© 2016 Nature America, Inc. All rights reserved. to to J.P.R. ( New York, New York, USA (K.K.Y.). Institute for Molecular Engineering, University of Chicago, Chicago, Illinois, USA (H.-L.T.) and Department of Urology, Icahn School of Medicine4 at Mount Sinai, San Francisco, California, USA. 1 ( domain (HF) histone-fold a as well as (PH) ogy domains N-terminal with Dbl homology (DH) and homolpleckstrin proteins adaptor transmembrane or receptors activated on motifs phosphotyrosine the in motifs PxxP of (PR) domain association proline-rich with Grb2, in which turn C-terminal binds via occur to recruitment thought membrane is SOS initial of The Ras. and by modulation autoinhibition, of allosteric release recruitment, membrane involving cells stem development growth cell as such processes diverse for critical is SOS by Ras of activation The digital signaling kinase in Ras receptor-induced of results patterns bimodal) mechanism (i.e., allosteric an through activation full cancers human many for responsible is Ras of activation deactivation Ras promote thus and Ras of activity GTPase intrinsic the enhance that GTP cytoplasmic This with process is opposed nucleotide by Ras-GTPase-activating proteins Ras-bound (RasGAPs) of exchange the catalyze that (RasGEFs) factors guanine-nucleotide-exchange of Ras variety a by mediated is activation Ras state. (GTP-bound) active or (GDP-bound) inactive an in exist can Ras pathways. signaling many Ras is a membrane-anchored small GTPase that plays a central role in recruited measurements questions how single Grb2-independent multiple recruitment SOS Kamlesh K Yadav Sune M Christensen triggered Ras activation One-way membrane trafficking of SOS in receptor- nature structural & molecular biology molecular & structural nature Received 5 February; accepted 8 July; published online 8 August 2016; Department of Biochemistry, New York University School of Medicine, New York, New York, USA. Department Department of Chemistry, University of California, Berkeley, Berkeley, California, USA. Son of Sevenless (SOS) is a widely distributed RasGEF distributed widely a is (SOS) Sevenless of Son Receptor-triggered activation of SOS is a multilayered process process multilayered a is SOS of activation Receptor-triggered

is receptors

SOS a [email protected]

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, interactions 6

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membranes. These These authors contributed equally to this work. small 1 4

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SOS activity is governed by a combination of weak-to-moderate weak-to-moderate of combination a by governed is activity SOS recruitment, membrane Grb2-facilitated beyond that, demonstrated enzyme exhibit increased responsiveness compared with that of the full-length even or competent signaling fully are and stimulation receptor after to the membrane localize the PR still domain lacking SOS constructs truncated that by observations challenged is recruitment membrane exchange nucleotide in promoting consequently SOS and Ras membrane-anchored to proximity positioning thereby cytosol, the from complex SOS–Grb2 acces substrate on ( sibility based model membrane-recruitment simple SOS1. on solely focused we appears to be redundant somewhat with SOS1 in cells syndromes CFC and Noonan as such disorders developmental in implicated been have and behavior signaling altered in result functions latory SOS denote we which core, catalytic the form together SOS1 in domains domains at CDC25 and REM situated the of pocket rim the allosteric an to Ras of binding on contingent is various with lipids interactions membrane through released is autoinhibition this membranes, On solution. in assayed when activity SOS autoinhibits essentially

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2 on Historically, SOS activation has been rationalized in terms of a of terms in rationalized been has activation SOS Historically, Department Department of Anatomy, University of California, San Francisco, Cat

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Present addresses: Novozymes A/S, Bagsvaerd, Denmark (S.M.C.), thought

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© 2016 Nature America, Inc. All rights reserved. molecules remaining at the bilayer after a pulse ( pulse a after bilayer the at remaining molecules ability of SOS1 engaging Ras by directly counting the number of SOS1 prob the quantifying of means convenient a provided method This solution, becomes trapped after binding Ras at the catalytic site catalytic the at Ras binding after trapped becomes solution, in nucleotide free of absence the in and, Ras membrane-bound with ( profile concentration defined a with pulse transient a in SLBs Ras-functionalized the over observed the perturb not ( SOS of did activity labeling that showed experiments Control (TIRFM). microscopy fluorescence reflection internal total and tracking of individual SOS at molecules the membrane surface by ble and bright fluorophore (ATTO 647N) reliable facilitated counting Fig. Supplementary 1b of GTP-BODIPY; and (GDP- imaging labels nucleotide epifluorescence fluorescent via Ras-bound Ras of density surface local the to access provided spectroscopy correlation fluorescence with obtained activity SOS to respect with functional that Ras was mobile fully and bound laterally permanently yielding thus Methods), (Online lipid functionalized mutant, to to referred as at Ras) the bilayer a via C181 henceforth maleimide- C118S 1–181, (residues H-Ras coupled we configuration, (SLBs; bilayers lipid supported on Ras with SOS of interaction the study to assay imaging an We developed Supported-lipid-bilayer RESULTS activation. SOS triggering of spontaneous of inhibition strong receptor importance the and underscores by activation initiated Ras for that quantitative function the receptor input-response affects the substantially of phenomenon this state signal; the triggering the of regardless inactivated be or active remain may machinery Ras-activation The assumed generally been has that process Grb2-dependent This mechanism endocytosis. differs substantially from the reversible by as such removed, actively being until Ras activates continuously and membrane the on which trapped in remains SOS1 process membrane-recruited trafficking one-way essentially an reveal results cell-based quantitative with in (details assays signaling combination in system reconstituted- membrane a used studies These observations. kinetic bulk and measurements dwell-time single-molecule of series a through tion, contributions of the different domains of SOS1 to membrane associa individual the mapped we question, this Toaddress regulated? is it proteins fusion Grb2-SOS1 using approaches synthetic-biology in or activity, SOS of models computational and activation of Ras by SOS have not been captured in earlier mechanistic processivity and membrane irreversible recruitment essentially in the ( period residency membrane single a during proteins Ras of thousands activate processively to capacity be can resolved molecules directly SOS individual of activity catalytic the which in platform supported-lipid-bilayer fluid micropatterned a using membranes through Ras- and lipid-binding interactions. Additionally, domain are recruited successfully to reconstituted Ras-functionalized ( function SOS for model plified oversim an is Grb2 via receptors integral the membrane to recruitment by SOS mediated of domains interactions multiple protein-lipid and protein-protein s e l c i t r a  In this system, we initiated measurements by flowing purified SOS1 Does such extreme processivity of SOS occur in cells, and, if so, how PR Grb2-binding the lacking constructs SOS that observed We 22 Supplementary Fig. 1c Fig. Supplementary , 3 38 8 , we found that a single SOS molecule has the the has molecule SOS single a that found we , , 3 9 ( Supplementary Fig. 1a Fig. Supplementary Fig. 2 Fig. and ref.

SOS-activation 15 Supplementary Note 1 Note Supplementary , 24 a ). During such a pulse, SOS1 interacts interacts SOS1 pulse, a such During ). , 33 3 Supplementary Note 1 Supplementary , 8 3 ). Labeling ). of Labeling SOS with a photosta 5 . These studies suggest that the the that suggest studies These . Fig. 1c Fig. Fig. 2 Fig. ).

assay , d a ). Such high degrees of degrees high Such ). 8 ). In this experimental experimental this In ). , ). A calibration curve curve calibration A ). 1 5 . Fig. 2 Fig. ). Altogether, our our Altogether, ). b ). and Online Online and 6 advance online publication online advance , 7 , 10 , 16– 30 36 , 1 4 , 3 9 0 7 - - - - . .

probability from the adsorption traces (i.e., the probability of a SOS recruitment of the quantification permitted densities, Ras measured SOS of profile centration of phase the assay ( the pulse during bilayer domains lipid-binding any but lacks pocket Ras-binding allosteric the and site active the both tains SOS the examining by recruitment analyzed quantitatively first we Here, membrane. the to SOS recruit to nism membrane at the operating loop feedback positive RasGTP Ras of state nucleotide the on depends SOS termed core, domains motif in (REM) the catalytic and the Ras-exchanger CDC25 between located site, allosteric an to binding RasGTP by activation its is context cellular the in SOS1 of aspect functional important An Allosteric kinetics. desorption the of measurement permitting ber, thus SOS1 was removed cham from the that reaction ensured dissociated and ( molecules SOS1 activated successfully and recruited the by Ras of turnover sustained) (i.e., processive in resulted and reaction exchange the initiated nucleotide unlabeled with Chasing Methods). effect on SOS1 activity, presumably through steric obstruction of the obstruction steric on through activity, presumably SOS1 effect autoinhibitory an exert domains N-terminal the that shown have ies DH-PH cassette a and by an HF domain flanked ( is SOS1 of core catalytic the side, N-terminal of retention and SOS recruitment membrane whether known not is It Regulation feedback) positive as to referred (commonly achieved is well- the which by known mechanism accelerating ofeffect on RasGTP SOS mediated one Ras activation is site allosteric its via SOS of the to SOS of nucleotide stateactivity of Ras specific average the of insensitivity the with RasGTP at the stage of membrane recruitment. These results, together Collectively, the data demonstrated a distinct positive allosteric effect of ( assays lular Ras. bound by allosterically limited the predominantly was membrane the from SOS of release ( active SOS membrane-bound scale) (minute-to-hour SOS activated as successfully identified in curve the of (tail membrane the at SOS remained processive highly of population a nucleotide, with chasing of binding prevent to Ras allosteric known SOS1, in SOS mutation of point property W729E A this of determinant primary the is Ras for nucle free of ( absence otide the in site catalytic the at Ras membrane binding the after at trapped is SOS expected, as that, demonstrating site catalytic the SOS to of binding Ras abolish to shown previously Ras, ( RasGTP displaying SLBs on enhancement ~16-fold an observing Ras, of state nucleotide the to membrane the at Ras ( with collision after trapped being molecule Fig. 2 Fig. Allosteric binding of binding by Ras provides SOS an Allosteric also mecha alternate We substantiated our findings in the SLB experiments with cel with experiments SLB the in findings our substantiated We SOS of recruitment membrane that found We Supplementary Fig. 1d Supplementary Cat d Fig. 2 Fig. are influenced by its flanking lipid-binding domains. At the the At domains. lipid-binding flanking its by influenced are 4 Supplementary Fig. 1d Fig. Supplementary Y64A and Online Methods)). Online and 2 rsle i ol taset erimn o SOS of recruitment transient only in resulted ,

activation

of , thus indicating that the allosteric binding pocket pocket binding allosteric the that indicating thus , Supplementary Note 2 Supplementary c 8 ). Nucleotide-dependent recruitment was preserved preserved was recruitment Nucleotide-dependent ).

