Ras oncogene at 85D


Table of contents

Ras interaction with AF-6

The dynamic rearrangement of cell-cell junctions (such as tight junctions and adherens junctions) is a critical step in various cellular processes, including establishment of epithelial cell polarity and developmental patterning. Tight junctions contain associated proteins such as occludin and its associated ZO-1 and ZO-2 (homologs of Drosophila Polychaetoid) and adherens junctions contain associated adhesion proteins such as cadherin and its associated catenins. The transformation of epithelial cells by activated Ras results in the perturbation of cell-cell contacts. The ALL-1 fusion partner from chromosome 6 (AF-6) has been identified as a Ras target. AF-6, a homolog of Drosophila Canoe) has the PDZ domain, which is thought to localize AF-6 at the specialized sites of plasma membranes, such as cell-cell contact sites. The roles of Ras and AF-6 were investigated in the regulation of cell-cell contacts and it was found that AF-6 accumulates at the cell-cell contact sites of polarized MDCKII epithelial cells and has a distribution similar to that of ZO-1 but somewhat different from that of catenins. Immunoelectron microscopy reveales a close association between AF-6 and ZO-1 at the tight junctions of MDCKII cells. Native and recombinant AF-6 interacts with ZO-1 in vitro. ZO-1 interacts with the Ras-binding domain of AF-6, and this interaction is inhibited by activated Ras. AF-6 accumulates with ZO-1 at the cell-cell contact sites in cells lacking tight junctions, such as Rat1 fibroblasts and PC12 rat pheochromocytoma cells. The overexpression of activated Ras in Rat1 fibroblasts results in the perturbation of cell-cell contacts, followed by a decrease of the accumulation of AF-6 and ZO-1 at the cell surface. These results indicate that AF-6 serves as one of the peripheral components of tight junctions in epithelial cells, and cell-cell adhesions in nonepithelial cells, and that AF-6 may participate in the regulation of cell-cell contacts, including tight junctions, via direct interaction with ZO-1 downstream of Ras (Yamamoto, 1997).

Mammalian Ras proteins associate with multiple effectors, including Raf, Ral guanine nucleotide dissociation stimulator, phosphoinositide 3-kinase and AF-6. In the nematode Caenorhabditis elegans, LIN-45/Raf has been identified genetically as an effector of LET-60/Ras. To search for other effectors in C. elegans, a yeast two-hybrid screening was carried out for LET-60-associating proteins. The screening identified a novel protein, designated Ce-AF-6, which exhibits a strong structural homology with human AF-6, rat Afadin and Drosophila Canoe and possesses both a Ras-associating (RA) domain and a PSD-95/DlgA/ZO-1 (PDZ) domain. Ce-AF-6 associates with human Ha-Ras in a GTP-dependent manner, with an efficiency comparable to that of human Raf-1 Ras-binding domain. When the effects of mutations of the Ras effector region residues were examined for associations with various effectors, Ce-AF-6 was found to possess a distinct and the most rigorous requirement for the effector region residues. These results strongly suggest that Ce-AF-6 is a putative effector of Ras that possesses a distinct recognition mechanism for association with Ras (Watari, 1998).

Ras interaction with RalGDS

Oncogenic Ras transforms cells through the activation of multiple downstream pathways mediated by separate effector molecules, one of which is Raf. A second ras-binding protein has been identified that can induce cellular transformation in parallel with activation of the Raf/mitogen-activated protein kinase cascade. The Ral guanine nucleotide dissociation stimulator (RalGDS) was isolated from a screen for Ras-binding proteins that specifically interact with a Ras effector-loop mutant, ras(12V,37G), which uncouples Ras from activation of Raf1. RalGDS, like ras(12V, 37G), cooperates synergistically with mutationally activated Raf to induce foci of growth and morphologically transformed NIH 3T3 cells. RalGDS does not significantly enhance MAP kinase activation by activated Raf, suggesting that the cooperativity in focus formation is due to a distinct pathway acting downstream of Ras and parallel to Raf (White, 1996).

