Cell growth, proliferation, differentiation and aging are tightly controlled by a complex network of signaling pathways. The RAS proteins and RAS signaling pathway play an essential role in normal cellular proliferation. Cancer occurs when proteins regulating these signaling pathways are over expressed or constitutively activated.
The Ras superfamily of small GTPases is composed of more than 150 members, which share a conserved structure and biochemical properties, acting as binary molecular switches turned on by binding GTP and off by hydrolyzing GTP to GDP. However, despite considerable structural and biochemical similarities, these proteins play multiple and divergent roles, being versatile and key regulators of virtually all fundamental cellular processes. Conversely, their dysfunction plays a crucial role in the pathogenesis of serious human diseases, including cancer and developmental syndromes.
The Ras GTPase superfamily includes low molecular weight (20-30 kDa), monomeric GTP-binding and hydrolyzing (GTPases) proteins that act as binary, GDP-/GTP-regulated, molecular switches in coupling extracellular signals to intracellular signaling networks that regulate a wide range of fundamental cellular processes, including proliferation, differentiation, morphology, polarity, adhesion, migration, survival, and apoptosis. Indeed, dysregulation of numerous Ras superfamily G-protein-dependent regulatory cascades underlies the development of human diseases. Ras proteins localize to discrete membrane microdomains where their cellular activity is cyclically regulated in response to either outside-in or inside-out signals by the opposing action of guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs), which promote the acquisition of the active GTP- and inactive GDP-bound conformation, respectively. Multiple GEFs and GAPs coexist in most cells, increasing the diversity of signals that regulate the activity of Ras proteins. In the active conformation, Ras proteins bind with differential affinities to a variety of effectors, which are defined as downstream signaling molecules that interact selectively with the GTP-bound form of Ras GTPases, and become activated as a consequence of this interaction. The existence of a large number of Ras effectors, as well as of extensive cross talk with multiple intracellular signaling pathways, further increases the signaling complexity of Ras GTPases.
Upstream signaling of RAS
When a growth factor (extracellular signal) binds to a tyrosine kinase receptor, it forms an active homodimer that undergoes autophosphorylation. This leads to binding of the adaptor proteins SHC and growth factor receptor bound protein 2 (GRB2). GRB2 then associates with GEFs through its SH3 domain. GEFs exchange bound GDP for GTP, thereby activating the RAS protein and initiating a signaling cascade. To terminate signaling, GAPs stimulate the intrinsic GTPase activity of RAS proteins, causing the hydrolysis of GTP to GDP, leading to deactivation of the RAS protein. Thus, upstream targets for KRAS inhibition include: (1) tyrosine kinases, (2) the GRB2/GEF interaction, and (3) GEFs.
The diversity of these exchange factors means that RAS is activated by a very varied collection of extracellular stimuli. This activation is opposed by the effects of the GAPs. These promote the hydrolysis of bound GTP by RAS by several orders of magnitude and normally ensure that RAS is rapidly inactivated after stimulation.
Downstream signaling of RAS
The activated GTP-bound RAS has several downstream effector enzymes, and it is through these kinase systems and the associated pathways that RAS controls cell proliferation, survival and other aspects of cell behaviour. Therapeutic interventions that target these enzymes might therefore be effective in treating tumours in which RAS is mutationally activated.
The most intensively studied protein among these effectors of RAS is the protein serine/threonine kinase-RAF. A combination of genetic and biochemical evidence showed that GTP-bound RAS binds to, and contributes to the activation of, the three closely related RAF proteins, c-RAF1, BRAF and ARAF. This interaction causes RAF to be relocated to the plasma membrane, thereby initiating the RAS-Raf-MEK-ERK signal transduction cascade, in which case, activated Raf phosphorylates and activates mitogen-activated protein kinase kinases 1 and 2 (MEK1 and MEK2)-dualspecificity kinases that are capable of phosphorylating and activating the mitogen-activated protein kinases (MAPKs) ERK1 and ERK2 (extracellular signal-regulated kinases 1 and 2). Substrates for ERK1/2 include cytosolic and nuclear proteins, which can be transported into the nucleus following activation. The full range of effects of activating these kinases is yet to be determined, but most attention has, so far, focused on the regulation of transcription factors. ERK phosphorylates ETS family transcription factors such as ELK1, which forms part of the serum response factor that regulates the expression of FOS; in addition, ERK phosphorylates c-JUN. This leads to activation of the AP1 transcription factor, which is made up of FOS–JUN heterodimers. As a result of stimulating these transcriptional regulators, key cell-cycle regulatory proteins, such as D-type cyclins, are expressed, which enables the cell to progress through the G1 phase of the cell cycle. So, RAF activation can promote cell-cycle progression, at least in conjunction with other signals.
Another clearly identified pathway downstream of RAS is PI3K pathway. RAS can interact directly with the catalytic subunit of type I phosphatidylinositol 3-kinases (PI3Ks), leading to activation of the lipid kinase as a result of its translocation to the membrane and conformational changes. PI3K phosphorylates phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2) to produce phosphatidylinositol-3,4,5-trisphosphate (PtdIns(3,4,5)P3) which functions as a second messenger that binds to a large number of proteins through the pleckstrin homology and other domains. PIP3 activates numerous target proteins, the best characterized of which are the kinases PDK1 (3-phosphoinositide-dependent protein kinase-1) and AKT, (also known as PKB). PDK1 is important for the activation of a large number of protein kinases of the AGC family, including AKT/PKB, some PKCs, p70S6K and RSK. AKT/PKB are a family of three closely related serine/threonine protein kinases containing amino-terminal pleckstrin homology domains that bind to the lipid products of phosphatidylinositol 3-kinase (PI3K). They are activated on PI3K stimulation and provide anti-apoptotic signals to the cell by phosphorylating various targets. In addition, PI3K activation leads to stimulation of RAC, a RHO family protein that is involved in the regulation not only of the actin cytoskeleton but also of transcription-factor pathways, most notably NF-κB.
A third well-studied effector family for RAS is described as RALGDS: RAL guanine nucleotide dissociation stimulator. RALGDS proteins are guanine nucleotide exchange factors (GEFs) for RAL, a RAS-related protein. Its downstream targets include Forkhead transcription factors--A large superfamily of transcription factors, of which one family, FoxO, is phosphorylated and inhibited by AKT.
In addition to the pathway above, Phospholipase Cε is another recognized RAS downstream effector. Phospholipase Cε (PLCε) catalyses the hydrolysis of phosphatidylinositol-4,5-bisphosphate to diacylglycerol and inositol trisphosphate, resulting in protein kinase C (PKC) activation and calcium mobilization from intracellular stores.
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