1Ras-Raf-MAPK SOS1 SHC Grb2 Ras Raf1 MEK1 MEK2 ERK1 ERK2 The Ras-Raf-MEK-ERK pathway is activated by a range of growth factor receptors, including epidermal growth factor receptor, platelet-derived growth factor receptor, type-1 insulin-like growth factor receptor, and fibroblast growth factor receptors. The pathway can also be activated by cytokines, steroid hormones, and several agonists that act via G-protein-coupled receptors.Growth factor stimulation leads to a signal transduction cascade, which involves sequential activation of a number of cytoplasmic and nuclear targets.In this cascade, the adaptor protein SHC becomes phosphorylated and acts as a binding site for the second adaptor protein, growth factor receptor-bound protein 2 (Grb2).1Grb2 then recruits the Ras guanine nucleotide exchange factor SOS (son of sevenless) and mediates Ras activation.1Activated Ras leads to the activation of Raf proteins. Raf serine/threonine kinase activity phosphorylates MEK, which activates MEK catalytic activity.1,2MEK1/2 is a dual-specific kinase that is essential to the Ras-Raf-MEK-ERK pathway. All three Raf isoforms share Ras as a common upstream activator, whereas MEK is the only commonly accepted downstream substrate.1References1. Kolch W. Biochem J 2000; 351: 289–3052. Harmer SL, DeFranco AL. J Biol Chem 1999; 274: 12183–12191MEK2PERK1ERK2Grb2, growth factor receptor-bound protein 2
2Ras-Raf-MAPK Transcription Transcription factors SOS1 SHC Grb2 Ras MEK1PMEK1/2 serves to amplify signals to ERK1/2 (also known as MAPK1/2) through phosphorylation of key tyrosine and threonine residues, thereby activating its catalytic activity.Activation of ERK1/2 via MEK1/2 is a critical step in growth factor signaling, as active ERK mediates a range of cellular effects, eg cell proliferation.1Active ERK phosphorylates and activates numerous other kinases (eg RSK) and also transcription factors (eg SRF).1The activation of Ras, therefore, induces a sequential kinase cascade that includes Raf, MEK, and ERK.2References1. Kolch W. Biochem J 2000; 351: 289–3052. Harmer SL, DeFranco AL. J Biol Chem 1999; 274: 12183–12191MEK2PERK1PTranscriptionSRFERK2PTranscription factorsGrb2, growth factor receptor-bound protein 2; SRF, serum response factor
3Attivazione costitutiva di Ras-Raf-MAPK MEK1PConstitutive activation of the Ras-Raf-MEK-ERK pathway has been implicated in many cancers, including pancreatic, colon, and melanoma. This is caused by cancer-associated, mutational activation of B-Raf and Ras proteins.Mutant Ras and Raf proteins are key activators of the Ras-Raf-MEK-ERK pathway:Oncogenic Ras has been found in ~20-30% of human cancers, including ~25% of lung cancers, 50% of colon cancers, and >90% of pancreatic cancers.1B-Raf mutations have been identified in >60% of malignant melanomas and ~70% of papillary thyroid cancers.2,3In addition, uncontrolled growth factor signaling, eg through receptor gene amplification or ligand overexpression, is also a key constitutive activator of this pathway.4References1. Oliff A. Biochim Biophys Acta 1999; 1423: C19–C302. Davies H et al. Nature 2002; 417: 949–9543. Cohen Y et al. J Natl Cancer Inst 2003; 95: 625–6274. Kolch W. Biochem J 2000; 351: 289–305MEK2PERK1PTranscriptionSRFERK2P
4Signalling upstream of RAS Signalling upstream of RAS. The activation state of RAS is controlled by the cycle of hydrolysis of bound GTP, which is catalysed by GTPase activating proteins (GAPs), and the replacement of bound GDP with fresh GTP, which is catalysed by guanine nucleotide exchange factors (GEFs). The best-studied activation mechanism involves the assembly of complexes of activated, autophosphorylated growth-factor-receptor tyrosine kinases with the GEF SOS through the adaptor protein GRB2, and possibly SHC, resulting in the recruitment of SOS to the plasma membrane, where RAS is located. Several other GEFs exist that have distinct regulatory mechanisms. In addition, a wide range of GAPs have now been identified for RAS, some of which are also subject to regulation. RAS is also activated through GEFs in response to activation of a wide range of G-protein-coupled receptors.
