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One-way membrane trafficking of SOS in receptor-triggered Ras activation

Nature Structural & Molecular Biology volume 23, pages 838–846 (2016)Cite this article

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Abstract

SOS is a key activator of the small GTPase Ras. In cells, SOS-Ras signaling is thought to be initiated predominantly by membrane recruitment of SOS via the adaptor Grb2 and balanced by rapidly reversible Grb2-SOS binding kinetics. However, SOS has multiple protein and lipid interactions that provide linkage to the membrane. In reconstituted-membrane experiments, these Grb2-independent interactions were sufficient to retain human SOS on the membrane for many minutes, during which a single SOS molecule could processively activate thousands of Ras molecules. These observations raised questions concerning how receptors maintain control of SOS in cells and how membrane-recruited SOS is ultimately released. We addressed these questions in quantitative assays of reconstituted SOS-deficient chicken B-cell signaling systems combined with single-molecule measurements in supported membranes. These studies revealed an essentially one-way trafficking process in which membrane-recruited SOS remains trapped on the membrane and continuously activates Ras until being actively removed via endocytosis.

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Figure 1: The catalytic core of SOS is stably and functionally recruited to Ras-decorated SLBs in vitro, independently of Grb2 and lipid-binding domains.
Figure 2: Stopped-flow SLB assay probing SOS recruitment and desorption.
Figure 3: The N terminus of SOS suppresses bilayer recruitment while prolonging dwell time in the active membrane-bound state.
Figure 4: Multiparameter assay of SOS-RAS-ERK pathway activity reveals the functional importance of SOS flanking domains in a cell-signaling context.
Figure 5: SOSCat flanking domains block spontaneous activation in the basal state but promote RAS-ERK signal transduction after receptor stimulation.
Figure 6: PR-domain-dependent localization of SOS to BCR microclusters and SOS depletion from the central BCR cluster formed between B cells and SLBs decorated with BCR-cross-linking antibody.
Figure 7: PR-domain-dependent SOS endocytosis mediates signal attenuation.

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Acknowledgements

The authors thank the members of the laboratories of J.P.R., J.T.G., J. Kuriyan, and D.B.-S. for helpful comments and suggestions. The authors thank J. Kuriyan for insightful comments on the manuscript. In addition, the authors thank W.-C. Lin and L. Iversen for assistance with initial SLB experiments. This research was supported by a P01 Program grant from NIH-NIAID (AI091580 – to J.P.R. and J.T.G.). Further support came from R01-CA187318 NIH-NCI and R01-AI104789 (both to J.P.R.) and ARRA stimulus supplement GM078266 (to D.B.-S. and K.K.Y.) as well as a grant from the Danish Council for Independent Research, Natural Sciences (to S.M.C.). We thank T. Kurosaki (RIKEN) for providing wild-type and SOS1−2− DT40 B cells.

Author information

Author notes

  1. Sune M Christensen, Hsiung-Lin Tu & Kamlesh K Yadav

    Present address: Present addresses: Novozymes A/S, Bagsvaerd, Denmark (S.M.C.), Institute for Molecular Engineering, University of Chicago, Chicago, Illinois, USA (H.-L.T.) and Department of Urology, Icahn School of Medicine at Mount Sinai, New York, New York, USA (K.K.Y.).,

  2. Sune M Christensen, Hsiung-Lin Tu and Jesse E Jun: These authors contributed equally to this work.

  3. Jeroen P Roose and Jay T Groves: These authors jointly supervised this work.

Authors and Affiliations

  1. Department of Chemistry, University of California, Berkeley, Berkeley, California, USA.,

    Sune M Christensen, Hsiung-Lin Tu, Steven Alvarez, Meredith G Triplet & Jay T Groves

  2. Department of Anatomy, University of California, San Francisco, San Francisco, California, USA.,

    Jesse E Jun & Jeroen P Roose

  3. Department of Molecular and Cell Biology, Howard Hughes Medical Institute, University of California, Berkeley, Berkeley, California, USA.,

    Jeffrey S Iwig

  4. Department of Biochemistry, New York University School of Medicine, New York, New York, USA

    Kamlesh K Yadav & Dafna Bar-Sagi

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Contributions

S.M.C., H.-L.T., and J.E.J. performed experiments and analyzed data. S.A. and M.G.T. assisted with live-cell experiments. J.S.I. purified proteins. K.K.Y. performed COS1 cell experiments under supervision of D.B.-S. J.T.G., J.P.R., S.M.C., H.-L.T., and J.E.J. conceptualized and designed experiments. S.M.C., H.-L.T., J.E.J., J.P.R., and J.T.G. wrote the paper. J.T.G. and J.P.R. supervised the project. All authors discussed and commented on the results.

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Correspondence to Jeroen P Roose or Jay T Groves.

