTP- rotation of with incubated detection and (for Rac2), GST–PAK-CRIB and Rac1 or bound 20–50 GTP-RhoG) of approximately detection containing 750 20 beads, A to Rac. added 4B or was RhoG supernatant of remaining levels the total assess to taken aliquots Mg qiirtdt 37 to equilibrated rt L)wt M n nue ih02m PGfr1 t20 at h 16 for IPTG mM 0.2 with induced and AMP with (LB) broth (DE3) BL21 Rosetta2 hr ramn ihihbtr a eurd niio ramnswere treatments Inhibitor 37 required. at performed was all inhibitors with treatment where hoielss(3 MNH mM (130 ammonium by lysis removed erythrocytes chloride remaining the and from harvested interface (Amersham). were 62%–55% cytospin) Percoll by assessed 55% pure, and (70–95% Neutrophils 62% comprising gradients discontinuous (1640 centrifugation by separated (HBSS++), BSA free (326 acid centrifuged endotoxin/fatty and 0.25% and HEPES mM 15 with ftm ecie.Clswr upne na1m ia oueo ice- of mM 100 volume MgCl 7.4, final mM pH ml 2 Tris-HCl 1 NP40, mM 1% a 50 NaCl, glycerol, in (10% suspended buffer were GST-FISH cold Cells described. time of 6–0 ue sesdb yopn eete ahdoc (326 once washed HBSS++–Percoll then Cells were lysis. the cytospin) 4 chloride by ammonium assessed from by pure, (60–80% removed removed Non- erythrocytes Percoll. and were 58% interface and leukocytes HBSS++ of granulocytic comprised gradients discontinuous eoebigprfe sn -lHsrpFs lwclm (GE column Flow tobacco Fast with HisTrap incubated 5-ml were a fractions P-Rex1-containing using Healthcare). purified being before o esrmnso PtdIns(3,4,5) of measurements For mouse from neutrophils marrow primary bone of preparation and Isolation ARTICLE RESEARCH 2598 (residues frame His in P-Rex1 (Invitrogen) N-terminal of HTb an pProEx domains vector with the DH-PH into the subcloned was encoding 40–405) fragment cDNA A assays Guanine-nucleotide-exchange (1 neutrophils activity primary Rac2 and Rac1 Mouse RhoG, endogenous of Measurement were above, described as isolated 37 neutrophils, to equilibrated primary Mouse PtdIns(3,4,5) endogenous of Measurement wc ihHS+ n newt ubcosPScnann Ca containing PBS Dulbecco’s with once and HBSS++ with twice n eaae ycnrfgto (1640 HBSS++ in cold centrifugation GTP-RhoG ice in of by suspended measure was separated marrow reproducible bone and a Mouse obtain cells. we resting to which factors, necessary paracrine and/or autocrine found by procedures activation isolation basal neutrophil reduce our to refined we translocation, DOCK2 (per and (see mice experiment independent more mice. an or individual as legends). treated between two figure was variation preparation from reduce pooled to marrow Each combined bone was possible, genotype) the on depending Where D-PBS++ of assay. volume appropriate an in re-suspended and .4m CaCl mM 1.54 KH mM 0.176 c.Lstswr etiue (13,000 centrifuged were Lysates ice. ocnrtoso MPo 5,adicbtda 37 at incubated and C5a, or fMLP of concentrations upnin eeicbtdfr2mna 37 at min 2 for incubated were suspensions lLtuclnBfr1 min. 10 for B Latrunculin ml arwwssseddi