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The Ras Superfamily of Small GTPases: The Unlocked Secrets

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 1120))

Abstract

The Ras superfamily of small GTPases is composed of more than 150 members, which share a conserved structure and biochemical properties, acting as binary molecular switches turned on by binding GTP and off by hydrolyzing GTP to GDP. However, despite considerable structural and biochemical similarities, these proteins play multiple and divergent roles, being versatile and key regulators of virtually all fundamental cellular processes. Conversely, their dysfunction plays a crucial role in the pathogenesis of serious human diseases, including cancer and developmental syndromes.

Fuelled by the original identification in 1982 of mutationally activated and transforming human Ras genes in human cancer cell lines, a variety of powerful experimental techniques have been intensively focused on discovering and studying structure, biochemistry, and biology of Ras and Ras-related small GTPases, leading to fundamental research breakthroughs into identification and structural and functional characterization of a huge number of Ras superfamily members, as well as of their multiple regulators and effectors.

In this review we provide a general overview of the major milestones that eventually allowed to unlock the secret treasure chest of this large and important superfamily of proteins.

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References

  1. Wennerberg K, Rossman KL, Der CJ (2005) The Ras superfamily at a glance. J Cell Sci 118:843–846

    Article  PubMed  CAS  Google Scholar 

  2. Bourne HR, Sanders DA, McCormick F (1990) The GTPase superfamily: a conserved switch for diverse cell functions. Nature 348:125–132

    Article  PubMed  CAS  Google Scholar 

  3. Cox AD, Der CJ (2010) Ras history: the saga continues. Small GTPases 1:2–27

    Article  PubMed  PubMed Central  Google Scholar 

  4. Rojas AM, Fuentes G, Rausell A et al (2012) The Ras protein superfamily: evolutionary tree and role of conserved amino acids. J Cell Biol 196:189–201

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  5. Haigis KM, Kendall KM, Wang Y et al (2008) Differential effects of oncogenic K-Ras and N-Ras on proliferation, differentiation and tumor progression in the colon. Nat Genet 40:600–608

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  6. Karnoub AE, Weinberg RA (2008) Ras oncogenes: split personalities. Nat Rev Mol Cell Biol 9:517–531

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  7. Heasman SJ, Ridley AJ (2008) Mammalian Rho GTPases: new insights into their functions from in vivo studies. Nat Rev Mol Cell Biol 9:690–701

    Article  PubMed  CAS  Google Scholar 

  8. Grosshans BL, Ortiz D, Novick P (2006) Rabs and their effectors: achieving specificity in membrane traffic. Proc Natl Acad Sci U S A 103:11821–11827

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  9. Zerial M, Mcbride H (2001) Rab proteins as membrane organizers. Nat Rev Mol Cell Biol 2:107–117

    Article  PubMed  CAS  Google Scholar 

  10. Donaldson JG, Jackson CL (2011) ARF family G proteins and their regulators: roles in membrane transport, development and disease. Nat Rev Mol Cell Biol 12:362–375

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  11. Moore MS (1998) Ran and nuclear transport. J Biol Chem 273:22857–22860

    Article  PubMed  CAS  Google Scholar 

  12. Biou V, Cherfils J (2004) Structural principles for the multispecificity of small GTP-binding proteins. Biochemistry 43:6833–6840

    Article  PubMed  CAS  Google Scholar 

  13. Vetter IR, Wittinghofer A (2001) The guanine nucleotide-binding switch in three dimensions. Science 294:1299–1304

    Article  PubMed  CAS  Google Scholar 

  14. Colicelli J (2004) Human RAS superfamily proteins and related GTPases. Sci STKE 2004:RE13

    PubMed  PubMed Central  Google Scholar 

  15. Herrmann C (2003) Ras-effector interactions: after one decade. Curr Opin Struct Biol 13: 122–129

    Article  PubMed  CAS  Google Scholar 

  16. Pai EF, Kabsch W, Krengel U et al (1989) Structure of the guanine-nucleotide-binding domain of the Ha-ras oncogene product p21 in the triphosphate conformation. Nature 341: 209–214