, 4 membrane 3 , essentially abrogated recruitment ( recruitment abrogated , essentially Cat

(ref. (ref. nature structural & molecular biology molecular & structural nature

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via Fig. 2 Fig. and ref. ref. and 2

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by and d

). A Y64A point mutation in in mutation point Y64A A ). N-terminal Cat 4 1 Cat Supplementary Fig. 2a Fig. Supplementary

and is thought to enable a enable to thought is and membrane molecules. The long-lived The long-lived molecules. 3 was recruited to the Ras Ras the to recruited was Fig. 2 Fig. 8 Cat ), thus indicating that that indicating thus ), module, which con which module, Cat c Fig. 2 Fig. ). The known ). con The known

was catalytically catalytically was domains Cat Fig. 2c Fig.

recruitment was sensitive sensitive was Cat c 8 ), which we we which ), , ( 22 , Fig. 2 Fig. , 8 Cat 4 d , 1 9 ). After ). After Fig. 2 Fig. . . , thus thus , – d d Cat Cat ). ). ). ). a ------

© 2016 Nature America, Inc. All rights reserved. tion associated with Noonan syndrome (SOS syndrome Noonan with associated tion structures in observed site Ras-binding allosteric of the hindrance steric by the evidenced as membrane, the to recruitment initial its hindering by activation SOS1 of spontaneous is N down-modulation the terminus that clearly demonstrated a major of observations These property the ( evident was binding Ras-specific struct, ( (SOS of the full N terminus (construct comprising HF-DH-PH-Cat domains to Inclusion by the membrane recruitment approximately three-fold. (SOS core catalytic the to unit DH-PH the Appending to domains SOS the of N-terminal adding after SOS1 recruitment unclear. are mechanisms underlying the but autoinhibition, of release the in role charged lipids negatively for sites interaction additional several contains 4,5-bisphos (PIP phate phosphatidylinositol with interacts domain PH The structures crystal in observed as pocket, Ras-binding allosteric nature structural & molecular biology molecular & structural nature membrane surface. This experiment was repeated five times. five repeated was experiment This surface. membrane the from dissociating without manner a sustained in Ras activating SOS of copies individual which to corrals membrane indicate channel GDP the in depicted assay single-molecule SOS membrane-recruited and channel) (red ( SLBs. fluid Ras-functionalized micropatterned on based assay activity SOS ( LAB. protein adaptor the on motifs phosphotyrosine to Grb2 of binding via receptors B-cell activated of downstream membrane plasma the to recruited is SOS example, shown the In pathway. transduction ( Grb2. with interact which motifs, PxxP indicate domain PR C-terminal the in boxes Yellow domains. regulatory flanking the with together depicted is (Cat) unit catalytic ( domains. SLBs Ras-decorated to 1 Figure Fig. 3 Fig. c a We observed a pronounced damping effect on initial membrane membrane initial on effect damping pronounced a Weobserved H-Ras Glass Recruited SOSCat (counts ×103) Lipid membrane HDPC 23 100 150 200 Cat 50 d b 0 ,

2 ) Representative overlay image of fluorescent GDP bound to Ras Ras to bound GDP fluorescent of image overlay ) Representative ). Even in the case of the highly autoinhibited HDPC con HDPC autoinhibited highly the of case the in Even ). were recruited and exhibited highly processive SOS processive highly exhibited and recruited were 4 The catalytic core of SOS is stably and functionally recruited recruited functionally and stably is SOS of core catalytic The 0 . These lipid interactions are generally believed to play a play to believed generally are interactions lipid These . a )) damped recruitment by ~66-fold relative to that of SOS 23 2 ) The domain architecture of full-length (FL) hSOS1. The The hSOS1. (FL) full-length of architecture domain ) The ) lipids ) Time (s) , 4 3 100 Recruitment . Interestingly, a gain-of-function R552G point muta point R552G gain-of-function a Interestingly, . SOS 35 , Cat 4 0 in vitro in 4 and phosphatidic acid phosphatidic and 1 b Time (s) ) Classical model of the SOS-Ras-ERK signal- SOS-Ras-ERK the of model ) Classical SOS pulse 400 1 10

% of maximum lipid-binding and Grb2 of , independently 0 0 SO 0 S c Cat 50 Time (s . Darker areas with depleted signal in signal depleted with areas . Darker 800 pulse 100 ) 2 15 0 GDP-GTP chase 2 Cat Ras GDP-GTP chase 0 (green channel) in the the in channel) (green RasY64A Supplementary Fig. 2e Fig. Supplementary GT P -SOS 3 HDPC R552 HDPC Ras 3 400 Time (s)

Desorptio , and the HF domain domain HF the and , SOS GTP Cat W729 GTP advance online publication online advance E n GxP 800 DPC ) 6 Cat , compared compared , activity, activity, c ) reduced reduced ) ) Single ) Single d Recruitment probability 10 10 10 10 b 23 –4 –3 –2 –1 Cat , Cat 4 3 ). ). SOS 2 - - - . .

µ m Ra Cat W729E on bilayers displaying RasGTP ( RasGTP displaying bilayers on ( regulated membrane tightly step. recruitment for As SOS observed 3a Fig. Supplementary SOS with determined from reconstituted-SLB assays—on cellular SOS1-Ras SOS1-Ras cellular assays—on reconstituted-SLB properties—as from SOS1 determined chemical intrinsic of effects the establish To Multicomponent ( response potent a to pled an in rare to which events are activation dual interesting functionality cou rise gives time dwell and probability recruitment membrane between anticorrelation This state. membrane-bound active the in the initial recruitment probability and enhancement of the dwell time of inhibition two thus domains major mediate functions: N-terminal scale; hour the on period residency mean a (with bilayers functionalized SOS s Fig. Fig. 2c SO c a 1 1 hSOS1 Although the N-terminal domains inhibited initial recruitment, recruitment, initial inhibited domains N-terminal the Although H-Ra HF S Glass Regulatory uni Ca DPC Ra SOS t –ATTO 647N s 98 Fig. 3 Fig. s Y64A , Cat DH and SOS and d Ra Allosteri HDPC ), ), the longer constructs also exhibited increased recruitment

s SOS Diffusion barrier SOS c 44 Ra PH , , t 6 0 c s Cat Ra Ra Supplementary Fig. 3b Fig. Supplementary , caused a slight relief of such inhibition ( inhibition such of relief slight a caused , Ca Lipid membrane s s t RE GD GT HDPC 05 Catalytic Ra FL Catalytic uni

P P analysis M Ca 776 CDC2 exhibited extremely long dwell times on Ras- on times dwell long extremely exhibited t ( assay. the of phases two the of representation ( desorption. and recruitment SOS 2 Figure receptor ligation B-cell (BCR) after transduction signal ERK SOS1-Ras-MAPK digital characterize that system we B-cell have to used previously (SOS1 double-deficient SOS2- and SOS1- a optimized we signaling, available online. available are graphs and plots for data Source condition. each for shown data the of average the indicate lines horizontal Black sample. a SLB from data represents triangle assay. Each stopped-flow the 1 of phase from quantified probabilities ( enzymes. labeled to unlabeled of ratio applied the to according × 55 55 of view of a field for are counts indicated The SOS. of site active Ras and pocket; allosteric abolished an with mutant SOS used Experiments ( SOS labeled c b GD GTP , hs mhszn te motne f a of importance the emphasizing thus ), 5 ) Traces from the stopped-flow assay. stopped-flow ) Tracesthe from ) Single-molecule tracking of ATTOof 647N– tracking ) Single-molecule s t Supplementary Fig. 3d Fig. Supplementary PxxP motifs Grb2-bindin

P 1068 Y64A of PR

binding SOS-Ras-ERK Grb2 , a construct deficient in binding to the the to binding in deficient , a construct

Supplementary Fig. 3a Fig. Supplementary Stopped-flow SLB assay probing probing assay SLB Stopped-flow 1333 g Cat diffusing at the bilayer. bilayer. the at diffusing b , c 8 Ag , and Online Methods). The The Methods). Online and 45 d GRB2 P P ) Membrane recruitment recruitment ) Membrane GRB2 , 4 d RasGDP density 6

