Mechanism of SOS PR-domain autoinhibition revealed by single-molecule assays on native protein from lysate

Lee, Young Kwang; Low-Nam, Shalini T.; Chung, Jean K.; Hansen, Scott D.; Lam, Hiu Yue Monatrice; Alvarez, Steven; Groves, Jay T.

doi:10.1038/ncomms15061

Title: Mechanism of SOS PR-domain autoinhibition revealed by single-molecule assays on native protein from lysate

Journal Article · Sat Apr 01 00:00:00 EDT 2017 · Nature Communications

DOI:https://doi.org/10.1038/ncomms15061· OSTI ID:1623887

Lee, Young Kwang ^[1]; Low-Nam, Shalini T. ^[2]; Chung, Jean K. ^[2]; Hansen, Scott D. ^[2]; Lam, Hiu Yue Monatrice ^[2]; Alvarez, Steven ^[2]; Groves, Jay T. ^[1]

Univ. of California, Berkeley, CA (United States). Dept. of Chemistry; Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Molecular Biophysics and Integrated Bioimaging Div.
Univ. of California, Berkeley, CA (United States). Dept. of Chemistry

The guanine nucleotide exchange factor (GEF) Son of Sevenless (SOS) plays a critical role in signal transduction by activating Ras. Here we introduce a single-molecule assay in which individual SOS molecules are captured from raw cell lysate using Ras-functionalized supported membrane microarrays. This enables characterization of the full-length SOS protein, which has not previously been studied in reconstitution due to difficulties in purification. Our measurements on the full-length protein reveal a distinct role of the C-terminal proline-rich (PR) domain to obstruct the engagement of allosteric Ras independently of the well-known N-terminal domain autoinhibition. This inhibitory role of the PR domain limits Grb2-independent recruitment of SOS to the membrane through binding of Ras·GTP in the SOS allosteric binding site. More generally, this assay strategy enables characterization of the functional behaviour of GEFs with single-molecule precision but without the need for purification.

View Accepted Manuscript (DOE)

Cite

Export

Save

Research Organization:: Lawrence Berkeley National Lab (LBNL), Berkeley, CA (United States)

Sponsoring Organization:: USDOE Office of Science (SC); National Institutes of Health (NIH) National Cancer Institute (NCI)

Grant/Contract Number:: AC02-05CH11231; U01CA202241

OSTI ID:: 1623887

Journal Information:: Nature Communications, Vol. 8, Issue 1; ISSN 2041-1723

Publisher:: Nature Publishing GroupCopyright Statement

Country of Publication:: United States

Language:: English

References (63)