    Article  PubMed  CAS  Google Scholar 

  17. Tong L, Milburn MV, de Vos AM et al (1989) Structure of ras proteins. Science 245:244

    Article  PubMed  CAS  Google Scholar 

  18. Milburn MV, Tong L, deVos AM et al (1990) Molecular switch for signal transduction: structural differences between active and inactive forms of protooncogenic ras proteins. Science 247:939–945

    Article  PubMed  CAS  Google Scholar 

  19. Schlichting I, Almo SC, Rapp G et al (1990) Time-resolved X-ray crystallographic study of the conformational change in Ha-Ras p21 protein on GTP hydrolysis. Nature 345:309–315

    Article  PubMed  CAS  Google Scholar 

  20. Boriack-Sjodin PA, Margarit SM, Bar-Sagi D et al (1998) The structural basis of the activation of Ras by Sos. Nature 394:337–343

    Article  PubMed  CAS  Google Scholar 

  21. Scheffzek K, Ahmadian MR, Kabsch W et al (1997) The Ras-RasGAP complex: structural basis for GTPase activation and its loss in oncogenic Ras mutants. Science 277:333–338

    Article  PubMed  CAS  Google Scholar 

  22. Ahearn IM, Haigis K, Bar-Sagi D et al (2011) Regulating the regulator: post-translational modification of RAS. Nat Rev Mol Cell Biol 13:39–51

    Article  PubMed  Google Scholar 

  23. Willumsen BM, Christensen A, Hubbert NL et al (1984) The p21 ras C-terminus is required for transformation and membrane association. Nature 310:583–586

    Article  PubMed  CAS  Google Scholar 

  24. Srivastava SK, Lacal JC, Reynolds SH et al (1985) Antibody of predetermined specificity to a carboxy-terminal region of H-ras gene products inhibits their guanine nucleotide-binding function. Mol Cell Biol 5:3316–3319

    PubMed  CAS  PubMed Central  Google Scholar 

  25. Bernards A, Settleman J (2004) GAP control: regulating the regulators of small GTPases. Trends Cell Biol 14:377–385

    Article  PubMed  CAS  Google Scholar 

  26. Cherfils J, Zeghouf M (2013) Regulation of small GTPases by GEFs, GAPs, and GDIs. Physiol Rev 93:269–309

    Article  PubMed  CAS  Google Scholar 

  27. Etienne-Manneville S, Hall A (2002) Rho GTPases in cell biology. Nature 420:629–635

    Article  PubMed  CAS  Google Scholar 

  28. Schmidt A, Hall A (2002) Guanine nucleotide exchange factors for Rho GTPases: turning on the switch. Genes Dev 16:1587–1609

    Article  PubMed  CAS  Google Scholar 

  29. Wolfman A, Macara IG (1990) A cytosolic protein catalyzes the release of GDP from p21ras. Science 248:67–69

    Article  PubMed  CAS  Google Scholar 

  30. West M, Kung HF, Kamata T (1990) A novel membrane factor stimulates guanine nucleotide exchange reaction of ras proteins. FEBS Lett 259:245–248

    Article  PubMed  CAS  Google Scholar 

  31. Yamamoto T, Kaibuchi K, Mizuno T et al (1990) Purification and characterization from bovine brain cytosol of proteins that regulate the GDP/GTP exchange reaction of smg p21s, ras p21-like GTP-binding proteins. J Biol Chem 265:16626–16634

    PubMed  CAS  Google Scholar 

  32. Downward J, Riehl R, Wu L et al (1990) Identification of a nucleotide exchange-promoting activity for p21ra. Proc Natl Acad Sci U S A 87:5998–6002

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  33. Burstein ES, Macara IG (1992) Characterization of a guanine nucleotide-releasing factor and a GTPase-activating protein that are specific for the ras-related protein p25rab3A. Proc Natl Acad Sci U S A 89: 1154–1158

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  34. Bourne HR, Sanders DA, McCormick F (1991) The GTPase superfamily: conserved structure and molecular mechanism. Nature 349:117–127