Cat signaling . Here, we introduced introduced we Here, . Single SO ; ; SOS SOS µ − SO m 2 Ras receptor (BCR) Stimulated s e l c i t r a S , − 2 Plasma membrane e and were scaled scaled were and ) DT40 chicken chicken DT40 ) S GD Cat W729E Cat ). Ca P t 0 a ). ) Cartoon ) Cartoon Fig. 3 Fig. Ras

, , a Ras µ pERK MEK Raf m 5 b –2 GT µ and and m P 900 Cat  -

© 2016 Nature America, Inc. All rights reserved. traces in in traces ( levels ( traces. shown the to corresponding assay the of space parameter 3D the SOS and SOS of concentrations protein increasing across level pERK basal of ( (MFI). intensity fluorescence mean as reported is level ( SOS for BCR of stimulation after time and level expression SOS increasing of a function as activation ( intensity. int., units; SOS human expressing B cells model in pathway SOS-RAS-ERK the of analysis SOS1 ( context. signaling a cell- in domains flanking SOS of importance functional the reveals 4 Figure the of representations 3D We depicted levels. expression SOS1 with quantitative along level the individual-cell the at revealed responses Ras-ERK of Methods), magnitudes (Online assay p-FLOW the ( cytometry flow and (pERK) phospho-ERK to antibody an using by kinase ERK the of activation or microscopy fluorescence by tion localiza EGFP-SOS monitored and ligation, BCR induced or cells lated these into (hSOS1) SOS1 entirely devoid of endogenous SOS1 and SOS2, left the cells unstimu human of variants EGFP-tagged s e l c i t r a  online. available are graphs and plots for data Source s.e.m. bars, Error experiments. p-FLOW and cultures cell independent seven on based plots and graphs are available online. available are graphs and plots for data Source samples. indicated the over average an for coefficient n SOS samples: SLB of number following ( traces desorption fitting by obtained was construct each for period residency mean assay. The stopped-flow ( SOS for (data s.e.m. bars, Error positions. different 15 least at in imaged was sample Each SOS SLB: one from data reflects bar each which for HDPC, for except samples, SLB of number following the for collected data of average the represents bar assay. Each stopped-flow the from obtained constructs SOS of ( bilayer. the on RasGDP with conducted were shown experiments assay. All SLB stopped-flow the in tested constructs ( state. membrane-bound active the in time dwell prolonging 3 Figure f d a c Fig. 4a Fig. = 2; HDPC R552G, R552G, HDPC = 2;

, ) Membrane residence time of SOS constructs obtained from the the from obtained constructs SOS of time residence ) Membrane ) cells. Arrowheads indicate the time of BCR activation. The pERK pERK The activation. BCR of time the indicate Arrowheads ) cells. g Intracellular stainingofpERK ) Comparative plots representing the dynamic change in BCR-induced pERK as a function of stimulation time in cells expressing superphysiological superphysiological expressing cells in time stimulation of a function as pERK BCR-induced in change dynamic the representing plots ) Comparative Stimulation withanti-BCR − 2 f for flowcytometry ) and intermediate levels ( levels intermediate ) and −

FL , DT40 B cells. [SOS], SOS concentration. ( concentration. SOS [SOS], B cells. DT40 e–g transfection Transient Multiparameter assay of SOS-RAS-ERK pathway activity activity pathway SOS-RAS-ERK of assay Multiparameter The N terminus of SOS suppresses bilayer recruitment while while recruitment bilayer suppresses SOS of N terminus The b . The yellow plane on the cube indicates the subspace of subspace the indicates cube the on plane yellow . The ). The latter experimental platform, henceforth denoted denoted henceforth platform, experimental latter The ). FL DT40 cells . Red fill indicates increased activity of SOS of activity increased indicates fill . Red SOS1 C-terminally fused to an EGFP label. A.U., arbitrary arbitrary A.U., label. EGFP an to fused C-terminally Cat , , plasmids SOS-encoding n – 2 a = 4; DPC, DPC, = 4; – ) p-FLOW assay of pERK in transiently transfected transfected transiently in pERK of assay ) p-FLOW Cat c n are replotted from from replotted are , = 3. Error bars, estimated s.d. of the fit fit the of s.d. estimated bars, Error = 3. d ) BCR-induced SOS-RAS-ERK pathway pathway SOS-RAS-ERK ) BCR-induced [SOS ] n Stimulated b = 4; HDPC, HDPC, = 4; Cat Resting g (5 min) (0 min) -expressing ( -expressing ) of SOS ) of Supplementary Fig. 3b Fig. Supplementary Cat , , Cells (counts) Cells (counts) Figure n

hSOS1-EGFP Cells (counts) Cat 1 pERK intensity(A.U.) 1 = 5; DPC, DPC, = 5; pERK intensity(A.U.) n 1 int. (A.U.) b = 2; HDPC R552G, R552G, HDPC = 2; +anti-BCR 2 and SOS and 2 2 3 ) Recruitment probability probability ) Recruitment 3 c 4 3 5 ) and SOS ) and 2 4 4 6 for comparison). comparison). for 7 5 5 8 b 9 6 6 ) Multiparameter ) Multiparameter 7 7 FL n e 8 8 Cat = 4; HDPC, HDPC, = 4; ) Comparison ) Comparison . . ( 9 9 FL as compared to SOS to compared as h a ) from the the ) from – -expressing -expressing ) SOS ) SOS j

) Ratios of pERK observed in SOS in observed pERK of ) Ratios SOS advance online publication online advance

n FL level Expression

level Expression = 3.

Cat

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20 e ). ).

© 2016 Nature America, Inc. All rights reserved. Yellow planes on the cubes to the left indicate the corresponding subspace of the 3D parameter space in the p-FLOW assay (c.f. (c.f. assay p-FLOW the in space parameter 3D the of subspace corresponding the indicate left the to cubes the on planes Yellow mutant. KR-EE the in interaction lipid membrane of disruption showing variants, SOS1 wild-type and (**) mutant PH-domain KR-EE the in activation ( domain DH-PH ( state). (basal cells unstimulated for concentration SOS increasing ( domain PR and ( 5 Figure SOS for not but SOS of anchoring membrane irreversible essentially the that suggests cells SOS outperformed SOS min.), (10–20 stimulation BCR after points time later at decreased signals attenuation. was Whereas SOS the signal difference SOS in activation Even under these conditions, BCR stimulation ERK increased further ( stimulation receptor of absence the in ERK of activation SOS expressing cells with compared 4c Fig. Supplementary SOS by triggered those from substantially differed that patterns signaling Ras-ERK triggered domains, lipid-interacting SOS nature structural & molecular biology molecular & structural nature online. available are graphs and plots for data Source s.e.m. bars, Error experiments. independent three on based a

– pERK pERK

c pERK

[SOS] [SOS] [SOS] ) p-FLOW assays showing inhibition of spontaneous ERK activation after addition of SOS of addition after activation ERK spontaneous of inhibition showing assays ) p-FLOW Cat BCR stimulated BCR stimulated +anti-BCR +anti-BCR Cat , lacking the Grb2-binding domain as well as the N-terminal N-terminal the as well as domain Grb2-binding the lacking ,

SOS observed in reconstituted assays may exist in cells as well, well, as cells in exist may assays reconstituted in observed Basal Cat Cat Tim Tim Tim continued to signal in a sustained manner, SOS and a in sustained to signal continued flanking domains block spontaneous activation in the basal state but promote RAS-ERK signal transduction after receptor stimulation. stimulation. receptor after transduction signal RAS-ERK promote but state basal the in activation spontaneous block domains flanking d c g FL e (KR/EE) e e ), HF domain ( domain HF ), ) (depicted schematically in domain diagrams at the top). The ratio of pERK MFI for longer to shorter SOS variants is plotted against against plotted is variants SOS shorter to longer for MFI pERK of ratio The top). the at diagrams domain in schematically ) (depicted DPC DPC . Cat DPC FL Cat -containing cells ( cells -containing ( d a Ratio of pERK MFI Ratio of pERK MFI Fig. 4f HF HF HF HF ,

Ratio of pERK MFI 0.5 1.0 1.5 d 0.5 1.0 1.5 2.0 0.5 1.0 1.5 ). Cells expressing high levels of SOS of levels high expressing Cells ). DH DH DH DH , i e ). The sustained signaling from SOS ). signaling The sustained ), and PR domain ( domain PR and ), 0 0 ∆ PH PH ** PH PH DPC(KR-EE)/DPC DPC/Cat 3 3 FL REM REM Time (min) REM REM Time (min) DPC/Cat , exhibited more spontaneous spontaneous more exhibited , Fig. 4c Fig. 5 5 CDC2 CDC2 CDC2 CDC2 10 5 5 10 5 5 [SOS] , f PR PR

PR PR , f i ) (depicted schematically in domain diagrams at the top). ( top). the at diagrams domain in schematically ) (depicted 20 ). Another notable notable Another ). advance online publication online advance 20 FL FL -induced pERK -induced ( Fig. 4c Fig. (KR/EE) h HDPC HDPC Fig. 4e Fig. HDPC DPC b e d , d

