Membrane-dependent signal integration by the Ras activator Son of sevenless

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1 Memrne-dependent signl integrtion y the Rs ctivtor Son of sevenless Jodi Guresko 1,6, Willim J Glush 2,6, Sen Boykevisch 3, olger Sondermnn 1,5, Dfn Br-Sgi 3, Jy T Groves 2,4 & John Kuriyn 1,4 The kinetics of Rs ctivtion y Son of sevenless (SOS) chnges profoundly when Rs is tethered to memrnes, insted of eing in solution. SOS hs two inding sites for Rs, one of which is n llosteric site tht is distl to the ctive site. The ctivity of the SOS ctlytic unit (SOS ct ) is up to 5-fold higher when Rs is on memrnes compred to rtes in solution, ecuse the llosteric Rs site nchors SOS ct to the memrne. This effect is locked y the N-terminl segment of SOS, which occludes the llosteric site. We show tht SOS responds to the memrne density of Rs molecules, to their stte of GTP loding nd to the memrne concentrtion of phosphtidylinositol-4,5-isphosphte (PIP 2 ), nd tht the integrtion of these signls potentites the relese of utoinhiition. Signl trnsduction y growth fctor receptors proceeds through the recruitment to the plsm memrne of signling proteins tht re normlly in the cytoplsm 1. This memrne locliztion cn, y itself, ring out profound chnges in signling ctivity, ecuse colocliztion t the memrne increses the effective concentrtions of proteins y s much s 1,-fold, therey driving protein-protein interctions tht would otherwise not occur 2,3. A now clssic exmple of such signling switch is the ctivtion of Rs y growth fctor receptors, which relies on the phosphotyrosine-dependent recruitment of dptor proteins, such s growth fctor receptor ound protein 2 (Gr2), tht ring the nucleotide exchnge fctor SOS to the memrne 4,5. Memrne locliztion of SOS results in interction with Rs nd its consequent conversion to the ctive GTP-ound stte 6. Artificil trgeting of SOS to the plsm memrne, y fusing it to myristoyltion or frnesyltion sequences, results in sustined receptor-independent Rs ctivity in cells 7. SOS hs two inding sites for Rs, which cn oth, in principle, e occupied simultneously y memrne-ound Rs 8. One of these is the ctive site, t which empty Rs is ound trnsiently, leding to nucleotide exchnge 6. The other site inds to nucleotide-loded Rs. Occuption of the second site y Rs stimultes the nucleotide exchnge ctivity of SOS llostericlly, y cusing conformtionl chnges t the ctive site tht llow sustrte Rs to ind 8 1. By crrying out studies of humn Rs nd SOS proteins in solution, we hve shown previously tht ccess to the llosteric site is controlled y regultory domins tht re present in SOS, nd tht Rs-GTP inding to the llosteric site sets up positive-feedck loop for Rs ctivtion in cells 1,11. The importnce of the llosteric site ws lso demonstrted y studies on the ctivtion of Rs y SOS in T cells, which depends on the priming of Rs-GTP y nother Rs-specific nucleotide exchnge fctor, Rs-GRP1 (ref. 12). SOS hs three min functionl segments (Fig. 1). The ctlytic segment, which we refer to s SOS ct, contins the domin (nmed for the Rs-ctivtor protein in yest) nd the Rs exchnger motif () domin. An N-terminl regultory segment contins domin with two histone folds (the histone domin), followed y Dl-homology () nd pleckstrin-homology () domins. The domin of SOS physiclly occludes the llosteric Rs inding site in crystl structures of SOS 1. The domin is closely ssocited with the domin 13 nd intercts with PIP 2 (refs ) nd phosphtidic cid 18. It hs recently een shown tht the domin couples SOS ctivtion to the mitogen-dependent genertion of phosphtidic cid 18. We do not, in this pper, consider the C-terminl segment, which contins inding sites for dptor proteins such s Gr2 (refs ). The histone domin, locted upstrem of the - unit, inds tightly to the rest of SOS y docking onto helicl linker tht connects the domin to the domin 23,24. Muttion of Arg552 in this helicl linker disrupts the internl docking of the histone domin 24. The importnce of this interction in suppressing SOS ctivity hs een highlighted y genetic study of Noonn syndrome, developmentl disorder chrcterized y lerning prolems, skeletl nomlies nd congenitl hert defects 25. One of the Noonn syndrome ssocited muttions tht mp to humn SOS1 involves 1 Deprtment of Moleculr nd Cell Biology, Deprtment of Chemistry, nd owrd ughes Medicl Institute, QB3 Institute, 176 Stnley ll, University of Cliforni, Berkeley, Cliforni 9472, USA. 2 Deprtment of Chemistry, University of Cliforni, Berkeley, Cliforni 9472, USA. 3 Deprtment of Biochemistry, New York University School of Medicine, New York, New York 116, USA. 4 Physicl Biosciences Division, Lwrence Berkeley Ntionl Lortory, Berkeley, Cliforni 9472, USA. 5 Present ddress: Deprtment of Moleculr Medicine, College of Veterinry Medicine, Cornell University, Ithc, New York 14853, USA. 6 These uthors contriuted eqully to this work. Correspondence should e ddressed to J.K. (kuriyn@erkeley.edu), J.T.G. (jtgroves@ll.gov) or D.B.-S. (dfn.r-sgi@nyumc.org). Received 26 Ferury; ccepted 2 Mrch; pulished online 4 My; corrected fter print 15 My 28; doi:1.138/nsm VOLUME 15 NUMBER 5 MAY 28 NATURE STRUCTURAL & MOLECULAR BIOLOGY

2 elicl linker istone folds Memrne PIP 2 hedgroup Noonn syndrome muttion R552G elicl linker - unit Ctlytic unit SOS DPC SOS DPC istone SOS ct Allosteric Rs Gr2 inding Rs t ctlytic site Figure 1 SOS structure. () Domin orgniztion of SOS. () Model for SOS locliztion t the memrne 24. The two sic residues in the domin tht re crucil for PIP 2 inding 17 nd mutted in this work (K456 nd R459) re indicted y gry spheres. replcement of Arg552 y glycine, resulting in enhnced ctivtion of Rs nd extrcellulr signl regulted kinse 2 (ERK) fter epiderml growth fctor (EGF) stimultion 26,27. Other muttions ssocited with Noonn syndrome mp to the regions of SOS tht interct with the llosteric Rs molecule or re predicted to help destilize the utoinhiited conformtion. Memrne-ound Rs could, in principle, mintin SOS t the memrne y engging the llosteric site. Such colocliztion could increse the ility of SOS to ctlyze nucleotide exchnge on Rs drmticlly, y incresing the proility of encounters etween SOS nd Rs, nd potentilly short-circuit the controls tht mke SOS sensitive to input signls. To ddress this issue, we studied the kinetics of humn Rs ctivtion y SOS when Rs is tethered to memrnes. Simply constrining the physicl dimensionlity of the rection to surfce y coupling Rs (ut not SOS) to phospholipid memrnes leds to drmtic increse (up to B5-fold) in the ctivity of SOS ct. This effect requires Rs inding to the llosteric site of SOS, nd is further enhnced y the conversion of Rs-GDP to Rs-GTP. The unchecked ctivtion of Rs is prevented y the N-terminl domins of SOS, which lock the locliztion of SOS to the memrne y llosteric Rs. A key insight is provided y investigtion of the Noonn syndrome ssocited muttion R552G, which is found to ctivte SOS only in memrne context nd only in the presence of PIP 2. The ctivity of utoinhiited SOS is stimulted y incresing Rs density t the memrne, y the replcement of Rs-ound GDP y GTP nd y incresing PIP 2 concentrtions in the