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Integrated supplementary information

Supplementary Figure 1 Quantification of two-dimensional Ras fluidity and surface density on SLBs and benchmarking of labeled SOS constructs.

(a) Fluorescence recovery after photobleaching (FRAP) experiment showing fluorescence from nucleotide loaded Ras at 0 s and 120s s after bleaching a circular region of a supported lipid bilayer. The recovery of the bleached signal demonstrates that Ras is laterally mobile at the membrane. The bottom panel shows the corresponding line-profiles of the fluorescence intensity across the bleached spot as indicated. FRAP analysis to verify lateral fluidity of the membrane was carried out for every supported lipid bilayer (SLB) sample employed in this work.

(b) Calibration curve to convert intensity extracted from microscopy images to Ras density. The curve was constructed from FCS measurements of Ras density (via the BODIPY labeled nucleotide) at a number of supported lipid bilayers combined with EPI imaging (plot ordinate) of the same sample positions.

(c) Labeling SOS with ATTO-647N does not perturb its enzymatic activity in the stopped-flow assay. The activities of the shown SOS constructs were benchmarked by performing stopped flow nucleotide exchange assays on Ras functionalized supported lipid bilayers with comparable Ras densities and using 100 nM of the respective SOS construct. For SOSCat, DPC and HDPC no significant alteration of the activity was observed upon labeling. The data indicates a slightly decreased activity for the labeled HDPC(R552G) construct. Of note, the minor decrease in activity for labeled HDPC(R552G) would not affect the conclusions on the data presented in Figure 3b,c. The activity for each construct was evaluated by fitting the fluorescence decay (see caption for d below) in the chase phase of the stopped flow assay with a monoexponential decay function. The decay rate observed for the respective unlabeled construct was set to 1 and the rate for the corresponding labeled variant was accordingly normalized. The bars represent the fitted decay coefficients (one trace for each condition) except for SOSCat where the bars indicate the average of two such measurements. Error bars indicate the estimated standard deviations of the fit coefficients.

(d) Processive turnover of Ras by SOSCat in the stopped flow assay. Shown intensity traces were collected immediately after the initiation of the chase phase of the stopped flow assay where unlabeled nucleotide is flowed through the reaction chamber (GDP or GTP, as indicated, see also Figure 2a, right). The shown traces were collected after first populating the bilayers with SOSCat and washing away unbound SOS molecules. It is evident that the recruited SOSCat is highly processive at the membrane surface, as evidenced by sustained Ras turnover throughout the nucleotide chase (resulting in decline of fluorescence as labeled nucleotides are exchanged for unlabeled nucleotides).

Source data for plots and graphs are available online.

Source data

Supplementary Figure 2 Analysis of surface CD69 expression in transfected Jurkat T cells and spontaneous localization of SOSCat to the membrane in resting COS-1 cells.

(a) Analysis of Ras-signal dependent CD69 expression in transfected Jurkat T cells. The indicated SOSCat construct or empty vector were cotransfected with a GFP-expression construct and 20 hours later CD69 expression was assessed in unstimulated Jurkat T Cells. Surface CD69 (sCD69) MFI’s (mean fluorescence intensity) are plotted as a function of GFP expression level. Source data available online.

(b) Raw data for the panel in a. Jurkat T cells were transiently transfected for 20 hours with 10 µg of wild-type or allosteric mutant (W729E) SOSCat- encoding plasmid together with 10 µg of GFP plasmid. The activity of Ras-ERK pathway was measured by FACS staining of surface CD69 (sCD69) together with GFP intensity measurement. GFP-positive cells were sub-gated into 9 fractions. Geometric mean fluorescence of CD69 level was determined for each fraction. Data shown in a and b are representative of five independent experiments.

(c) Analysis of SOSCat (red) and H-Ras(A59G D38E) (green) localization in unstimulated, transfected COS-1 cells. The panels to the right show data for a mutant impaired in allosteric Ras binding (SOSCat-L687E R688A). H-Ras(A59G D38E) is a mutant that only binds the allosteric site on SOS. The data show that spontaneous membrane-localization of SOSCat requires an intact allosteric pocket. Data in figure 2c are a reproduction of previous published work (Gureasko, J. et al., Nat. Struct. Mol. Biol. 15, 452-61, 2008).

(d) Model of SOSCat membrane recruitment via its allosteric pocket.

(e) HDPC recruitment, as measured in the stopped-flow assay, with and without Ras at the membrane. Relative recruitment was calculated by normalizing the SOS counts at the membrane to the concentration of SOS injected during the pulse. Data, as well as averages (red lines) for two SLB samples for each condition are shown. Note that other constructs in this study are recruited at least six times (in the case of HDPC(R552G)) more avidly compared to HDPC (Fig. 3b) and in this regard the control establish a baseline for the reported observations. Source data available online.