Allosteric gating of Son of sevenless activity by the histone domain Yadav, Kamlesh K.; Bar-Sagi, Dafna Proceedings of the National Academy of Sciences, Vol. 107, Issue 8 https://doi.org/10.1073/pnas.0914315107	journal	February 2010
Monitoring the Waiting Time Sequence of Single Ras GTPase Activation Events Using Liposome Functionalized Zero-Mode Waveguides Christensen, Sune M.; Triplet, Meredith G.; Rhodes, Christopher Nano Letters, Vol. 16, Issue 4 https://doi.org/10.1021/acs.nanolett.6b00969	journal	March 2016
Structural Analysis of Autoinhibition in the Ras Activator Son of Sevenless Sondermann, Holger; Soisson, Stephen M.; Boykevisch, Sean Cell, Vol. 119, Issue 3 https://doi.org/10.1016/j.cell.2004.10.005	journal	October 2004
Grb2 mediates the EGF-dependent activation of guanine nucleotide exchange on Ras Gale, Nicholas W.; Kaplan, Susan; Lowenstein, Eve J. Nature, Vol. 363, Issue 6424 https://doi.org/10.1038/363088a0	journal	May 1993
Electrofluidic Lipid Membrane Biosensor Lee, Young Kwang; Nam, Jwa-Min Small, Vol. 8, Issue 6 https://doi.org/10.1002/smll.201102093	journal	January 2012
Ras regulation and function in lymphocytes Genot, Elisabeth; Cantrell, Doreen A. Current Opinion in Immunology, Vol. 12, Issue 3 https://doi.org/10.1016/s0952-7915(00)00089-3	journal	June 2000
Regulation of Epidermal Growth Factor Receptor Signaling by Phosphorylation of the Ras Exchange Factor hSOS1 Porfiri, Emilio; McCormick, Frank Journal of Biological Chemistry, Vol. 271, Issue 10 https://doi.org/10.1074/jbc.271.10.5871	journal	March 1996
Epidermal growth factor regulates the exchange rate of guanine nucleotides on p21ras in fibroblasts. Buday, L.; Downward, J. Molecular and Cellular Biology, Vol. 13, Issue 3 https://doi.org/10.1128/MCB.13.3.1903	journal	March 1993
Subunit counting in membrane-bound proteins Ulbrich, Maximilian H.; Isacoff, Ehud Y. Nature Methods, Vol. 4, Issue 4 https://doi.org/10.1038/nmeth1024	journal	March 2007
N Terminus of Sos1 Ras Exchange Factor: Critical Roles for the Dbl and Pleckstrin Homology Domains Qian, Xiaolan; Vass, William C.; Papageorge, Alex G. Molecular and Cellular Biology, Vol. 18, Issue 2 https://doi.org/10.1128/mcb.18.2.771	journal	February 1998
Targeted Sos1 deletion reveals its critical role in early T-cell development Kortum, R. L.; Sommers, C. L.; Alexander, C. P. Proceedings of the National Academy of Sciences, Vol. 108, Issue 30 https://doi.org/10.1073/pnas.1104295108	journal	July 2011
Dark-Field-Based Observation of Single-Nanoparticle Dynamics on a Supported Lipid Bilayer for In Situ Analysis of Interacting Molecules and Nanoparticles Lee, Young Kwang; Kim, Sungi; Nam, Jwa-Min ChemPhysChem, Vol. 16, Issue 1 https://doi.org/10.1002/cphc.201402529	journal	October 2014
Molecular mechanisms in signal transduction at the membrane Groves, Jay T.; Kuriyan, John Nature Structural & Molecular Biology, Vol. 17, Issue 6 https://doi.org/10.1038/nsmb.1844	journal	May 2010
Germ Line Gain of Function with SOS1 Mutation in Hereditary Gingival Fibromatosis Jang, Shyh-Ing; Lee, Eun-Jin; Hart, P. Suzanne Journal of Biological Chemistry, Vol. 282, Issue 28 https://doi.org/10.1074/jbc.m701609200	journal	May 2007
In vivo functional analysis of the Ras exchange factor son of sevenless Karlovich, C.; Bonfini, L.; McCollam, L. Science, Vol. 268, Issue 5210 https://doi.org/10.1126/science.7725106	journal	April 1995
GEFs and GAPs: Critical Elements in the Control of Small G Proteins Bos, Johannes L.; Rehmann, Holger; Wittinghofer, Alfred Cell, Vol. 129, Issue 5 https://doi.org/10.1016/j.cell.2007.05.018	journal	June 2007