BS(ihu Ca (without HBSS in suspended was marrow eiaie n nlsdb CEIM sdsrbdpeiul (Clark previously described extracted, 2011). as lipids al., LC-ESI/MS and et by lysed, then analysed were and Cells derivatised time. of periods indicated the ed eewse he ie nGTFS ufradsseddin suspended and blotting. and SDS-PAGE western 12% buffer by by separated GST-FISH analysed were in Samples buffer. times sample three Laemmli washed were Beads ˚ )i BS+adr-upne t1 at re-suspended and HBSS++ in C) o esrmnso hGGP d4-T,FatnadRSlevels ROS and F-actin Cdc42-GTP, RhoG-GTP, of measurements For 2+ upeetdwt / lcs n MNaHCO mM 4 and glucose g/l 1 with supplemented , 2 2 PO 34m NaHCO mM 13.4 , ˚ n 1 and C 4 ˚ . Mguoe MMgCl mM 1 glucose, mM 5.5 , sflos 0 Mwrmni o i,20 min, 5 for wortmannin nM 100 follows: as C ˚ n 2 and C g .coli E. tro eprtr) el eer-upne and re-suspended were Cells temperature). room at 6 a.PRx HP rti a xrse in expressed was protein DH-PH P-Rex1 tag. 6 10 el EDBocecs rw nlysogeny in grown Biosciences) (EMD cells 2 6 0m a)adicbtdfr3mnon min 3 for incubated and NaF) mM 20 , 4 6 l MKl .8m Na mM 0.78 KCl, mM 5 Cl, 10 el tmltdwt 10 with stimulated cells 6 g 6 P 10 3 tro eprtr)fr3 i on min 30 for temperature) room at 3 o – i.Clswr washed were Cells min. 1–2 for ) el tmltd ihindicated with stimulated, cells 7 n a-T ees os bone mouse levels, Rac-GTP and g el/li -B+)were D-PBS++) in cells/ml o i t4 at min 3 for 6 ˚ m g ro osiuainexcept stimulation to prior C 2+ fglutathione–Sepharose- of l 10 m t4 at n Mg and fGTEM1(for GST–ELMO1 of g 7 P m nDPS+ Cell D-PBS++. in /ml 3 levels ˚ 2 )fr3 i on min 30 for C) .8m MgSO mM 0.28 , ˚ 2+ o h periods the for C ,supplemented ), ˚ m )ad50 and C) 3 m MPfor fMLP M (D-PBS++), oueof volume l 2 HPO 2+ g and m ˚ ˚ C. m g/ at C 4 4 l , , sae ndpiaeadtetda ehia replicates. technical were as Samples treated (HRP). and peroxidase duplicate horseradish in U/ml assayed 26.7 of presence the agdPRx prfe rmbclvrsifce f el)and cells) Sf9 EE- baculovirus-infected full-length recombinant from using (purified experiments [ of P-Rex1 number tagged small a in monitored. thvrspoes vrih orlaeteHis the release to overnight protease virus etch n2 MTi-C H80 0 MNC,1m DA MDTT at stored mM and 2 N2 EDTA, liquid protein in mM DH-PH frozen 1 P-Rex1 snap concentrated, NaCl, purified pooled, containing mM were Fractions 200 glycerol. 8.0, 10% pH and Tris-HCl equilibrated Healthcare) mM (GE 20 column exclusion in size S-200 an onto loaded atrduiga no XnE-C 9 aea a mg data image Ti-E Raw Hilversum (SVI, Nikon camera. software NL). 897 Professional a and Huygens EM-CCD on using unit, iXon deconvolved disk acquired were Andor spinning an were CSU-10 using Yokogawa Images captured a movie. with microscope each confocal of minute first 10 a which during ietdot h ls oesi neto niaigrn and ring imaging an of insert coverslip 37 1 were glass of cells to presence D-PBS++, the the equilibrated in in onto washing ice, After on pipetted (Invitrogen). min, Orange 5 Mask for Cell incubated and BSA 0.1% xrse n uiidRoTae