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Ras subfamily

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Ras subfamily

H-Ras structure PDB 121p, surface colored by conservation in Pfam seed alignment: gold, most conserved; dark cyan, least conserved.
Identifiers
Symbol Ras
Pfam PF00071
InterPro IPR013753
PROSITE PDOC00017
SCOP 5p21
SUPERFAMILY 5p21
OPM protein 1uad
CDD cd04138

Ras is a small GTPase, and are involved in transmitting signals within cells (cellular signal transduction). Ras is the prototypical member of the Ras superfamily of proteins, which are all related in 3D structure and regulate diverse cell behaviours.

When Ras is 'switched on' by incoming signals, it subsequently switches on other proteins, which ultimately turn on genes involved in cell growth, differentiation and survival. As a result, mutations in ras genes can lead to the production of permanently activated Ras proteins. This can cause unintended and overactive signaling inside the cell, even in the absence of incoming signals.

Because these signals result in cell growth and division, overactive Ras signaling can ultimately lead to cancer.[1] The 3 Ras genes in humans (H-ras, K-ras, and N-ras) are the most common oncogenes in human cancer; mutations that permanently activate Ras are found in 20% to 25% of all human tumors and up to 90% in certain types of cancer (e.g., pancreatic cancer).[2] For this reason, Ras inhibitors are being studied as a treatment for cancer, and other diseases with Ras overexpression.

Contents

  • History 1
  • Structure 2
  • Function 3
    • Activation and deactivation 3.1
    • Membrane attachment 3.2
  • Members 4
  • Ras in cancer 5
    • Inappropriate activation 5.1
    • Constitutively active Ras 5.2
    • Ras-targeted cancer treatments 5.3
  • References 6
  • Further reading 7
  • External links 8

History

The first two ras genes, H-ras and K-ras, were first identified[3] from studies of two cancer-causing viruses, the Harvey sarcoma virus and Kirsten sarcoma virus, by Edward M. Scolnick and colleagues at the National Institutes of Health (NIH).[4] These viruses were discovered originally in rats during the 1960s by Jennifer Harvey[5] and Werner Kirsten,[6] respectively, hence the name Rat sarcoma.[3] In 1982, activated and transforming human ras genes were discovered in human cancer cells by Geoffrey M. Cooper at Harvard,[7] Mariano Barbacid and Stuart A. Aaronson at the NIH,[8] Robert Weinberg at MIT,[9] and Michael Wigler at Cold Spring Harbor Laboratory.[10] A third ras gene was subsequently discovered by researchers in the group of Robin Weiss at the Institute of Cancer Research,[11][12] and Michael Wigler at Cold Spring Harbor Laboratory,[13] named NRAS, for its initial identification in human neuroblastoma cells.

The three human ras genes encode extremely similar proteins made up of chains of 188 to 189 amino acids, designated H-Ras, N-Ras and K-Ras4A and K-Ras4B (the two K-Ras proteins arise from alternative splicing).

Structure

H-Ras structure PDB 121p, ribbon showing strands in purple, helices in aqua, loops in gray. Also shown are the bound GTP analog and magnesium ion.

Ras contains six strands of beta sheet and five alpha helices:[14] It consists of two domains, a G domain of 166 amino acids, about 20kDa, that binds guanosine nucleotides, and a C terminal membrane targeting region (CAAX-COOH, also known as CAAX box) which is lipid-modified by farnesyl transferase, RCE1 and ICMT.

The G domain contains five G motifs that bind GDP/GTP directly. The G1motif, or the P-loop, binds the beta phosphate of GDP and GTP. The G2 motif, also called Switch I, contains threonine35, which binds the terminal phosphate (γ-phosphate) of GTP and the divalent magnesium ion bound in the active site. The G3 motif, also called Switch II, has a DXXGQ motif. The D is aspartate57, which is specific for guanine versus adenine binding, and Q is glutamine61, the crucial residue that activates a catalytic water molecule for hydrolysis of GTP to GDP. The G4 motif contains a LVGNKxDL motif, and provides specific interaction to guanine. The G5 motif contains a SAK consensus sequence. The A is alanine146, which provides specificity for guanine rather than adenine.

The two switch motifs G2 and G3 are the main parts of the protein that move upon activation by GTP. This conformational change by the two switch motifs is what mediates the basic functionality as a molecular switch protein. This GTP bound state of Ras is the "on" state, and the GDP bound state is the "off" state.

Ras also binds a magnesium ion which helps to coordinate nucleotide binding.

Function

Overview of signal transduction pathways involved in apoptosis.

Ras proteins function as binary molecular switches that control intracellular signaling networks. Ras-regulated signal pathways control such processes as actin cytoskeletal integrity, proliferation, differentiation, cell adhesion, apoptosis, and cell migration. Ras and ras-related proteins are often deregulated in cancers, leading to increased invasion and metastasis, and decreased apoptosis.