– Ratio of pERK MFI Ratio of pERK MFI f and and 0.8 1.0 1.2 1.4 1.5 0.5 1.0 ) Time dependence of the pERK MFI ratio after BCR stimulation, plotted for the the for plotted stimulation, BCR after ratio MFI pERK the of ) Time dependence , Cat HF HF h Cat ∆ Cat HF Ratio of pERK MFI HF 0.5 1.0 1.5 ). ). ,

DH DH DH DH 0 mechanisms appear to be contradictory be to proposed appear mechanisms several but activity, SOS1 affect that mechanisms regu latory established have studies cellular and structural of number A SOS SOS cells DT40 ( wild-type in expressed level physiological the matches levels hSOS1 intermediate with reconstitution that suggesting thus DT40 cells, for wild-type to that observed were those nearly identical SOS intermediate this SOS than expressing cells of that SOS intermediate levels revealed SOS examination selective However, output. signal 0 Supplementary Fig. 4b Fig. Supplementary Domains flanking SOS flanking Domains HDPC(KR-EE)/HDPC PH PH PH PH ** 5 3 3 HDPC/DPC HDPC/DPC Cat

Time (min) autoinhibition 3 Time (min) RE RE have both positive and negative regulatory roles. regulatory negative and positive both have RE RE 5 M M M M

Cat 5 CDC2 CDC2 CDC2 CDC2 in response to BCR stimulation ( stimulation BCR to response in 10 10 Cat 10 5 5 [SOS] 5 5 flanking domains: DH-PH domain ( domain DH-PH domains: flanking 20 20 PR PR PR PR 20

prevents FL level resulted in rescued pERK responses responses pERK rescued in resulted level ). The data revealed that domains flanking flanking domains that revealed data The ). Cat i (KR/EE) might initially appear to merely dampen might initially HDPC DPC Cat-PR

spontaneous g c f Cat –

i Ratio of pERK MFI Ratio of pERK MFI ) Comparison of BCR-induced ERK ERK BCR-induced of ) Comparison 1.5 0.5 1.0 0.8 1.0 1.2 1.4

Ratio of pERK MFI HF HF 0.5 1.0 1.5 HF HF DH DH 22– 0 FL DH DH 0 24 signaled more signaled efficiently

activation PH PH HDPC(KR-EE)/DPC ** , 4 3 Cat-PR/Cat PH Cat-PR/Cat PH Fig. 4c Fig. 3 Time (min) Fig. 4g Fig. 3 . To understand how how To understand . Time (min) RE RE a ), HF domain ( domain HF ), RE RE s e l c i t r a M M 5 M M 5 CDC2 CDC2 , d CDC2 CDC2 , 10 ). Data are are Data ). j ). Moreover, ). 10 5 5 [SOS] 5 5 PR PR 20 PR PR 20 ∆ b ), ),  - © 2016 Nature America, Inc. All rights reserved. Next we investigated SOS1 regulation in BCR-stimulated cells express Positive SOS catalytic the of activity the curbing in potency similar have domains SOS to relative domain to of DH-PH that the comparable was PR domain HF the by the conferred of of inhibition magnitude The domains. effect DH-PH and autoinhibitory the of independent was effect this domain in restricting ligand-independent activation of SOS1 ( onto SOS domain PR the only Grafting Grb2. via receptors SOS to activated role in tory connecting assays SLB earlier our in nation SOS1 including full-length the PR domain, thus preventing its exami despite considerable effort, it has not been feasible to purify functional with our p-FLOW results for the resting cell state ( closed SOS a of conformation stabilizing by and activation allosteric blocking by bition Ras from GDP dislodge or allosteric RasGDP and accommodate fully cannot pocket autoinhibition catalytic the activation, DH-mediated of removal without and pocket, allosteric the at binding Ras limits domain DH the that Figure 3 SOS than SOS than SOS i.e., core; catalytic the ing flank of domains number the with manner in an incremental scaled SOS ( of levels high expressing cells in Ras-ERK of activation neous cells resting SOS on focused first we interface, membrane the near activation allosteric in spontaneous signaling cells yet SOS1 restricts allows for controlled online. available are graphs and plots for data Source experiments). five over imaged cells (95 cells in shown cell the for synapse bilayer ( trajectories. microcluster out ( lapse in channels image SOS and separate as plotted also are overlays displayed The cluster. BCR central a of formation the (right) after and (left) before signals fluorescence green) (EGFP, SOS and 32/1. ( DPC-PR, SOS 17/2; R552G, FL 21/2; FL, 34/2; HDPC-SH2, 30/2; HDPC, 28/5; SOS are: samples cells/SLB of Numbers average indicate lines horizontal Red cell. one from data represents graph the on dot Each microclusters. BCR and variants SOS in given is replicates of number The shown. are bilayer the contacting after point time (~5-min) SOS expressing Cells microclusters. BCR and SOS of localization spatial illustrating images ( antibody. linking BCR-cross- with decorated SLBs and cells B between formed cluster BCR central the from depletion SOS and microclusters BCR to SOS 6 Figure s e l c i t r a  ( levels SOS1-EGFP intermediate ing Fig. 5a Fig.

The C-terminal PR domain is most noted for its positive regula positive its for noted most is domain PR C-terminal The SOS to domains N-terminal of Addition Cat Cat Cat Supplementary Movie 1 Movie Supplementary (left) and SOS and (left) core in resting cells. resting in core ( ± Cat

, FL

b Fig. 5a Fig. b regulation s.e.m. for the shown scatter data. data. scatter shown the for s.e.m. PR-domain-dependent localization of of localization PR-domain-dependent DPC Cat . Structural . and Structural studies biochemical on SOS1 demonstrated (green) movement at the cell-bilayer interface. The trajectories were obtained by tracking individual BCR and SOS clusters in a time time a in clusters SOS and BCR individual tracking by obtained were trajectories The interface. cell-bilayer the at movement (green) and and b flanking domains in the basal state ( state basal the in domains flanking . ( . 6 signaling ( signaling , ( 7 b . ) Colocalization of different different of Colocalization ) Fig. 5 Fig. c Supplementary Fig. 5a Fig. Supplementary , ) Overlay ) of Overlay (Cy5, red) anti-BCR c a ), thus demonstrating that the N- and C-terminal C-terminal and N- the that demonstrating thus ), ) Representative TIRFM TIRFM Representative )

of FL b 23 ). These results corroborate the SLB results in in results SLB the corroborate results These ). (right) at an early early an at (right)

SOS , Fig. 5 Fig. 4 7 . These structural findings are consistent consistent are findings structural These . 22

activity ) of the cell shown in in shown cell the of ) e , 4 ) Normalized time traces of the fluorescence intensity of SOS of intensity fluorescence the of traces time Normalized ) Cat a 3 Cat ), and SOS and ), . The HF strengthens SOS autoinhi SOS strengthens HF The . 3 DPC 8 revealed an inhibitory effect of this of this effect an inhibitory revealed -PR, 9/2; 9/2; -PR, . c

. The phenomenon of SOS depletion from the central BCR cluster was observed in 69% of SOS of 69% in observed was cluster BCR central the from depletion SOS of phenomenon The . signaling was more restrained restrained more was signaling

in Fig. 5d Fig. Cat

– stimulated

, d ). The inhibitory potential potential inhibitory The ).

HDPC – Cat

f ). It has been reported reported ). It been has blocked the sponta the blocked was more inhibited inhibited more was c Fig. Fig. 5a b Fig. 5a Fig. . Each tracked position of a microcluster is indicated by a dot. Chains of connected dots draw draw dots connected of Chains dot. a by indicated is microcluster a of position tracked Each . a

cells Colocalization (%) ~5 min Anti-BCR (Cy5) SOS 20 40 60 80

SOS 0

Cat Cat – – c HDPC ). ). Notably, c advance online publication online advance 3

), i.e., in in i.e., ), HDPC-SH2 Fig. 5 µ m c SOS (EGFP) ); ------FL R552GFL

associated with the mutant and wild-type SOS1 variants ( variants SOS1 wild-type and mutant the with associated interaction PIP the disrupting thereby domain, PH the within mutation) (KR-EE R459E and K456E of mutation combined ity, introduced we possibil this To test system. cell the in PH of binding phospholipid the counteracting truncation HF the from arise may disparity This COS-1 cells and differentiation in mouse embryonic-stem-cell in Ras of loading GTP regulate positively to reported been has tion inhibitory effect of ( DH-PH was because unexpected PH-lipid assays interac SLB the in observed time dwell increased the ( SOS membrane-recruited of time residence the with agreement in are ( domain PR con the SOS taining for stimulation BCR after HF of role regulatory positive ( output signaling increased in resulted of of inclusion inclusion with the the DH-PH, HF in domain SOS of SOS that to relative effect inhibitory purely a had alone domain DH-PH Ras binding allosteric allowing thus lipids, membrane with domain PH the of interaction that autoinhibition by the DH domain can be by released electrostatic the ability to simultaneously dampen SOS activity in the basal basal the in activity SOS SOS of dampen sides simultaneously both to ability on the domains flanking ( the assays SLB our in measurements lipid through membrane. at the SOS1 domains, active stabilize PH cooperatively interactions, and HF the that indicate collectively ( domain HF of effect posi the regulatory negating tive thereby DPC, wild-type shorter the of those to interaction phospholipid-PH through SOS1 membrane-targeted stabilizing in of HF requirement the thus supporting out duration, assay entire the through activation SOS1 antagonized markedly HDPC in mutation effect, ( in pERK small decrease in a small resulting relatively a had format DPC in mutation KR-EE The SOS For the DH-PH, our results from stimulated cells conflicted with with conflicted cells stimulated from results our DH-PH, the For In sum, p-FLOW results ( results p-FLOW sum, In Cat -PR Cat Anti-BCR (Cy5)

