The RIT1 C-terminus associates with lipid bilayers via charge complementarity

RIT1 is a member of the Ras superfamily of small GTPases involved in regulation of cellular signaling. Mutations to RIT1 are involved in cancer and developmental disorders. Like many Ras subfamily members, RIT1 is localized to the plasma membrane. However, RIT1 lacks the C-terminal prenylation that helps many other subfamily members adhere to cellular membranes. We used molecular dynamics simulations to examine the mechanisms by which the C-terminal peptide (CTP) of RIT1 associates with lipid bilayers. We show that the CTP is unstructured and that its membrane interactions depend on lipid composition. While a 12-residue region of the CTP binds strongly to anionic bilayers containing phosphatidylserine lipids, the CTP termini fray from the membrane allowing for accommodation of the RIT1 globular domain at the membrane-water interface.

[1]  I. Vetter,et al.  The Guanine Nucleotide-Binding Switch in Three Dimensions , 2001, Science.

[2]  M. Dasso.  The Ran GTPase: Theme and Variations , 2002, Current Biology.

[3]  S. Miyano,et al.  Novel recurrent mutations in the RAS-like GTP-binding gene RIT1 in myeloid malignancies , 2013, Leukemia.

[4]  P. Mourrain,et al.  Rasl11b Knock Down in Zebrafish Suppresses One-Eyed-Pinhead Mutant Phenotype , 2008, PloS one.

[5]  Angela N. Brooks,et al.  Mapping the Hallmarks of Lung Adenocarcinoma with Massively Parallel Sequencing , 2012, Cell.

[6]  E. Guccione,et al.  Chromatin association and regulation of rDNA transcription by the Ras‐family protein RasL11a , 2010, The EMBO journal.

[7]  Berk Hess,et al.  GROMACS: High performance molecular simulations through multi-level parallelism from laptops to supercomputers , 2015 .

[8]  F. McCormick,et al.  SHOC2–MRAS–PP1 complex positively regulates RAF activity and contributes to Noonan syndrome pathogenesis , 2018, Proceedings of the National Academy of Sciences.

[9]  Raul Rabadan,et al.  The integrated landscape of driver genomic alterations in glioblastoma , 2013, Nature Genetics.

[10]  S. Moores,et al.  Sequence dependence of protein isoprenylation. , 1991, The Journal of biological chemistry.

[11]  Berk Hess,et al.  P-LINCS:  A Parallel Linear Constraint Solver for Molecular Simulation. , 2008, Journal of chemical theory and computation.

[12]  H. Waldmann,et al.  The role of G-domain orientation and nucleotide state on the Ras isoform-specific membrane interaction , 2012, European Biophysics Journal.

[13]  C. Balduini,et al.  Targeting of the small GTPase Rap2b, but not Rap1b, to lipid rafts is promoted by palmitoylation at Cys176 and Cys177 and is required for efficient protein activation in human platelets. , 2008, Cellular signalling.

[14]  C. Marshall,et al.  A polybasic domain or palmitoylation is required in addition to the CAAX motif to localize p21 ras to the plasma membrane , 1990, Cell.

[15]  J. Paton,et al.  Rasd1, a small G protein with a big role in the hypothalamic response to neuronal activation , 2016, Molecular Brain.

[16]  M. Zerial,et al.  Association of Rap1a and Rap1b proteins with late endocytic/phagocytic compartments and Rap2a with the Golgi complex. , 1994, Journal of cell science.

[17]  Val J. Watts,et al.  Dexamethasone-Induced Ras Protein 1 Negatively Regulates Protein Kinase C δ: Implications for Adenylyl Cyclase 2 Signaling , 2006, Molecular Pharmacology.

[18]  M. Matter,et al.  R‐Ras interacts with filamin a to maintain endothelial barrier function , 2011, Journal of cellular physiology.

[19]  C DeLisi,et al.  The detection and classification of membrane-spanning proteins. , 1985, Biochimica et biophysica acta.

[20]  M. Gelb,et al.  Posttranslationally processed structure of the human platelet protein smg p21B: evidence for geranylgeranylation and carboxyl methylation of the C-terminal cysteine. , 1990, Proceedings of the National Academy of Sciences of the United States of America.

[21]  K. Cao,et al.  Cloning and characterization of a novel small monomeric GTPase, RasL10B, with tumor suppressor potential , 2006, Biotechnology Letters.