5This illustration of the Ras signalling pathway highlights proteins affected by mutations in developmental disorders and cancer. Growth factor binding to cell-surface receptors results in activated receptor complexes, which contain adaptors such as SHC (SH2-containing protein), GRB2 (growth-factor-receptor bound protein 2) and Gab (GRB2-associated binding) proteins. These proteins recruit SHP2 and SOS1, the latter increasing Ras–guanosine triphosphate (Ras–GTP) levels by catalysing nucleotide exchange on Ras. The GTPase-activating protein (GAP) neurofibromin (NF1) binds to Ras–GTP and accelerates the conversion of Ras–GTP to Ras–GDP (guanosine diphosphate), which terminates signalling. Several Ras–GTP effector pathways have been described, and some of the key effectors are depicted here. The BRAF–mitogen-activated and extracellular-signal regulated kinase kinase (MEK)–extracellular signal-regulated kinase (ERK) cascade often determines proliferation and becomes deregulated in certain cancers and in developmental disorders such as cardio-facio-cutaneous syndrome. Ras also activates the phosphatidylinositol 3-kinase (PI3K)– 3-phosphoinositide-dependent protein kinase 1 (PDK1)–Akt pathway that frequently determines cellular survival. RALGDS, RALGDS-like gene (RGL), RGL2 and TIAM1 are exchange factors of Ral and Rac, respectively. Among the effectors of Ral is phospholipase D (PLD) an enzyme that regulates vesicle trafficking. Rac regulates actin dynamics and, therefore, the cytoskeleton. Ras also binds and activates the enzyme phospholipase C (PLC ), the hydrolytic products of which regulate calcium signalling and the protein kinase C (PKC) family. P, phosphate; Y, receptor tyrosine.
6Regulation of Ras. Ras proteins are GTPases that cycle between an activated GTP-bound and an inactivated GDP-bound form. Guanine nucleotide exchange factors (GEFs) induce dissociation of GDP, which allows binding of GTP. GTPase-activating proteins trigger the hydrolysis of bound GTP. Oncogenic Ras remains in the active GTP-bound form, because the GAP-induced GTP hydrolysis is completely abrogated.
7HRAS, NRAS, KRAS4A and KRAS4B are highly homologous throughout the G domain (amino acids 1–165). The first 85 amino acids are identical in all four proteins and specify binding to guanosine diphosphate (GDP) and guanosine triphosphate (GTP). This includes the P loop (phosphate-binding loop, amino acids 10–16), which binds the -phosphate of GTP, and switch I (amino acids 32–38) and II (amino acids 59–67) which regulate binding to Ras regulators and effectors. The next 80 amino acids (85–165) show 85–90% sequence identity. The C-terminal hypervariable domain (amino acids 165–188/189) specifies membrane localization through post-translational modifications that include the farnesylation of each isoform on the C-terminal CAAX motif (CVLS, CVVM, CIIM and CVIM, respectively) and palmitoylation of key cysteines on HRAS, NRAS and KRAS4A; these cysteines are highlighted below each representation (C). Membrane localization of KRAS4B is facilitated by a stretch of lysines (KKKKKK) proximal to the CVIM motif. To highlight the degree of homology, a box at the bottom of each isoform representation shows the conserved residues in magenta and the variable residues in pink. Somatic RAS mutations found in cancer introduce amino-acid substitutions at positions 12, 13 and 61
9RAS processing and association with the plasma membrane RAS processing and association with the plasma membrane. The four RAS proteins (HRAS, KARAS, KBRAS and NRAS) are synthesized initially as cytosolic, inactive proteins. They undergo a rapid series of post-translational modifications signalled by the carboxy-terminal CAAX tetrapeptide sequence. FTase uses farnesyl diphosphate (FPP) and catalyses the covalent addition of the 15-C farnesyl isoprenoid (F) to the cysteine residue of the CAAX sequence, followed by endoprotease (RCE1) cleavage of the AAX residues and, finally, carboxylmethylation (ICMT) of the now terminal farnesylated cysteine residue. Inhibitors of all three steps have been developed, but only FTase inhibitors (FTIs) have advanced to evaluation in clinical trials. KRAS and NRAS proteins undergo alternative prenylation when FTase activity is blocked by FTIs, resulting in their modification by geranylgeranyl transferase I (GGTase I), which uses geranylgeranyl pyrophosphate (GGPP) and catalyses the addition of the 20-C geranylgeranyl isoprenoid (GG) to the cysteine residue. So, whereas RAS proteins are not normally modified by GG, the GG-modified forms of RAS retain membrane association and transforming activity.