DPC-PR SOS ~5 min under conditions of BCR stimulation ( of stimulation BCR conditions under FL 2 15 4 ( , 3 5 Fig. 5 Fig. , and compared the BCR-stimulated ERK activation activation ERK BCR-stimulated the compared and , Supplementary Figure 7c Figure Supplementary FL d -EGFP and BCR at the center of the cell-supported cell-supported the of center the at BCR and -EGFP

h nature structural & molecular biology molecular & structural nature 15 ). The KR-EE HDPC signals were comparable comparable were signals HDPC KR-EE The ). SOS (EGFP) , 22 2.5 Supplementary Fig. 5e Fig. Supplementary , 3 in vitro in 3 µ . . In our p-FLOW assay, we found that the m Fig. Fig. SOS Anti-BCR 5 observation that HF enhances the the enhances HF that observation ) combined with single-molecule single-molecule with combined ) c t FL 0 Anti-BCR Figs. 2 2 Figs. Fig. 5 Fig. Fig. 5 Fig. . ( . Fig. 5 Fig. e d

) Trajectories of BCR (red) (red) BCR of Trajectories ) Normalized intensity (A.U.) 0.5 0.6 0.7 0.8 0.9 1.0 g Fig. 3 Fig. i SOS and e ). ). However, KR-EE the ). These observations observations These ). ). We also observed a observed We ). also 0 Fig. 5 Fig. – FL

g Cat 3 4 c Time (min) ). These findings findings These ). t ) indicated that that indicated ) 0 ). +8mi have evolved evolved have FL d 8 Fig. 3 Fig. -expressing -expressing ). ). In contrast 12 n Fig. 5g Fig. SO Anti-BCR c 16 15 S 3 ). The The ). FL 2 , µ 22 -PH -PH m DPC – , 3 i 3 ). ). - - - - - .

© 2016 Nature America, Inc. All rights reserved. and and the SLB. Approximately 15–20 min after cell a landing, large cen cells B the between formed synapses the of center the toward moved 4 Note Supplementary ( colocalization SOS1-BCR bled SOS to domain PR the of addition contrast, In ( microclusters BCR of sites SOS onto grafted Grb2 of domain SH2 SOS chimeric any expression, of SOS devoid endogenous system B-cell our In function. SOS1 and complexes 7b Fig. ( clusters BCR the with colocalize not ( Ras presum allosteric membrane, binding the through to ably localize did it although clusters, SOS BCR whereas clusters, BCR of sites to a of imaging ( TIRFM antibody the on label Cy5 through observed as microclusters, BCR of SOS of activation with BCR the and activates recognizes that functionalized antibody SLBs on spread were SOS1-EGFP human ing antigen-presenting with ( cells interacting cells B of geometry signaling platform SLB we live-cell experiment, hybrid the this used For microscopy. confocal TIRFM by spinning-disc cells and living in SOS1-EGFP imaged we SOS1, of ficking targeting. membrane artificial resembling phenotype cellular and a thus molecular produces of recruitment, mode membrane primary 6 Fig. membrane the to SOS1 targets artificially which with a C-terminally grafted farnesylation signal sequence from H-Ras, functionalized when truncation SOS of characteristics these SOS mimics Interestingly, signaling. induced of points time late at ation and in cellular p-FLOW assays are less sensitive than SOS SOS Regulation (further in stimulation discussed receptor after activity SOS enhance but state 10 bar, Scale experiment). each for condition per counted cells (25 experiments independent two of average an represent results The graph. bar the (SOS pocket allosteric SOS1 an with compared is SOS EGFP-tagged cells. COS-1 EGF-stimulated in vesicles endocytic 10 bar, Scale channels. image in plotted also is image merged the in inset enlarged The experiment). per analyzed cells (25 experiments independent three in cells the of >75% in observed pattern colocalization the of representative are shown EGF. Images with min 25 for stimulated cells COS-1 in marker endosomal Rab5 the with SOS1 internalized of 10 bar, Scale points. time indicated the for EGF ( samples. SLB across s.d. bars, Error 78/3/2. HDPC-SH2, 75/3/2; in 3 bar, Scale landing). cell after min (~10–30 points time late at zone contact cell-bilayer the from away appearing structures vesicle-like ( attenuation. 7 Figure nature structural & molecular biology molecular & structural nature ‘BCR as to referred commonly phenomenon a appeared, cluster tral c ) Localization of EGFP-tagged SOS1 EGFP-tagged of Localization ) µ B-cell activation from the supported membrane led to formation formation to led membrane supported the from activation B-cell To and investigate membrane traf recruitment subsequent further Over time, the initially scattered BCR clusters concatenated and and concatenated clusters BCR scattered initially the time, Over a m. ( m. . Numbers of cells/SLB samples/cell cultures are: FL, 97/4/2; HDPC, HDPC, 97/4/2; FL, are: cultures samples/cell cells/SLB of Numbers . Cat Supplementary Fig. 7a Fig. Supplementary ). Deletion of the Grb2-binding domain of SOS1, its putative putative its SOS1, of domain Grb2-binding the of Deletion ). b , , SOS ). Contrasting reports have addressed the roles of signaling signaling of roles the addressed have reports Contrasting ).

) Statistics of cells displaying internal SOS puncta, as shown shown as puncta, SOS internal displaying cells of Statistics ) PR-domain-dependent SOS endocytosis mediates signal signal mediates endocytosis SOS PR-domain-dependent

of DPC a Supplementary Note 3 Note Supplementary µ ) Confocal data in 3D rendering, showing SOS showing rendering, 3D in data Confocal )

m. Source data for plots and graphs are available online. available are graphs and plots for data Source m. superprocessive , , and SOS 54 , 5 5 FL L687E R688A L687E FL (Online Methods). (Online ). FL HDPC molecule with a functionally impaired impaired functionally a with molecule Fig. 6 Fig. µ Fig. 6 Fig. ). SOS-deficient DT40 B cells express cells B DT40 SOS-deficient ). m. ( m. are all highly processive in SLB processive assays are highly all

Fig. 6 Fig. SOS ). Representative images accompany accompany images Representative ). Supplementary Figure 7d Figure Supplementary a e ). SOS ). ) Kinetics of SOS localization to to localization SOS of Kinetics ) b FL and and ).

in COS-1 cells stimulated with with stimulated cells COS-1 in by b Fig. 6a Fig. HDPC , , Fig. 6 Fig.

49– Cat Supplementary Fig. 7b Fig. Supplementary endocytosis FL Supplementary Fig. 7b Fig. Supplementary did not colocalize with with colocalize not did 5 was efficiently recruited recruited efficiently was 2 HDPC , did not colocalize with with colocalize not , did to simulate the native native the simulate to b µ , b Cat and and m. ( m. 5

). SOS ). 3 4 -SH2, with a single single a with -SH2, , thereby triggering triggering , thereby advance online publication online advance or to SOS to or 8 d ( ) Colocalization Colocalization ) Supplementary Supplementary Supplementary Supplementary HDPC FL FL as separate separate as to attenu -enriched -enriched also did did also DPC

ena

and FL

FL ). ). ------

it was depleted from the central BCR cluster ( cluster BCR from it the central was depleted but we at points that found time BCR, later activated the with moved capping’ are removed from the plasma membrane via endocytosis ( endocytosis via membrane plasma the from removed are (ref. Rab5 protein marker ( structures vesicular to perinuclear had most localized SOS molecules membrane 10 min after EGF stimulation. By 30 min after stimulation, We observed prominent membrane recruitment of SOS1 at the plasma stimulation. (EGF) factor growth epidermal before SOS1 distributed SOS EGFP-tagged cyto larger much plasmic volume than a that of DT40 B with cells. Visualization of transfected cells COS-1 used we membrane, plasma terminus. C the requires process BCR-signal-dependent a in suggest These observations that removal of SOS1 from the membrane ( antibody BCR-activating the displaying bilayers on only SOS for ( cles vesi of endocytic reminiscent were and cells the inside located were microscopy revealed the appearance of punctate SOS structures, ( which points time later at signaling ERK also correlates with attenuation this occurrence of SOS leaves the plasma membrane at the site of the central BCR cluster, and Fig. 7 Fig. e d c a SOS o oe eiiiey drs dsperne f SOS of disappearance address definitively more To Fig. 7 Fig. FL L687ER688 c FL ). The vesicular SOS1 colocalized with the early endosomal endosomal early the with colocalized SOS1 vesicular The ). SOS in endocytic 5 EGF (min) Anti-BCR functionalized SOS 6

EGF (min) but not for SOS for not but vesicles (% cells) ( a FL 100 Fig. 6 Fig. ). Moreover, these vesicle-like structures appeared only only appeared structures vesicle-like these Moreover, ). 20 40 60 80 FL 0 A

bilayer outline Cell 0 FL c and and SOS SOS revealed predominantly cytoplasmic and evenly cytoplasmic predominantly revealed SOS SOS 0 0 FL L687ER688A FL 5 FL Supplementary Movie 1 Movie Supplementary HDPC 5 7 ), thus indicating that SOS1 molecules molecules SOS1 that indicating thus ), 10 or the chimeric SOS chimeric the or Time (min) HDPC Fig. 4 Fig. 15 Rab5 5 1 0 g ). Confocal fluorescence fluorescence Confocal ). 20 Fig. Fig. 6d s e l c i t r a b ). SOS ). 25 Cells with internal , e HDPC FL