Ras activates several pathways, of which the mitogen-activated protein (MAP) kinase cascade has been well-studied. This cascade transmits signals downstream and results in the transcription of genes involved in cell growth and division.[15] There is a separate AKT pathway that inhibits apoptosis.

Activation and deactivation

Ras is a G protein, or a guanosine-nucleotide-binding protein. Specifically, it is a single-subunit small GTPase, which is related in structure to the Gα subunit of heterotrimeric G proteins (large GTPases). G proteins function as binary signaling switches with "on" and "off" states. In the "off" state it is bound to the nucleotide guanosine diphosphate (GDP), while in the "on" state, Ras is bound to guanosine triphosphate (GTP), which has an extra phosphate group as compared to GDP. This extra phosphate holds the two switch regions in a "loaded-spring" configuration (specifically the Thr-35 and Gly-60). When released, the switch regions relax which causes a conformational change into the inactive state. Hence, activation and deactivation of Ras and other small G proteins are controlled by cycling between the active GTP-bound and inactive GDP-bound forms.

The process of exchanging the bound nucleotide is facilitated by inactivates Ras by activating its GTPase activity. Thus, GAPs accelerate Ras inactivation.

GEFs catalyze a "push and pull" reaction which releases GDP from Ras. They insert close to the P-loop and magnesium cation binding site and inhibit the interaction of these with the gamma phosphate anion. Acidic (negative) residues in switch II "pull" a lysine in the P-loop away from the GDP which "pushes" switch I away from the guanine. The contacts holding GDP in place are broken and it is released into the cytoplasm. Because intracellular GTP is abundant relative to GDP (approximately 10 fold more[15] GTP predominantly re-enters the nucleotide binding pocket of Ras and reloads the spring. Thus GEFs facilitate Ras activation.[14] Well known GEFs include Son of Sevenless (Sos) and cdc25 which include the RasGEF domain.

The balance between GEF and GAP activity determines the guanine nucleotide status of Ras, thereby regulating Ras activity.

In the GTP-bound conformation, Ras has high affinity for numerous effectors which allow it to carry out its functions. These include PI3K. Other small GTPases may bind adaptors such as arfaptin or second messenger systems such as adenylyl cyclase. The Ras binding domain is found in many effectors and invariably binds to one of the switch regions, because these change conformation between the active and inactive forms. However, they may also bind to the rest of the protein surface.

Other proteins exist which may augment the activity of Ras family proteins. One example is GDI (GDP Disassociation Inhibitor); These function by slowing the exchange of GDP for GTP and thus, prolonging the inactive state of Ras family members. Other proteins that further augment this cycle may exist.

Membrane attachment

Ras is attached to the cell membrane owing to its prenylation and palmitoylation (HRAS and NRAS) or the combination of prenylation and a polybasic sequence adjacent to the prenylation site (KRAS). The C-terminal CaaX box of Ras first gets farnesylated at its Cys residue in the cytosol, allowing Ras to loosely insert into the membrane of the endoplasmatic reticulum and other cellular membranes. The Tripeptide (aaX) is then cleaved from the C-terminus by a specific prenyl-protein specific endoprotease and the new C-terminus is methylated by a methyltransferase. K-Ras procession is completed at this stage. Dynamic electrostatic interactions between its positively charged basic sequence with negative charges at the inner leaflet of the plasma membrane account for its predominant localization at the cell surface at steady-state. NRAS and HRAS are further processed on the surface of the Golgi apparatus by palmitoylation of one or two Cys residues, respectively, adjacent to the CaaX box. The proteins thereby become stably membrane anchored (lipid-rafts) and are transported to the plasma membrane on vesicles of the secretory pathway. Depalmitoylation eventually releases the proteins from the membrane, allowing them to enter another cycle of palmitoylation and depalmitoylation.[16] This cycle is believed to prevent the leakage of NRAS and HRAS to other membranes over time and to maintain their steady-state localization along the Golgi apparatus, secretory pathway, plasma membrane and inter-linked endocytosis pathway.