10| HRAS, NRAS and KRAS4A are prenylated (HRAS is only farnesylated, whereas NRAS and KRAS4A can be farnesylated or geranylgeranylated) before undergoing proteolytic removal of the AAX tripeptide by RAS converting enzyme 1 (RCE1) and carboxymethylation by isoprenylcysteine carboxymethyltransferase (ICMT) in the endoplasmic reticulum (ER). Subsequently, they are palmitoylated in the Golgi and transferred to the plasma membrane (PM) to which they attach through their farnesyl (F) or geranylgeranyl (GG), and palmitoyl moieties. b | KRAS4B can be farnesylated or geranylgeranylated and then undergoes proteolytic removal of the AAX tripeptide by RCE1 and carboxymethylation by ICMT in the ER. It does not undergo palmitoylation, but attaches to the PM through its farnesyl moiety and a polybasic, lysine-rich sequence located near the terminal cysteine. FPP, farnesyl pyrophosphate; FTase, farnesyltransferase; GGPP, geranylgeranyl pyrophosphate; GGTase I, geranylgeranyltransferase I; SAM, S-adenyosyl methionine.
11Fungi, mammals and archaebacteria exclusively use the mevalonate pathway for biosynthesis of isoprenoids. 3-hydroxy 3-methylglutaryl-CoA (HMG-CoA) is converted to mevalonate by HMG-CoA reductase, the rate-limiting enzyme at the apex of the mevalonate pathway. Mevalonate is then converted to isopentenyl pyrophosphate (isopentenyl-PP; the 5-carbon basic isoprene unit), which is subsequently converted to farnesyl pyrophosphate (farnesyl-PP; a 15-carbon isoprenoid) through a series of enzymatic reactions. After addition of isopentenyl-PP, farnesyl-PP can be converted to geranylgeranyl pyrophosphate (geranylgernayl-PP; a 20-carbon isoprenoid), or alternatively farnesyl-PP can be converted to cholesterol or dolichyl phosphate (dolichyl-P), which is used for N-glycosylation of growth factor receptors such as insulin-like growth factor receptor. HMG-CoA reductase is the target of the cholesterol-lowering statins, whereas isopentenyl-PP isomerase and farnesyl-PP synthase are targets of bisphosphonates. Importantly, in normal cells, cholesterol and isoprenoid products suppress HMG-CoA reductase via post-translational downregulation. Conversely, tumour cells are resistant to cholesterol-mediated suppression, although they remain sensitive to isoprenoid-mediated suppression. Addition of isoprenoids to statins may prevent upregulation of HMG-CoA reductase, which might occur as a resistance mechanism to statin therapy. This explains the synergistic activity of statins and isoprenoids. CAAX, C denotes cysteine, A represents any aliphatic amino acid and X may be any amino acid; FTase, farnesyltransferase; GGTase, geranylgeranyltransferase.
13Ras-INDEPENDENT EFFECT FOR FTIs EVIDENCE FOR ARas-INDEPENDENT EFFECT FOR FTIsLack of correlation between susceptibility and Ras statusDifferences in the kinetics of development of the effectsInhibition of K-Ras- and N-Ras-dependent cell growth
14Farnesyltransferase inhibitors (FTIs) inhibit FTase modification of RAS and other proteins. FTIs are effective inhibitors of HRAS farnesylation and function. FTIs also prevent the farnesylation of KRAS and NRAS; however, these RAS isoforms then undergo alternative prenylation and modification by geranylgeranylation. Experimental evidence supports the possibility that RHOB proteins are important targets for the antitumour activity of FTIs. The increased formation of RHOB-GG, which might have a growth-inhibitory function, is proposed to be the mechanism for this inhibition. However both RHOB-F and RHOB-GG have been shown to inhibit tumour growth in vitro and in vivo51. Other candidate targets for proteins that mediate the antitumour effects of FTIs include other RAS-family GTPases (RHEB and RND), CENP and PRL. Some farnesylated RAS-family proteins (RIG and NOEY2) seem to function as tumour suppressors; so, the loss of function that is caused by FTI treatment might promote, rather than prevent, proliferation in some tissues.