). ). Thus, SOS SOS puncta (%) 10 20 30 40 0 -driven Ras- -driven FL 15 3 Merg FL 0 Fig. 7a Fig. -SH2, and and -SH2, from the the from 30 FL Fig. 7 Fig. initially initially HDPC HDPC-SH2 e , d b FL ). ). ). ).  - -

© 2016 Nature America, Inc. All rights reserved. tional boost from receptor-mediated Grb2 recruitment to trigger a trigger to recruitment Grb2 receptor-mediated from boost tional addi the requiring thus activity, GAP background of context the in inconsequential as to slow be sufficiently must be activation taneous spon this levels, expression endogenous Under concentration. with scales that event a probabilistic and is simply Chatelier’s Le principle of by SOS is driven activation spontaneous the Essentially, sion level. expres SOS with scales of SOS activation spontaneous that revealed cells, B SOS-deficient in SOS1 of reconstitution by SOS-Ras-ERK enabled cascade, the of activity the of mapping activation. of multiparameter rate Our the increases clearly domain PR the by binding state. basal the in of SOS to inhibition contributes PR the domain In addition, process. signaling the of phase the on depending terminator, signal a or tator facili a signal as either acting cells, dual in receptor-stimulated functions performs domain PR the that revealed stimulation), receptor after time and activity, pathway level, expression (i.e., transduction signal of aspects multifactorial the considers Our which assay, p-FLOW signaling. SOS in role regulatory negative even positive, or a plays redundant, domain PR C-terminal the whether unclear been on results matter.this In it apparently with conflicting has particular, nals maintain control of SOS via Grb2 binding. The literature abounds sig receptor-mediated how to shifts question the SOS, of acteristics PIP as such messengers second lipidic bind which domains, HF and PH lipid-interacting by the augmented further is of Grb2 independently more increasingly RasGTP. This ability to respond to receptor stimuli produces SOS recruited the as feedback positive strong by fueled is tors that produce RasGTP and facilitate SOS recruitment; this process for example, of because of the activity or RasGRP other exchange fac activation, receptor after primed are levels RasGTP context, cellular a In triggering. receptor sensing of capable are domain PR binding Grb2- the lacking constructs SOS how explaining RasGDP,thereby to relative by RasGTP accelerated is strongly Ras of by SOS allosteric timescales. at signaling-relevant quasi-irreversible of is SOS recruitment membrane is present; equilibrium no dynamic molecules Ras of thousands activate sively sustained association of SOS with the membrane, where it can proces for sufficient is anchoring) membrane SOS of mechanisms other of independently (i.e., alone mechanism this systems, tuted-membrane lipid-membrane In reconsti pocket. binding a at allosteric the Ras by engaging with surface associates stably SOS that shown have we cells in competent signaling remain domain PR Grb2-binding the lacking mutants SOS-truncation that observation results have challenged this the model, classical recurring particularly thus explaining how signals are relayed downstream. However, several atbalance the RasGEF-RasGAP membrane in favor of Ras activation, the tip to presumed is SOS of localization membrane increased the membrane via the adaptor protein Grb2. In its classical interpretation, accepted to involve recruitment of SOS from the cytosol to the plasma commonly is pathway Ras the to receptors from propagation Signal DISCUSSION SOS1. of endocytosis the counteract to ( (SOS binding Ras allosteric in impaired mutant A SOS1 pocket. Ras-binding allosteric We found that the of kinetics was by SOS1 influenced endocytosis the s e l c i t r a  productive Ras signal (extended discussion in Fig. Fig. 7

From of the perspective receptor-mediated activation of SOS, Grb2 char activating irreversible and nearly In of spontaneous the light Here probability that we the membrane recruitment demonstrated 2 and phosphatidic acid. phosphatidic and e ). ). Binding of SOS1 to Ras via its pocket thus allosteric appears FL L687E R688A L687E FL ) exhibited accelerated endocytosis endocytosis accelerated exhibited ) 3 8 Supplementary Supplementary Note 5 . Strikingly, essentially essentially Strikingly, . 29– 3 4 . More recently, More . advance online publication online advance ------;

hyperplastic syndromes such as hereditary gingival fibromatosis gingival hereditary as such syndromes hyperplastic syndrome, developmental Noonan to linked are which domain, PR several found we databases, public Mining date. to unsuccessful been have produce functional full-length SOS1 protein including the PR domain to transition state the activated of to efforts SOS is because unknown, basis The for structural PR-domain-facilitated autoinhibition and the in features autoimmune to leads and domain of this roles regulatory both perturbs EF hand, a point-mutated with induced activation calcium- for allowing in simultaneously while RasGEF state this autoinhibited keeping the in role dual a play RasGRP1 in hands EF the that Weestablished effect. biological profound a have RasGEFs shutdown the as mechanism. domain PR the via membrane the from removal active with circuit a trafficking follows one-way SOS activation state, in discussed levels (further activation ERK sustained exhibit they are and domain PR endocytosed, the not lacking constructs SOS molecules. Ras new to remove SOS from the plasma membrane, cutting effectively off access to mechanism regulated actively an provides that attenuation signal 8a Fig. Supplementary 4. 3. 2. 1. reprints/index.htm at online available is information permissions and Reprints The authors declare no competing interests.financial project. All authors and discussed commented on the results. H.-L.T., J.E.J., J.P.R., and J.T.G. wrote the paper. J.T.G. and J.P.R. the supervised S.M.C., H.-L.T., and J.E.J. conceptualized and experiments. designed S.M.C., performed COS1 cell experiments under of supervision D.B.-S. J.T.G., J.P.R., and M.G.T. assisted with live-cell experiments. J.S.I. proteins.purified K.K.Y. S.M.C., H.-L.T., and J.E.J. performed experiments and analyzed data. S.A. andtype SOS1 Natural (to Sciences S.M.C.). We thank T. Kurosaki (RIKEN) for providing wild- and K.K.Y.) as well as a grant from the Danish Council for Independent Research, AI104789 (both to J.P.R.) and ARRA stimulus supplement GM078266 (to D.B.-S. and J.T.G.). Further support came from NIH-NCI R01-CA187318 and R01- was supported by a P01 Program grant from NIH-NIAID (AI091580 – to J.P.R. W.-C. Lin and L. Iversen for assistance with initial SLB experiments. This research for insightful comments on the manuscript. In addition, the authors thank and D.B.-S. for helpful comments and suggestions. The authors thank J. Kuriyan The authors thank the members of the laboratories of J.P.R., J.T.G., J. Kuriyan, ve Note: Any Supplementary Information and Source Data files are available in the the in v available are references associated any and Methods M disease. human to contribute and effects tle point mutations in the PR domain may biological have substantial and various cancers ( C AUTH A e ck O

rsion of the pape ethods r Recently, it has become clear that single–amino acid variants in in variants acid single–amino that clear become has it Recently, MPETI Stephen, A.G., Esposito, D., Bagni, R.K. & McCormick, F. Dragging ras back in the in F.back McCormick, ras & Dragging R.K. Bagni, D., Esposito, A.G., Stephen, personalities. split oncogenes: Ras R.A. Weinberg, & A.E. Karnoub, cascades. signalling kinase MAP Mammalian M. Karin, & L. Chang, Increasing Der,C.J. & G.J. Clark, K.L., Rossman, Khosravi-Far,R., S.L., Campbell, ring. Biol. Cell (2001). 37–40 signaling. Ras of complexity s i now o O n Cancer Cell Cancer R R C

SOS1 o f N ledgme

t ON G G FI 9 h − , 517–531 (2008). 517–531 , e 2 variants with point mutations or stop codons in the the in codons stop or mutations point with variants TRIBUTI

− p N DT40 B cells. r l . . a A

p 25 N n e 5

r CIAL CIAL I 8 , 272–281 (2014). 272–281 , ts . . . A single–amino acid variant allele, nature structural & molecular biology molecular & structural nature Supplementary Fig. 8e Supplementary ON Supplementary Note 6 Note Supplementary – d N S Oncogene ). We propose endocytosis as a method of method a as Weendocytosis ). propose TERESTS

17 , 1395–1413 (1998). 1395–1413 , ). ). It is plausible that sub ). Thus, in its natural natural its in Thus, ). Rasgrp1 http://www.nature.c Rasgrp1 Anaef Nat. Rev. Mol. Rev. Nat. Nature mice