Using patterned supported lipid membranes to investigate the role of receptor organization in intercellular signaling Nair, Pradeep M.; Salaita, Khalid; Petit, Rebecca S. Nature Protocols, Vol. 6, Issue 4 https://doi.org/10.1038/nprot.2011.302	journal	March 2011
Single-Molecule Analysis of Lipid–Protein Interactions in Crude Cell Lysates Arauz, Edwin; Aggarwal, Vasudha; Jain, Ankur Analytical Chemistry, Vol. 88, Issue 8 https://doi.org/10.1021/acs.analchem.5b04127	journal	April 2016
Digital Signaling and Hysteresis Characterize Ras Activation in Lymphoid Cells Das, Jayajit; Ho, Mary; Zikherman, Julie Cell, Vol. 136, Issue 2 https://doi.org/10.1016/j.cell.2008.11.051	journal	January 2009
Membrane targeting of the nucleotide exchange factor Sos is sufficient for activating the Ras signaling pathway Aronheim, Ami; Engelberg, David; Li, Nanxin Cell, Vol. 78, Issue 6 https://doi.org/10.1016/0092-8674(94)90271-2	journal	September 1994
Micropatterning Fluid Lipid Bilayers on Solid Supports Groves, J. T. Science, Vol. 275, Issue 5300 https://doi.org/10.1126/science.275.5300.651	journal	January 1997
Structural landscape of the proline-rich domain of Sos1 nucleotide exchange factor McDonald, Caleb B.; Bhat, Vikas; Kurouski, Dmitry Biophysical Chemistry, Vol. 175-176 https://doi.org/10.1016/j.bpc.2013.02.008	journal	May 2013
Sos-mediated cross-activation of wild-type Ras by oncogenic Ras is essential for tumorigenesis Jeng, Hao-Hsuan; Taylor, Laura J.; Bar-Sagi, Dafna Nature Communications, Vol. 3, Issue 1 https://doi.org/10.1038/ncomms2173	journal	January 2012
Tracking single molecules at work in living cells Kusumi, Akihiro; Tsunoyama, Taka A.; Hirosawa, Kohichiro M. Nature Chemical Biology, Vol. 10, Issue 7 https://doi.org/10.1038/nchembio.1558	journal	June 2014
Regulation of Sos Activity by Intramolecular Interactions Corbalan-Garcia, Senena; Margarit, Steluta M.; Galron, Dalia Molecular and Cellular Biology, Vol. 18, Issue 2 https://doi.org/10.1128/mcb.18.2.880	journal	February 1998
One-way membrane trafficking of SOS in receptor-triggered Ras activation Christensen, Sune M.; Tu, Hsiung-Lin; Jun, Jesse E. Nature Structural & Molecular Biology, Vol. 23, Issue 9 https://doi.org/10.1038/nsmb.3275	journal	August 2016
Mammalian Son of Sevenless Guanine Nucleotide Exchange Factors: Old Concepts and New Perspectives Rojas, J. M.; Oliva, J. L.; Santos, E. Genes & Cancer, Vol. 2, Issue 3 https://doi.org/10.1177/1947601911408078	journal	March 2011
Regulation of Small GTPases by GEFs, GAPs, and GDIs Cherfils, Jacqueline; Zeghouf, Mahel Physiological Reviews, Vol. 93, Issue 1 https://doi.org/10.1152/physrev.00003.2012	journal	January 2013
Guanine-nucleotide-releasing factor hSos1 binds to Grb2 and links receptor tyrosine kinases to Ras signalling Li, N.; Batzer, A.; Daly, R. Nature, Vol. 363, Issue 6424 https://doi.org/10.1038/363085a0	journal	May 1993
Membrane-dependent signal integration by the Ras activator Son of sevenless Gureasko, Jodi; Galush, William J.; Boykevisch, Sean Nature Structural & Molecular Biology, Vol. 15, Issue 5 https://doi.org/10.1038/nsmb.1418	journal	May 2008
SOS1 mutations in Noonan syndrome: molecular spectrum, structural insights on pathogenic effects, and genotype-phenotype correlations Lepri, Francesca; De Luca, Alessandro; Stella, Lorenzo Human Mutation, Vol. 32, Issue 7 https://doi.org/10.1002/humu.21492	journal	April 2011
The Grb2 binding domain of mSos1 is not required for downstream signal transduction Wang, Wei; Fisher, Elizabeth M. C.; Jia, Qi Nature Genetics, Vol. 10, Issue 3 https://doi.org/10.1038/ng0795-294	journal	July 1995