memrne. Our results suggest tht full ctivtion of SOS requires the integrtion of multiple memrne-dependent signls, s well s further nchorge of SOS to the memrne, such s y the coupling to ctivted receptors. RESULTS Covlent ttchment of Rs to memrne surfces We ttched the conserved core of Rs to lipid memrnes covlently using thiol-mleimide crosslinking 28. This strtegy voids the difficulties inherent in purifying lipid-modified Rs. A previous report hs suggested tht the lipid modifiction of Rs cn lter its ehvior s sustrte for SOS 29, ut these studies were done with oth Rs nd SOS in solution nd its relevnce to the interction of SOS with memrne-ound Rs is uncler. We quntified SOS ctivity y monitoring the displcement of fluorescently leled GDP nlogs ound to Rs y excess unleled GDP or GTP 8,3 32. To ensure tht our results were roust, we tethered Rs to oth smll unilmellr lipid vesicles (verge dimeter B1 nm) nd plnr supported lipid ilyers 33. Direct oservtion of fluorescently leled nucleotides ound to Rs molecules coupled to the surfce of supported lipid ilyers verified tht the memrne components were fluid nd homogenously mixed nd tht ggregtion ws not fctor. The vesicle system, on the other hnd, llows more rpid ulk experimentl redout. By using oth methods, we explored greter rnge of Rs-SOS concentrtions thn would e possile with either one lone, nd we hve unified our results with single quntittive kinetic model. Different fluorescent nucleotides were used in the two experimentl configurtions (Methods). Rs surfce densities for lipid vesicles re in the rnge of B8 to B17, molecules per mm 2 (Supplementry Tle 1 online nd Methods). For the supported ilyers, the Rs surfce densities rnge from B5 to B4,5 Rs molecules per mm 2 (Methods). The numer of Rs molecules on the surfce of n NI3T3 cell is estimted to e in the rnge of 1, 5, (ref. 34). Assuming dimeter of 1 mm nd sphericl shpe, this corresponds to surfce density of uniformly distriuted Rs in the rnge of 3 15 Rs molecules per mm 2. -Rs nd K-Rs re not uniformly distriuted, ut insted form dynmiclly exchnging nnoclusters, with out 3% of the Rs molecules in these nnoclusters 35,36. The rdius of nnocluster is estimted to e 6 12 nm, with surfce density of B4, to B16, Rs molecules per mm 2. Thus, our experimentl Rs surfce densities re in the rnge of densities within these nnoclusters, the formtion of which is essentil for Rs ctivtion 35,36. For comprison, the mximl close-pcked density of Rs molecules on surfce is estimted to e B1, molecules per mm 2. Enhnced ctivity of SOS ct when Rs is on memrnes We compred the SOS ct -ctlyzed nucleotide exchnge rte in solution to the rte otined for rection t the sme overll Rs nd SOS concentrtions y volume, except tht ll the Rs molecules were tethered to lipid vesicles. For consistency with previous solution studies 1,37, we kept the ulk concentrtions of oth Rs nd SOS t 1 mm nd exchnged mnt-dgdp 31 for unleled GDP. In oth cses, overll nucleotide exchnge rtes showed pseudo first-order kinetics, ecuse the fluorescently leled nucleotide tht is detected ws replced y unleled nucleotide. The exchnge rte is 19 ± 1 s 1 with oth SOS ct nd Rs in solution, wheres nerly 5-fold increse to.84 ± 6 s 1 is mesured for memrnetethered Rs (surfce density B5,3 molecules per mm 2 ; Fig. 2). To ensure tht memrne nchorge of Rs did not lter the intrinsic rte of nucleotide relese, we took dvntge of n isolted domin construct of the SOS homolog Rs gunine nucleotide relesing fctor 1 (RsGRF1). In contrst to SOS, which is ctive only when Rs inds to the llosteric site tht ridges the nd domins, the isolted domin of RsGRF1 is ctive NATURE STRUCTURAL & MOLECULAR BIOLOGY VOLUME 15 NUMBER 5 MAY

3 on its own 9,37. The rte of RsGRF1-ctlyzed nucleotide relese ws 71 ± 1 s 1 with oth RsGRF1 nd Rs in solution, nd no considerle difference ws oserved when Rs ws memrne-ound (the relese rte is 86 ± 2 s 1 for Rs surfce density of B2,6 molecules per mm 2 on vesicles; Supplementry Fig. 1 online). Dependence of Rs ctivtion on memrne surfce density The pronounced ccelertion in the rte of SOS ct -ctlyzed nucleotide exchnge upon restricting Rs to memrnes suggested tht the llosteric Rs inding site might e loclizing SOS to the memrne, where the proility of encounters with sustrte Rs molecules is incresed gretly. In support of this hypothesis, the oserved SOS ct -ctlyzed nucleotide relese rte incresed s function of Rs surfce density on the memrne for oth lipid vesicles nd plnr lipid ilyers (Fig. 3 shows dt for GDP replcing Rs-ound nucleotide). This dependency on Rs surfce density is roust nd is quntittively predicted y the following rection model over wide rnge of experimentl prmeters: Rs +SOS Ð Rs SOS+Rs Ð Rs SOS Rs! Rs SOS Rs ðpreferred pthwyþ Rs + SOS Ð SOS Rs +Rs Ð Rs SOS Rs! Rs SOS Rs ere, Rs * nd Rs refer to Rs ound to leled nd unleled nucleotide, respectively (see Supplementry Discussion online for complete description of the rection scheme). RsSOS, SOSRs nd RsSOSRs represent Rs ound t the llosteric site, the ctlytic site nd t oth sites, respectively. All Rs molecules re ttched to the memrne, nd ll SOS molecules re either in solution or ound to one or two Rs molecules. The model does not include ny direct interction etween SOS nd the memrne nd considers only GDP replcing Rs-ound nucleotide. The predicted vlues of the overll exchnge rtes for this simplified rection scheme re superimposed onto the experimentl dt presented in Figure 3. As expected, much of the rte ccelertion Fluorescence k =.84 ± 6 s 1 Rs k = 19 ± 1 s SOS ct SOS ct Figure 2 The rte of SOS ct -ctlyzed nucleotide exchnge is incresed drmticlly when Rs is tethered to memrnes. The rte of fluorescently leled mnt-dgdp relese from Rs in solution (lck) nd Rs tethered to lipid vesicles (green) in the sence nd presence of SOS ct is compred (mnt-dgdp exchnged for GDP). The ulk volume concentrtion of Rs nd SOS (when present) is 1 mm in ll rections. The Rs surfce density is B5,3 molecules per mm 2. is predicted to rise from the ility of the llosteric Rs inding site to recruit nd loclize SOS ct to the memrne. Binding of Rs t the llosteric site converts SOS ct from n inctive conformtion to n ctive one, ut these oservtions of kinetic rte enhncements s function of Rs surfce density indicte tht memrne recruitment hs mjor role in ddition to ny such direct llostery. For the supported ilyers, only smll mount of Rs is present, ecuse the memrne is restricted to single surfce of the rection chmer. A low concentrtion of SOS (1 nm) ws therefore used in these experiments. Even so, SOS in the rection chmer is in excess nd its concentrtion in solution remins essentilly constnt throughout the rection. The vesicle experiments were done with oth high (1 mm) nd low (1 nm) SOS concentrtions, ut in oth cses SOS ws limiting nd the concentrtion of free SOS in solution decresed s the rection proceeds. Nonetheless, the rection scheme depicted ove ccurtely predicts ll of the dt from oth systems on the sis of single set of kinetic prmeters. Rs Rs Rs mnt-dgdp Dissocition rte (s 1 ) , 4, 6, 8, Rs per µm 2 1, 2, 3, 4, Rs per vesicle Rs per µm 2 1 µm SOS 1:1 SOS: Rs Figure 