Members

The clinically most notable members of the Ras subfamily are HRAS, KRAS and NRAS, mainly for being implicated in many types of cancer.[17]

However, there are many other members of this subfamily as well:[18] DIRAS1; DIRAS2; DIRAS3; ERAS; GEM; MRAS; NKIRAS1; NKIRAS2; NRAS; RALA; RALB; RAP1A; RAP1B; RAP2A; RAP2B; RAP2C; RASD1; RASD2; RASL10A; RASL10B; RASL11A; RASL11B; RASL12; REM1; REM2; RERG; RERGL; RRAD; RRAS; RRAS2

Ras in cancer

Mutations in the Ras family of proto-oncogenes (comprising H-Ras, N-Ras and K-Ras) are very common, being found in 20% to 30% of all human tumours.[17] it is reasonable to speculate that a pharmacological approach that curtails Ras activity may represent a possible method to inhibit certain cancer types. Ras point mutations are the single most common abnormality of human proto-oncogenes.[19] Ras inhibitor trans-farnesylthiosalicylic acid (FTS, Salirasib) exhibits profound anti-oncogenic effects in many cancer cell lines.[20][21]

Inappropriate activation

Inappropriate activation of the gene has been shown to play a key role in signal transduction, proliferation and malignant transformation.[15]

Mutations in a number of different genes as well as RAS itself can have this effect. Oncogenes such as p210BCR-ABL or the growth receptor erbB are upstream of Ras, so if they are constitutively activated their signals will transduce through Ras.

The tumour suppressor gene NF1 encodes a Ras-GAP – its mutation in neurofibromatosis will mean that Ras is less likely to be inactivated. Ras can also be amplified, although this only occurs occasionally in tumours.

Finally, Ras oncogenes can be activated by point mutations so that the GTPase reaction can no longer be stimulated by GAP – this increases the half life of active Ras-GTP mutants.[22]

Constitutively active Ras

Constitutively active Ras (RasD) is one which contains mutations that prevent GTP hydrolysis, thus locking Ras in a permanently 'On' state.

The most common mutations are found at residue G12 in the P-loop and the catalytic residue Q61.

  • The glycine to valine mutation at residue 12 renders the GTPase domain of Ras insensitive to inactivation by GAP and thus stuck in the "on state". Ras requires a GAP for inactivation as it is a relatively poor catalyst on its own, as opposed to other G-domain-containing proteins such as the alpha subunit of heterotrimeric G proteins.
  • Residue 61[23] is responsible for stabilizing the transition state for GTP hydrolysis. Because enzyme catalysis in general is achieved by lowering the energy barrier between substrate and product, mutation of Q61 to K necessarily reduces the rate of intrinsic Ras GTP hydrolysis to physiologically meaningless levels.

See also "dominant negative" mutants such as S17N and D119N.

Ras-targeted cancer treatments

Reovirus was noted to be a potential cancer therapeutic when early studies on reovirus suggested it reproduces well in certain cancer cell lines. It has since been shown to replicate specifically in cells that have an activated Ras pathway (a cellular signaling pathway that is involved in cell growth and differentiation).[24] Reovirus replicates in and eventually kills Ras-activated tumour cells and as cell death occurs, progeny virus particles are free to infect surrounding cancer cells. This cycle of infection, replication and cell death is believed to be repeated until all tumour cells carrying an activated Ras pathway are destroyed. Another tumor lysing virus that specifically targets tumor cells with an activated Ras pathway is a type II herpes simplex virus (HSV-2) based agent, designated FusOn-H2.[25] Activating mutations of the Ras protein and upstream elements of the Ras protein may play a role in more than two thirds of all human cancers, including most metastatic disease. Reolysin, a formulation of reovirus, and FusOn-H2 are currently in clinical trials or under development for the treatment of various cancers.[26] In addition, a treatment based on siRNA anti mutated K-RAS (G12D) called siG12D LODER is currently in clinical trials for the treatment of locally advanced pancreatic cancer (NCT01188785, NCT01676259).[27]