    Article  PubMed  CAS  Google Scholar 

  35. Seabra MC, Wasmeier C (2004) Controlling the location and activation of Rab GTPases. Curr Opin Cell Biol 16:451–457

    Article  PubMed  CAS  Google Scholar 

  36. Saraogi I, Akopian D, Shan SO (2011) A tale of two GTPases in cotranslational protein targeting. Protein Sci 20:1790–1795

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  37. Ferro E, Trabalzini L (2010) RalGDS family members couple Ras to Ral signalling and that’s not all. Cell Signal 22:1804–1810

    Article  PubMed  CAS  Google Scholar 

  38. Malumbres M, Barbacid M (2003) RAS oncogenes: the first 30 years. Nat Rev Cancer 3: 459–465

    Article  PubMed  CAS  Google Scholar 

  39. Pylayeva-Gupta Y, Grabocka E, Bar-Sagi D (2011) RAS oncogenes: weaving a tumorigenic web. Nat Rev Cancer 11:761–774

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  40. Casey PJ (1992) Biochemistry of protein prenylation. J Lipid Res 33:1731–1740

    PubMed  CAS  Google Scholar 

  41. Downward J (2003) Targeting RAS signalling pathways in cancer therapy. Nat Rev Cancer 3:11–22

    Article  PubMed  CAS  Google Scholar 

  42. Kelley GG, Reks SE, Ondrako JM et al (2001) Phospholipase C(epsilon): a novel Ras effector. EMBO J 20:743–754

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  43. Peterson SN, Trabalzini L, Brtva TR et al (1996) Identification of a novel RalGDS-related protein as a candidate effector for Ras and Rap1. J Biol Chem 271:29903–29908

    Article  PubMed  CAS  Google Scholar 

  44. Cox AD, Hisaka MM, Buss JE et al (1992) Specific Isoprenoid Modification Is Required for Function of Normal, but Not Oncogenic Ras Protein. Mol Cell Biol 12:2606–2615

    PubMed  CAS  PubMed Central  Google Scholar 

  45. Mochizuki N, Yamashita S, Kurokawa K et al (2001) Spatio-temporal images of growth-factor-induced activation of Ras and Rap1. Nature 411:1065–1068

    Article  PubMed  CAS  Google Scholar 

  46. Retta SF, Balzac F, Avolio M (2006) Rap1: a turnabout for the crosstalk between cadherins and integrins. Eur J Cell Biol 85:283–293

    Article  PubMed  CAS  Google Scholar 

  47. Balzac F, Avolio M, Degani S et al (2005) E-cadherin endocytosis regulates the activity of Rap1: a traffic light GTPase at the crossroads between cadherin and integrin function. J Cell Sci 118:4765–4783

    Article  PubMed  CAS  Google Scholar 

  48. Campbell SL, Khosravi-Far R, Rossman KL et al (1998) Increasing complexity of Ras signaling. Oncogene 17:1395–1413

    Article  PubMed  CAS  Google Scholar 

  49. Cai W, Shi GX, Andres DA (2013) Putting the Rit in cellular resistance: Rit, p38 MAPK and oxidative stress. Commun Integr Biol 6:e22297

    Article  PubMed  PubMed Central  Google Scholar 

  50. Shi GX, Cai W, Andres DA (2013) Rit subfamily small GTPases: regulators in neuronal differentiation and survival. Cell Signal 25: 2060–2068

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  51. Sahai E, Marshall CJ (2002) Rho-GTPases and cancer. Nat Rev Cancer 2:133–142

    Article  PubMed  Google Scholar 

  52. Madaule P, Axel R (1985) A novel ras-related gene family. Cell 41:31–40

    Article  PubMed  CAS  Google Scholar 

  53. Ridley AJ (2012) Historical overview of Rho GTPases. Methods Mol Biol 827:3–12

    Article  PubMed  CAS  Google Scholar 

  54. Didsbury J, Weber RF, Bokoch GM et al (1989) Rac, a novel ras-related family of proteins that are botulinum toxin substrates. J Biol Chem 264:16378–16382

    PubMed  CAS  Google Scholar 

  55. Munemitsu S, Innis MA, Clark R et al (1990) Molecular cloning and expression of a G25K cDNA, the human homolog of the yeast cell cycle gene CDC42. Mol Cell Biol 10: 5977–5982