Micropattern Formation in Supported Lipid Membranes Groves, Jay T.; Boxer, Steven G. Accounts of Chemical Research, Vol. 35, Issue 3, p. 149-157 https://doi.org/10.1021/ar950039m	journal	March 2002
Abl-dependent tyrosine phosphorylation of Sos-1 mediates growth-factor-induced Rac activation Sini, Patrizia; Cannas, Angela; Koleske, Anthony J. Nature Cell Biology, Vol. 6, Issue 3 https://doi.org/10.1038/ncb1096	journal	February 2004
Regulation of Ras Signaling Dynamics by Sos-Mediated Positive Feedback Boykevisch, Sean; Zhao, Chen; Sondermann, Holger Current Biology, Vol. 16, Issue 21 https://doi.org/10.1016/j.cub.2006.09.033	journal	November 2006
Massively Parallel and Highly Quantitative Single-Particle Analysis on Interactions between Nanoparticles on Supported Lipid Bilayer Lee, Young Kwang; Kim, Sungi; Oh, Jeong-Wook Journal of the American Chemical Society, Vol. 136, Issue 10 https://doi.org/10.1021/ja501225p	journal	February 2014
How is SOS activated? Let us count the ways Findlay, Greg M.; Pawson, Tony Nature Structural & Molecular Biology, Vol. 15, Issue 6 https://doi.org/10.1038/nsmb0608-538	journal	June 2008
H-Ras forms dimers on membrane surfaces via a protein-protein interface Lin, W. -C.; Iversen, L.; Tu, H. -L. Proceedings of the National Academy of Sciences, Vol. 111, Issue 8 https://doi.org/10.1073/pnas.1321155111	journal	February 2014
Role of the histone domain in the autoinhibition and activation of the Ras activator Son of Sevenless Gureasko, Jodi; Kuchment, Olga; Makino, Debora Lika Proceedings of the National Academy of Sciences, Vol. 107, Issue 8 https://doi.org/10.1073/pnas.0913915107	journal	February 2010
Single Cell GFP-Trap Reveals Stoichiometry and Dynamics of Cytosolic Protein Complexes Wedeking, Tim; Löchte, Sara; Richter, Christian P. Nano Letters, Vol. 15, Issue 5 https://doi.org/10.1021/acs.nanolett.5b01153	journal	April 2015
Activation of Extracellular Signal-Regulated Kinase but Not of p38 Mitogen-Activated Protein Kinase Pathways in Lymphocytes Requires Allosteric Activation of SOS Jun, J. E.; Yang, M.; Chen, H. Molecular and Cellular Biology, Vol. 33, Issue 12 https://doi.org/10.1128/mcb.01593-12	journal	April 2013
Gain-of-function SOS1 mutations cause a distinctive form of Noonan syndrome Tartaglia, Marco; Pennacchio, Len A.; Zhao, Chen Nature Genetics, Vol. 39, Issue 1 https://doi.org/10.1038/ng1939	journal	December 2006
Real-time single-molecule co-immunoprecipitation analyses reveal cancer-specific Ras signalling dynamics Lee, Hong-Won; Kyung, Taeyoon; Yoo, Janghyun Nature Communications, Vol. 4, Issue 1 https://doi.org/10.1038/ncomms2507	journal	February 2013
Single Molecule Diffusion of Membrane-Bound Proteins: Window into Lipid Contacts and Bilayer Dynamics Knight, Jefferson D.; Lerner, Michael G.; Marcano-Velázquez, Joan G. Biophysical Journal, Vol. 99, Issue 9 https://doi.org/10.1016/j.bpj.2010.08.046	journal	November 2010
Ras activation by SOS: Allosteric regulation by altered fluctuation dynamics Iversen, Lars; Tu, Hsiung-Lin; Lin, Wan-Chen Science, Vol. 345, Issue 6192 https://doi.org/10.1126/science.1250373	journal	July 2014
Single-molecule pull-down for studying protein interactions Jain, Ankur; Liu, Ruijie; Xiang, Yang K. Nature Protocols, Vol. 7, Issue 3 https://doi.org/10.1038/nprot.2011.452	journal	February 2012
Structural Evidence for Feedback Activation by Ras·GTP of the Ras-Specific Nucleotide Exchange Factor SOS Margarit, S. Mariana; Sondermann, Holger; Hall, Brian E. Cell, Vol. 112, Issue 5 https://doi.org/10.1016/S0092-8674(03)00149-1	journal	March 2003
