Ras Pathway
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Ras Pathway
Normal cell function, and its contribution to overall physiology, depends on the proper response of cells to extracellular stimulus. Ras, a legendary cellular and biochemical signaling molecule that displays dynamic nature of signal transduction across the membrane in determining cellular responses to external stimuli, is associated to an ever increasing list of signaling pathways with immense gene expression strategies. In both normal as well as in tumorigenic cell lines, Ras, as a Biotimer, plays a central role in integration and transduction of mitogenic and metabolic signals elicited by membrane receptors to some of the most vital biological pathways such as the MAPK (Mitogen-Activated Protein Kinase)/ERK (Extracellular Signal-Regulated Kinase) Pathway, the Akt (v-Akt Murine Thymoma Viral Oncogene Homolog)/PKB (Protein Kinase-B) Pathway and the Rho Family GTPases, which are implicated in Cell Proliferation, Survival, and Stress Response respectively (Ref.1). The ubiquitous protein Ras accomplishes a diversity of functions through the expression of different Ras gene products, viz. H-Ras, K- Ras, and N-Ras, in cell type and developmentally restricted manners (Ref. 2). Out of the three highly conserved and identical Ras genes, H-Ras has a high profile activity. Ras is attached to the cell membrane by Prenylation and after further Palmityolation, it exists in a normal dynamic equilibrium between Lipid Rafts and other Noncholesterol-Dependent Microdomains (Ref.3, 4 & 5). The regulatory G-protein Ras has an intrinsic GTP Hydrolase activity and Ras biological activity is controlled by a catalyzed GDP/GTP cycle which facilitates signaling convergence and divergence. The conformational changes monitoring Ras activities are catalyzed by two different sets of Ras regulators. Ras is activated to a GTP-bound state by a group of regulatory factors known as GEFs (Guanine Nucleotide Exchange Factors), which are themselves activated by mitogenic signals and through feedback from Ras. All RasGEFs have a common CDC25 (Cell Division Cycle-25) cell homology catalytic domain also called the RasGEF domain, and an adjacent Amino-terminal REM (Ras Exchange Motif). Different downstream Ras signal progressions are activated or initiated by different sets of GEFs. On the other hand, another group of regulators, the GAPs (GTPase Activating Proteins), increase the rate of GTP hydrolysis and stimulate the return of Ras to an inactive GDP bound state (Ref.3, 6 & 7).

Ras is a signaling hub activated by convergent signaling pathways initiated by extracellular stimulus, such as Growth Factors, Cytokines, Hormones and Neurotransmitters that excite apposite cell surface receptors viz., RTKs (Receptor Tyrosine Kinases), Hematopoietic Growth Factor Receptors, Cytokine Receptors, and Heterotrimeric GPCR (G-Protein Coupled Serpentine Receptors) that cause stimulation of associated NRTKs (Nonreceptor Tyrosine Kinases). These signals are then integrated into signaling complexes and sent to other locations in the cell by binding to different downstream Ras Effector Proteins in association with other scaffold proteins (Ref.8 & 9). Ligand binding to the extracellular domain of RTKs causes receptor dimerization, stimulation of Protein Tyrosine Kinase Activity, and their autophosphorylation. The Tyrosine autophosphorylation sites in Growth Factor Receptors (eg, EGFR (Epidermal Growth Factor Receptor) and PDGFR (Platelet-Derived Growth Factor Receptor)) function as high-affinity binding sites for SH2 (Src Homology 2) domains of signaling molecules such as GRB2 (Growth Factor Receptor-Bound Protein-2), SHC (SHC (Src Homology 2 Domain-Containing) Transforming Protein) and SOS (Son of Sevenless) (Ref.3). In Hematopoietic cells and other related cell lines, the Heterotrimeric GPCR stimulate the Ras-MAPK pathway. Following ligand stimulation, the PT (Pertussis Toxin)-sensitive heterotrimeric Gi protein is released from the receptor with its concomitant dissociation into GN-AlphaI (Guanine Nucleotide-Binding Protein G (i) Subunit-Alpha) and GN-Beta (Guanine Nucleotide-Binding Protein-Beta)-GN-Gamma (Guanine Nucleotide-Binding Protein-Gamma) subunits followed by their corresponding activations. The activated G-Protein subunits carry-out Tyrosine phosphorylation of SHC (Ref.10). In T lymphocytes, Ras is activated by two Ras exchange factors, SOS and RasGRP (Ras Guanyl Nucleotide Releasing Protein), the later being recruited through the adapter LAT (Linker for Activation of T-Cells) via the second-messenger DAG (Diacylglycerol). The TCR (T-Cell Receptor) bound Vav (Oncogene Vav), and the TCR-associated complex ZAP70 (Zeta-Chain-Associated Protein Kinase)-Lck (Lymphocyte-Specific Protein-Tyrosine Kinase)-Fyn (Fyn Oncogene Related to Src, FGR, YES)-SYK (Spleen Tyrosine Kinase) communicate with the intracellular signaling molecule PLC (Phospholipase-C), which generates membrane DAG. DAG recruits PKC (Protein Kinase-C) and RasGRP family members to the membrane and contributes to their activation. GN-Beta-GN-Gamma subunit also possesses a similar ability to activate PLC-DAG-PKC channel. Calmodulin prevents Ras activation by PKC (Ref.3 & 11). RAPGEFs (Rap Guanine Nucleotide Exchange Factors) also function as RasGEFs as they convey the Ras activating signals from Adrenaline via ADR-Beta (Adrenergic Receptor-Beta) and its associated protein MAGI (Membrane-Associated Guanylate Kinase Inverted). Activating signals from Calcium Channelsand GPCRs stimulate RasGRF (Ras Protein-Specific Guanine Nucleotide Releasing Factor) family members, comprising another set of RasGEFs, which are again unique in activating Ras on the membrane of the Endoplasmic Reticulum (Ref.4).