15Statins, biphosphonates and isoprenoids, alone or in combinations, target the mevalonate pathway, whereas farnesyltransferase inhibitors (FTIs), geranylgeranyltransferase inhibitors (GGTIs) and dual prenylation inhibitors (DPIs) target the prenylation process. Several strategies have been developed for targeting the functional regulation of the RAS superfamily of GTPases. These focus either on compounds that inhibit the interaction between the RAS superfamily of GTPases and regulatory proteins (for example, interfacial inhibitors), or on drugs that target individual RAS superfamily GTPases (for example, bacterial toxins, GTP analogues, small-interfering RNA (siRNA) inhibitors), or regulatory proteins (siRNA inhibition), thereby blocking GDP/GTP exchange and inhibiting activation of downstream effectors. GAP, GTPase-activating protein; GDI, guanine nucleotide dissociation inhibitor; GEF, guanine nucleotide exchange factor; ICMT, isoprenylcysteine carboxymethyltransferase; RCE1, RAS converting enzyme.
17Fig. 2. Attenuation of reovirus by viral and cellular adaptation during persistent reovirus infection. Co-adaptation of reovirus and its cellularhost resulted in mutation and truncation of the viral S1 gene coding sequence. This in turn caused an attenuation of viral apoptoticpotential in healthy cells and tissues (Kim et al., 2007a) while retaining the ability to eliminate tumors in vivo. The mouse on the left wasinjected in the flank with tumor cells showing massive proliferation, whereas the mouse on the right was injected with tumor cells plusattenuated virus, completely eliminating tumor growth.
18Attivazione costitutiva di Ras-Raf-MAPK MEK1PConstitutive activation of the Ras-Raf-MEK-ERK pathway has been implicated in many cancers, including pancreatic, colon, and melanoma. This is caused by cancer-associated, mutational activation of B-Raf and Ras proteins.Mutant Ras and Raf proteins are key activators of the Ras-Raf-MEK-ERK pathway:Oncogenic Ras has been found in ~20-30% of human cancers, including ~25% of lung cancers, 50% of colon cancers, and >90% of pancreatic cancers.1B-Raf mutations have been identified in >60% of malignant melanomas and ~70% of papillary thyroid cancers.2,3In addition, uncontrolled growth factor signaling, eg through receptor gene amplification or ligand overexpression, is also a key constitutive activator of this pathway.4References1. Oliff A. Biochim Biophys Acta 1999; 1423: C19–C302. Davies H et al. Nature 2002; 417: 949–9543. Cohen Y et al. J Natl Cancer Inst 2003; 95: 625–6274. Kolch W. Biochem J 2000; 351: 289–305MEK2PERK1PTranscriptionSRFERK2P
19Structure of the RAF proteins Structure of the RAF proteins. The RAF-isoforms, A-RAF, B-RAF and C-RAF, share three conserved regions: CR1 (yellow), CR2 (orange) and CR3 (red). The amino acids that are highlighted below the individual isoforms refer to known phosphorylation sites. CR1 contains the RAS-binding domain (RBD) and the cysteine-rich domain (CRD), which are both required for membrane recruitment. Phosphorylation of S43, just N-terminal to this region, blocks C-RAF binding to RAS, probably through steric hindrance. CR2 contains one of the binding sites, which encompasses S259 (the numbering refers to C-RAF). The other two binding sites are centred on S233 and S621. CR3 contains the catalytic domain (the activation segment is highlighted in pink). The negative-charge regulatory region (N-region) is just upstream of CR3 and contains residue Y341, which is conserved in A-RAF (Y302), but is replaced by D448 in B-RAF (shown as a grey box). S338 is conserved in all RAF proteins (S299 in A-RAF and S445 in B-RAF), but it is constitutively phosphorylated in B-RAF (blue star). The catalytic domain contains the two activation-segment phosphorylation sites T491 and S494, which are conserved in A-RAF (T452 and T455) and B-RAF (T598 and S601).
20SORAFENIB (Nexavar®): Low molecular weight inhibitor of VEGFR, PDGFR, KIT, RET and Raf-1 kinase activity
21Mechanism of action of sorafenib Sorafenib blocks receptor tyrosine kinase signalling (VEGFR, PDGFR, c-Kit and RET) and inhibits downstream Raf serine/threonine kinase activity to prevent tumour growth by anti-angiogenic, antiproliferative and/or pro-apoptotic effects. ERK, extracellular signal-regulated kinase; GDNF, glial-derived neurotrophic factor; MEK, mitogen-activated protein kinase kinase; PDGFR, platelet-derived growth factor receptor; SCF, stem cell factor; VEGFR, vascular endothelial growth factor receptor.