a are u ihaFluoroMax- 20 a at with out (Horiba) carried spectrometer was 4 RhoGTPases purified and expressed 2007). al., et (Mitin previously auoiu-netdS9clsuigTio 14,a described as X114), Triton using 2002). al., et cells (Welch previously Sf9 baculovirus-infected eete ahdtiei -B+ ih2 S,sseddi 200 in suspended again. BSA, 2% Cells with centrifuged temperature. D-PBS++ in room twice at ng/ml washed and min 50 200 then 15 and were for in X-100 BSA incubated Triton and re-suspended 1% TRITC-phalloidin) containing D-PBS++/2% cells BSA (D-PBS++/0.1% re- in discarded, buffer were ml diluted 1 Cells were discarded. were Supernatants supernatants (500 in cells and centrifuged temperature) Fixed BSA, suspended room 0.1% temperature. at with with min D-PBS++ room time, 4 of at of volume excess min periods an 15 indicated for the final) for stimulated, 10 were sample per B+ ih01 S n ihriae ne wide-field under imaged either and BSA Cell 0.1% (Olympus epifluorescence with PBS++ O rdcinb TadRhoG and WT by activity production oxidase ROS NADPH neutrophil of Measurement hn io akr(IIaigFclt)frhsassac ihDOCK2–GFP with assistance his deconvolution. for and Facility) also capture We Imaging image support. (BI technical Walker their the for Simon Unit and thank Services spectrometry) Biological Keith mass Babraham the and (BI at construct Clark team GST–ELMO1 Jonathan the Kulkarni, supplying Suhasini for Davidson, Burridge Keith thank We Acknowledgements 4 at isolated were neutrophils Mouse translocation DOCK2 of Imaging 4 at isolated accumulation were F-actin neutrophils polarised Mouse and total of Measurement 37 at D-PBS++ in cells/ml H75 0 MNC,5m MgCl mM Tris-HCl 5 mM NaCl, 20 mM containing 200 7.5, mixtures pH assay Exchange-reaction 2007). oaiainefcec E)wsdtrie sdsrbdi i.7. and Fig. in polarisation described for as determined scored was were (EP) efficiency Cells polarisation cytometer. flow FACSCalibur ylmnldpnetceiuiecnei lt uioee,as luminometer, (2.5 neutrophils Mouse plate 2005). al., a et (Condliffe in previously described chemiluminescence luminol-dependent by rprdadalwdt qiirt ihcniuu trig After 200 stirring. at ( added continuous was intensity with protein equilibrate fluorescence DH-PH to P-Rex1 allowed equilibration, and 2 and prepared (Invitrogen) mant-GTP nM 400 -B+ ih01 S n qiirtdfr2mna 37 at min 2 for equilibrated and BSA 0.1% with D-PBS++ His 35 natraie muorcptto-ae sa a loperformed also was assay immunoprecipitation-based alternative, An -ehlnhaiol(at-T noprto nobacterially into incorporation (mant)-GTP N-methylanthraniloyl S]GTP m 6 tge hGPsswr xrse n uiida described as purified and expressed were RhoGTPases -tagged MPadfxdi neulvlm f4 aaomleye(2% paraformaldehyde 4% of volume equal an in fixed and fMLP M ora fCl cec 21)17 5920 doi:10.1242/jcs.153049 2589–2600 127, (2014) Science Cell of Journal c -odn fE-agd ii-oiidRo prfe from (purified RhoG lipid-modified EE-tagged, of S-loading m ˚ o i.Clswr hniae o min, 7 for imaged then were Cells min. 5 for C MPwsapida ahsiuaindrn the during