[22]  D. Goeddel,et al.  Heterologous expression and characterization of the human R-ras gene product , 1987, Molecular and cellular biology.

[23]  M. Ikura,et al.  Biochemical Classification of Disease-associated Mutants of RAS-like Protein Expressed in Many Tissues (RIT1)* , 2016, The Journal of Biological Chemistry.

[24]  Harald Stenmark,et al.  The Rab GTPase family , 2001, Genome Biology.

[25]  Theresa M. Grana,et al.  Rit, a non-lipid-modified Ras-related protein, transforms NIH3T3 cells without activating the ERK, JNK, p38 MAPK or PI3K/Akt pathways , 2000, Oncogene.

[26]  K. Geering,et al.  Regulation of Ca2+ channel expression at the cell surface by the small G-protein kir/Gem , 2001, Nature.

[27]  W. Hunziker,et al.  Nuclear Localization of Endogenous RGK Proteins and Modulation of Cell Shape Remodeling by Regulated Nuclear Transport , 2007, Traffic.

[28]  Maurine E. Linder,et al.  Protein lipidation , 2007 .

[29]  P. Casey,et al.  Prenylation of CaaX-type proteins: Basic principles through clinical applications , 2002 .

[30]  W. L. Jorgensen,et al.  Comparison of simple potential functions for simulating liquid water , 1983 .

[31]  F. Tamanoi,et al.  Differential Requirement of CAAX-mediated Posttranslational Processing for Rheb Localization and Signaling , 2009, Oncogene.

[32]  S. A. Ong,et al.  Rasd1 Modulates the Coactivator Function of NonO in the Cyclic AMP Pathway , 2011, PloS one.

[33]  Gordon B Mills,et al.  ARHI is a Ras-related small G-protein with a novel N-terminal extension that inhibits growth of ovarian and breast cancers , 2003, Oncogene.

[34]  T. Kataoka,et al.  RA-GEF-1, a Guanine Nucleotide Exchange Factor for Rap1, Is Activated by Translocation Induced by Association with Rap1·GTP and Enhances Rap1-dependent B-Raf Activation* , 2001, The Journal of Biological Chemistry.

[35]  Donavan T. Cheng,et al.  Mutational Landscape of Metastatic Cancer Revealed from Prospective Clinical Sequencing of 10,000 Patients , 2017, Nature Medicine.

[36]  R. Ghirlando,et al.  Structural basis of recognition of farnesylated and methylated KRAS4b by PDEδ , 2016, Proceedings of the National Academy of Sciences.

[37]  John D McPherson,et al.  Next-generation sequencing identifies rare variants associated with Noonan syndrome , 2014, Proceedings of the National Academy of Sciences.

[38]  W. Kabsch,et al.  Dictionary of protein secondary structure: Pattern recognition of hydrogen‐bonded and geometrical features , 1983, Biopolymers.

[39]  Long Yu,et al.  Cloning and characterization of the human gene RAP2C, a novel member of Ras family, which activates transcriptional activities of SRE , 2007, Molecular Biology Reports.

[40]  P. Casey,et al.  The COOH-terminal domain of the Rap1A (Krev-1) protein is isoprenylated and supports transformation by an H-Ras:Rap1A chimeric protein , 1991, Molecular and cellular biology.

[41]  The Plasma Membrane as a Competitive Inhibitor and Positive Allosteric Modulator of KRas4B Signaling. , 2020, Biophysical journal.

[42]  C. Der,et al.  The Ras branch of small GTPases: Ras family members don't fall far from the tree. , 2000, Current opinion in cell biology.

[43]  Mingming Jia,et al.  COSMIC: exploring the world's knowledge of somatic mutations in human cancer , 2014, Nucleic Acids Res..

[44]  J. Furuhjelm,et al.  The C-terminal end of R-Ras contains a focal adhesion targeting signal , 2003, Journal of Cell Science.

[45]  Matthias Buck,et al.  Computational Modeling Reveals that Signaling Lipids Modulate the Orientation of K-Ras4A at the Membrane Reflecting Protein Topology. , 2017, Structure.

[46]  F. McCormick,et al.  The molecular functions of RIT1 and its contribution to human disease. , 2020, The Biochemical journal.