3 The memrne-dependent increse in therteofsos ct -ctlyzed Rs exchnge is function of the surfce density of Rs. () The rte for SOS ct -ctlyzed nucleotide exchnge for Rs coupled to lipid vesicles is shown s function of Rs surfce density (Rs per mm 2 ). Although the surfce density of Rs is vried, the ulk volume concentrtions of SOS nd Rs re the sme for ech mesurement (1 mm). MntdGDP is displced y unleled GDP. Experimentl rtes re shown s points. The solid line represents the rtes predicted from the kinetic scheme (Supplementry Discussion). () The rte for SOS ct -ctlyzed nucleotide exchnge for Rs coupled to supported lipid ilyers is shown s function of Rs surfce density. In these experiments there is lrge excess of SOS molecules versus memrneound Rs ([SOS] ¼ 1 nm). BODIPY-GDP is used s the fluorescent nucleotide. Experimentl dt re indicted y points, nd the solid line represents the rtes predicted y the kinetic model, using the sme set of prmeters s in. Two dt points, etween densities of 3, 4, Rs per mm 2,hve pprently errnt vlues, ut re included for completeness. Rs BODIPY-GDP Dissocition rte (s 1 ) nm SOS >2: 1 SOS: Rs Rs 454 VOLUME 15 NUMBER 5 MAY 28 NATURE STRUCTURAL & MOLECULAR BIOLOGY

4 Figure 4 Memrne locliztion of SOS ct y llosteric Rs inding in cells. () Left, colocliztion of SOS ct nd Rs (A59G D38E), Rs vrint tht inds only to the llosteric site of SOS, t the plsm memrne. Immunofluorescence studies revel tht when SOS ct (red, ove left) nd Rs (A59G D38E) (green, elow left) re co-trnsfected into COS1 cells, oth proteins coloclize t the plsm memrne (yellow, inset). Right, the redistriution of SOS to the memrne is not seen when mutnt form of SOS ct, SOS ct(l687e R688A), is coexpressed with Rs (A59G D38E) (green, elow right). SOS ct(l687e R688A) is impired in inding to Rs t the llosteric site. () SOSct(L687E R688A) is defective in ERK MAP kinse ctivtion, ut trgeting SOS ct(l687e R688A) to the memrne using the Rs-derived memrne nchoring sequence, CAAX, fully restores SOS signling SOS ct ctivity. Error rs in the r grph represent the s.d. from three independent experiments. The levels of ERK ctivtion, s represented y ERK phosphoryltion (P-ERK; green (ppers yellow due to overlp)), were quntified y densitometry nd normlized to levels of totl ERK (ERK; red). P-ERK nd ERK comigrte, so the ppernce of P-ERK gives n pprently yellow color..u., ritrry units; WCL, whole cell lyste. Locliztion of SOS ct to the plsm memrne in cells Next we investigted the ility of Rs to loclize SOS ct to the memrne in cellulr context y trnsfecting cells with SOS ct nd oserving the effects of muttions in the llosteric Rs inding site. COS1 cells were trnsfected with SOS ct constructs nd Rs vrint (Rs (A59G D38E) ) tht inds to the llosteric site of SOS ut does not stimulte downstrem signling ecuse it hs reduced ffinity for effector proteins, such s Rf 11. In the presence of Rs (A59G D38E), wildtype SOS ct shows pronounced memrne locliztion (Fig. 4). In contrst, SOS ct vrint contining two muttions tht weken the inding of Rs to the llosteric site (SOS ct(l687e R688A) ) 1,11 fils to loclize to the memrne (Fig. 4). These results demonstrte tht the interction of Rs with the llosteric site is necessry to promote the stle interction of SOS ct with the plsm memrne. To ssess the functionl significnce of this recruitment mechnism we mesured the signling output of SOS ct using ERK MAP kinse ctivtion s redout. As seen previously, expression of SOS ct in cells leds to roust ctivtion of ERK2 (ref. 1). A mutnt of SOS ct in which interction with Rs t the llosteric site is wekened, SOS ct(l687e R688A), is defective in ERK2 kinse ctivtion 1 (Fig. 4). This defect is fully rescued y constitutive memrne trgeting of SOS ct(l687e R688A) using Rs-derived memrne-nchoring sequence (Fig. 4). Becuse Rs inding t the llosteric site is required for SOS ctivity 9, we infer tht the strong interction etween the memrne nd lipid-modified SOS, which increses the locl concentrtion of SOS t the memrne, drives the inding of Rs to the llosteric site despite the impednce rising from muttions t the llosteric site. Role of the llosteric site in SOS ctivtion t the memrne The cell-sed studies descried ove show tht muttions t the llosteric site prevent the memrne locliztion of SOS ct. Such muttions should lso prevent the memrne-dependent enhncement of SOS ct ctivity in vitro. Indeed, memrne-dependent stimultion of the nucleotide exchnge rte is sustntilly reduced in SOS ct vrint tht hs reduced ffinity for llosteric Rs (SOS ct(w729e) ). This mutnt form of SOS hs essentilly the sme properties s SOS ct(l687e R688A), used in the cell-sed experiments 1, nd the in vitro dt for SOS ct(w729e) re discussed lter. Interprettion (A59G D38E) Rs ct(l687e R688A) SOS (A59G D38E) Rs ERK phosphoryltion (.u.) IP: A WCL IB: P-ERK (yellow) IB: ERK (red) IB: T7 (SOS ct ) Vector SOS ct ct(l687e R688A) SOS SOS ct CAAX SOS ct(l687e R688A) CAAX of the reduced ctivity of SOS ct(w729e) in terms of memrne locliztion is, however, complicted y the fct tht Rs inding to the llosteric site is required for SOS ctivtion even when Rs is in solution 1. We therefore crried out dditionl experiments tht test the importnce of the llosteric site in memrne nchorge, using SOS ct with n intct llosteric site. These experiments rely on Rs mutnt (Rs Y64A ) tht inds to only the llosteric site 8,1. Rs-GTP inds out tenfold more tightly to the llosteric site thn does Rs-GDP 1. When ll rection components re in solution, the ddition of Rs Y64A ound to the nonhydrolyzle GTP nlog GppNp (Rs Y64A -GppNp) stimultes the ctivity of SOS ct (refs. 8,1). The nucleotide exchnge rte increses from 19 ± 1 s 1 (without Rs Y64A -GppNp) to 85 ± 2 s 1 (tenfold excess of Rs Y64A -GppNp; 1 mm Rs-GDP nd SOS ct, GDP exchnged for GDP; Fig. 5). The results re different when the sustrte Rs-GDP molecules re tethered to lipid vesicles nd the llosteric ctivtor Rs Y64A -GppNp is dded in solution. Insted of incresing, the exchnge rte decreses slightly, from.15 ± 1 s 1 (for moderte Rs surfce density of 1,3 Rs per mm 2 nd ulk concentrtion of Rs nd SOS ct of 1 mm) to 62 ± 7 s 1 for the sme rection crried out in the presence of tenfold excess of Rs Y64A -GppNp in solution (Fig. 5). We interpret this to men tht ddition of Rs Y64A -GppNp in solution competes with memrne-ound Rs for the llosteric site of SOS ct, therey trpping some of the SOS ct in solution. These exchnge rections were crried out with excess unleled GDP. When excess unleled GTP is used insted, the Rs-GTP produced y the exchnge rection is ttched to the memrne, nd this results in n Btenfold increse in the rte of SOS ct -ctlyzed nucleotide relese for n equl surfce density of memrne-ound Rs (Supplementry Fig. 2 online). In nother experiment, we coupled the llosteric ctivtor Rs Y64A - GppNp to lipid vesicles ut kept sustrte Rs-GDP in solution. For comprison, when Rs Y64A -GppNp, Rs-GDP (sustrte Rs) nd SOS ct re ll in solution, the rte of SOS ct -ctlyzed nucleotide relese from Rs is 27 ± 1 s 1 (1:1:1 stoichiometric rtio of Rs-GDP: SOS ct : Rs Y64A -GppNp, ll t 1 mm concentrtion). In contrst, when Rs Y64A -GppNp is memrne-tethered, nd Rs- GDP nd SOS ct re oth in solution, the rte decreses to 11 ± 9 s 1 (ll components t 1 mm ulk concentrtion; Fig. 5c). We NATURE STRUCTURAL & MOLECULAR BIOLOGY VOLUME 15 NUMBER 5 MAY