stimulation bath a as applied was fMLP M l ˚ emission )wr nuae ih160 with incubated were C) ˚ sdsrbdpeiul Mtne al., et (Mitin previously described as C ‘ ,60 R, 6 nm; 360 , 2 / ˚ 2 ,ssedd(t5 (at suspended C, 2 MDT %(/)glycerol, (v/v) 5% DTT, mM 1 , ˚ 6 m os etohl a measured was neutrophils mouse ,sseddi -B+ with D-PBS++ in suspended C, fterlvn Taewere GTPase relevant the of M betv)o nlsdo a on analysed or objective) l excitation 6 a n subsequently and tag 6 4 m was nm) 440 , m 10 uio in Luminol M 6 m ˚ .10 C. m n the and M el/l in cells/ml) staining l 2 6 m 80 6 g g/ml cells 10 ˚ for C. m 6 l

Journal of Cell Science uiaa . iei,A . l,F . aco . rw,T,Ho,J., Hoog, T., Brown, R., Faccio, W., F. Alt, V., A. Miletic, K., S., M. Fujikawa, Kumar, B., Reason, R., George, K., Fritsch, I., Krijger, de R., Fritsch, ie . oaok,C,Kua,S,S,T,Sa,L . arr . okn,B., Hopkins, M., Maurer, H., L. Saal, T., Su, S., D. Koujak, C., Wu, Hodakoski, and B., Fine, Z. Li, C., Guo, G., Bokoch, Z., Mo, X., Dong, Andrews, A., S. Walker, A., Segonds-Pichon, I., Fyfe, T., Humby, S., Donald, Coadwell, John S., Krugmann, R., Barnouin, C., Lecureuil, K., Hill, S., Donald, iae,M C. M. Dinauer, Co odif,A . eb .M,Fruo,G . aisn . unr . Vigorito, M., Turner, K., Davidson, J., G. Ferguson, M., L. Webb, M., A. Condliffe, egr .F,Hds . efra,T . eie .L,Lwec,M S., M. Lawrence, L., Y. Deribe, P., T. Heffernan, E., Hodis, A., F., Byron, M. D., Berger, J. Humphries, D., R. Nunan, C., R. Williamson, D., M. Bass, Co T., Crabbe, D., C. Ellson, E., K. Anderson, K., Davidson, M., A. J., Condliffe, M. Wakelam, F., Karpe, S., T. Smith, V., Juvin, E., K. Anderson, J., Clark, Tosello-Trampont, F., S. Walk, M., Lu, C., Grimsley, L., and Haney, C. E., Gauthier-Rouvie`re, Brugnera, A., Debant, S., Schmidt, E., Vignal, A., Blangy, P-Rex12 OK–F iefrtesuy ..sple h Vav123 the supplied H.W. study; the the supplied Y.F. for measurements; mice PIP3 K.A. DOCK2–GFP P-Rex; analysed full-length and of with performed assays assays designed, GEF GEF performed analysed C.L. and incorporation; from performed mant-GTP cells designed, involving K.R. experiments mice; experiments; analysed DOCK2–GFP analysed and and performed manuscript; performed designed, the designed, L.G. co-wrote manuscript, and the project co-wrote the supervised G.D. and conceived L.S. and P.H. contributions Author interests. competing no declare authors The interests Competing ARTICLE RESEARCH abr .A,Dnl,S,Tee,S,Adro,K . hln .adWelch, and M. Thelen, E., K. Anderson, S., Thelen, S., Donald, A., M. Barber, in Deposited References studentship. PhD BBSRC months. a 6 of after receipt release P05/1). in for number each PMC [grant were Foundation C.L. Lung and grant British G.D. project the and and BB/J004456/1 BB/1008489/1]; number Research number Sciences grant Biological [programme and (BBSRC) Biotechnology Council the by supported was work This Funding eindadsprie E saso atGPincorporation. mant-GTP of assays GEF supervised and designed ˆte ˆte rdrcs . ih,S,Mlie,S,Moe,S .e al. et L. S. 3-kinase