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ho C, u G, kwoe, . Fomn MA & a-ai D Phospholipase D. Bar-Sagi, & M.A. Frohman, K., Skowronek, G., Du, C., Zhao, Wang,W. L. McCollam, Corbalan-Garcia,Margarit,S., S.M.,Galron,Yang, D.,Bar-Sagi, S.S.& Regulation D. C.A. Karlovich, Kholodenko, B.N., Hoek, J.B. & Westerhoff, H.V. Why cytoplasmic signalling signalling cytoplasmic Why H.V. Westerhoff, & J.B. Hoek, B.N., Kholodenko, SOS1 novel A M. Mizuguchi, Y.& Mizuno, J., Takita, M., Saitoh, M., Tumurkhuu, A.E. Roberts, S.M. Margarit, the by activity sevenless of Son of gating Allosteric D. Bar-Sagi, & K.K. Yadav, J. Gureasko, J. Gureasko, P.Chardin, H. Waterman, and SH2 The D. Bowtell, T.& Pawson, J., Wade, Fernley,R., M., Rozakis-Adcock, N. Li, Grb2 D. Bar-Sagi, & J. Schlessinger, E.J., Lowenstein, S., Kaplan, N.W., Gale, the through p21ras regulates factor growth Epidermal J. Downward, & L. Buday, Findlay, G.M. Wang, D.Z. Baltanás,F.C. R.L. Kortum, lymphocytes. in function and regulation Ras D.A. Cantrell, & E. Genot, S.E. Egan, cellular and factors exchange ras of J.P.Regulation Roose, & I. Rubio, J.E., Jun, the J. Das, at transduction signal in mechanisms Molecular J. Kuriyan, & J.T. Groves, ways. the count us Let activated? SOS is How T. Pawson, & G.M. Findlay, on. Ras turn F.receptors McCormick, how transduction: Signal Nat. Cell Biol. Cell Nat. D2-generated phosphatidic acid couples EGFR stimulation to Ras activation by Sos. transduction. signal (1995). Son-of-sevenless. factor exchange Ras the in activityintramolecularSosbyof interactions. sevenless. (2000). membranes. cell to recruited be should PI3K proteins and RAS both in signaling affects pathways. syndrome Costello/CFC in mutation syndrome. SOS. factor exchange nucleotide Ras-specific domain. histone (2010). of the Ras activator Son of Sevenless. sevenless. of GRB2. to binds (2002). signaling. negative in Grb2 for role a unveils endocytosis activator Ras the to receptor mSos1. EGF the couple Grb2 mammalian of domains SH3 signalling. Ras to kinases tyrosine receptor Ras. on exchange Nature nucleotide guanine of activation EGF-dependent the mediates nucleotide Sos and protein, adapter Grb2 factor. receptor, exchange of complex a of formation fate. cell stem embryonic of control Dev. development. and signaling EGFR in Sos1 for requirement a demonstrating survival.homeostasisorganismaland development. Immunol. (1993). n yoie iae inl rndcin n transformation. and transduction signal kinase tyrosine in cells. T (2013). in 239 messengers lipid by activation ras of localization cells. membrane. Biol. Mol. Struct. (1993).

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© 2016 Nature America, Inc. All rights reserved. nioyfntoaie spotd ii blyr fr iecl imaging. live-cell for bilayers lipid supported Antibody-functionalized after recovery fluorescence with sample photobleaching (FRAP). each for confirmed was Ras and lipids of fluidity two-dimensional The flow. constant under 7.4) pH TCEP, 3 mL reaction buffer (40 mM HEPES, 100 mM NaCl, 5 mM MgCl with washing by removed was nucleotide fluorescent unbound any and ture, cent nucleotides to the SLB. Ras incubation showed no detectable nonspecific binding of the applied fluores 488–GppNp. A control experiment in which samples underwent all steps except in this study included BODIPY-GDP, BODIPY-GTP,HEPES, 100 mM NaCl, and 5 mM ATTOMgCl, pH 7.4). Fluorescent488–GDP, nucleotides used and ATTO 10 with samples NaCl,immediatelystepThiswas7.4). pH followed byovernight incubation of tion with EDTA in loading buffer (50 mM EDTA, 40 mM HEPES, and 150 mM incuba 20-min a in stripped was Ras nativenucleotidetothe bound 7.4); pH 4 °C and washed with 3 mL loading buffer (40 mM HEPES and 150 mM NaCl, throughout the sample preparation for liquid injections and washing steps. for 10 min. A motorized syringe pump (PHD 2000, Harvard Apparatus)acted MCC waswas usedquenched by treatment with 2- was then incubated for 2.5 h with Ras inBioptechs). PBS (1 mg/ml).The sampleAfter Ras chambers,wasincubation, flow (FCS2 chambersincubated flow in mountedslides unre piranha-etchedglass with casein in PBS (2.5 mg/ml) for 10 min and and the remainder Egg-PC. (Avestin times TxRed-DHPE, 0.01% MCC-DOPE, 3% DOPS, 3% compositionlipidwas The 11 extruded Millipore). was membranes; polycarbonate suspension 30-nm-pore-diameter Miniextruder, vesicle The 7.45). (pH PBS in film lipidrehydration dried byformedwere (SUVs)vesiclesthe lamellar of by rotary evaporation evaporated(40 °C, 10 min) followed was by N Solvent flask. round-bottomed described a in mixed previously were chloroform as prepared were bilayers for bilayers lipid supported Ras-decorated system ( assay stopped-flow the indicated that in labeling had constructs a negligible labeled effect on and the unlabeled of comparison ments were conducted to ascertain that labeling did not alter enzyme behavior; needed in the interpretation of related results. Here, nucleotide-exchange experi efficiencies exceeded 100%. andSOS SOS (NanoDrop 2000, Thermo Scientific), yielding 90% for SOS (GE Healthcare). The degree of labeling was columns determinedPD-10 with byremoved fluorophoreswere UnreactedUV-vis °C. 23 spectroscopyat h 2 for by reaction of 1:10 molar ratio of unlabeled protein with ATTOProtein 647N–maleimidelabeling and benchmarking. mation for commercial antibodies is available on the manufacturers’ websites. Cy5-labeled streptavidin was from Life Technologies (43-4316). Validationand (SAB3700240), SigmaBiotinylated frompurchasedanti-chicken wasIgM infor purchased from Sigma-Aldrich, and GDP was purchased from MP Biomedicals. (nonhydrolyzable analog of GTP were purchased from Jena Bioscience. GTP wasguanosine diphosphate (EDA–GDP–ATTO 488) and EDA–GppNp–ATTO 488 werepurchased from Invitrogen. ATTO 647N–maleimide, ATTO 488–labeled Lipids were purchased from Avanti. TR-DHPE,SOS1 were expressed inBODIPY-GDP, and BODIPY-GTP (residues 1–1049), and SOS tions: C838A, C635A, C980S, and E718C), SOS termed Ras herein), SOS bilayer,the couplingtofor used C181 positionat cysteine single a with 1–181 reagents.Proteinsand ONLINE nature structural & molecular biology molecular & structural nature Bilayers for live-cell experiments were prepared as described above, with a lipid Immediately before microscopy, samples were brought to room tempera room to brought were samples microscopy, before Immediately Forloading of fluorescent nucleotide onto samples Ras, were equilibrated at SLBs were formed by incubation of the SUV suspension for 30 min on cleaned always is caution and behavior,protein alter potentially can labeling Dye DPC , 106% for SOS for106% , Supplementary Fig. 1c HDPCR552G

METHODS µ M fluorescent nucleotide analog in reaction buffer (40 mM (40 buffer reaction in analognucleotidefluorescent M contained multiple cysteines,thus explaining whylabeling HDPC H-Ras Cat Escherichia coli Cys-lite (residues 566–1049 with the following muta HDPC R552G , and 118% for SOS for 118% and , C118S C181 C118S ). SOS constructs were fluorescently labeled (residues 1–1049 with R552G) of human and purified as previously described (H-Ras construct containingresiduesconstruct (H-Ras DPC β -mercaptoethanol in PBS (5 mM) n vitro in HDPC R552G HDPC (residues 198–1049), SOS in vitro 22 2 flow (20 min). Small uni , 3 8 assays. . Lipids dissolved in in dissolved Lipids . activity of SOS in our Cat . SOS . Cys-lite, 119% for Ras-decorated Ras-decorated DPC 2 , , and 1 mM , SOS , HDPC HDPC 2 2 ------.