Unusual Interplay of Two Types of Ras Activators, RasGRP and SOS, Establishes Sensitive and Robust Ras Activation in Lymphocytes Roose, Jeroen P.; Mollenauer, Marianne; Ho, Mary Molecular and Cellular Biology, Vol. 27, Issue 7 https://doi.org/10.1128/mcb.01882-06	journal	February 2007
Single-molecule imaging reveals mechanisms of protein disruption by a DNA translocase Finkelstein, Ilya J.; Visnapuu, Mari-Liis; Greene, Eric C. Nature, Vol. 468, Issue 7326 https://doi.org/10.1038/nature09561	journal	November 2010
Human Sos1: a guanine nucleotide exchange factor for Ras that binds to GRB2 Chardin, P.; Camonis, J.; Gale, N. Science, Vol. 260, Issue 5112 https://doi.org/10.1126/science.8493579	journal	May 1993
Interaction Domains of Sos1/Grb2 Are Finely Tuned for Cooperative Control of Embryonic Stem Cell Fate Findlay, Greg M.; Smith, Matthew J.; Lanner, Fredrik Cell, Vol. 152, Issue 5 https://doi.org/10.1016/j.cell.2013.01.056	journal	February 2013
Grb2 Is a Negative Modulator of the Intrinsic Ras-GEF Activity of hSos1 Zarich, Natasha; Oliva, José Luis; Martínez, Natalia Molecular Biology of the Cell, Vol. 17, Issue 8 https://doi.org/10.1091/mbc.e05-12-1104	journal	August 2006
Mammalian Grb2 Regulates Multiple Steps in Embryonic Development and Malignant Transformation Cheng, Alec M.; Saxton, Tracy M.; Sakai, Ryuichi Cell, Vol. 95, Issue 6 https://doi.org/10.1016/s0092-8674(00)81702-x	journal	December 1998
Why cytoplasmic signalling proteins should be recruited to cell membranes Kholodenko, Boris N.; Hoek, Jan B.; Westerhoff, Hans V. Trends in Cell Biology, Vol. 10, Issue 5 https://doi.org/10.1016/s0962-8924(00)01741-4	journal	May 2000
Integrative Analysis of Complex Cancer Genomics and Clinical Profiles Using the cBioPortal Gao, J.; Aksoy, B. A.; Dogrusoz, U. Science Signaling, Vol. 6, Issue 269 https://doi.org/10.1126/scisignal.2004088	journal	March 2013
How receptors turn Ras on McCormick, Frank Nature, Vol. 363, Issue 6424 https://doi.org/10.1038/363015a0	journal	May 1993
Conformational dynamics of a class C G-protein-coupled receptor Vafabakhsh, Reza; Levitz, Joshua; Isacoff, Ehud Y. Nature, Vol. 524, Issue 7566 https://doi.org/10.1038/nature14679	journal	August 2015
Association of Sos Ras exchange protein with Grb2 is implicated in tyrosine kinase signal transduction and transformation Egan, Sean E.; Giddings, Barton W.; Brooks, Mary W. Nature, Vol. 363, Issue 6424 https://doi.org/10.1038/363045a0	journal	May 1993
The SH2 and SH3 domains of mammalian Grb2 couple the EGF receptor to the Ras activator mSos1 Rozakis-Adcock, Maria; Fernley, Ross; Wade, John Nature, Vol. 363, Issue 6424 https://doi.org/10.1038/363083a0	journal	May 1993
Covalent Ras Dimerization on Membrane Surfaces through Photosensitized Oxidation Chung, Jean K.; Lee, Young Kwang; Lam, Hiu Yue Monatrice Journal of the American Chemical Society, Vol. 138, Issue 6 https://doi.org/10.1021/jacs.5b12648	journal	February 2016
Epidermal Growth Factor Regulates the Exchange Rate of Guanine Nucleotides on p21^ras in Fibroblasts Buday, László; Downward, Julian Molecular and Cellular Biology, Vol. 13, Issue 3 https://doi.org/10.1128/mcb.13.3.1903-1910.1993	journal	March 1993
The structural basis of the activation of Ras by Sos Boriack-Sjodin, P. Ann; Margarit, S. Mariana; Bar-Sagi, Dafna Nature, Vol. 394, Issue 6691 https://doi.org/10.1038/28548	journal	July 1998
A complex of Grb2 adaptor protein, Sos exchange factor, and a 36-kDa membrane-bound tyrosine phosphoprotein is implicated in ras activation in T cells. Buday, L.; Egan, S. E.; Rodriguez Viciana, P. Journal of Biological Chemistry, Vol. 269, Issue 12 https://doi.org/10.1016/S0021-9258(17)37070-9	journal	March 1994