Moores, S., Mildiner, phosphoinositide S., Nishi, J., distinct Fredericks, regulate J. directly Downward, GTPases isoforms. and of G. families Stamp, M., Diefenbacher, 1261-1265. ncne hog niiino TNb xhnefco P-REX2a. factor exchange by PTEN of al. inhibition et through S. cancer Mense, in L., M. Sulis, M., Keniry, neutrophils. mouse in factor exchange Biol. nucleotide guanine coordination. Rac2 primary motor and C. USA H. morphology Sci. Welch, dendrite Acad. and S. Natl. cell L. Proc. Purkinje Wilkinson, P., regulates al. Emson, J., P-Rex2 et W. Coadwell, R. R., L. S. Stephens, T., P. Rac. for Hawkins, factor A., exchange guanine-nucleotide 572 S. new Walker, a P-Rex2, R., (2004). S. Andrews, W., Hematol. Opin. vltoaiycnevdPdn(,,)3bnigdmi sncsayfor necessary is domain PtdIns(3,4,5)P3-binding signalling. DOCK180 conserved evolutionarily uefml fDC10rltdpoen ihgaiencetd exchange nucleotide guanine with proteins activity. DOCK180-related of T. P. superfamily Hawkins, and R. oxidase. L. NADPH Stephens, neutrophil the R., regulates E. RhoG Chilvers, M., Manifava, E., 1432-1440. 21) eaoagnm eunigrvasfeun RX mutations. al. PREX2 et frequent P. Ghosh, reveals E., sequencing Nickerson, Nature genome R., I. Melanoma Watson, (2012). E., Ivanova, A., Protopopov, integrin RhoG-regulated and caveolin- J. through M. healing Humphries, endocytosis. wound and P. initiates Martin, trigger R., M. Morgan, 29967-29976. h rmdrsiaoybrto ua u o uieneutrophils. murine not but human of burst P. respiratory M. primed Wymann, L., the Webb, M., al. Turner, et B., Vanhaesebroeck, K., Okkenhaug, spectrometry. T. mass P. by tissues Hawkins, and 267-272. cells and in species R. molecular Dock180-ELMO L. the Stephens, through mediated S. K. is Ravichandran, and activity R. complex. G. Fink, Rac-GEF H., Madhani, Unconventional G., I. Macara, C., A. rhoG. of activation direct the through P. Fort, eaam uuisadpoponstd 3-kinase. phosphoinositide and subunits betagamma C. H. ,J . ooaa .B,Bs,J .adVoi K. Vuori, and A. J. Bush, B., A. Motoyama, F., J. ´, ,J .adVoi K. Vuori, and F. J. ´, 172-176. , 15 20) eunilatvto fcasI n ls AP3 sipratfor important is PI3K IA class and IB class of activation Sequential (2005). 2 20) ebaetasoaino -e1i eitdb protein G by mediated is P-Rex1 of translocation Membrane (2007). 485 1874-1879. , / .Cl Sci. Cell J. 2 20) roE1cnrl a-adCc2dpnetcl structures cell Cdc42-dependent and Rac- controls TrioGEF1 (2000). a.Cl Biol. Cell Nat. Cell n Rac2 and 502-506. , e.Cell Dev. 20) euaino etohlfnto yRcGTPases. Rac by function neutrophil of Regulation (2003). 153 10 1050-1063. , 8-15. , 115 2 / a.Cl Biol. Cell Nat. 2 21 4901-4913. , iefrtesuy n dtdtemnsrp;C.D. manuscript; the edited and study, the for mice 4 574-582. , 681-693. , 20) dniiaino neouinrl conserved evolutionarily an of Identification (2002). 105 4483-4488. , 7 .Cl Sci. Cell J. 797-807. , 20) ciaino h IKpathway PI3K the of Activation (2009). 21) uniiaino PtdInsP3 of Quantification (2011). .Immunol. J. 113 21) ydcn4hair syndecan-4 A (2011). 