Ras regulates a diversity of downstream cytoplasmic signaling cascades, the most important and prominent among which is the activation of the ‘Multiple Kinase Cascade’-MAPK/ERK Signaling Pathway. Ras phosphorylates and activates Raf which activate MEKs that in turn activate MAPK/ERK. Some Akt isoforms phosphorylate Raf1 to create an auto-inhibitory conformation state maintained by 14-3-3 Dimers, blocking the MAPK Pathway. Downstream, activated ERK regulates Growth Factor-responsive targets in the cytosol like SOS, RSKs (Ribosomal S6 Kinases), MNKs (MAPK-Interacting Kinases), cPLA2 (Cytosolic Phospholipase-A2) and the translation factors such as eIF4EBP (Eukaryotic Translation Initiation Factor-4E-Binding Protein). ERK phosphorylation also promotes self homodimerization and translocation into the nucleus where it phosphorylates a number of transcription factors like the members of the Ets (v-Ets Avian Erythroblastosis Virus E26 Oncogene Homolog) Family and a family of RSK-Related Kinases- the MSKs (Mitogen- and Stress-Activated Protein Kinases), thereby regulating gene expression. These factors are involved in ternary complex formation at the SRE (Serum Response Elements), which regulate the expression of immediate-early genes, such as the c-Jun, c-Fos,c-Myc, ATF2, Jun-B, and HBEGF(Heparin-Binding EGF-Like Growth Factor Precursor) genes and eventually contribute to Cell Proliferation. The ERK pathway demonstrates a direct link of Growth Factor Signaling to Ribosome Biosynthesis via ERK-dependent phosphorylation of BRF1 (B-Related Factor-1) and UBF (Upstream Binding Factor). ERKs also mediate gene expression through the phosphorylation of CREB(cAMP Response Element-Binding Protein) to activate its transactivation potential. The Ras-to-MAPK Pathway is an essential shared element of mitogenic signaling and is important in controlling Development, Differentiation and Cell Cycle Control (Ref.3, 7 & 10).

Ras phosphorylates the p110 catalytic domain of PI3K (Phosphatidylinositde-3 Kinase), the second major Ras Effector that responds to signals transmitted via GPCRs and mediates some apparently distinct cellular functions, including Mitogenic Signaling (DNA Synthesis), protection against Apoptosis, Intracellular Vesicle Trafficking and Secretion, Dendrite Morphogenesis and Regulation of Ras-dependent Actin and Integrin Cytoskeletal Remodeling. PI3K induces Cyclin-D1 transcription and E2F(E2F Transcription Factor) activity and along with MEK, dephosphorylates Cofilin, an Actin-remodeling protein (Ref.12). PI3K executes its normal functions using distinct Phosphoinositide products and Akt. In response to signals received by GPCRs, Cytokine Receptors and Growth Factor Receptors, which communicate through SOS, RasGRF, RasGRP and Vav, PI3K catalyzes the phosphorylation of the lipid second-messenger molecule PIP2 (Phosphatidylinositol-4,5-bisphosphate) to yield PIP3 (Phosphatidylinositol 3,4,5-trisphosphate). PIP3 in turn activates the PDKs (Phosphoinositide-Dependent Kinases), which then activate Akt. Akt executes its primary responsibility in the Akt/PKB Pathway linked to several Inflammatory and Neoplastic Pathways and monitors the regulation of cellular Apoptosis. As a Ras Effector, on one hand, it inhibits the activity of Caspase9 and on the other it activates the IKKs (Inhibitor of Kappa Light Chain Gene Enhancer in B-Cells Kinases), which steer NF-KappaB(Nuclear Factor-KappaB) towards the nucleus for triggering the expression of several Anti-Apoptotic as well as Neuro-protective genes (Ref.10 & 13).