5 c 1 2 No exchnge fctor No exchnge fctor No exchnge fctor k = 19 ± 1 s 1 Rs Rs k = 85 ± 2 s 1 k = 62 ± 7 s 1 k =.152 ± 13 s 1 k = 11 ± 9 s 1 (1 ) Rs Y64A k = 27 ± 1 s 1 Rs (1 ) interpret this result to men tht SOS ct is trpped y memrneound Rs Y64A nd is less ccessile to Rs-GDP in solution. The N-terminl regultory segment inhiits SOS The ility of memrne-tethered Rs to stimulte the ctivity of SOS ct rises the question of how the ctivity of SOS is suppressed until n ctivting signl is received. Insight is provided y nlysis of SOS constructs tht contin the N-terminl domins in ddition to SOS ct (SOS DPC nd SOS DPC ; Fig. 1). The llosteric Rs inding site is inccessile in crystl structures of SOS DPC nd SOS DPC (ref. 1, nd O. Kuchment nd J.K., unpulished dt). For n equl surfce density of memrne-ound Rs, the rte of nucleotide relese for SOS DPC nd SOS DPC is etween 1 times nd 6 times slower thn for SOS ct (rections crried out with 1:1 stoichiometric rtio of SOS nd memrne-coupled Rs-GDP, t ulk concentrtion of 1 mm; Figs. 3 nd 6). We conclude tht Rs Rs (1 ) (1 ) Rs Y64A Rs Rs Y64A Rs Y64A Figure 6 Activity of SOS constructs contining N-terminl regultory domins. () The rtes for SOS ct(w729e) -, SOS DPC -ndsos DPC -ctlyzed nucleotide relese from Rs-coupled vesicles re shown s function of Rs surfce density. Although the Rs density is vried, the ulk volume concentrtions of Rs nd SOS re the sme for ech mesurement (1 mm). Error rs indicte the mens with stndrd error from t lest two independent experiments. () The ctivities of SOS DPC (gry), SOS DPC (purple) nd SOS ct (green) towrd Rs coupled to vesicles when mntdgdp is exchnged for either unleled GDP or unleled GTP re compred for n equl surfce density of Rs (B1,3 Rs per mm 2 ;ulk concentrtion of Rs nd SOS is 1 mm). Note tht the replcement of GDP for GTP on memrne-ound Rs results in strong enhncement in the ctivity of ll SOS constructs. Figure 5 The sustrte Rs molecule nd the ctivting Rs molecule oth need to e tethered to the memrne for mximl SOS ctivity. A mutnt form of Rs, Rs Y64A, which inds to the llosteric site of SOS, ut not to the ctlytic site 8, is used in these experiments. () All components re in solution. () Rs mnt-dgdp molecules re tethered to lipid memrnes, nd Rs Y64A -GppNp (llosteric ctivtor) nd SOS ct re oth in solution. (c) Rs Y64A -GppNp is tethered to lipid memrnes, nd Rs mnt-dgdp (sustrte Rs) nd SOS ct re oth in solution. n importnt role of the histone domin nd the - unit of SOS is to inhiit the locliztion of SOS to the memrne y llosteric Rs. The inhiitory effect of the - unit ws lso mnifested when fluorescently leled GDP ound to Rs ws replced with GTP, despite the enhnced ffinity of Rs-GTP for the llosteric site 8,1 (Supplementry Fig. 2). For exmple, Figure 6 compres the ctivities of SOS DPC,SOS DPC nd SOS ct towrd memrne-ound Rs when leled GDP is exchnged for either unleled GDP or unleled GTP (ulk concentrtion of Rs nd SOS is 1 mm). The rections re compred for the sme level of Rs surfce density (B1,3 Rs molecules per mm 2 ), nd the replcement of GDP y GTP results in mrked increse in the rection rte of ll SOS constructs (Fig. 6). Nevertheless, in comprison to SOS ct, the presence of the - unit in SOS DPC nd SOS DPC suppresses the exchnge ctivity sustntilly (Fig. 6). PIP 2 -dependent ctivtion of SOS The crystl structure of construct of SOS contining intct regultory nd ctlytic segments (SOS DPC ) hs een determined recently nd indictes tht the histone domin intercts with the domin, the domin nd the helicl - linker (Olg K. nd J.K., unpulished dt). A modeled loction of the histone domin, which is sed on smll-ngle X-ry scttering dt 24 nd is correct in generl terms lthough not in detil, is shown in Figure 1. Muttion of Arg552 in the helicl linker connecting the domin of SOS to the domin (R552G) releses the histone domin from the rest of mnt-dgdp Dissocition rte (s 1 ) , SOS DPC SOS DPC SOS ct(w729e) 1 µm SOS 4, 6, 8, Rs per µm , 4, 6, 8, Rs per µm , No exchnge fctor (excess GDP, excess GTP) Rs mnt-dgdp SOS DPC Rs mnt-dgdp 4, Rs per µm Rs mnt-dgdp Rs-GDP SOS ct Rs mnt-dgdp Rs-GTP 1: 1 SOS: Rs 6, 8, 1 µm SOS 1: 1 SOS: Rs Rs-GDP SOS DPC Rs-GTP 456 VOLUME 15 NUMBER 5 MAY 28 NATURE STRUCTURAL & MOLECULAR BIOLOGY

6 c 1 µm SOS DPC, SOS DPC, SOS DPC(R552G), no exchnge fctor µm SOS ct Rs mnt-dgdp solution Rs-GDP Rs mnt-dgdp solution Rs-GTP Rs on supported ilyers Rs BODIPY-GDP Rs-GDP.5 No exchnge fctor SOS DPC (no PIP 2 ) 1. SOS DPC (2% PIP 2 ) SOS ct (2% PIP 2 ) 1 nm concentrtion SOS 24 nd leds to incresed Rs ctivtion 26,27, suggesting tht the docking of the histone domin is crucil for inhiition. We therefore wondered whether the ility of the R552G muttion to ctivte SOS is due to relese of the lockge of the llosteric site y the N-terminl segment. We first compred the properties of three SOS constructs, two with the histone domin (SOS DPC nd SOS DPC(R552G) ) nd one without (SOS DPC ), in experiments where oth Rs nd SOS re present in solution, t 1 mm concentrtion. All three SOS constructs show low levels of ctivity compred to SOS ct, even when the rection is crried out in n excess of unleled GTP (Fig. 7). These results demonstrte tht the Noonn syndrome ssocited muttion hs no noticele effect on SOS ctivity when Rs nd SOS re oth off the memrne. Next we compred the sme three SOS constructs, ut this time with Rs loclized to lipid vesicles. We used ulk SOS concentrtion of 1 nm, which is 1-fold lower thn tht used in the first set of experiments nd resulted in SOS ct ctivity tht ws roughly the sme s the solution rte (compre Fig. 7,). Agin, SOS DPC, SOS DPC(R552G) nd SOS DPC showed low levels of ctivity, essentilly indistinguishle from ckground (Fig. 7). Thus, the Noonn syndrome ssocited muttion in SOS does not llow Rs to ccess the llosteric site on these memrnes, which contin phosphtidylcholine (PC) nd phosphtidylserine (PS) (see Methods for detiled description of the memrne composition). The domin of SOS inds to PIP 2 in vesicles with reltively high ffinity (K d E1 mm) (lso see Supplementry Fig. 3 online). In ddition, role for phosphtidic cid in ctivting SOS hs een reported recently 18,38. We therefore wondered whether lipid inding to the domin of SOS could potentite the effect of the R552G muttion. Figure 7 shows the results of experiments where Rs is coupled to lipid vesicles nd mnt-dgdp is displced y unleled PIP % PIP Rs on lipid vesicles Rs mnt-dgdp Rs-GTP 3 3 SOS DPC( mutnt), SOS DPC, SOS DPC, SOS DPC(R552G), no exchnge fctor SOS ct SOS DPC( mutnt), SOS DPC, no exchnge fctor SOS DPC(R552G) SOS DPC SOS ct 1 nm concentrtion 1 nm concentrtion Figure 7 PIP 2 -dependent ctivtion of SOS. () The ctivities of SOS DPC,SOS DPC( mutnt), SOS DPC,SOS DPC(R552G) nd SOS ct towrd Rs in solution when mnt-dgdp is exchnged for unleled GDP nd GTP re compred (ulk concentrtion of Rs nd SOS is 1 mm). SOS DPC( mutnt) is mutnt form of SOS DPC tht contins muttions (K456E nd R459E) tht olish inding to PIP 2.SOS DPC(R552G) contins the muttion ssocited with Noonn syndrome 26,27.