the analysis (i.e., SOS bound to defects in the bilayer). This is a crucial aspect of Theten images of SOSat each position allowed usto discard immobile SOSin avoidbleaching. to chamber flow the in positiondifferent a imaged we point, fluorescent nucleotide on Ras and ten images of the labeled SOS. For each time kinetics were quantified at different time points by acquisition of an image of the of nucleotide (120 instead of from pulse the plateau SOS (data in the during binding peak observed the from inferred was binding transientmembraneamanner ( engagedthe in SOS absencethebilayerof nucleotide.intothefree Ras via For experiments,Y64A We experimentally confirmed that SOS in our system ( probabilitywas recruitment indeed the stablyinfer tetheredto used and level nucleotide) free of absence the in Ras SOS molecules remaining on the bilayer after the pulse (due100 nM and flowed overto the bilayer captureas a transient pulse. The number byof labeled catalytic structs were mixed at the desired ratio (typically 1:20) at a total concentration of Stopped-flowsupportedlipid bilayer assay. followed by copious washing with PBS. chicken IgM (62 nM; SAB3700240, Sigma) for 30 min. Each incubation step was streptavidin (18.8 nM) for 30 min and was then incubated with biotinylated suspensionanti- (1 mg/mL) for 30 min. The sample was then treated with Cy5-labeledin a microscopy chamber (A-7816, Life Technologies) was incubated with SUV remainderEgg-PC. Apiranha-etched glass slide (1, Fisher Scientific) mounted composition of 5% DOPS, 0.1% biotinyl cap PE, 0.005% TxRed-DHPE, and the and 0.1% BSA. previouslydescribed as performed also weretechniques previouslyoutas described werecarried nance, plasmid transfection, and stimulation BCR of chicken DT40 B cell lines Maintenance and transfection of DT40 and Jurkat cell lines. ( ( ver ( chase the of seconds few first the during species desorbingrapidlyof fraction SOS For chase. nucleotide the of initiation the at observed ( structs RasGTP ( displayingmembraneson period prolongedresidency a as well probabilityas sensitivity to the nucleotide state of Ras with consistently increased recruitment Ras and SOS came from the observation that all SOS constructs tested exhibited that are laterally mobile. SOS through single-molecule tracking, we were able to focus entirely on species proteinbilayercountingadheretoByconstructsto defects. membrane-bound various of tendencies differential possible as well as defects of number the in the experimental design because it avoids bias from sample-to-sample variation 2.5 mM KCl, 0.35 mM Na free RPMI at 37 °C. Cells were imaged in pH 7.40, 10 mM HEPES, 68 mM NaCl, 5-min centrifugation at 500 from cell culture medium to 1 mL of serum-free RPMI by pelleting cells through imaging. Live-cell blotting with anti-GFP antibody (Cell Signaling, 2956) ( (SRGGR)between SOS1 and EGFP CDS. Expression was confirmed by western (Clontech).resultingThe SOS1-EGFP construct five–aminobearsa acidlinker PCR-amplifiedwith Xba I- and Not I-flanked primers from pEGFP-N1 plasmid ACCC and were maintained according to the provided guidelines. ment shown in flow cytometry and by BCR-induced pERK2 measurement similar to the experi by confirmed was receptor(BCR) B-cell expressionauthentication,of surface confirmed to be free of mycoplasma contamination. For routine cell functional SOS1 B cells were generated in T. Kurosaki’s laboratory (RIKEN). Both wild-type and Fig. 2 Fig. 3 The nucleotide-exchange reaction was initiated by providing a continuous flow For specific comparison of desorption for successfully activated SOS con SOS activated successfully for desorption of comparison specific For between interactions specific probed assay the that demonstration clear A Togenerate EGFP-tagged hSOS1 variants, EGFP coding sequence (CDS) was Supplementary Fig. 1d Fig. Supplementary − c 2 c ). This fast-desorbing fraction did not contribute to processive Ras turno and − Fig. 3 Fig. DT40 B cells were gifts from T. Kurosaki. The obtained cell lines were Fig. 2 Supplementary Fig. 3b c Supplementary Figure 4 d ), traces were normalized to the SOS count at the membrane the at count SOS the to normalized were traces ), and µ For live-cell microscopy, 2.5 million cells were exchanged were cells million microscopy,2.5 live-cell For M GDP or GTP). SOS desorption and nucleotide-exchange Supplementary Fig. 3a 2 HPO g ; this was followed by 30-min incubation in serum- ), and for the comparisons with other constructs other with comparisons the for and ), 4 , 3 mM Fig. 2 , c ), we cropped the first 10 s of the trace. 30 , d 4 4 . Jurkat T cells were obtained from the d 0 6 -glucose, 1 mM CaCl was counted at the single-molecule the atcounted was . Jurkatculturetransfection. andcell ). – Labeled and unlabeled SOS conandSOSunlabeled Labeled c ). Fig. 2 Fig. Supplementary Note 7 Note Supplementary Supplementary Fig. 4a doi: 8 . The SOS1 The . c 10.1038/nsmb.3275 ), and the extentofthe and ), Cat Culture mainte , we observed a observed we , 2 , 2 mM MgCl − 2 − DT40 ). 2 ). - - - - - ,

© 2016 Nature America, Inc. All rights reserved. ACCC. In brief, cells grown on cover slips were transfected with either the the either pCGT-T7-SOS with transfected were slips cover on grown cells brief, In ACCC. culturedandtreated previouslyas described transfections cell immunofluorescenceand COS1 staining. ferent expression levels. acquired events were disregarded for fair comparison of SOS1 variants with dif 100 than fewer with Subsetsexpression level. SOS their accordingto intervals equal of bins nine into sorted were cells activation, ERK of analysisFor ware. FlowJosoft FACSCaliburwith analyzed a and with point (BD) machine time IgG conjugated with APC (Jackson Immunochemicals, 711-136-152). Signaling, 9101). pERK was visualized by secondary staining with goatthree anti-rabbittimes with FACS wash buffer and stained for permeabilizedpERK with withprechilled 90% rabbitmethanol overnight.antisera Cells were (Cellthen washed subsequently EDTA)and mM 10 and BSA, 1% (PBS, buffer FACSwash with were fixed for 20 min at room temperature. Fixed cells were washed three times Stimulation was then stopped by addition of 4% paraformaldehyde-PBS,mouse IgM (clone M4, Southernand Biotech, 8300-01)cells for the desired time period. to established procedures cellular staining of BCR-induced ERK phosphorylation was performed according determined for each fraction. was CD69 of fluorescence mean geometric The fractions. nine into subgated were cells GFP-positive measurements. intensity GFP with together 555531) way was measured by FACS staining of surface CD69 (sCD69, BD Pharmingen, plasmid together with 10 for 20 h with 10 analysis. data and cytometry Flow the microscope chamber. micrographsRICMandbrightfield werenumberacquiredpositionsof ata in after cells were added to the chamber, 488-nm and 640-nm TIRF, together with ~1–5 min to follow the kinetics of the signaling every reaction. acquired Approximatelywere images TIRF 30cells, min selected few a For SLBs. on antibody BCR-engagedfor used was channelSOS-EGFP, 640-nm for the used and was channel 488-nm The cells. of additionbefore °C 37 to heated wasbilayer The SAB3700240). (Sigma, anti-BCR with functionalized SLBs to cells of addition heater(ChamlideTechnology).TC-A,Quorum Experimentswere initiated by doi: ForFACS acquisition, minimuma of 100,000events were collectedfor each For quantitative and qualitative assays of the RAS-ERK signal module, intra Live-cell imaging was performed with a stage-top incubator and an objective 10.1038/nsmb.3275 Cat µ g of wild-type or allosteric mutant (W729E) SOS r SOS or 4 µ 6 . In brief, cells were stimulated with BCR cross-linking Cat-L687ER688A g of GFP plasmid. The activity of the Ras-ERK path Jurkat T cells were transientlytransfectedwere cells JurkatT osrc tgte wt GFP-tagged with together construct 2 2 . COS1 cellswereCOS1 obtained. from COS1 cells werecells COS1 Cat -encoding ­ - - -

Andor). The microscope was controlled with theobjective mode, and images were collected on an EMCCD (iXon ultra 897, control module (OBIS scientific remote). The TIRF setup was operated through 633-nm lasers (all from the OBIS product line, Coherent) controlled via a laser NA). The microscope had a custom-built laser launch with 488-nm, 561-nm,Technical and Instruments) equipped with a Nikon Apo TIRF 100x oil specifications:ing inverted(Nikon microscope objectivebody EclipseTi (TiHUBC/A), (1.49 was operated with MetaMorph software (Molecular Devices). HQ515/30 and HQ700/75 (Chroma Technology),were respectively.images TIRF The microscope 647-nm and 488- for filters emission Band-pass imaging. TIRF objective the through for used were lasers Crystalaser) (RCL-050-640; 647-nm and Coherent) HP; (Sapphire 488-nm illumination. epifluorescence and an EMCCD camera (Andor iXon 597DU). A NA)mercury (1.49 arcobjective immersion lampoil 100× was TIRF usedApo Nikon for a with microscope inverted Ti Eclipse Nikon a on performed was microscopy fluorescence tion platforms. microscopy Optical Axiovert 200M microscope. Zeiss a on conducted was Imaging Invitrogen. from obtained was EGF protein.GFP-fusion a as expressed was protein Rab5 R-6393). (Cappel, antibody rhodamine-conjugatedanti-mouse by followed AB3790), Millipore, Expressed SOS proteins were visualized by staining withTriton (v/v)X-100. anti-T70.1% with antibodypermeabilized formaldehydeand (v/v) (EMD3.7% in H-Ras 62. 61. data analysis procedures that can be implemented in a given coding language. Code availability. imaging experiments can be found in Data analysis. was controlled with sion objective (1.49 NA) and an EMCCD (Andor iXon3 888), systemand the microscope Confocal microscopy was performed on a custom-built spinning-disk confocal For live-cell experiments, an additional TIRF setup was used with the follow

Greene, A.C. control Computer N. Stuurman, & R. Vale, K., Hoover, N., Amodaj, A., Edelstein, trans-endocytosis of ephrinA1. of trans-endocytosis using microscopes of 5G D38E A59G 6 2 . Briefly, images were captured with a Nikon Apo TIRF 100× oil-immer A detailed description of the data analysis procedures relating to et al. –encoding plasmid. After 24 h, transfected cells were fixed fixed were cells transfected h, 24 After plasmid. –encoding

Supplementary Note 7 Spatial organization of EphA2 at the cell-cell interface modulates µ Manager µ nature structural & molecular biology molecular & structural nature Manager. 6 1 Biophys. J. Biophys. . The axial slice step size was 0.5 pfursec ad oa itra reflec internal total and Epifluorescence Curr. Protoc. Mol. Biol. Mol. Protoc. Curr. Supplementary Note 7 provides a detailed description of the

106 µ Manager , 2196–2205 (2014). 2196–2205 ,

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