References

  1. ^ Goodsell DS (1999). "The molecular perspective: the ras oncogene". Oncologist 4 (3): 263–4.  
  2. ^ Downward J (January 2003). "Targeting RAS signalling pathways in cancer therapy". Nat. Rev. Cancer 3 (1): 11–22.  
  3. ^ a b Malumbres M, Barbacid M (June 2003). "RAS oncogenes: the first 30 years". Nat. Rev. Cancer 3 (6): 459–65.  
  4. ^ Chang EH, Gonda MA, Ellis RW, Scolnick EM, Lowy DR (August 1982). "Human genome contains four genes homologous to transforming genes of Harvey and Kirsten murine sarcoma viruses". Proc. Natl. Acad. Sci. U.S.A. 79 (16): 4848–52.  
  5. ^ Harvey JJ (December 1964). "An unidentified virus which causes the rapid production of tumours in mice". Nature 204 (4963): 1104–5.  
  6. ^ Kirsten WH, Schauf V, McCoy J (1970). "Properties of a murine sarcoma virus". Bibl Haematol (36): 246–9.  
  7. ^ Cooper GM (August 1982). "Cellular transforming genes". Science 217 (4562): 801–6.  
  8. ^ Santos E, Tronick SR, Aaronson SA, Pulciani S, Barbacid M (July 1982). "T24 human bladder carcinoma oncogene is an activated form of the normal human homologue of BALB- and Harvey-MSV transforming genes". Nature 298 (5872): 343–7.  
  9. ^ Parada LF, Tabin CJ, Shih C, Weinberg RA (June 1982). "Human EJ bladder carcinoma oncogene is homologue of Harvey sarcoma virus ras gene". Nature 297 (5866): 474–8.  
  10. ^ Taparowsky E, Suard Y, Fasano O, Shimizu K, Goldfarb M, Wigler M (December 1982). "Activation of the T24 bladder carcinoma transforming gene is linked to a single amino acid change". Nature 300 (5894): 762–5.  
  11. ^ Marshall CJ, Hall A, Weiss RA (September 1982). "A transforming gene present in human sarcoma cell lines". Nature 299 (5879): 171–3.  
  12. ^ Hall A, Marshall CJ, Spurr NK, Weiss RA (1983). "Identification of transforming gene in two human sarcoma cell lines as a new member of the ras gene family located on chromosome 1". Nature 303 (5916): 396–400.  
  13. ^ Shimizu K, Goldfarb M, Perucho M, Wigler M (January 1983). "Isolation and preliminary characterization of the transforming gene of a human neuroblastoma cell line". PNAS 80 (2): 383–7.  
  14. ^ a b Vetter IR, Wittinghofer A (November 2001). "The guanine nucleotide-binding switch in three dimensions". Science 294 (5545): 1299–304.  
  15. ^ a b c Lodish H, Berk A, Zipursky SL, Matsudaira P, Baltimore D, Darnell J (2000). "Chapter 25, Cancer". Molecular cell biology (4th ed.). San Francisco: W.H. Freeman.  
  16. ^ Rocks O, Peyker A, Bastiaens PI (2006). "Spatio-temporal segregation of Ras signals: one ship, three anchors, many harbors". Current Opinion in Cell Biology 18 (4): 351–7.  
  17. ^ a b Bos J (1989). "ras oncogenes in human cancer: a review". Cancer Res 49 (17): 4682–9.  
  18. ^ Wennerberg K, Rossman KL, Der CJ (March 2005). "The Ras superfamily at a glance". J. Cell. Sci. 118 (Pt 5): 843–6.  
  19. ^ Robbins and Cotran (2010). Pathologic Basis of Disease 8th ed. p. 282. 
  20. ^ Rotblat B, Ehrlich M, Haklai R, Kloog Y (2008). "The Ras inhibitor farnesylthiosalicylic acid (Salirasib) disrupts the spatiotemporal localization of active Ras: a potential treatment for cancer.". Methods Enzymol 439: 467–89.  
  21. ^ Roy Blum, yoel kloog (2005). "Ras Inhibition in Glioblastoma Down-regulates Hypoxia-Inducible Factor-1, Causing Glycolysis Shutdown and Cell Death". Cancer Research 65 (3): 999–1006.  
  22. ^ Reuter C, Morgan M, Bergmann L (2000). "Targeting the Ras signaling pathway: a rational, mechanism-based treatment for hematologic malignancies?". Blood 96 (5): 1655–69.  
  23. ^ Omim - Neuroblastoma Ras Viral Oncogene Homolog; Nras
  24. ^ Lal R, Harris D, Postel-Vinay S, de Bono J (October 2009). "Reovirus: Rationale and clinical trial update". Curr. Opin. Mol. Ther. 11 (5): 532–9.  
  25. ^ Fu, Xinping; Prigge-J, Cai-R; Xiaoliu Zhang. "A mutant type 2 herpes simplex virus deleted for the protein kinase domain of the ICP10 gene is a potent oncolytic virus". Molecular Therapy 13 (5): 882–890.  
  26. ^ Thirukkumaran C, Morris DG (2009). "Oncolytic viral therapy using reovirus". Methods Mol. Biol. 542: 607–34.  
  27. ^ "ClinicalTrials.gov". 

Further reading

  • Agrawal AG, Somani RR (June 2009). "Farnesyltransferase inhibitor as anticancer agent". Mini Rev Med Chem 9 (6): 638–52.  
  • Agrawal G, Somani RR (2011). "Farnesyltransferase Inhibitor in Cancer Treatment, Current Cancer Treatment". In Özdemir Ö. Current Cancer Treatment - Novel Beyond Conventional Approaches. InTech.  

External links

  • "Brain tumour findings offer hope of new strategy Canadian Cancer Society says" at ncic.cancer.ca
  • "Novel cancer treatment gets NCI support" at arstechnica.com
  • ras Proteins at the US National Library of Medicine Medical Subject Headings (MeSH)
  • ras Genes at the US National Library of Medicine Medical Subject Headings (MeSH)
  • - The Interactive FlyRas oncogene at 85D Drosophila
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