() Nucleotide exchnge y the indicted SOS constructs is shown in the sence nd presence of PIP 2 in Rs-coupled lipid vesicles. (Rs surfce densities of B4,55 molecules per mm 2 nd B5, molecules per mm 2, respectively; [SOS] ¼ 1 nm, tht is, 1-fold lower thn in ; [Rs] (y volume) ¼ 1 mm). In these rections, mnt-dgdp is displced y unleled GTP in solution. Note tht inclusion of 3% PIP 2 into Rs-coupled vesicles results in sustntil increse in the ctivity of the Noonn syndrome mutnt (SOS DPC(R552G) )ndsos DPC towrd memrne-ound Rs. (c) Inclusion of 2% PIP 2 into Rs-coupled supported ilyers results in mrked increse in the ctivity of SOS DPC ([SOS] ¼ 1 nm; Rs BODIPY-GDP exchnged for GDP). Dt for SOS ct in the presence of 2% PIP 2 is shown for comprison. GTP in the presence of 1 nm SOS DPC,SOS DPC nd SOS DPC(R552G). Inclusion of PIP 2 in memrnes results in mrked increse in the ctivity of SOS constructs contining the Noonn syndrome ssocited muttion (SOS DPC(R552G) ) or in which the histone domin is entirely deleted (SOS DPC ) (Fig. 7). For exmple, the rte of nucleotide relese y SOS DPC(R552G) is 126 ± 7 s 1, compred to 19 ± 3 s 1 for SOS DPC, oth in the presence of 3% PIP 2.The rte for SOS DPC is 1 ± 1 s 1, tht is, the sme s tht for the unstimulted rection (Fig. 7). Similr results re otined using supported ilyers insted of vesicles, s shown in Figure 7c for the effect of PIP 2 on SOS DPC Rs mnt-dgdp Rs-GTP No exchnge fctor SOS DPC(R552G) 1% PIP 2 k = 28 s 1 (~6, Rs per µm 2 ) 3% PIP 2 k = 117 s 1 (~4,81 Rs per µm 2 ) 36 1 nm SOS 1: 1 SOS: Rs Figure 8 The Noonn syndrome mutnt, SOS DPC(R552G), is responsive to the memrne density of PIP 2. The nucleotide exchnge rtes of 1 nm SOS DPC(R552G) towrd Rs tethered to lipid vesicles contining 1% nd 3% PIP 2 re compred when mnt-dgdp is displced y unleled GTP (B6, Rs per mm 2 nd B4,81 Rs per mm 2, respectively; [Rs] (y volume) ¼ 1 mm). NATURE STRUCTURAL & MOLECULAR BIOLOGY VOLUME 15 NUMBER 5 MAY

7 Figure 9 The integrtion of severl memrne-locliztion signls in the ctivtion of Rs nd SOS. () At low Rs-GDP (ornge) surfce densities nd low surfce concentrtions of PIP 2 (purple circles), the N-terminl regultory segment mintins SOS in n inctive stte y inhiiting the locliztion of SOS to the memrne y llosteric Rs. Rs inding to the llosteric site cuses conformtionl chnge t the ctive site, promoting sustrte enggement 9.() The ctivity of utoinhiited SOS is stimulted y incresing Rs density t the memrne, the replcement of Rs-ound GDP y GTP (green) nd y incresing PIP 2 concentrtions in the memrne. (c) Further nchorge of SOS to the memrne, such s y the coupling to ctivted receptors, in comintion with high levels of Rs density, the genertion of Rs-GTP nd high levels of PIP 2, results in effective relese of utoinhiition. The PIP 2 -dependent ctivity of SOS DPC is olished if the domin of SOS is mutted so tht it cnnot ind to PIP 2 (SOS DPC(K456E R459E) ) (Fig. 7 nd Supplementry Fig. 3). Thus, the effect of PIP 2 is medited through the domin. The simplest explntion for the ility of PIP 2 to stimulte SOS ctivity is tht it engges the domin nd therey provides n dditionl tether for SOS t the memrne. The resulting enhncement in the locl concentrtion of SOS would fcilitte entry of Rs to the llosteric site, overcoming the resistnce fforded y the N-terminl segment. Notly, the inclusion of phosphtidic cid in memrnes, either lone or with PIP 2, hs no effect on the rtes of nucleotide exchnge in our ssys for the constructs tested (dt not shown). Another interesting point is the mechnism y which the histone domin impedes the ctivity of SOS DPC.SOS DPC inds to PIP 2 -contining vesicles (Supplementry Fig. 3), suggesting tht the histone domin does not directly occlude the domin ut might insted lock the simultneous enggement of the memrne y the domin nd thetworsindingsitesofsos. Integrtion of memrne-dependent signls y SOS In the preceding sections, the ctivities of different SOS constructs were compred under similr conditions, emphsizing the inhiitory ction of the N-terminl segment. A different perspective emerges, however, when the ctivity of ny one prticulr construct of SOS is monitored s the rection conditions re chnged. Our results show tht incresing the surfce density of Rs on memrnes results in sustntil increses in SOS ctivity. Although the highest levels of ctivity were seen with SOS ct, ll the constructs of SOS tht we studied responded in similr wy to Rs surfce density (Figs. 3 nd 6). Even for SOS DPC, which hd the lowest ctivity in our ssys, the rte of nucleotide relese incresed y out 65-fold s the Rs surfce density incresed from 83 Rs per mm 2 to 8,2 Rs per mm 2 for rections in which leled GDP is replced y unleled GDP (ulk concentrtion of Rs nd SOS is 1 mm) (Fig. 6). Superimposed on this Rs density dependence is the effect of Rs-GTP production, which incresed the ctivity of SOS ct, SOS DPC nd SOS DPC y roughly nother fctor of ten, for the sme surfce density of Rs (Fig. 6 nd Supplementry Fig. 2). It ws shown recently tht the ctivtion of Rs is coupled to the formtion of nnoclusters tht re crucil for switch-like response to the input signl 35. The locl concentrtion of Rs within these nnoclusters is likely to e out two orders of mgnitude greter thn if Rs were distriuted uniformly throughout the plsm memrne, s noted erlier. Thus, the dependence of SOS ctivity on oth Rs surfce density nd the stte of GTP loding on Rs could provide one mechnism for mking Rs ctivtion contingent on the formtion of Rs nnoclusters. c Rs Rs Rs Rs GDP GTP GDP GTP GDP PIP 2 is present in iologicl memrnes t levels of 1 5% totl lipid 39. The level of PIP 2 is elevted in el cells following EGF stimultion nd is responsive to the ctivtion of phospholipse D nd the production of phosphtidic cid 4. SOS is responsive to the memrne density of PIP 2. For exmple, Figure 8 compres the ctivity of the Noonn syndrome ssocited mutnt (SOS DPC(R552G), 1 nm ulk concentrtion) when Rs ws tethered to memrnes contining 1% nd 3% PIP 2. Rections using vesicles with higher PIP 2 density showed higher levels of SOS ctivity (117 s 1 versus 28 s 1 ), even though the Rs surfce density ws lower for the vesicles with higher PIP 2 (B4,8 moelcules per mm 2 versus 6, molecules per mm 2 ). Our procedure for cross-linking Rs to vesicles did not llow us to precisely control the Rs surfce densities, which were determined only fter the vesicles were prepred. A more thorough mpping of the responsiveness of SOS to the surfce densities of Rs nd PIP 2 wits further study. DISCUSSION The clssicl model for the ctivtion of Rs y SOS involves the prtitioning of SOS from the cytoplsm to the plsm memrne in response to growth fctor induced receptor ctivtion. Such mechnism leves open the possiility tht chnce encounters etween SOS nd Rs would led to the erroneous ctivtion of Rs, with disstrous consequences for the cell. We hd shown previously tht Rs Rs Rs Rs Rs Rs Additionl memrne nchorge Rs Rs Rs Rs Rs Rs Rs GTP 458 VOLUME 15 NUMBER 5 MAY 28 NATURE STRUCTURAL & MOLECULAR BIOLOGY