729-739. , 21) A n RHO and RAS (2013). .Bo.Chem. Biol. J. 2 20) Vav1/2/3-null (2003). 20) oe and novel A (2005). 20) -e1i a is P-Rex1 (2005). / 2 176 P-Rex1 , a.Methods Nat. 5314-5320. , Science Blood ESLett. FEBS 2 / 2 (2008). (2002). (2006). , Curr. Curr. 325 106 282 8 , , , , ii,N,Bts . oe .E,Dr .J,Sne,J n osa,K L. K. Rossman, and J. Sondek, J., C. Der, E., M. Yohe, L., J., Betts, Sondek, N., M., Mitin, Hall, C., Grimsley, L., K. Rossman, M., J. Kinchen, M., Lu, Brugnera, C., deBakker, C., Grimsley, L., K. Rossman, M., J. Kinchen, M., Lu, L., R. Mort, F., Rambow, J., W. Faller, D., A. Campbell, S., Lawn, D. R., Wu, C. Lindsay, and V. A. Smrcka, Z., Zhang, W., Xie, H., Jiang, Z., Li, asn . ec,C,Mnga-esn . an .M n urde K. Burridge, and M. K. Hahn, E., Monaghan-Benson, C., Welch, T., Samson, J. Sondek, and J. C. Der, L., K. Rossman, Spaetti, B., Petryniak, R., Kapur, B., J. Lowe, L., Zhen, C., Kim, W., A. Roberts, Cao, H., Mihara, S., Takasuga, T., Hongu, W., and Su, H., K. Fukuhara, Nagashima, A., H., Nishikimi, Sawa, M., Tsuda, A., Oda, M., Maeda, H., Nishihara, C. H. Welch, and T. D. Patton, E., K. Anderson, S., A., Donald, Inayoshi, D., C. M., Lawson, Noda, R., Takii, Y., Tanaka, A., Nishikimi, Y., Kunisaki, aai . reSsk,J,Jns .G,Oier-o-ats .J,Stanford, J., A. Oliveira-dos-Santos, G., R. Jones, J., Irie-Sasaki, T., Sasaki, Sahai, S., Pinner, P., Marra, H., Paterson, J., Ahn, G., Gadea, V., Sanz-Moreno, ukri . ag . hn,Z n afr,D. Barford, and Z. Zhang, J., Yang, K., Kulkarni, i,C,Mrhl .C,Pnigr .adDnur .C. M. Dinauer, and J. Penninger, O. C., Hoeller, C. and Marchal, D. C., Traynor, P., Kim, Langridge, R., R. Kay, ug .C,Ce,S . al .D,Srk,K . o .C,Yn,J .and T. J. Yang, M. C., Negishi, Y. and Lo, H. M., Katoh, K. Stroka, D., C. Paul, H., S. Chen, C., W. Hung, oi,E,Wto,I . ruo,G . rl,S . meisi . Theurillat, M., Imielinski, T., S. Arold, V., G. Kryukov, R., I. Watson, E., and J. Hodis, S. D’Souza, S., K. Ravichandran, G., Lajoie, J., G. Vilk, T., Irvine, E., Ho, ish . aaav .L,Grad,C,Azln,O,Prl,L,Slno L., Silengo, L., Pirola, O., S., Azzolino, C., Fujimoto, Garlanda, K., L., V. Katanaev, Harada, E., Hirsch, S., Ueda, S., Takeuchi, N., Hiramoto-Yamaki, H. Katoh, and M. Negishi, K., Hiramoto, il . rgan . nrw,S . odel .J,Fnn . Welch, P., Finan, J., W. Coadwell, R., S. Andrews, S., Krugmann, K., Hill, Dock180. 20) ees fatihbto fAE yAClast D4 activation CDC42 to leads APC by ASEF suppression. of tumor autoinhibition and of Release (2007). of S. family Dock180 K. the via Ravichandran, exchange nucleotide and GEFs. of V. regulation for Yajnik, model inhibition O., al. M. et Hengartner, J. Sondek, D., functions Klingele, ELMO B., of L. domain Haney, PH C., (2004). A. Tosello-Trampont, metastasis. E., melanoma and al. et migration A. melanoblast R. Commun. efficient Ridgway, P., for Cammareri, required A., Li, P., signal Timpson, chemoattractant-mediated in PI3Kgamma and transduction. -beta3 and PLC-beta2 21) noeosRo srpdyatvtdatreiemlgot factor growth factors. epidermal exchange after guanine-nucleotide activated Cell multiple