Ras oncogenes: split personalities

Abstract

Extensive research on the Ras proteins and their functions in cell physiology over the past 30 years has led to numerous insights that have revealed the involvement of Ras not only in tumorigenesis but also in many developmental disorders. Despite great strides in our understanding of the molecular and cellular mechanisms of action of the Ras proteins, the expanding roster of their downstream effectors and the complexity of the signalling cascades that they regulate indicate that much remains to be learnt.

Timeline. Key events in the field of Ras research

GAP, GTPase-activating proteins; GEF, guanine nucleotide-exchange factors; MAPK, mitogen-activated protein kinase; PI3K, phosphoinositide 3-kinase; PIK3A, phosphatidyl-inositol-4,5-bisphosphate 3-kinase catalytic subunit-α; PLC, phospholipase C; RalGDS, Ral guanine nucleotide-dissociation stimulator; SV40, simian virus-40;TERT, telomerase reverse transcriptase; TIAM1, T-cell lymphoma invasion and metastasis-1.

Figure 1. The ras family of small gTPases

a | The primary structure of Ras proteins. The Ras family of GTPases encompasses 36 genes, which encode 39 Ras proteins (20–29 kDa) in the human genome. Most of the Ras family proteins have been characterized and found to regulate important cellular processes, such as growth, cytoskeletal rearrangements, adhesion, motility and differentiation. Secondary structural elements are shown as arrows (for β-sheets, yellow) and cylinders (for α-helices, blue). Ras family members share substantial primary sequence homology in their N termini, particularly in the phosphate-binding loop (P-loop) and the nucleotide-sensitive switch I and II regions. The C terminus contains the membrane-targeting CAAX sequences. H-Ras isoform-1 numbering indicated. b | Overall sequence comparison among Ras proteins. The Ras family radial tree was generated using matrices derived from a ClustalW multiple sequence alignment of the various Ras family members. a.a., amino acids.

Figure 2. C-terminal processing of ras proteins

a | C-terminal sequences. The C terminus is highly divergent among the Ras family members and contains the membrane-targeting sequences. The C-terminal sequences of Kirsten- (K-), Harvey- (H-) and neuroblastoma- (N-) Ras proteins are shown. Cys residues in red or green are farnesylated or palmitoylated, respectively. b | C-terminal processing. A farnesyl pyrophosphate moiety (orange) is covalently attached to the newly-synthesized cytoplasmic Ras proteins by the enzyme farnesyltransferase (FTase)^–. This reaction is followed, in the endoplasmic reticulum, by the proteolytic cleavage of the last three amino-acid residues (AAX) by Ras-converting enzyme-1 (RCE1), and by the carboxymethylation of the last Cys residue by ICMT1 (for example, see REF. 224). The C-terminal Lys residues in K-Ras-4B are sufficient to anchor it in the membrane, whereas H-, N- and K-Ras-4A require a palmitoylation step (by a palmitoyltransferase (PTase)) in which a palmitoyl moiety (red) is attached to the C-terminal upstream Cys residues before their insertion in the membrane is stabilized. In the presence of FTase inhibitors, both K-Ras-4A and N-Ras can become prenylated by geranylgeranyl transferase.

Figure 3. Recycling of ras proteins

Cytoplasmic Ras proteins traffic through the endoplasmic reticulum (ER) where they are post-translationally processed by farnesyltransferase. Farnesylated Kirsten (K)-Ras-4B is then transported directly to the plasma membrane through unknown mechanisms^,, whereas Harvey- (H-), neuroblastoma- (N-), and K-Ras-4A transit through the Golgi apparatus and undergo a palmitoylation step before homing to plasma-membrane invaginations enriched with the cholesterol-binding protein caveolin (see REF. 228). Efficient activation of these Ras proteins might require their localization to specific plasma-membrane microdomains (lipid raft or non-raft domains) that regulate the nature of the recruited Ras effectors (and hence the identities of the signalling pathways that are activated downstream of Ras) and the amplitude of the Ras signal activation. Endocytosed and de-palmitoylated H- and N-Ras are shuttled back from the plasma membrane to the Golgi where they are recycled. Alternatively, ubiquitylated plasma-membrane-tethered H- or N-Ras (representing ~2% of total cellular H- or N-Ras levels) are de-ubiquitylated and targeted to endocytic vesicles. The nature and function of the signalling circuitry that Ras proteins execute in endosomes and other cytoplasmic organelles is an emerging area of intense investigation.