8 SOS is inctive unless Rs is ound to the llosteric site, nd tht the enhnced ffinity of Rs-GTP for this site mkes SOS sensitive to the ctivtionstteofrs.wenowdemonstrtethtrsisletoloclize the SOS ctlytic unit to the memrne nd strongly potentite its ctivity. The N-terminl segment of SOS locks the enggement of the llosteric site y Rs, providing thermodynmic resistnce to direct ctivtion of SOS y Rs. But, ecuse Rs is t the memrne, this mechnism lso enles the ctivtion of SOS in response to severl cues tht cn ct synergisticlly to overcome this resistnce (Fig. 9). A key finding in this regrd is tht Noonn syndrome ssocited muttion in SOS (R552G), which wekens the utoinhiition of SOS, results in ctivtion only in memrne-dependent context. The presence of the llosteric Rs inding site mkes ll the SOS constructs tht we hve studied, even those tht re utoinhiited, responsive to the surfce density of Rs. The incresed density of Rs in nnoclusters 35 is therefore expected to strongly enhnce SOS ctivity. The genertion of n initil urst of Rs-GTP, either through the coupling of SOS to ctivted receptors, or through the ction of gents such s phosphtidic cid 18 or priming exchnge fctor such s Rs-GRP1 12, will lso result in sustntil stimultion of SOS ctivity. SOS is responsive to the surfce concentrtion of PIP 2,nd we hve shown tht the comintion of high Rs surfce density, the presence of Rs-GTP nd high levels of PIP 2 results in incresed SOS ctivity despite the presence of the utoinhiitory segments. SOS seems to hve evolved the cpility of integrting severl signls s condition for the ctivtion of Rs signling. This property of SOS is reminiscent of the memrne-dependent ctivtion of the Wiscott-Aldrich syndrome protein (WASP), which is controlled y Rho GTPses nd other signls, such s the memrne density of PIP 2, tht re integrted to result in ctivtion 41,42. This complexity in the input-output response functions of proteins tht re t key nodes in cellulr signl trnsduction is likely to emerge s generl theme tht ensures fidelity in cellulr responses to input signls. METODS Protein preprtion. SOS DPC (residues 1 149), SOS DPC (residues ) nd SOS ct (residues ) of humn SOS1 were expressed nd purified s descried 1,24. We generted point mutnts using the Quikchnge site-directed mutgenesis kit (Strtgene) nd confirmed y DNA sequencing. Mutnt proteins were expressed nd purified s for the wild-type proteins. The typicl 166-residue -Rs construct used in previous studies 8,1 ws extended to include C-terminl cysteine t position 181. Cys118 of humn -Rs ws mutted to serine, leving one cysteine t position 181. Rs (C181,C118S) (residues 1 181, C118S) ws cloned into pproex T vector (Invitrogen) using BmI nd EcoRI site. Rs (C181,C118S) ws produced in Escherichi coli nd purified using n N-terminl hexhistidine tg. Following elution, uffers were exchnged using Fst Deslting Column (GE elthcre) equilirted in 25 mm Tris-Cl (p 8.2), 5 mm NCl nd 4 mm DTT, followed y either size-exclusion chromtogrphy on Superdex 75 column (GE elthcre) tht ws equilirted in gel-filtrtion uffer (25 mm EPES- NO (p 7.4), 1 mm NCl, 1% (w/v) glycerol nd 2 mm Tris (2-croxyethyl) phosphine (TCEP)) or treted with the tocco etch virus (TEV) protese overnight t 4 1C efore gel-filtrtion chromtogrphy. Frctions of Rs (C181,C118S) were pooled nd frozen t finl concentrtion of out 1 mg ml 1. MS nlysis confirmed the identity of the proteins. Preprtion of mleimide-functionlized memrnes. Phospholipids nd nlogs including 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2- dioleoyl-sn-glycero-3-[phospho-l-serine] (DOPS), 1,2-dioleoyl-sn-glycero-3- phosphte (DOPA), phosphtidylinositol-4,5-isphosphte (PIP 2; swine rin), 1,2-dioleoyl-sn-glycero-3-phosphoinositol-3,4,5-trisphosphte (PIP 3 ) nd 1,2-diplmitoyl-sn-glycero-3-phosphoethnolmine-N-[4-(p-mleimidomethyl) cyclohexne-croxmide] (MCC-PE) were purchsed from Avnti Polr Lipids. The fluorescent lipid nlogs Texs Red 1,2-dihexdecnoylsn-glycero-3-phosphoethnolmine (TR-PE) nd Mrin Blue 1,2-dihexdecnoyl-sn-glycero-3-phosphoethnolmine (MB-PE) were purchsed from Invitrogen. Mleimide-functionlized vesicles were prepred y drying mixture of DOPC, DOPS, MCC-PE nd TR-PE (vesicle experiments) or MB-PE (supported ilyer experiments) in chloroform. Mixtures contined 1 mol% (molr percentge) DOPS, 1 mol% MCC-PE,.3 mol% TR-PE or 2 mol% MB-PE, nd the lnce consisted of DOPC. Some experiments lso included DOPA, PIP 2 or PIP 3 where noted. Dried lipid films were plced under vcuum or gentle nitrogen strem for t lest 1 h nd resuspended in degssed uffer (25mM EPES-NO (p 7.4), 1 mm NCl nd 1% (w/v) glycerol). The hydrted films were sujected to repeted freeze-thw cycles, nd vesicles formed y extrusion through 1 nm polycronte filters or y proe soniction for PIP 2 -contining supported ilyer experiments. Vesicles were then immeditely conjugted to Rs (C181,C118S) (overnight, 4 1C), or used to form supported ilyers y vesicle rupture followed y Rs (C181,C118S) linkge (overnight, 4 1C). Preprtion of Rs-coupled lipid vesicles. Rs (C181,C118S) ws coupled to vesicles contining etween 1 1 mol% mleimide-derivtized lipid in rection crried out under rgon for 2 h t room temperture. Mleimidelipid ws present in ten-fold molr excess over Rs (C181,C118S). Coupling rections were terminted nd excess mleimide-lipid quenched y ddition of 5 mm -mercptoethnol. Unmodified Rs (C181,C118S) protein ws seprted from Rs (C181,C118S) -conjugted vesicles y size-exclusion chromtogrphy using n XK-16 column (GE elthcre) contining out 3 ml of Sephrose CL-4B resin (Sigm) equilirted in gel-filtrtion uffer (25 mm EPES- NO (p 7.4), 1 mm NCl, 1% (w/v) glycerol nd 1 mm DTT). Vesicle dimeter (1 14 nm) nd monodispersity oth efore nd fter Rs conjugtion ws confirmed y dynmic light scttering (Wytt DynPro Titn). MS confirmed the formtion of stle thioether linkge etween Rs (C181,C118S) nd mleimide-derivtized lipids. The lipid concentrtion fter Rs (C181,C118S) conjugtion ws determined y TR-PE sornce. Protein concentrtion nd conjugtion efficiency ws mesured y Brdford ssy of Rs (C181,C118S) -contining vesicles, where the Brdford regent signl due to lipids ws removed y ssying equivlent vesicle solutions with no protein. We used SDS-PAGE to confirm protein conjugtion mesurements y creting solutions of equl Rs concentrtion y volume, ut with different Rs conjugtion efficiencies. Nucleotide exchnge experiments using Rs-coupled lipid vesicles. Nucleotide exchnge ctivity ws mesured s descried 8,1. We used mnt-dgdp (Jen Biosciences) insted of mnt-gdp to void rtifcts cused y isomeriztion of the fluorescent lel 31. Rs-coupled vesicles were incuted with ten-fold molr excess of mnt-dgdp in the presence of.2 mm EDTA in gelfiltrtion uffer. Rections were stopped with 4 mm MgCl 2, nd free nucleotide ws removed y gel filtrtion using Np-1 columns (GE elthcre) equilirted with rection uffer (4 mm EPES-NO (p 7.4), 4 mm MgCl 2 nd 1 mm DTT). The finl concentrtion of Rs mnt-dgdp coupled vesicles ws otined using the Texs Red dilution fctor, s descried ove. Rections were initited y rpid mixing of 1 ml of2mm (or 2 nm) SOS with 1 ml of Rs mnt-dgdp conjugted vesicles using stopped-flow pprtus (RX2; Applied Photophysics) linked to Fluoromx-3 fluorimeter (ORIBA Join Yvon). The surfce density of Rs ws vried, ut the totl concentrtion of Rs stock efore mixing ws kept constnt t 2 mm. Rection progress ws monitored y fluorescence intensity t 43 nm with 37 nm excittion. Rections were performed t 25 1C in rection uffer supplemented with 2 mm unleled GDP or GTP nucleotide. Dt were otined y repeting rections on different dys with different protein smples nd different Rs-coupled vesicle preprtions. The dt were fit to either single- or doule-exponentil decy function using the progrm Prism 4 (Grphpd Prism, Inc.). For disply purposes, dt were normlized etween 1 nd the minimum fluorescence vlue oserved for given preprtion of vesicles. Preprtion of Rs-conjugted supported plnr ilyers. Supported ilyer experiments were performed in glss-ottomed 96-well plte (Nlge Nunc NATURE STRUCTURAL & MOLECULAR BIOLOGY VOLUME 15 NUMBER 5 MAY