RalGDS is a GDP/GTP exchange protein for ral p24, a member of the small GTP-binding protein superfamily. RalGDS interacts directly with the GTP-bound active form of ras p21 through the effector loop of ras p21 in vitro, in insect cells and in the yeast two-hybrid system. These results suggest that RalGDS functions as an effector protein of ras p21. RalGDS interacts with ras p21 in mammalian cells in response to an extracellular signal. Epidermal growth factor (EGF) induces the interaction of c-ras p21 and RalGDS in COS cells expressing both proteins, but not in the cells expressing RalGDS and c-ras p21T35A (an effector loop mutant of ras p21). Cyclic AMP-dependent protein kinase (protein kinase A) regulates the selectivity of ras p21-binding to either RalGDS or Raf-1. Protein kinase A phosphorylates RalGDS as well as (1-149)Raf (amino acid residues 1-149). Although the phosphorylated (1-149)Raf has a lower affinity for ras p21 than the unphosphorylated (1-149)Raf, both the phosphorylated and unphosphorylated RalGDS have the similar affinities for ras p21. The phosphorylation of RalGDS does not affect its ability to stimulate the GDP/GTP exchange of ral p24. Pretreatment of COS cells with forskolin further stimulates the interaction of ras p21 and RalGDS induced by EGF under conditions in which EGF-dependent Raf-1 activity is inhibited. These results indicate that ras p21 distinguishes between RalGDS and Raf-1 according to their phosphorylation by protein kinase A (Kikuchi, 1996).

Ras mutants with the ability to interact with different effectors have played a critical role in the identification of Ras-dependent signaling pathways. Two mutants, RasS35 and RasG37, which differ in their ability to bind Raf-1, were used to examine Ras-dependent signaling in thyroid epithelial cells. Wistar rat thyroid cells are dependent upon thyrotropin (TSH) for growth. Although TSH-stimulated mitogenesis requires Ras, TSH activates protein kinase A (PKA) and downregulates signaling through Raf and the mitogen-activated protein kinase (MAPK) cascade. Cells expressing RasS35 (a mutant that binds Raf) or RasG37 (a mutant that binds RalGDS) exhibit TSH-independent proliferation. RasS35 stimulates morphological transformation and anchorage-independent growth. RasG37 stimulates proliferation but not transformation as measured by these indices. TSH exerts markedly different effects on the Ras mutants and transiently represses MAPK phosphorylation in RasS35-expressing cells. In contrast, TSH stimulates MAPK phosphorylation and growth in cells expressing RasG37. The Ras mutants, in turn, exert differential effects on TSH signaling. RasS35 abolishes TSH-stimulated changes in cell morphology and thyroglobulin expression, while RasG37 has no effect on these activities. Together, the data indicate that cross talk between Ras and PKA discriminates between distinct Ras effector pathways (Miller, 1998).

In response to epidermal growth factor in COS cells, Ral GDP dissociation stimulator (RalGDS), a putative effector protein of Ras, stimulates the GDP/GTP exchange reaction of the post-tanslationally lipid-modified (but not the unmodified) form of Ral. The RalGDS action on Ral is enhanced by an active form of Ras but not a Ras mutant, which is not post-translationally modified in the cells. The RalGDS activity is inhibited by acidic membrane phospholipids, such as phosphatidylinositol and phosphatidylserine but not by phosphatidylcholine or phosphatidylethanolamine in vitro. The post-translationally modified form but not unmodified form of Ras, Ral, and Rap were incorporated in liposomes consisting of these phospholipids. When Ral is incorporated alone in the liposomes, RalGDS does not stimulate the dissociation of GDP from Ral. When Ral is incorporated with the GTP-bound form of Ras in the liposomes, RalGDS stimulates the dissociation of GDP from Ral, while the GDP-bound form of Ras does not affect the RalGDS action. The Ras-dependent Ral activation through RalGDS requires the Ras-binding domain of RalGDS. Rap, which shares the same effector loop as Ras, also stimulates the dissociation of GDP from Ral through RalGDS in the liposomes, although Rap does not enhance the RalGDS action in COS cells. Taken together with observations that Ras recruits RalGDS to the membrane, these results indicate that the post-translational modifications of Ras and Ral are important for Ras-dependent Ral activation through RalGDS and that colocalization of Ras and Ral on the membrane is necessary for Ral activation in intact cells (Kishida, 1997).

Rlf is a ubiquitously expressed distinct relative of RalGDS that interacts with active Ras in vitro. Rlf, when co-expressed with Ras mutants, associates in vivo with RasV12 and the effector-domain mutant RasV12G37, but not with RasV12E38 or RasV12C40. Rlf exhibits guanine nucleotide exchange activity towards the small GTPase Ral and, importantly, Rlf-induced Ral activation is stimulated by active Ras. In addition, RasV12 and RasV12G37 synergize with Rlf in the transcriptional activation of the c-fos promoter. Rlf, when targeted to the plasma membrane using the Ras farnesyl attachment site (Rlf-CAAX), is constitutively active, inducing both Ral activation and c-fos promoter activity. Rlf-CAAX-induced gene expression is insensitive to dominant negative Ras and the MEK inhibitor PD98059, and involves activation of the serum response element. Expression of Rlf-CAAX is sufficient to induce proliferation of NIH 3T3 cells under low-serum conditions. These data demonstrate that Rlf is an effector of Ras, and Ras functions as an exchange factor for Ral. Rlf mediates a distinct Ras-induced signalling pathway to gene induction. It has been determined that a constitutively active form of Rlf can stimulate transcriptional activation and cell growth (Wolthuis, 1997).

