A computationally engineered RAS rheostat reveals RAS/ERK signaling dynamics

Synthetic protein switches controlled with user-defined inputs are powerful tools for studying and controlling dynamic cellular processes. To date, these approaches have relied primarily on intermolecular regulation. Here, we report a computationally-guided framework for engineering intramolecular regulation of protein function. We utilize this framework to develop Chemically Inducible Activator of RAS (CIAR), a single-component RAS rheostat that directly activates endogenous RAS in response to a small molecule. Using CIAR, we show that direct RAS activation elicits markedly different RAS/ERK signaling dynamics compared to growth factor stimulation, and that these dynamics differ between cell types. We also found that the clinically-approved RAF inhibitor vemurafenib potently primes cells to respond to direct wild-type RAS activation. These results demonstrate the utility of CIAR for quantitatively interrogating RAS signaling. Finally, we demonstrate the general utility of our approach to design intramolecularly-regulated protein tools by applying this methodology to the Rho Family GEFs.

[1]  Chao Zhang,et al.  RAF inhibitors transactivate RAF dimers and ERK signaling in cells with wild-type BRAF , 2010, Nature.

[2]  Alfred Wittinghofer,et al.  GEFs and GAPs: Critical Elements in the Control of Small G Proteins , 2007, Cell.

[3]  J. Eng,et al.  Comet: An open‐source MS/MS sequence database search tool , 2013, Proteomics.

[4]  Jing Wang,et al.  WEB-based GEne SeT AnaLysis Toolkit (WebGestalt): update 2013 , 2013, Nucleic Acids Res..

[5]  U. Rapp,et al.  Differential regulation of Raf isozymes by growth versus differentiation inducing factors in PC12 pheochromocytoma cells , 1996, FEBS letters.

[6]  Giulio Superti‐Furga,et al.  Serine and tyrosine phosphorylations cooperate in Raf‐1, but not B‐Raf activation , 1999, The EMBO journal.

[7]  J. Reis-Filho,et al.  Kinase-Dead BRAF and Oncogenic RAS Cooperate to Drive Tumor Progression through CRAF , 2010, Cell.

[8]  I. Verma,et al.  Production and purification of lentiviral vectors , 2006, Nature Protocols.

[9]  D. Schadendorf,et al.  RAS mutations are associated with the development of cutaneous squamous cell tumors in patients treated with RAF inhibitors. , 2012, Journal of clinical oncology : official journal of the American Society of Clinical Oncology.

[10]  I. Amit,et al.  EGR1 and the ERK‐ERF axis drive mammary cell migration in response to EGF , 2012, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[11]  Wang Shen,et al.  A small-molecule inhibitor of Bcl-XL potentiates the activity of cytotoxic drugs in vitro and in vivo. , 2006, Cancer research.

[12]  Tobias Meyer,et al.  An inducible translocation strategy to rapidly activate and inhibit small GTPase signaling pathways , 2005, Nature Methods.

[13]  Toru Komatsu,et al.  Organelle-Specific, Rapid Induction of Molecular Activities and Membrane Tethering , 2010, Nature Methods.

[14]  N. Dumaz,et al.  ERK and PDE4 cooperate to induce RAF isoform switching in melanoma , 2011, Nature Structural &Molecular Biology.

[15]  P. Yaswen,et al.  A Versatile Viral System for Expression and Depletion of Proteins in Mammalian Cells , 2009, PloS one.

[16]  Jayanta Debnath,et al.  Morphogenesis and oncogenesis of MCF-10A mammary epithelial acini grown in three-dimensional basement membrane cultures. , 2003, Methods.

[17]  T. Veenstra,et al.  Protein Phosphatase 2A Positively Regulates Ras Signaling by Dephosphorylating KSR1 and Raf-1 on Critical 14-3-3 Binding Sites , 2003, Current Biology.

[18]  Wendell A Lim,et al.  Engineering synthetic signaling proteins with ultrasensitive input/output control , 2007, Nature Biotechnology.

[19]  Pradeep Kota,et al.  Engineered allosteric activation of kinases in living cells , 2010, Nature Biotechnology.

[20]  John Kuriyan,et al.  The structural basis of the activation of Ras by Sos , 1998, Nature.

[21]  W. Kolch,et al.  Cyclic AMP-Dependent Kinase Regulates Raf-1 Kinase Mainly by Phosphorylation of Serine 259 , 2002, Molecular and Cellular Biology.

[22]  Ariana Peck,et al.  Structure of the BRAF-MEK complex reveals a kinase activity independent role for BRAF in MAPK signaling. , 2014, Cancer cell.

[23]  Michael J Ackerman,et al.  Gain-of-function RAF1 mutations cause Noonan and LEOPARD syndromes with hypertrophic cardiomyopathy , 2007, Nature Genetics.

[24]  M. Therrien,et al.  Regulation of RAF protein kinases in ERK signalling , 2015, Nature Reviews Molecular Cell Biology.

[25]  Carla Mattos,et al.  A comprehensive survey of Ras mutations in cancer. , 2012, Cancer research.

[26]  Natalie I. Tasman,et al.  A guided tour of the Trans‐Proteomic Pipeline , 2010, Proteomics.

[27]  Dustin J Maly,et al.  A small molecule-regulated guanine nucleotide exchange factor. , 2010, Journal of the American Chemical Society.