9 Interntionl). Mleimide-functionlized vesicles (5 ml, 1 mg ml 1 totl lipid concentrtion) in 5 mm Tris-Cl (p 7.4) nd 75 mm NCl were dded to 5 ml 2 O in NO-clened well, equilirted for 1 min, nd rinsed with 5 ml.1% (w/v) BSA in 1 mm Tris-Cl (p 7.4) nd 15 mm NCl. Rs (C181,C118S) protein ws dded to finl concentrtion of 8 mg ml 1 nd the conjugtion ws crried out overnight t 4 1C. Bilyers were wshed with 5 ml loding uffer (4 mm EPES-NO (p 7.4)) supplemented with 5 mm EDTA followed y incution for 1.5 h t 4 1C. Wells were re-equilirted into loding uffer nd incuted with 1 mm BODIPY-GDP or BODIPY-GTP (Invitrogen) with 5 mm MgCl 2 for 1 h t 4 1C. Unound nucleotide ws wshed wy with loding uffer supplemented with 5 mm MgCl 2. Fluorescence recovery showed tht oth MB-PE lipids nd BODIPY-leled nucleotides on Rs were lterlly moile (Supplementry Fig. 4 online). The surfce density of Rs in ech well ws mesured fter exchnge rections were complete y mouse nti pn-rs IgG (EMD Biosciences) inding followed y BODIPY got nti-mouse IgG (Invitrogen) inding. The fluorescence intensity of nti-mouse IgG (djusted for leling efficiency nd sorption-fluorescence chrcteristics) ws compred to ilyer stndrds with known BODIPY-PE lipid density. Nucleotide exchnge experiments using Rs-coupled supported lipid ilyers. Rections on supported ilyers were conducted using Nikon TE-3 microscope equipped with Photometrics Coolsnp Q CCD cmer. Exchnge rections were initited y rpid ddition nd mixing to give finl concentrtion of 1 nm SOS, 2 mm GDP (or GTP) in rection uffer (4 mm EPES-NO (p 7.4), 5 mm MgCl 2 ). Rection progress ws monitored y the emission intensity of the ilyer minus the intensity of scrtched region of re glss in the sme frme to correct for solution signl (Supplementry Fig. 5 online). The resulting intensity ws fit to sum of two exponentil decys using Prism 4 (Grphpd Prism, Inc.). Fluorescence vlues were normlized to 1 for disply purposes. Cell culture, trnsfections nd immunofluorescence leling. COS1 cells were cultured in DMEM (GIBCO-BRL) supplemented with 5% (v/v) FBS (GIBCO-BRL) in humidified incutor with 5% CO 2 t 37 1C. Cell trnsfections were performed using the Fugene 6 trnsfection regent (Roche) s per mnufcturer s directions. COS1 cells were grown on cover slips nd trnsfected with GFP-tgged -Rs (A59G D38E) nd either the SOS ct or SOS ct(l687e R688A) construct cloned into the pcgt expression vector. After 24 h of expression, cells were fixed in 3.7% (v/v) formldehyde in PBS for 1 h t room temperture. Cells were then permeilized with.1% (v/v) Triton X-1 for 3 min t room temperture, then wshed five times with PBS, nd susequently incuted in 2% (w/v) BSA-PBS for 1 min. Anti-T7 ntiody (Novgen; 1:5) ws diluted in 2% (w/v) BSA-PBS nd incuted with the cells for 1 h t 37 1C. The cells were wshed five times with PBS nd incuted with rhodmine-conjugted got nti-mouse ntiody (Cppel; 1:1) for 1 h t 37 1C. Coverslips were mounted in Immunomount (Shndon) nd exmined using Zeiss Axiovert 2M microscope. Genertion of SOS ct -Rs lipid til fusion constructs. A PCR frgment contining the lst 25 mino cids of pcgn -Rs with 5 KPNI nd 3 BmI engineered restriction sites ws digested with the respective enzymes. This frgment ws cloned in frme into pcgt-sos ct or pcgt-sos ct(l687e R688A) digested with KPNI nd BmI, creting SOS ct -Rs fusion contining mino cids of SOS fused to the lst 25 mino cids of -Rs. ERK MAP kinse ctivtion ssy. ERK ctivtion ws determined y cotrnsfecting el cells with hemgglutinin (A)-tgged ERK MAP kinse nd the indicted T7-tgged SOS constructs. After 24 h of expression, the cells were serum strved for 16 h. Cells were wshed twice with ice-cold PBS nd lysed in 4 ml ice-cold uffer contining 1 mm Tris (p 7.6), 15 mm NCl, 1 mm EDTA, 1% (w/v) glycerol, 1% (v/v) Triton X-1, 1 mm N 3 VO 4, 1 mm NF, 1 mm phenyl-methnesulfonyl fluoride, 1 mg ml 1 pepsttin, 1 mg ml 1 protinin, 1 mg ml 1 leupeptin nd 1 mm enzmidine. The lystes were clrified t 14, g for 15 min nd then incuted with nti-a ntiody (12CA5) for 1 h t 4 1C. The immune complexes were incuted with protein A Sephrose eds (Sigm) for 45 min t 4 1C. Immune complexes were wshed four times with ice-cold lysis uffer nd eluted with SDS smple uffer. Smples were run on SDS-PAGE gels nd trnsferred to nitrocellulose memrnes (Schleicher & Schuell). The memrnes were incuted with either nti- T7 (Novgen, 1:1,) or nti-erk2 (Upstte Biotechnology, 1:1,) nd phospho-erk1/2 (Cell Signling, 1:1,) ntiodies. Susequently, memrnes were incuted with IRDye 8 conjugted got nti-rit (Rocklnd, 1:1,) nd Alex-Fluor 68 got nti-mouse (Moleculr Proes, 1:1,) ntiodies nd visulized using the Odyssey Infrred Imging System (LiCor). Reltive ERK phosphoryltion ws quntified using Odyssey softwre nd normlized to totl ERK expression. Note: Supplementry informtion is ville on the Nture Structurl & Moleculr Biology wesite. ACKNOWLEDGMENTS We thnk T. Freedmn, O. Kuchment, N. Endres, X. Zhng nd M. Lmers for helpful discussions; nd D. King for MS. J.G. is supported y the Moleculr Biophysics US Ntionl Institutes of elth (NI) grnt T32 GM8295. J.T.G. nd W.J.G. re supported y Chemicl Sciences, Geosciences nd Biosciences Division, Office of Bsic Energy Sciences of the US Deprtment of Energy under Contrct No. DE_AC3-76SF98, nd D.B.-S. y NI GM Pulished online t Reprints nd permissions informtion is ville online t reprintsndpermissions 1. Pwson, T. & Scott, J.D. Signling through scffold, nchoring, nd dptor proteins. Science 278, (1997). 2. Kholodenko, B.N., oek, J.B. & Westerhoff,.V. Why cytoplsmic signlling proteins should e recruited to cell memrnes. Trends Cell Biol. 1, (2). 3. Kuriyn, J. & Eisenerg, D. The origin of protein interctions nd llostery in colocliztion. Nture 45, (27). 4. Schlessinger, J. Cell signling y receptor tyrosine kinses. Cell 13, (2). 5. Quillim, L.A. New insights into the mechnisms of SOS ctivtion. Sci. STKE 27, pe67 (27). 6. Borick-Sjodin, P.A., Mrgrit, S.M., Br-Sgi, D. & Kuriyn, J. The structurl sis of the ctivtion of Rs y Sos. Nture 394, (1998). 7. Aronheim, A. et l. Memrne trgeting of the nucleotide exchnge fctor Sos is sufficient for ctivting the Rs signling pthwy. Cell 78, (1994). 