    PubMed  CAS  PubMed Central  Google Scholar 

  56. Ridley A (2013) GTPase switch: Ras then Rho and Rac. Nat Cell Biol 15:337

    Article  PubMed  CAS  Google Scholar 

  57. Ridley AJ, Hall A (1992) The small GTP-binding protein rho regulates the assembly of focal adhesions and actin stress fibers in response to growth factors. Cell 70:389–399

    Article  PubMed  CAS  Google Scholar 

  58. Ridley AJ, Hall A (2004) Snails, Swiss, and serum: the solution for Rac ‘n’ Rho. Cell 116:S23–5, 2 p following S25

    Article  PubMed  CAS  Google Scholar 

  59. Ridley AJ, Hall A (1992) The small GTP-binding protein rac regulates growth factor-induced membrane ruffling. Cell 70:401–410

    Article  PubMed  CAS  Google Scholar 

  60. Nobes CD, Hall A (1995) Rho, rac, and cdc42 GTPases regulate the assembly of multimolecular focal complexes associated with actin stress fibers, lamellipodia, and filopodia. Cell 81:53–62

    Article  PubMed  CAS  Google Scholar 

  61. Gonzalez-Ramos M, Mora I, de Frutos S et al (2012) Intracellular redox equilibrium is essential for the constitutive expression of AP-1 dependent genes in resting cells: studies on TGF-beta1 regulation. Int J Biochem Cell Biol 44:963–971

    Article  PubMed  CAS  Google Scholar 

  62. Karin M, Gallagher E (2005) From JNK to pay dirt: jun kinases, their biochemistry, physiology and clinical importance. IUBMB Life 57:283–295

    Article  PubMed  CAS  Google Scholar 

  63. Pinkus R, Weiner LM, Daniel V (1996) Role of oxidants and antioxidants in the induction of AP-1, NF-kappaB, and glutathione S-transferase gene expression. J Biol Chem 271:13422–13429

    Article  PubMed  CAS  Google Scholar 

  64. Liu M, Bi F, Zhou X et al (2012) Rho GTPase regulation by miRNAs and covalent modifications. Trends Cell Biol 22:365–373

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  65. Gallegos ME, Balakrishnan S, Chandramouli P et al (2012) The C. elegans rab family: identification, classification and toolkit construction. PLoS One 7:e49387

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  66. Pasqualato S, Renault L, Cherfils J (2002) Arf, Arl, Arp and Sar proteins: a family of GTP-binding proteins with a structural device for ‘front-back’ communication. EMBO Rep 3:1035–1041

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  67. Stewart M (2007) Molecular mechanism of the nuclear protein import cycle. Nat Rev Mol Cell Biol 8:195–208

    Article  PubMed  CAS  Google Scholar 

  68. Weis K (2003) Regulating access to the genome: nucleocytoplasmic transport throughout the cell cycle. Cell 112:441–451

    Article  PubMed  CAS  Google Scholar 

  69. Yudin D, Fainzilber M (2009) Ran on tracks—cytoplasmic roles for a nuclear regulator. J Cell Sci 122:587–593

    Article  PubMed  CAS  Google Scholar 

  70. Guttler T, Gorlich D (2011) Ran-dependent nuclear export mediators: a structural perspective. EMBO J 30:3457–3474

    Article  PubMed  PubMed Central  Google Scholar 

  71. Gibbs JB, Schaber MD, Marshall MS et al (1987) Identification of guanine nucleotides bound to ras-encoded proteins in growing yeast cells. J Biol Chem 262:10426–10429

    PubMed  CAS  Google Scholar 

  72. Satoh T, Kaziro Y (1995) Measurement of Ras-bound guanine nucleotide in stimulated hematopoietic cells. Methods Enzymol 255:149–155

    Article  PubMed  CAS  Google Scholar 

  73. Herrmann C, Martin GA, Wittinghofer A (1995) Quantitative analysis of the complex between p21ras and the Ras-binding domain of the human Raf-1 protein kinase. J Biol Chem 270:2901–2905