Source data

Supplementary Figure 3 Additional SOS recruitment data.

(a) Recruitment probability for DPC, HDPC and HDPC(R552G) on bilayers containing RasGDP or RasGTP. RasGDP data are replotted from Figure 3b for comparison. Note the generally increased recruitment probabilities across the panel of SOS constructs for binding to RasGTP. Each bar represents the average of data collected at N SLB samples, for RasGDP;RasGTP: DPC N=3;N=3, HDPC N=2;N=2, HDPC(R552G) N=3;N=3. Each sample was imaged at least at 15 different positions. Error bars represent SEM. For HDPC the individual data points from the two samples are shown instead of an average.

(b) Desorption traces of SOS constructs on RasGDP displaying bilayers. The shown traces represent averages over N SLB samples: SOSCat N=5, DPC N=4, HDPC N=2, HDPC(R552G) N=3. The data were fitted (solid lines) in accordance with a monoexponential decay process to extract the mean residency period for each construct (data presented in main Fig. 3c). In the case of SOSCat we observed a fraction of rapidly desorbing species during the first few seconds of the chase (Fig. 2c). This fast desorbing fraction was not contributing substantially to processive Ras turnover (Supplementary Fig. 1d) and for the comparison with other constructs shown here we cropped the first 10 s of the trace.

(c) Desorption traces of SOS constructs on RasGTP displaying bilayers. The shown traces represent averages over N SLB samples: SOSCat N=3, DPC N=2, HDPC N=2, HDPC(R552G) N=3. Same data format as in b. Note the generally slower desorption kinetics when SOS is bound via RasGTP (compare with RasGDP data presented in b).

(d) The single molecule SOS activity assay reported in Iversen et al. (Iversen, L. et al., Science. 345, 50-4, 2014) was adapted with the modification that here SOS was continuously incubated over the bilayers during imaging (as opposed to populating bilayers with SOS by providing a transient pulse through the flow cell). SOSHDPC (unlabeled) was incubated at a Ras-functionalized patterned supported lipid bilayer (3x3 µm corrals) at 12.5 pM. Ras was initially loaded with fluorescent nucleotide analog. The nucleotide label was imaged before and after incubation with SOS. After incubation a few corrals exhibited a depleted nucleotide signal relative to the neighboring corrals, indicating that a SOS molecule got activated at these particular sites of the membrane.

(e) The same experiment as in d, but with fluorescently labeled SOSDPC at 100 pM and dual imaging of the SOS and nucleotide labels. The 3 panels display time-traces of SOS and nucleotide on Ras for selected corrals. Step increases in the SOS channel signifies stable association of a SOS molecule with the membrane in the particular corral. As expected, arrival of SOS is followed by depletion of the initially labeled nucleotides on Ras as these are exchanged for unlabeled counterparts. In trace 1 and 3 the single dye label on SOS undergoes step photobleaching. The data illustrates that robust Ras activation requires stable association of SOS with the membrane and that catalysis takes place in a highly processive manner.

Source data

Supplementary Figure 4 Multiparameter p-FLOW assay of the Ras-ERK pathway.

(a) Western blot for GFP confirming correct expression size of EGFP fusion SOS1 variants.

(b) Transfected cells expressing intermediary level of SOS1-EGFP exhibit signaling kinetics comparable to wild-type DT40 cells. BCR-induced fold increase in mean fluorescence intensity (MFI) of p-ERK relative to resting level (t= 0 min) is plotted for wild-type DT40 and full-length hSOS1-transfected SOS1-2- DT40 cells. Alternating subsets out of a total of 9 bins are compared to whole population of WT DT40 cells. Each data point represents four independent experiments (mean +/- SEM).

(c) SOS-EGFP can rescue ERK activation defect in SOS1-2- DT40. Representative scattered (left most column) and gated FACS data (right columns) at different time points after M4 stimulation are shown. Note that gate 9 has higher noise due to fewer acquired events (< 100 events).

(d) Two dimensional representation of p-ERK versus hSOS1-EGFP intensity across various time points after BCR stimulation comparing SOSFL (top row) and SOSCat (bottom row).

Source data

Supplementary Figure 5 SOSCat flanking domains block spontaneous activation in the basal state but promote Ras-ERK signal transduction after receptor stimulation.

(a,b) Incremental suppression by additional N-terminal domains (see also Fig. 5a-c).