[47]  Toshihiko Ogura,et al.  Gain-of-function mutations in RIT1 cause Noonan syndrome, a RAS/MAPK pathway syndrome. , 2013, American journal of human genetics.

[48]  I. Herman,et al.  Indirect association of ezrin with F-actin: isoform specificity and calcium sensitivity , 1995, The Journal of cell biology.

[49]  S. Sligar,et al.  PIP2 Influences the Conformational Dynamics of Membrane bound KRAS4b , 2019, bioRxiv.

[50]  A. Ridley,et al.  Rho family proteins: coordinating cell responses. , 2001, Trends in cell biology.

[51]  Alexander D. MacKerell,et al.  Update of the CHARMM all-atom additive force field for lipids: validation on six lipid types. , 2010, The journal of physical chemistry. B.

[52]  R. Bast,et al.  RAS-related GTPases DIRAS1 and DIRAS2 induce autophagic cancer cell death and are required for autophagy in murine ovarian cancer cells , 2018, Autophagy.

[53]  Mitsuhiko Ikura,et al.  Oncogenic and RASopathy-associated K-RAS mutations relieve membrane-dependent occlusion of the effector-binding site , 2015, Proceedings of the National Academy of Sciences.

[54]  K Schulten,et al.  VMD: visual molecular dynamics. , 1996, Journal of molecular graphics.

[55]  P. Sigler,et al.  Structure of the high affinity complex of inositol trisphosphate with a phospholipase C pleckstrin homology domain , 1995, Cell.

[56]  H. Cavé,et al.  Activating Mutations of RRAS2 Are a Rare Cause of Noonan Syndrome. , 2019, American journal of human genetics.

[57]  C. Marshall,et al.  All ras proteins are polyisoprenylated but only some are palmitoylated , 1989, Cell.

[58]  Frank McCormick,et al.  The GTPase superfamily: a conserved switch for diverse cell functions , 1990, Nature.

[59]  W. Im,et al.  Automated Builder and Database of Protein/Membrane Complexes for Molecular Dynamics Simulations , 2007, PloS one.

[60]  Alfonso Valencia,et al.  The Ras protein superfamily: Evolutionary tree and role of conserved amino acids , 2012, Journal of Cell Biology.

[61]  J Wala,et al.  Oncogenic RIT1 mutations in lung adenocarcinoma , 2014, Oncogene.

[62]  Priyanka Prakash,et al.  Oncogenic K-Ras Binds to an Anionic Membrane in Two Distinct Orientations: A Molecular Dynamics Analysis. , 2016, Biophysical journal.

[63]  M. Arpin,et al.  Ezrin contains cytoskeleton and membrane binding domains accounting for its proposed role as a membrane-cytoskeletal linker , 1993, The Journal of cell biology.

[64]  E. Obersztyn,et al.  Contribution of RIT1 mutations to the pathogenesis of Noonan syndrome: Four new cases and further evidence of heterogeneity , 2014, American journal of medical genetics. Part A.

[65]  Gert Vriend,et al.  A series of PDB related databases for everyday needs , 2010, Nucleic Acids Res..

[66]  S. Paradis,et al.  The GTPase Rem2 regulates synapse development and dendritic morphology , 2011, Developmental neurobiology.

[67]  Ken Matsumoto,et al.  Novel small GTPase M-Ras participates in reorganization of actin cytoskeleton , 1997, Oncogene.

[68]  R. Ochs,et al.  A mechanism for the partial insertion of protein kinase C into membranes. , 2001, Biochemical and biophysical research communications.

[69]  J. Yewdell,et al.  Gem: an induced, immediate early protein belonging to the Ras family. , 1994, Science.

[70]  S. Endo,et al.  Rap2 function requires palmitoylation and recycling endosome localization. , 2009, Biochemical and biophysical research communications.

[71]  E. Lapetina,et al.  Prenyl group identification of rap2 proteins: a ras superfamily member other than ras that is farnesylated. , 1993, Biochemical Journal.

[72]  T. Elston,et al.  Divergent Roles of CAAX Motif-signaled Posttranslational Modifications in the Regulation and Subcellular Localization of Ral GTPases* , 2015, The Journal of Biological Chemistry.

[73]  T. Meyer,et al.  PI(3,4,5)P3 and PI(4,5)P2 Lipids Target Proteins with Polybasic Clusters to the Plasma Membrane , 2006, Science.

