Ras GTPase

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This article is about p21/Ras protein. For the p21/waf1 protein, see p21.
SCOP2
5p21 / SCOPe / SUPFAM
CDDcd04138
Available protein structures:
Pfam  structures / ECOD  
PDBRCSB PDB; PDBe; PDBj
PDBsumstructure summary

Ras, from "Rat sarcoma virus", is a family of related proteins that are expressed in all animal cell lineages and organs. All Ras protein family members belong to a class of protein called 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 three-dimensional 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

differentiation, and survival
. Mutations in Ras genes can lead to the production of permanently activated Ras proteins, which 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 three Ras genes in humans (HRAS, KRAS, and NRAS) 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.

History

The first two Ras genes,

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. Their gene symbols are HRAS, NRAS and KRAS, the latter of which produces the K-Ras4A and K-Ras4B isoforms from alternative splicing.[citation needed]

Structure

HRas 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

alpha helices.[14]
It consists of two domains: a G domain of 166
farnesyl transferase, RCE1, and ICMT.[citation needed
]

The G domain contains five G motifs that bind GDP/GTP directly. The G1 motif, or the P-loop, binds the beta phosphate of GDP and GTP. The G2 motif, also called Switch I or SW1, 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 or SW2, 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 (SW1) and G3 (SW2), are the main parts of the protein that move when GTP is hydrolyzed into GDP. 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. The two switch motifs have a number of conformations when binding GTP or GDP or no nucleotide (when bound to SOS1, which releases the nucleotide).[15]

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

cell 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

transcription of genes involved in cell growth and division.[16] Another Ras-activated signaling pathway is the PI3K/AKT/mTOR pathway
, which stimulates protein synthesis and cellular growth, and inhibits apoptosis.

Activation and deactivation

Ras is a

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

nucleophilic
attack on the gamma-phosphate of GTP. An inorganic phosphate is released and the Ras molecule is now bound to a GDP. Since the GDP-bound form is "off" or "inactive" for signaling, GTPase Activating Protein 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

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)[16] 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 a high affinity for numerous

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

Membrane attachment

Ras is attached to the

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.[18]

However, there are many other members of this subfamily as well:[19]

DIRAS3; ERAS; GEM; MRAS; NKIRAS1; NKIRAS2; 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 tumors.[18] 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.[20]
Ras inhibitor trans-farnesylthiosalicylic acid (FTS, Salirasib) exhibits profound anti-oncogenic effects in many cancer cell lines.[21][22]

Inappropriate activation

Inappropriate activation of the gene has been shown to play a key role in improper signal transduction, proliferation and malignant transformation.[16]

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.[citation needed]

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.[23]

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[24] 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 (Glutamine to Lysine) 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 studies suggested it reproduces well in certain cancer cell lines. It replicates specifically in cells that have an activated Ras pathway (a cellular signaling pathway that is involved in cell growth and differentiation).[25] 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.[citation needed
]

Another tumor-lysing virus that specifically targets tumor cells with an activated Ras pathway is a type II

Reolysin, a formulation of reovirus, and FusOn-H2 are currently in clinical trials or under development for the treatment of various cancers.[27] 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).[28]

In glioblastoma mouse models SHP2 levels were heightened in cancerous brain cells. Inhibiting SHP2 in turn inhibited Ras dephosphorylation. This reduced tumor sizes and accompanying rise in survival rates.[29][30]

Other strategies have attempted to manipulate the regulation of the above-mentioned localization of Ras.

farnesylation of Ras and therefore weaken its affinity to membranes.[2] Other inhibitors are targeting the palmitoylation cycle of Ras through inhibiting depalmitoylation by acyl-protein thioesterases, potentially leading to a destabilization of the Ras cycle.[31]

A novel inhibitor finding strategy for mutated Ras molecules was described in.[32] The Ras mutations in the 12th residue position inhibit the bound of the regulatory GAP molecule to the mutated Ras, causing uncontrolled cell growth. The novel strategy proposes finding small glue molecules, which attach the mutated Ras to the GAP, prohibiting uncontrolled cell growth and restoring the normal function. For this goal a theoretical Ras-GAP conformation was designed with a several Å gap between the molecules, and a high-throughput in silico docking was performed for finding gluing agents. As a proof of concept, two novel molecules were described with satisfying biological activity.

In other species

In most of the cell types of most species, most Ras is the GDP type. This is true for Xenopus oocytes and mouse fibroblasts.[33]

Xenopus laevis

As mentioned above most X. oocyte Ras is the GDP conjugate. Mammal Ras induces

p120Ras GAP in this pathway.[33]

Drosophila melanogaster

Expressed in all tissues of Drosophila melanogaster but mostly in neural cells. Overexpression is somewhat lethal and, during development, produces eye and wing abnormalities. (This parallels - and may be the reason for - similar abnormalities due to mutated receptor tyrosine kinases.) The D. genes for rases in mammals produce abnormalities.[33]

Aplysia

Most expression in Aplysia spp. is in neural cells.[33]

Caenorhabditis elegans

The gene in C. elegans is let 60. Also appears to play a role in receptor tyrosine kinase formation in this model. Overexpression yields a multivulval development due to its involvement in that region's normal development; overexpression in effector sites in lethal.[33]

Dictyostelium discoideum

Essential in

Adenylate cyclase activity is unaffected by ras.[33]

References

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Further reading

External links
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