    Article  PubMed  CAS  Google Scholar 

  74. Benard V, Bohl BP, Bokoch GM (1999) Characterization of rac and cdc42 activation in chemoattractant-stimulated human neutrophils using a novel assay for active GTPases. J Biol Chem 274:13198–13204

    Article  PubMed  CAS  Google Scholar 

  75. Ren XD, Kiosses WB, Schwartz MA (1999) Regulation of the small GTP-binding protein Rho by cell adhesion and the cytoskeleton. EMBO J 18:578–585

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  76. Suryavanshi N, Ridley AJ (2013) Determining Rho GTPase Activity by an Affinity-Precipitation Assay. Methods Mol Biol 1046:191–202

    Article  PubMed  Google Scholar 

  77. Garcia-Mata R, Wennerberg K, Arthur WT et al (2006) Analysis of activated GAPs and GEFs in cell lysates. Methods Enzymol 406:425–437

    Article  PubMed  CAS  Google Scholar 

  78. Serebriiskii I, Khazak V, Golemis EA (1999) A two-hybrid dual bait system to discriminate specificity of protein interactions. J Biol Chem 274:17080–17087

    Article  PubMed  CAS  Google Scholar 

  79. Han L, Colicelli J (1995) A human protein selected for interference with Ras function interacts directly with Ras and competes with Raf1. Mol Cell Biol 15:1318–1323

    PubMed  CAS  PubMed Central  Google Scholar 

  80. Van Aelst L, Barr M, Marcus S et al (1993) Complex formation between RAS and RAF and other protein kinases. Proc Natl Acad Sci U S A 90:6213–6217

    Article  PubMed  PubMed Central  Google Scholar 

  81. White MA, Vale T, Camonis JH et al (1996) A role for the Ral guanine nucleotide dissociation stimulator in mediating Ras-induced transformation. J Biol Chem 271:16439–16442

    Article  PubMed  CAS  Google Scholar 

  82. Miyawaki A (2003) Visualization of the spatial and temporal dynamics of intracellular signaling. Dev Cell 4:295–305

    Article  PubMed  CAS  Google Scholar 

  83. Pollok BA, Heim R (1999) Using GFP in FRET-based applications. Trends Cell Biol 9:57–60

    Article  PubMed  CAS  Google Scholar 

  84. Tsien RY, Miyawaki A (1998) Seeing the machinery of live cells. Science 280:1954–1955

    Article  PubMed  CAS  Google Scholar 

  85. Hodgson L, Shen F, Hahn K (2010) Biosensors for characterizing the dynamics of rho family GTPases in living cells. Curr Protoc Cell Biol, Chapter 14, Unit 14.11: 1–26

    Google Scholar 

  86. Nakamura T, Matsuda M (2009) In vivo imaging of signal transduction cascades with probes based on Forster Resonance Energy Transfer (FRET). Curr Protoc Cell Biol, Chapter 14, Unit 14.10

    Google Scholar 

  87. Bos JL (2001) Glowing switches. Nature 411:1006–1007

    Article  PubMed  CAS  Google Scholar 

  88. Pertz O, Hodgson L, Klemke RL et al (2006) Spatiotemporal dynamics of RhoA activity in migrating cells. Nature 440:1069–1072

    Article  PubMed  CAS  Google Scholar 

  89. Machacek M, Hodgson L, Welch C et al (2009) Coordination of Rho GTPase activities during cell protrusion. Nature 461:99–103

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  90. Lam AJ, St-Pierre F, Gong Y et al (2012) Improving FRET dynamic range with bright green and red fluorescent proteins. Nat Methods 9:1005–1012

    Article  PubMed  CAS  PubMed Central  Google Scholar 

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Goitre, L., Trapani, E., Trabalzini, L., Retta, S.F. (2014). The Ras Superfamily of Small GTPases: The Unlocked Secrets. In: Trabalzini, L., Retta, S. (eds) Ras Signaling. Methods in Molecular Biology, vol 1120. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-791-4_1

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  • DOI: https://doi.org/10.1007/978-1-62703-791-4_1

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