[74]  C. Balduini,et al.  Identification and biochemical characterization of Rap2C, a new member of the Rap family of small GTP-binding proteins. , 2006, Biochimie.

[75]  M. Dasso,et al.  The Ran GTPase regulates mitotic spindle assembly , 1999, Current Biology.

[76]  Hongyan Chen,et al.  LZTR1 inactivation promotes MAPK/ ERK pathway activation in glioblastoma by stabilizing oncoprotein RIT1 , 2020 .

[77]  P. Casey,et al.  Protein lipidation in cell signaling. , 1995, Science.

[78]  C J Marshall,et al.  A CAAX or a CAAL motif and a second signal are sufficient for plasma membrane targeting of ras proteins. , 1991, The EMBO journal.

[79]  S. Gabriel,et al.  Genomic Correlates of Immune-Cell Infiltrates in Colorectal Carcinoma , 2016, Cell reports.

[80]  Catherine A. Shang,et al.  Whole-genome landscapes of major melanoma subtypes , 2017, Nature.

[81]  M. Parrinello,et al.  Polymorphic transitions in single crystals: A new molecular dynamics method , 1981 .

[82]  S. Sligar,et al.  Membrane-Bound Ras as a Conformational Clock. , 2020, Biophysical journal.

[83]  L. Hesson,et al.  RRP22 is a farnesylated, nucleolar, Ras-related protein with tumor suppressor potential. , 2005, Cancer research.

[84]  J. Camonis,et al.  Localization of RalB signaling at endomembrane compartments and its modulation by autophagy , 2019, Scientific Reports.

[85]  K. Levental,et al.  Plasma membranes are asymmetric in lipid unsaturation, packing, and protein shape , 2020, Nature Chemical Biology.

[86]  T. Rudel,et al.  The Tumor Suppressor DiRas3 Forms a Complex with H-Ras and C-RAF Proteins and Regulates Localization, Dimerization, and Kinase Activity of C-RAF* , 2012, The Journal of Biological Chemistry.

[87]  C. Der,et al.  Lipid Modification of Ras Superfamily GTPases: Not Just Membrane Glue , 2011 .

[88]  O. Migita,et al.  Spectrum of mutations and genotype–phenotype analysis in Noonan syndrome patients with RIT1 mutations , 2016, Human Genetics.

[89]  C. Kahn,et al.  Overexpression of Rad Inhibits Glucose Uptake in Cultured Muscle and Fat Cells* , 1996, The Journal of Biological Chemistry.

[90]  D. Mitter,et al.  Genotype and phenotype in patients with Noonan syndrome and a RIT1 mutation , 2016, Genetics in Medicine.

[91]  A. Gorfe,et al.  Ras membrane orientation and nanodomain localization generate isoform diversity , 2010, Proceedings of the National Academy of Sciences.

[92]  D. Zack,et al.  Rin, a Neuron-Specific and Calmodulin-Binding Small G-Protein, and Rit Define a Novel Subfamily of Ras Proteins , 1996, The Journal of Neuroscience.

[93]  M. Ikura,et al.  Calmodulin disrupts plasma membrane localization of farnesylated KRAS4b by sequestering its lipid moiety , 2020, Science Signaling.

[94]  A. Garcia,et al.  Chapter 5 Simulations of Temperature and Pressure Unfolding of Peptides and Proteins with Replica Exchange Molecular Dynamics , 2006 .

[95]  P. Bastiaens,et al.  Imaging Activation of Two Ras Isoforms Simultaneously in a Single Cell , 2005, Chembiochem : a European journal of chemical biology.

[96]  J. Pérez-Gil,et al.  Palmitoylation of R-Ras by human DHHC19, a palmitoyl transferase with a CaaX box. , 2010, Biochimica et biophysica acta.

[97]  F. McCormick,et al.  The RAS GTPase RIT1 compromises mitotic fidelity through spindle assembly checkpoint suppression , 2020, Current Biology.

[98]  Herbert Waldmann,et al.  An Acylation Cycle Regulates Localization and Activity of Palmitoylated Ras Isoforms , 2005, Science.

[99]  S. Yamanaka,et al.  Differential Membrane Localization of ERas and Rheb, Two Ras-related Proteins Involved in the Phosphatidylinositol 3-Kinase/mTOR Pathway* , 2005, Journal of Biological Chemistry.