Figure 4. Anatomy of ras regulation

a | The structure of Ras. The Ras three-dimensional fold is shown to consist of six β-sheets and five α-helices interconnected by a series of ten loops. Crystallographic structures of inactive RasGDP (2.0 Å resolution; Protein Data Bank code 4q21) and active RasGppNHp (1.35 Å resolution; PDB code 5p21) are shown, with the nucleotide-sensitive switch I and II regions depicted in red and green, respectively. The GDP and GTP nucleotides are shown as balls. b | Nucleotide-dependent structural rearrangements. The differences between the inactive GDP-bound and the active GTP-bound Ras reside mainly in two regions, termed switch I (~residues 30–40) and switch II (~residues 60–76), both of which are required for the interactions of Ras with its regulators and effectors. The γ-phosphate induces significant changes in the orientation of the switch II region through the interactions that it establishes with Thr35 and Gly60. Notably, these two residues are among the most conserved residues in the GTPase family, suggesting that the mechanisms of GTP binding (and hydrolysis) are essentially the same among various members. c | Ras in complex with its regulators. The crystal structures of Ras in complex with the DBL homology (DH)/pleckstrin homology (PH) domain of son of sevenless (SOS) (left; 2.8 Å resolution; PDB code 1bkd) and with p120 Ras GTPase-activating protein (p120GAP) are shown (right; 2.5 Å resolution; PDB code 1wq1). d | The Ras guanine nucleotide-exchange factor (RasGEF) binding interface. The insertion of the α-helical hairpin of SOS (green; specifically Leu938 and Glu942) into the nucleotide pocket of Ras (yellow) reorientates Ala59 and perturbs the phosphate-binding (P)-loop, facilitating guanine nucleotide ejection. e | The RasGAP binding interface. Similarly, the insertion of the catalytic Arg789 finger of GAP into the active site of Ras stabilizes Gln61 and contributes to GTP hydrolysis. All structures were visualized with DeepView-Swiss-PdbViewer, and images were generated with POV-Ray.

Figure 5. Ras signalling networks

Ras proteins function as nucleotide-driven switches that relay extracellular cues to cytoplasmic signalling cascades. The binding of GTP to Ras proteins locks them in their active states, which enables high affinity interactions with downstream targets that are called effectors. Subsequently, a slow intrinsic GTPase activity cleaves off the γ-phosphate, leading to Ras functional inactivation and thus the termination of signalling. This on–off cycle is tightly controlled by GTPase-activating proteins (GAPs) and guanine-nucleotide exchange factors (GEFs). GAPs, such as p120GAP or neurofibromin (NF1), enhance the intrinsic GTPase activity and hence negatively regulate Ras protein function. Conversely, GEFs (also known as GTP-releasing proteins/factors, termed GRPs or GRFs), such as RasGRF and son of sevenless (SOS), catalyse nucleotide ejection and therefore facilitate GTP binding and protein activation. The classical view of Ras signalling depicts Ras activation and recruitment to the plasma membrane following receptor Tyr kinase (RTK) stimulation by growth factors (GF). Activated Ras engages effector molecules — belonging to multiple effector families — that initiate several signal-transduction cascades. Outputs shown represent the main thrusts of the indicated pathways. Ras activation can also occur in endomembrane compartments, namely the endoplasmic reticulum and the Golgi. Activating mutations in the different components of the Ras–Raf–mitogen-activated protein kinase (MAPK) pathway are associated with the indicated developmental disorders, suggesting that MAPK-signal antagonism might be a rational approach to manage certain cardio-facio-cutaneous (CFC) syndromes. AF-6, acute lymphoblastic leukaemia-1 fused gene on chromosome 6; CD1, cadherin domain-1; CDC42, cell division cycle-42; ELK, ETS-like protein; ERK, extracellular signal-regulated kinase; ETS, E26-transcription factor proteins; Ins(1,4,5)P₃, inositol-1,4,5-trisphosphate; JNK, Jun N-terminal kinase; MEK, mitogen-activated protein kinase/ERK kinase; NF-κB, nuclear factor-κB; PI3K, phosphoinositide 3-kinase; PKB/C, protein kinase B/C; PLA/Cε/D, phospholipase A/Cε/D; RalBP1, Ral-binding protein-1; RASSF, Ras association domain-containing family; Rin1, Ras interaction/interference protein-1; SAPK, stress-activated protein kinase; SHP2, Src-homology-2 domain-containing protein Tyr phosphatase-2; TIAM1, T-cell lymphoma invasion and metastasis-1.