Oncogenic Ras inhibits the differentiation of skeletal muscle cells through the activation of multiple downstream signaling pathways, including a Raf-dependent, mitogen-activated or extracellular signal-regulated kinase kinase/mitogen-activated protein kinase (MEK/MAPK)-independent pathway. A non-Raf binding Ras effector-loop variant (H-Ras G12V,E37G), which retains interaction with the Ral guanine nucleotide dissociation stimulator (RalGDS), inhibits the conversion of MyoD-expressing C3H10T1/2 mouse fibroblasts to skeletal muscle. H-Ras G12V,E37G, RalGDS, and the membrane-localized RalGDS CAAX protein inhibit the activity of alpha-actin-Luc, a muscle-specific reporter gene containing a necessary E-box and serum response factor (SRF) binding site, while a RalGDS protein, defective for Ras interaction, has no effect on alpha-actin-Luc transcription. H-Ras G12V,E37G does not activate endogenous MAPK, but does increase SRF-dependent transcription. Interestingly, RalGDS, RalGDS CAAX, and RalA G23V inhibit H-Ras G12V, E37G-induced expression of an SRF-regulated reporter gene, demonstrating that signaling through RalGDS does not duplicate the action of H-Ras G12V,E37G in this system. As additional evidence for this, it is shown that H-Ras G12V,E37G inhibits the expression of troponin I-Luc, an SRF-independent muscle-specific reporter gene, whereas RalGDS and RalGDS CAAX do not. Although these studies show that signaling through RalGDS can interfere with the expression of reporter genes dependent on SRF activity (including alpha-actin-Luc), the studies also provide strong evidence that an additional signaling molecule(s) activated by H-Ras G12V,E37G is required to achieve the complete inhibition of the myogenic differentiation program (Ramocki, 1998).

In COS cells, Ral GDP dissociation stimulator (RalGDS)-induced Ral activation is stimulated by RasG12V or a Rap1/Ras chimera in which the N-terminal region of Rap1 was ligated to the C-terminal region of Ras but not by Rap1G12V or a Ras/Rap1 chimera in which the N-terminal region of Ras is ligated to the C-terminal region of Rap1, although RalGDS interacts with these small GTP-binding proteins. When RasG12V, Ral and the Rap1/Ras chimera are individually expressed in NIH3T3 cells, they localized to the plasma membrane. Rap1Q63E and the Ras/Rap1 chimera are detected in the perinuclear region. When RalGDS is expressed alone, it is abundant in the cytoplasm. When coexpressed with RasG12V or the Rap1/Ras chimera, RalGDS is detected at the plasma membrane, whereas when coexpressed with Rap1Q63E or the Ras/Rap1 chimera, RalGDS is observed in the perinuclear region. RalGDS, which is targeted to the plasma membrane by the addition of Ras farnesylation site (RalGDS-CAAX), activates Ral in the absence of RasG12V. Although RalGDS does not stimulate the dissociation of GDP from Ral in the absence of the GTP-bound form of Ras in a reconstitution assay using the liposomes, RalGDS-CAAX can stimulate it without Ras. RasG12V activates Raf-1 when they are coexpressed in Sf9 cells, whereas RasG12V does not affect the RalGDS activity. These results indicate that Ras recruits RalGDS to the plasma membrane and that the translocated RalGDS induces the activation of Ral, but that Rap1 does not activate Ral due to distinct subcellular localization (Matsubara, 1999).

Ras proteins have the capacity to bind to and activate at least three families of downstream target proteins: Raf kinases, phosphatidylinositol 3 (PI 3)-kinase, and Ral-specific guanine nucleotide exchange factors (Ral-GEFs). The Ras/Ral-GEF and Ras/Raf pathways oppose each other upon nerve growth factor stimulation, with the former promoting proliferation and the latter promoting cell cycle arrest. Moreover, the pathways are not activated equally. While the Ras/Raf/Erk signaling pathway is induced for hours, the Ras/Ral-GEF/Ral signaling pathway is induced for only minutes. This preferential down-regulation of Ral signaling is mediated, at least in part, by protein kinase C (PKC). In particular, PKC activation by phorbol ester treatment of cells blocks growth factor-induced Ral activation while it enhances Erk activation. Moreover, suppression of growth factor-induced PKC activation enhances and prolongs Ral activation. PKC does not influence the basal activity of the Ral-GEF designated Ral-GDS but suppresses its activation by Ras. Interestingly, Ras binding to the C-terminal Ras binding domain of Ral-GDS is not affected by PKC activity. Instead, suppression of Ral-GDS activation occurs through the region N terminal to the catalytic domain, which becomes phosphorylated in response to phorbol ester treatment of cells. At present it is not clear which PKC isoform is responsible for down-regulation of Ral-GDS in response to NGF and EGF. The fact that the effect can be seen after PMA treatment argues that it is through the phorbol-responsive conventional PKCs alpha, beta, or gamma and/or the novel PKCs delta, epsilon, eta, or theta but not the atypical PKCs zeta and lambda. It also remains to be determined how PKC becomes activated, because multiple types of signaling molecules can enhance PKC activity in cells. For example, some PKC family members can be activated by diacylglycerol and calcium generated by NGF or EGF receptor-induced phospholipase C activation. Alternatively, Ras-Ral-regulated PLD could conceivably activate PKC isoforms, since the PLD product, phosphatidic acid, is known to be converted to diacylglycerol. These findings identify a role for PKC in determining the specificity of Ras signaling by its ability to differentially modulate Ras effector protein activation (Rusanescu, 2001).