rapidly through is stimulation RhoG Endogenous (2010). factors. nucleotide-exchange Biol. guanine with GTPases defense. RHO host by and characterized is function al.Rac2 neutrophil et GTPase 196. B. family in G. Rho Bradford, abnormalities B., cell-specific neutrophil J. hematopoietic Borneo, during the D., J. al. Pollock, et A., two H. by Hasegawa, dynamics M., DOCK2 . Kanai, of F., regulation Sanematsu, Q., lines. cell leukemia S. Tanaka, formyl-methionyl-leucyl- of responses. regulation the neutrophil in phenylalanine-dependent cooperate Vav1 and P-Rex1 (2011). chemotaxis. neutrophil during polarity Biol. and Cell motility J. regulates al. that et activator T. Rac Sasaki, T., a Sasazuki, F., Sanematsu, K., Watanabe, .L,Bln . aea,A,Ii,A,Buhr,D,Kzeazi .e al. et I. Kozieradzki, D., Bouchard, A., Itie, A., Wakeham, B., Bolon, L., W. movement. cell tumor J. of C. Marshall, and E. pcfcCc2adRcpoenatvto yddctro yoiei (DOCK) cytokinesis of dedicator by activation factors. exchange protein nucleotide Rac and Cdc42 specific eooei h/a unn uloieecag atrVv regulates functions. Vav1 Immunol. J. factor neutrophil exchange nucleotide formyl-methionyl-leucyl-phenylalanine-activated guanine Rho/Rac hemopoietic chemotaxis. of study the in directions h ok8-idn rti Elmo. protein Dock180-binding the spaces. confined versus unconfined in K. Konstantopoulos, .P,Ncesn . uli,D,L,L,Pae .e al. et C. Place, melanoma. L., in Li, mutations D., driver Auclair, E., Nickerson, P., J. polarity. cell L. Dagnino, oefrGpoenculdpoponstd -iaegmai inflammation. in gamma P. 3-kinase M. phosphoinositide Wymann, protein-coupled and Science F. G Altruda, for A., role Mantovani, S., Sozzani, H. mechanism. RhoG-dependent Katoh, a through and migration M. Negishi, migration. cell Rac-dependent promotes hshtdlnstl(,,)tipopaeadGeaam subunits. R. Gbetagamma L. Chem. and Stephens, and (3,4,5)-trisphosphate T. P. phosphatidylinositol Hawkins, E., C. H. n ciainbtadfeeta eurmn nMP inln nTadBcells. B and T in signaling MAPK development in Med. requirement Exp. in differential J. proteins a family but Vav activation for and role essential an define mice 21 6 167-180. , ur Biol. Curr. 1629-1642. , ora fCl cec 21)17 5920 doi:10.1242/jcs.153049 2589–2600 127, (2014) Science Cell of Journal 280 287 a.Src.Ml Biol. Mol. Struct. Nat. 2 4166-4173. , 20) OK soitswt rLadrgltsRc nhuman in Rac1 regulates and CrkL with associates DOCK2 (2002). 555. , 174 1049-1053. , o.Bo.Cell Biol. Mol. 171 Science 20) nernlne iaeitrcin ihEM2modulate ELMO2 with interactions kinase Integrin-linked (2009). Science 198 647-652. , 4425-4430. , 1595-1608. , 15 Blood 371-377. , 324 287 21) itntsgaigmcaim euaemigration regulate mechanisms signaling Distinct (2013). 20) a ciainadiatvto oto plasticity control inactivation and activation Rac (2008). a.Src.Ml Biol. Mol. Struct. Nat. Cell 20) hGatvtsRc ydrc neato with interaction direct by Rac1 activates RhoG (2003). 384-387. , 1046-1049. , 100 20 .Bo.Chem. Biol. J. 135 3033-3043. , 3968-3974. , Cell 21) pei4adEh2mdaecell mediate EphA2 and Ephexin4 (2010). 