[100]  Ritika Kundra,et al.  Clinical Sequencing Defines the Genomic Landscape of Metastatic Colorectal Cancer. , 2018, Cancer cell.

[101]  T. Steitz,et al.  The spontaneous insertion of proteins into and across membranes: The helical hairpin hypothesis , 1981, Cell.

[102]  Tae-Min Kim,et al.  The mutational burdens and evolutionary ages of early gastric cancers are comparable to those of advanced gastric cancers , 2014, The Journal of pathology.

[103]  J. Colicelli,et al.  Human RAS Superfamily Proteins and Related GTPases , 2004, Science's STKE.

[104]  F. Tamanoi,et al.  A novel approach to tag and identify geranylgeranylated proteins , 2009, Electrophoresis.

[105]  B. Wojciak-Stothard,et al.  Dual Chemical Probes Enable Quantitative System-Wide Analysis of Protein Prenylation and Prenylation Dynamics , 2019, Nature Chemistry.

[106]  Alan Hall,et al.  Rho family GTPases. , 2012, Biochemical Society transactions.

[107]  T. Darden,et al.  A smooth particle mesh Ewald method , 1995 .

[108]  David Botstein,et al.  RERG Is a Novel ras-related, Estrogen-regulated and Growth-inhibitory Gene in Breast Cancer* , 2001, The Journal of Biological Chemistry.

[109]  J. Cherfils,et al.  ARF GTPases and their GEFs and GAPs: concepts and challenges , 2019, Molecular biology of the cell.

[110]  Alexander D. MacKerell,et al.  Optimization of the additive CHARMM all-atom protein force field targeting improved sampling of the backbone φ, ψ and side-chain χ(1) and χ(2) dihedral angles. , 2012, Journal of chemical theory and computation.

[111]  W. R. Bishop,et al.  K- and N-Ras Are Geranylgeranylated in Cells Treated with Farnesyl Protein Transferase Inhibitors* , 1997, The Journal of Biological Chemistry.

[112]  D. van der Spoel,et al.  GROMACS: A message-passing parallel molecular dynamics implementation , 1995 .

[113]  Jeffery B. Klauda,et al.  CHARMM-GUI Membrane Builder for mixed bilayers and its application to yeast membranes. , 2009, Biophysical journal.

[114]  A. Caflisch,et al.  Membrane localization and flexibility of a lipidated ras peptide studied by molecular dynamics simulations. , 2004, Journal of the American Chemical Society.

[115]  G. Shi,et al.  Rit subfamily small GTPases: regulators in neuronal differentiation and survival. , 2013, Cellular signalling.

[116]  E. Conibear,et al.  ABHD17 proteins are novel protein depalmitoylases that regulate N-Ras palmitate turnover and subcellular localization , 2015, eLife.

[117]  Steven J. M. Jones,et al.  Comprehensive molecular profiling of lung adenocarcinoma , 2014, Nature.

[118]  Tae-Min Kim,et al.  The chronological sequence of somatic mutations in early gastric carcinogenesis inferred from multiregion sequencing of gastric adenomas , 2016, Oncotarget.

[119]  D. Coppola,et al.  Geranylgeranyltransferase I inhibitor GGTI-2154 induces breast carcinoma apoptosis and tumor regression in H-Ras transgenic mice. , 2003, Cancer research.

[120]  J. Hancock,et al.  Ras trafficking, localization and compartmentalized signalling. , 2012, Seminars in cell & developmental biology.

[121]  Krister Wennerberg,et al.  The Ras superfamily at a glance , 2005, Journal of Cell Science.

[122]  J. Pereira-Leal,et al.  Evolution of the Rab family of small GTP-binding proteins. , 2001, Journal of molecular biology.

[123]  G. Spiegelman,et al.  Roles played by Ras subfamily proteins in the cell and developmental biology of microorganisms. , 2003, Cellular signalling.

[124]  Chun Zhang,et al.  Roles of palmitoylation and the KIKK membrane-targeting motif in leukemogenesis by oncogenic KRAS4A , 2015, Journal of Hematology & Oncology.

[125]  G. Clark,et al.  Rig is a novel Ras-related protein and potential neural tumor suppressor , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[126]  A. Cox,et al.  K-Ras4A splice variant is widely expressed in cancer and uses a hybrid membrane-targeting motif , 2015, Proceedings of the National Academy of Sciences.