The Ras signaling pathway is also linked directly to cellular stress responses that supervise the G1/S phase transition of the Cell Cycle and subsequent induction of DNA Synthesis in the Nucleus of quiescent cells. Ras accomplishes this function by activating the third bonafide catalytic cascade constituents; RacCDC42 (Cell Division Cycle-42) and Rho, which collectively form the Rho Family GTPases. Activation signals for these proteins are administered through the TCR, which either activates RasGRP, or recruites a complex comprising CrkL and C3G to activate the RasGRFs, which in turn convey the signals to the small GTPase Ral (v-Ral Simian Leukemia Viral Oncogene Homolog) following Ras activation on the membranes of the Golgi and the Endoplasmic Reticulum respectively (Ref.14). The Ras Effector RalGDS (Ral Guanine Nucleotide Dissociation Stimulator) is an exchange factor for Ral, which on activation regulates multiple processes such as Receptor Endocytosis, Cytoskeletal Changes and DNA Synthesis (Ref.15). PKA (Protein Kinase-A) regulates the selectivity of Ras binding to RalGDS or Raf1, both of which are involved in the synergistic activation of gene expression by Ras. Ral is required for Src (v-Src Avian Sacroma (Schmidt-Ruppin A-2)Viral Oncogene)- and Ras-dependent formation of a functional PLD (Phospholipase-D)-RalA (v-Ral Simian Leukemia Viral Oncogene Homolog-A)-ARF (ADP-Ribosylation Factor) Complex, which powers Golgi Trafficking and Vesicle Formation. Catalytically active Ral-GTP associates with RalBP1 (RalA Binding Protein-1), a GAP for CDC42 and Rac, the signaling intermediates between Ras and MEKK in the signaling cascade to the JNK (c-Jun Kinase) Pathway and the p38 Pathway through PAK (p21-Activated Kinase). They also contribute to the activation of RhoA GTPase, which acts synergistically with Rac1 and CDC42 through a JNK-independent pathway. The Ras activated Rho GTPases stimulate SRF (Serum Response Factor)-dependent transcription and activate the transcription factor NF-KappaB. Rho controls the assembly of Actin Stress Fibers and Focal Adhesion Complexes by the implementation of two families of RhoA Effectors-the protein kinases ROCKs (Rho-Associated Coiled-Coil-Containing Protein Kinases) and PRKs (Protein Kinase C-Related Kinases) (Ref.15). CDC42 stimulates the polymerization of Actin to Filopodia or Microspikes, while Rac regulates Actin Filament Accumulation at the Plasma Membrane to produce Lamellipodia and Membrane Ruffles. Ras has two different activation approaches for Rac. In one path, PI3K functions upstream of Rac and serves as a link between Ras and the Rho GTPases by activating RacGEFs (Rac Guanine Nucleotide Exchange Factors), like TIAM (T-Cell Lymphoma Invasion And Metastasis), while the other utilizes SOS. In both cases, the Rac activation is followed by activation of a cascade of GTPases including CDC42 and Rho (Ref.13).

Besides these cascade modules, several other proteins such as PKC-Zeta (Protein Kinase-C-Zeta), AF6 (ALL1 Fused Gene from Chromosome-6), Canoe, RASSF5 (Ras Association Domain-Containing Family Protein-5) and RIN1 (Ras and Rab Interactor-1) possess Ras Binding Domains and act as downstream Ras Effectors that evoke some precise and exclusive functions. PKC-Zeta is a Ras-dependent Anti-Apoptotic Serine/Threonine PKC atypical isoform insensitive to DAG and Calcium (Ref.3). AF6 binds to Ras-GTP and contains motifs involved in specific cell-cell interaction. RASSF5 is another true Ras Effector that associates with Ras following EGFR and PMA activation and increases the complexity of the Ras-dependent Apoptosis regulation as a potential tumor suppressor. The RAS effector RIN1 directly competes with Raf1 and is regulated by 14-3-3 proteins, which in turn are controlled by PKD (Protein Kinase-D) Phosphorylation. It exchanges signals from Abl1 (v-Abl Abelson Murine Leukemia Viral Oncogene Homolog-1) and Abl2 (v-Abl Abelson Murine Leukemia Viral Oncogene Homolog-2), which regulate cytoskeletal remodeling, and also acts as an activating GEF for Rab5A (Ras-Associated Protein Rab5A), facilitating Ras-activated Receptor Endocytosis (Ref. 8). Ras monitors the G1 progression by phosphorylating Rb (Retinoblastoma) with the help of Cyclin D-CDK4/6 and Cyclin E-CDK2Complexes and by simultaneous down-regulation of p27Kip1 (Ref. 15). The downstream Ras signaling cascades activate scores of Transcription Factors which in turn induce the expression of target genes. Ras activates the members of the Ets Family and the AP-1 (Activator Protein-1) Transcription Factor Complex thereby inducing the expression of endogenous genes. There is a wide applicability of the Ras activated expression of genes, but Ras functions stand highly dedicated towards the control of Cell Cycle (Ref.7, 10 & 13).