Cited By (5)

A molecular assembly phase transition and kinetic proofreading modulate Ras activation by SOS Huang, William Y. C.; Alvarez, Steven; Kondo, Yasushi Science, Vol. 363, Issue 6431 https://doi.org/10.1126/science.aau5721	journal	March 2019
Autoactivation of small GTPases by the GEF–effector positive feedback modules Goryachev, Andrew B.; Leda, Marcin F1000Research, Vol. 8 https://doi.org/10.12688/f1000research.20003.1	journal	January 2019
More from less – bottom-up reconstitution of cell biology Ganzinger, Kristina A.; Schwille, Petra Journal of Cell Science, Vol. 132, Issue 4 https://doi.org/10.1242/jcs.227488	journal	February 2019
Auto-regulation of Rab5 GEF activity in Rabex5 by allosteric structural changes, catalytic core dynamics and ubiquitin binding Lauer, Janelle; Segeletz, Sandra; Cezanne, Alice eLife, Vol. 8 https://doi.org/10.7554/elife.46302	journal	November 2019
Allosteric Structural Alterations and Auto-regulation of Rab5 GEF Activity in Rabex5 Lauer, Janelle; Cezanne, Alice; Guaitoli, Giambattista https://doi.org/10.1101/562504	posted_content	February 2019

Similar Records

Phosphotyrosine-mediated LAT assembly on membranes drives kinetic bifurcation in recruitment dynamics of the Ras activator SOS

Journal Article · Fri Jul 01 00:00:00 EDT 2016 · Proceedings of the National Academy of Sciences of the United States of America · OSTI ID:1623887

Huang, William Y. C.; Yan, Qingrong; Lin, Wan-Chen; +6 more

Structure of the C-terminal guanine nucleotide exchange factor module of Trio in an autoinhibited conformation reveals its oncogenic potential

Journal Article · Tue Feb 19 00:00:00 EST 2019 · Science Signaling · OSTI ID:1623887

Bandekar, Sumit J.; Arang, Nadia; Tully, Ena S.; +5 more

Human Sos1: A guanine nucleotide exchange factor for ras that binds to GRB2

Journal Article · Fri May 28 00:00:00 EDT 1993 · Science (Washington, D.C.); (United States) · OSTI ID:1623887

Chardin, P; Camonis, J; Gale, N W; +4 more

Related Subjects

Science & Technology - Other Topics

Title: Mechanism of SOS PR-domain autoinhibition revealed by single-molecule assays on native protein from lysate

Citation Formats

References (63)

Cited By (5)

Similar Records

Related Subjects