(c,d) Incremental suppression by additional N-terminal domains is consistent to similar variants lacking PR domain. The ratio of pERK MFI for longer to shorter SOS variants is plotted against increasing SOS concentration for unstimulated cells (basal state). Addition of SOSCat- flanking domains inhibits spontaneous activation of ERK: DH-PH domain (c) and HF domain (d) (see also Fig. 5a-c)

(e-g) Time dependence of pERK MFI ratio after BCR stimulation is plotted for indicated constructs: Full length with a truncated HF domain (f) and full length with a truncated C-terminal PR domain (g). In each structural diagram, truncated domains are greyed out. p-FLOW assay data in this figure were obtained from BCR stimulated cells with intermediate SOS expression level as indicated in e.

All shown data are based on three independent cell cultures and pFLOW experiments. Error bars represent SEM. Yellow plane on the cubes in a and e indicates the corresponding subspace of the 3D parameter space in the pFLOW assay (c.f., Fig. 4c,d). Δ highlights the difference between the compared constructs.

Source data

Supplementary Figure 6 Membrane-targeting lipid modification of full-length (FL) SOS1 allows spontaneous activation of ERK and prolonged response after BCR stimulation.

(a) Farnesylation tag engineered to FL WT hSOS1 (SOS1-F) and point-mutated inactive control (SOS1-F*).

(b) Western blotting shows mobility shift by lipid modification.

(c) Ligand-independent activation of pERK. To note, membrane-anchored full-length SOS1 signals to ERK independent of BCR stimulation as observed on SOSCat-expressing cells (see also, Fig 4e,h)

(d) Time trace of p-ERK response upon BCR stimulation at intermediary SOS1-expressing cells.

BCR-induced ERK activity is prolonged compared to unmodified SOS1 or inactive SOS1-F*.

Shown data are representative of two independent cell cultures and pFLOW experiments. Source data are available online.

Source data

Supplementary Figure 7 Additional hybrid live-cell SLB assay data and replotted overlays.

(a) The anti-BCR antibody was coupled to the bilayer via Cy5-labeled streptavidin thus providing a readout for the location of activated BCR microclusters whereas SOS was visualized via EGFP tagging.

(b) Representative microscopy images collected from live cell experiments for various SOS constructs. Bright field (top row), RICM (second row), EGFP (third row), and Cy5 (bottom row) channels of cells expressing different SOS-EGFP constructs (one cell for each column). The images were taken 20-40 minutes after cell landing and spread on the antibody-functionalized supported lipid bilayers. Information on number of samples for each construct is given in Figure 6b. Bar: 3 µm.

(c) Overlay micrographs from Figure 6c replotted as separate image channels. Bar: 3 µm.

(d) Overlay from the inset in Figure 7d replotted as separate image channels.

Supplementary Figure 8 Monomeric SH2 fusion of HDPC restores ERK activation at later time points, and interaction between SOS1 and Grb2 is prolonged during antigen-receptor-induced Ras-ERK activation and human cancer– and Noonan syndrome–associated alterations of SOS1.

(a,b) Time dependence of pERK MFI ratio after BCR stimulation is plotted for indicated constructs: Single SH2 domain from human GrRB2 was fused to C-terminus of HDPC (HDPC-SH2). p-FLOW assay data in this figure were obtained from BCR stimulated cells with intermediate SOS expression level. Shown data are based on four independent cell cultures and pFLOW experiments. Error bars represent SEM. Source data are available online.

(c,d) Mouse CD4+ T cell blasts and hSOS1 stably transfected DT40 B cells were stimulated with antigen receptor crosslinking antibodies for the indicated time. SOS1 was immunoprecipitated from resulting lysates. Immunoprecipitated SOS1 and associated Grb2 were visualized by Western blotting. Phosphorylated ERK was used to indicate the status of Ras-ERK pathway. For panel (d), the percentage to max pERK2 at the indicated time is calculated after normalization to total ERK2 amount. Shown results are representative of two independent replicate experiments of each cell type.

(e) Human cancer- and Noonan-syndrome associated alterations of SOS1. Each bar represents a SOS alteration detected in patients. Green bars indicate missense point mutations. Alterations disrupting the C-terminus are indicated with red bars (fs: frame shift, spl: aberrant splicing, *: STOP codon,). Yellow boxes indicate PxxP motifs in C-terminal interacting with Grb2. Modified from cBioPortal for Cancer Genomics Database (Gao, J. et al, Sci. Signal., 6, p. l1, 2013).

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Supplementary Figures 1–8 and Supplementary Notes 1–7 (PDF 2541 kb)

SOS-EGFP expressing B cell interacting with SLB presenting cross-linking antibody for the BCR

Time-lapse movie showing trajectories of BCR (red) and SOSFL (green) movement at the cell-bilayer interface. (AVI 284 kb)

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Christensen, S., Tu, HL., Jun, J. et al. One-way membrane trafficking of SOS in receptor-triggered Ras activation. Nat Struct Mol Biol 23, 838–846 (2016). https://doi.org/10.1038/nsmb.3275

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