Cells however, at times of ageing, injury and other overloaded stages, require Ras to remain in an inactivated form. This process of Ras inactivation is achieved by opting for GDP against GTP, which is made possible by a group of intracellular GAPs. Most extensively available RasGAPs in mammalian cells are: p120-GAP, NF1 (Neurofibromin-1), RasA2 (Ras p21 Protein Activator-2), RasA3 (Ras p21 Protein Activator-3) and an endocrine specific RasAL1 (Ras Protein Activator-Like-1). Recently, a novel RasGAP, IQGAP1 has been found to link Ras signaling to some Calmodulin-mediated process. All RasGAPs have a common Alpha-Helical Catalytic Domain, which interacts with Ras-GTP, enhancing its GTPase activity and resulting in its inactivation. Presence of several other domains such as the SH2, SH3 and PH (Pleckstrin Homology) domains that promote membrane association by binding to phospholipids and docking proteins, boost up the diversity in their functions. In addition to their well-established role as inhibitors of Ras, the GAPs also interact with the Ras Effector Domain and execute a myriad of functions as putative targets of the Effector System downstream of Ras (Ref.3). In neuronal cells, activated members of Eph Family, such as EphB2 (Ephrin Receptor), recruit p120-GAP, in a fashion that is associated with downregulation of the Ras-MAPK Pathway and Neurite Retraction. A physical alliance between p120-GAP and FLN-C (Filamin C) facilitates an interaction between the RNA Polymerase-II Kinase, CDK7 (Cyclin Dependent Kinase-7) and the RasGAP SH3 Domain-binding Protein G3BP, which are required for Myocyte Growth and Cardiac Hypertrophy. However, the most important role of p120-GAP is the connection between the Ras and Rho pathways by controlling the remodeling of the Cortical Actin Cytoskeleton following Growth Factor stimulation. The tumor suppressor NF1 is a functional homologue of p120-GAP and contains a GAP Related Domain responsible for stimulation of GTP-Hydrolysis on Ras. It links Neurofibromatosis to the regulation of the Ras-MAPK Pathway and its activation by oncogenes. NF1 also associates with microtubules and participates in structural reorganization of the cytoskeleton. I(1,3,4,5)P4 (1D-Myo-Inositol 1,3,4,5-Tetrakisphosphate) stimulation of the candidate receptors RasA2, RasA3 also contributes to negative regulation of Ras activity (Ref.16). The PTKs (Protein-Tyrosine Kinases), via their corresponding receptors, provide the activating stimulus for the RasGAPs. Downstream of PTKs, the RasGAP-binding protein DOK (Docking Protein) and its homologues are Tyrosine phosphorylated and act as a molecular platform for signal complex assembly. They have different PTB (Phosphotyrosine Binding) domains and Carboxyl-Terminal tails that potentially mediate different signal responses by recruiting distinct sets of SH2-containing GAPs. DOK proteins DOK1 (Docking Protein-1) and DOK2 (Docking Protein-2) respond to signals from GDNF (Glial Cell Line-Derived Neurotrophic Factor) and  Angpt1 (Angiopoietin-1) translocated into the cell through GDNFR (Glial Cell Line-Derived Neurotrophic Factor Receptor) and TIE2 (Protein Receptor Tyrosine Kinase-Epithelial-Specific-Tie2) respectively. DOKs also respond to the intracellular activation by BCR (Breakpoint Cluster Region) (Ref. 17). Ras is the central point for normal and tumorigenic cellular signal transduction and has enabled the establishment of an analytical understanding of tumor development in humans and their relation to a diverse array of signal transduction pathways in the cell. Germline Mutations in Ras and Ras-Related Proteins perturb human development and increase susceptibility to tumors leading to increased invasion and metastasis, and decreased Apoptosis. Activating mutations of Ras isoforms which impair GTPase activity and stabilize the GTP bound state are found in nearly one-third of all human cancers. H-Ras mutations are common in Bladder, Kidney, Thyroid and Cutaneous Squamous Cell Carcinomas, and in Costello Syndrome, a multiple congenital anomaly and mental retardation syndrome (Ref.18, 19 & 20). The growing number of Ras Regulators and Effectors is exponentially increasing the number of cellular processes from Kinase Cascades to Cell Cycle Progression, Apoptosis or Cell-to-Cell Interactions, where Ras plays active roles. The web of Ras interacting partners has different looks as per the cellular demands, but Ras is frequently in the middle of it and is a crucially important molecule for human health (Ref.1 & 2).