8. Mrgrit, S.M. et l. Structurl evidence for feedck ctivtion y RsGTP of the Rsspecific nucleotide exchnge fctor SOS. Cell 112, (23). 9. Freedmn, T.S. et l. A Rs-induced conformtionl switch in the Rs ctivtor Son of sevenless. Proc. Ntl. Acd. Sci. USA 13, (26). 1. Sondermnn,. et l. Structurl nlysis of utoinhiition in the Rs ctivtor Son of sevenless. Cell 119, (24). 11. Boykevisch, S. et l. Regultion of Rs signling dynmics y Sos-medited positive feedck. Curr. Biol. 16, (26). 12. Roose, J.P., Mollenuer, M., o, M., Kuroski, T. & Weiss, A. Unusul interply of two types of Rs ctivtors, RsGRP nd SOS, estlishes sensitive nd roust Rs ctivtion in lymphocytes. Mol. Cell. Biol. 27, (27). 13. Soisson, S.M., Nimnul, A.S., Uy, M., Br-Sgi, D. & Kuriyn, J. Crystl structure of the Dl nd pleckstrin homology domins from the humn Son of sevenless protein. Cell 95, (1998). 14. Zheng, J. et l. The solution structure of the pleckstrin homology domin of humn SOS1. A possile structurl role for the sequentil ssocition of diffuse B cell lymphom nd pleckstrin homology domins. J. Biol. Chem. 272, (1997). 15. Kuiseski, T.J., Chook, Y.M., Prris, W.E., Rozkis-Adcock, M. & Pwson, T. igh ffinity inding of the pleckstrin homology domin of msos1 to phosphtidylinositol (4,5)-isphosphte. J. Biol. Chem. 272, (1997). 16. Koshi, S. et l. The solution structure of the pleckstrin homology domin of mouse Son-of-sevenless 1 (msos1). J. Mol. Biol. 269, (1997). 17. Chen, R.., Corln-Grci, S. & Br-Sgi, D. The role of the domin in the signldependent memrne trgeting of Sos. EMBO J. 16, (1997). 18. Zho, C., Du, G., Skowronek, K., Frohmn, M.A. & Br-Sgi, D. Phospholipse D2- generted phosphtidic cid couples EGFR stimultion to Rs ctivtion y Sos. Nt. Cell Biol. 9, (27). 19. Budy, L. & Downwrd, J. Epiderml growth fctor regultes p21rs through the formtion of complex of receptor, Gr2 dpter protein, nd Sos nucleotide exchnge fctor. Cell 73, (1993). 2. Egn, S.E. et l. Assocition of Sos Rs exchnge protein with Gr2 is implicted in tyrosine kinse signl trnsduction nd trnsformtion. Nture 363, (1993). 21. Gle, N.W., Kpln, S., Lowenstein, E.J., Schlessinger, J. & Br-Sgi, D. Gr2 medites the EGF-dependent ctivtion of gunine nucleotide exchnge on Rs. Nture 363, (1993). 46 VOLUME 15 NUMBER 5 MAY 28 NATURE STRUCTURAL & MOLECULAR BIOLOGY

10 22. Li, N. et l. Gunine-nucleotide-relesing fctor hsos1 inds to Gr2 nd links receptor tyrosine kinses to Rs signlling. Nture 363, (1993). 23. Sondermnn,., Soisson, S.M., Br-Sgi, D. & Kuriyn, J. Tndem histone folds in the structure of the N-terminl segment of the rs ctivtor Son of Sevenless. Structure 11, (23). 24. Sondermnn,., Ngr, B., Br-Sgi, D. & Kuriyn, J. Computtionl docking nd solution X-ry scttering predict memrne-intercting role for the histone domin of the Rs ctivtor Son of sevenless. Proc. Ntl. Acd. Sci. USA 12, (25). 25. Trtgli, M. & Gel, B.D. Noonn syndrome nd relted disorders: genetics nd pthogenesis. Annu. Rev. Genomics um. Genet. 6, (25). 26. Roerts, A.E. et l. Germline gin-of-function muttions in SOS1 cuse Noonn syndrome. Nt. Genet. 39, 7 74 (27). 27. Trtgli, M. et l. Gin-of-function SOS1 muttions cuse distinctive form of Noonn syndrome. Nt. Genet. 39, (27). 28. McNew, J.A. et l. Close is not enough: SNARE-dependent memrne fusion requires n ctive mechnism tht trnsduces force to memrne nchors. J. Cell Biol. 15, (2). 29. Pechlivnis, M., Ringel, R., Popkirov, B. & Kuhlmnn, J. Prenyltion of Rs fcilittes hsos1-promoted nucleotide exchnge, upon Rs inding to the regultory site. Biochemistry 46, (27). 3. Lenzen, C., Cool, R.. & Wittinghofer, A. Anlysis of intrinsic nd CDC25-stimulted gunine nucleotide exchnge of p21rs-nucleotide complexes y fluorescence mesurements. Methods Enzymol. 255, (1995). 31. Guo, Z., Ahmdin, M.R. & Goody, R.S. Gunine nucleotide exchnge fctors operte y simple llosteric competitive mechnism. Biochemistry 44, (25). 32. Ahmdin, M.R., Wittinghofer, A. & errmnn, C. Fluorescence methods in the study of smll GTP-inding proteins. Methods Mol. Biol. 189, (22). 33. Groves, J.T. & Dustin, M.L. Supported plnr ilyers in studies on immune cell dhesion nd communiction. J. Immunol. Methods 278, (23). 34. Scheele, J.S., Rhee, J.M. & Boss, G.R. Determintion of solute mounts of GDP nd GTP ound to Rs in mmmlin cells: comprison of prentl nd Rs-overproducing NI 3T3 firolsts. Proc. Ntl. Acd. Sci. USA 92, (1995). 35. Tin, T. et l. Plsm memrne nnoswitches generte high-fidelity Rs signl trnsduction. Nt. Cell Biol. 9, (27). 36. Plowmn, S.J., Muncke, C., Prton, R.G. & ncock, J.F. -rs, K-rs, nd inner plsm memrne rft proteins operte in nnoclusters with differentil dependence on the ctin cytoskeleton. Proc. Ntl. Acd. Sci. USA 12, (25). 37. Lenzen, C., Cool, R.., Prinz,., Kuhlmnn, J. & Wittinghofer, A. Kinetic nlysis y fluorescence of the interction etween Rs nd the ctlytic domin of the gunine nucleotide exchnge fctor Mm. Biochemistry 37, (1998). 38. Mor, A. et l. The lymphocyte function-ssocited ntigen-1 receptor costimultes plsm memrne Rs vi phospholipse D2. Nt. Cell Biol. 9, (27). 39. McLughlin, S., Wng, J., Gmhir, A. & Murry, D. PIP(2) nd proteins: interctions, orgniztion, nd informtion flow. Annu. Rev. Biophys. Biomol. Struct. 31, (22). 4. ond, A. et l. Phosphtidylinositol 4-phosphte 5-kinse is downstrem effector of the smll G protein ARF6 in memrne ruffle formtion. Cell 99, (1999). 41. Ppynnopoulos, V. et l. A polysic motif llows N-WASP to ct s sensor of PIP(2) density. Mol. Cell 17, (25). 42. Buck, M., Xu, W. & Rosen, M.K. A two-stte llosteric model for utoinhiition rtionlizes WASP signl integrtion nd trgeting. J. Mol. Biol. 338, (24). NATURE STRUCTURAL & MOLECULAR BIOLOGY VOLUME 15 NUMBER 5 MAY

11 E R R AT U M Errtum: Memrne-dependent signl integrtion y the Rs ctivtor Son of sevenless Jodi Guresko, Willim J Glush, Sen Boykevisch, olger Sondermnn, Dgn Br-Sgi, Jy T Groves & John Kuriyn Nt. Struct. Mol. Biol. 15, (28); pulished online 4 My; corrected fter print 15 My 28 In the version of this rticle initilly pulished, the concentrtion units reported in Figure 7,c should e nm, not nm. The green dt series in Figure 7 should e leled SOS ct. The corrected figure pnels re shown elow: c PIP % PIP Rs on lipid vesicles Rs mnt-dgdp Rs-GTP 3 3 SOS DPC( mutnt), SOS DPC, SOS DPC, SOS DPC(R552G), no exchnge fctor SOS DPC( mutnt), SOS DPC, no exchnge fctor SOS DPC(R552G) SOS DPC SOS ct Rs on supported ilyers Rs BODIPY-GDP Rs-GDP 36 SOS ct No exchnge fctor SOS DPC (no PIP 2 ) SOS DPC (2% PIP 2 ) SOS ct (2% PIP 2 ) 1 nm concentrtion 1 nm concentrtion 1 nm concentrtion In ddition, on pge 452 of the rticle, the ffilition ddress for Willim J. Glush nd Jy T. Groves ws incorrect. Their correct ddress is Deprtment of Chemistry, University of Cliforni, Berkeley, Cliforni 9472, USA. Finlly, the lst sentence of the Acknowledgments listed incorrect funding informtion. The lst sentence should red, J.T.G. nd W.J.G. re supported y Chemicl Sciences, Geosciences nd Biosciences Division, Office of Bsic Energy Sciences of the US Deprtment of Energy under Contrct No. DE_AC3-76SF98, nd D.B.-S. y NI GM These errors hve een corrected in the TML nd PDF versions of the rticle. NATURE STRUCTURAL & MOLECULAR BIOLOGY