11 510-523. , 756-762. , Nature 150 a.Rv o.Cl Biol. Cell Mol. Rev. Nat. 20) ok srgltdb hGand RhoG by regulated is (2006). .Cl Biol. Cell J. 251-263. , ntrans in x.Cl Res. Cell Exp. 424 20) E en o unn on turning go: means GEF (2005). 286 20) euaino -e1by P-Rex1 of Regulation (2005). 14 461-464. , .Immunol. J. 25341-25351. , orglt a ciainvia activation Rac regulate to 21) utpefcosconfer factors Multiple (2011). 814-823. , .Cl Biol. Cell J. 202 807-824. , 21) adcp of landscape A (2012). 312 19) eiinyof Deficiency (1999). Immunity a.Rv o.Cell Mol. Rev. Nat. 20) Sequential (2009). 4205-4216. , 20) Changing (2008). 186 21) -e1is P-Rex1 (2011). 20) OK is DOCK2 (2006). 20) Steric- A (2005). 20) oe of Roles (2000). 9 190 20) Central (2000). 455-463. , 1467-1476. , 20) The (2003). 461-477. , o.Biol. Mol. 10 .Biol. J. 183- , 2599 Nat. N -

Journal of Cell Science tpes . in,L n akn,P. Hawkins, and L. Milne, L., Busillo, Stephens, A., M. Lemmon, H., Wang, C., Yang, C., Lopez-Haber, S., M. Sosa, EERHARTICLE RESEARCH 2600 a ul .D,Alnhm .J,Smo,T,Mle,J,Butr . Garcı E., Boulter, J., Meller, T., Samson, J., M. Allingham, D., J. Buul, van D. D. Billadeau, and M. Turner, .M,Lo . eoi,J . li-zno . ai .e al. et H. chemotaxis. Yagi, of A., understanding Klein-Szanto, signaling ErbB L., of mediator cancer. essential J. an breast as Benovic, in P-Rex1 Rac-GEF J., the of Luo, Identification M., activation, cell J. T development, migration. in neutrophil PI3Kgamma and of Function (2000). aa .adBrig,K. Burridge, and R. Mata, receptors. immune-recognition multi-subunit o.Cell Mol. Science 40 877-892. , 20) hGrgltsedteilaia cup apical endothelial regulates RhoG (2007). ur Biol. Curr. 20) A rtisa inlitgaosfor integrators signal as proteins VAV (2002). 287 1040-1046. , 18 R485-R494. , 20) oigtwrsabetter a towards Moving (2008). a.Rv Immunol. Rev. Nat. 2 476-486. , (2010). ´a- ioio . el . ees .J,Ryod,H,MAa,S,Esn .C., P. Emson, S., McAdam, H., Reynolds, J., B. Hebeis, S., Bell, E., Vigorito, ec,H .E,Cnlfe .M,Mle .J,Fruo,G . il . Webb, K., Hill, J., G. Ferguson, J., L. Milne, M., A. Condliffe, E., C. H. Welch, R., S. Andrews, J., G. Ferguson, D., C. Ellson, J., W. Coadwell, C., H. Welch, h a-eae TaeRhoG. GTPase Rac-related M. Turner, the and A. McKenzie, sebydwsra rmIA1eggmn n sivle nleukocyte in involved is and engagement migration. ICAM1 trans-endothelial from downstream assembly .M . kehu,K,Cawl,W . nrw,S . hln .e al. et M. Thelen, R., S. Andrews, function. J., neutrophil W. regulates Coadwell, P-Rex1 K., (2005). Okkenhaug, guanine- C., M. Gbetagamma-regulated L. R. and L. Rac. Stephens, for PtdIns(3,4,5)P3- factor and exchange a T. nucleotide P. P-Rex1, Hawkins, P., (2002). Tempst, H., Erdjument-Bromage, ora fCl cec 21)17 5920 doi:10.1242/jcs.153049 2589–2600 127, (2014) Science Cell of Journal .Cl Biol. Cell J. 20) muooia ucini ielacking mice in function Immunological (2004). o.Cl.Biol. Cell. Mol. Cell 178 108 1279-1293. , 809-821. , ur Biol. Curr. 24 719-729. , 15 1867-1873. ,

Journal of Cell Science