[28]  T. Kortemme,et al.  Differences in flexibility underlie functional differences in the Ras activators son of sevenless and Ras guanine nucleotide releasing factor 1. , 2009, Structure.

[29]  E. Nishida,et al.  Phosphoproteomics reveals new ERK MAP kinase targets and links ERK to nucleoporin-mediated nuclear transport , 2009, Nature Structural &Molecular Biology.

[30]  K. Moelling,et al.  Phosphorylation and regulation of Raf by Akt (protein kinase B). , 1999, Science.

[31]  S. Lemieux,et al.  Phosphoproteome dynamics reveal novel ERK1/2 MAP kinase substrates with broad spectrum of functions , 2013, Molecular systems biology.

[32]  M. Greenberg,et al.  Serum- and Glucocorticoid-inducible Kinase SGK Phosphorylates and Negatively Regulates B-Raf* , 2001, The Journal of Biological Chemistry.

[33]  D. Cameron,et al.  Raf-1 is the predominant Raf isoform that mediates growth factor-stimulated growth in ovarian cancer cells. , 2006, Carcinogenesis.

[34]  Brian Raught,et al.  Automated identification of SUMOylation sites using mass spectrometry and SUMmOn pattern recognition software , 2006, Nature Methods.

[35]  Yu Shyr,et al.  Survival in BRAF V600-mutant advanced melanoma treated with vemurafenib. , 2012, The New England journal of medicine.

[36]  Kam Y. J. Zhang,et al.  Clinical efficacy of a RAF inhibitor needs broad target blockade in BRAF-mutant melanoma , 2010, Nature.

[37]  J. Wolchok,et al.  Paradoxical Activation of T Cells via Augmented ERK Signaling Mediated by a RAF Inhibitor , 2013, Cancer Immunology Research.

[38]  Robert A. Weinberg,et al.  Ras oncogenes: split personalities , 2008, Nature Reviews Molecular Cell Biology.

[39]  V. Shingler,et al.  Serine 25 of oncoprotein 18 is a major cytosolic target for the mitogen-activated protein kinase. , 1993, The Journal of biological chemistry.

[40]  Y. Benjamini,et al.  Controlling the false discovery rate: a practical and powerful approach to multiple testing , 1995 .

[41]  W. Lim,et al.  Rewiring cellular morphology pathways with synthetic guanine nucleotide exchange factors , 2007, Nature.

[42]  Tobias Meyer,et al.  Rapid Chemically Induced Changes of PtdIns(4,5)P2 Gate KCNQ Ion Channels , 2006, Science.

[43]  Jared E. Toettcher,et al.  Using Optogenetics to Interrogate the Dynamic Control of Signal Transmission by the Ras/Erk Module , 2013, Cell.

[44]  Michael Z. Lin,et al.  Optical Control of Protein Activity by Fluorescent Protein Domains , 2012, Science.

[45]  A. Weiss,et al.  Unusual Interplay of Two Types of Ras Activators, RasGRP and SOS, Establishes Sensitive and Robust Ras Activation in Lymphocytes , 2007, Molecular and Cellular Biology.

[46]  M. Mann,et al.  Global, In Vivo, and Site-Specific Phosphorylation Dynamics in Signaling Networks , 2006, Cell.

[47]  B. Kuhlman,et al.  A genetically-encoded photoactivatable Rac controls the motility of living cells , 2009, Nature.

[48]  W. Lim,et al.  Reprogramming Control of an Allosteric Signaling Switch Through Modular Recombination , 2003, Science.

[49]  W. Kolch.  Coordinating ERK/MAPK signalling through scaffolds and inhibitors , 2005, Nature Reviews Molecular Cell Biology.

[50]  M. Pacher,et al.  Dephosphorylation of Ser-259 Regulates Raf-1 Membrane Association* , 2002, The Journal of Biological Chemistry.

[51]  D. Baker,et al.  RosettaRemodel: A Generalized Framework for Flexible Backbone Protein Design , 2011, PloS one.

[52]  D. Morrison,et al.  RAF Inhibitor-Induced KSR1/B-RAF Binding and Its Effects on ERK Cascade Signaling , 2011, Current Biology.

[53]  David R. Liu,et al.  Creating small-molecule-dependent switches to modulate biological functions. , 2005, Chemistry & biology.

[54]  J. Parsons,et al.  Extracellular Signal-Regulated Kinase 2 (ERK2) Phosphorylation Sites and Docking Domain on the Nuclear Pore Complex Protein Tpr Cooperatively Regulate ERK2-Tpr Interaction , 2008, Molecular and Cellular Biology.

[55]  Ming Zhou,et al.  Regulation of Raf-1 by direct feedback phosphorylation. , 2005, Molecular cell.

[56]  M. Belvin,et al.  RAF inhibitors prime wild-type RAF to activate the MAPK pathway and enhance growth , 2010, Nature.

[57]  Brian Kuhlman,et al.  Design of protein conformational switches. , 2006, Current opinion in structural biology.

[58]  Jared E. Toettcher,et al.  Light-based feedback for controlling intracellular signaling dynamics , 2011, Nature Methods.

[59]  P. Bastiaens,et al.  Growth factor-induced MAPK network topology shapes Erk response determining PC-12 cell fate , 2007, Nature Cell Biology.