Ras proteins transduce extracellular signals to intracellular signaling pathways by binding to and promoting the activation of at least three classes of downstream signaling molecules: Raf kinases, phosphoinositide-3-kinase (PI3-K) and Ral guanine nucleotide exchange factors (Ral-GEFs). Active Ral proteins can influence a variety of cellular processes by interacting with a distinct set of downstream target proteins. These include RalBP1 (or RLIP), which binds to a family of related EH domain proteins, Reps1 and POB1, and to the AP2 complex. These associations have implicated Ral function in the regulation of endocytosis. RalBP1 is also a GTPase activating protein (GAP) for Rac and CDC42. This, and the fact that Ral-GTP also binds to the actin cross-linking protein, filamin, connects Ral to the regulation of the actin cytoskeleton. Ral proteins associate constitutively with and participate in the regulation of phospholipase D1. This property may contribute to Ral’s effects on vesicle function, and also to its ability to promote cell proliferation. Ral proteins also mediate c-Src activation by EGF receptors, promote Ras-induced Jnk kinase activation, activate cyclin D expression and inhibit the forkhead transcription factor. Epidermal growth factor (EGF) activates Ral-GEFs, at least in part, by a Ras-mediated redistribution of the GEFs to their target, Ral-GTPases, in the plasma membrane. Ral-GEF stimulation by EGF involves an additional mechanism, PI3-K-dependent kinase 1 (PDK1)-induced enhancement of Ral-GEF catalytic activity. Remarkably, this PDK1 function is not dependent upon its kinase activity. Instead, the non-catalytic N-terminus of PDK1 mediates the formation of an EGF-induced complex with the N-terminus of the Ral-GEF, Ral-GDS, thereby relieving its auto-inhibitory effect on the catalytic domain of Ral-GDS. These results elucidate a novel function for PDK1 and demonstrate that two Ras effector pathways cooperate to promote Ral-GTPase activation (Tian, 2002).

The Ral guanine nucleotide-exchange factors (RalGEFs) serve as key effectors for Ras oncogene transformation of immortalized human cells. RalGEFs are activators of the highly related RalA and RalB small GTPases, although only the former has been found to promote Ras-mediated growth transformation of human cells. In the present study, it was determined whether RalA and RalB also had divergent roles in promoting the aberrant growth of pancreatic cancers, which are characterized by the highest occurrence of Ras mutations. Inhibition of RalA but not RalB expression universally reducea the transformed and tumorigenic growth in a panel of ten genetically diverse human pancreatic cancer cell lines. Despite the apparent unimportant role of RalB in tumorigenic growth, it was nevertheless critical for invasion in seven of nine pancreatic cancer cell lines and for metastasis as assessed by tail-vein injection of three different tumorigenic cell lines tested. Moreover, both RalA and RalB were more commonly activated in pancreatic tumor tissue than other Ras effector pathways. It is concluded that RalA function is critical to tumor initiation, whereas RalB function is more important for tumor metastasis in the tested cell lines; this argues for critical, but distinct, roles of Ral proteins during the dynamic progression of Ras-driven pancreatic cancers (Lim, 2006).

Ras interaction with leucine-rich repeat proteins

A novel gene, sur-8, has been identified and characterized that positively regulates Ras-mediated signal transduction during C. elegans vulval development. SUR-8 is a conserved Ras-binding protein with leucine-rich repeats. Reduction of sur-8 function suppresses an activated ras mutation and dramatically enhances phenotypes of mpk-1 MAP kinase and ksr-1 mutations, while increase of sur-8 dosage enhances an activated ras mutation. sur-8 appears to act downstream of or in parallel to ras but upstream of raf. sur-8 encodes a conserved protein that is composed predominantly of leucine-rich repeats. The SUR-8 protein interacts directly with Ras but not with the Ras(P34G) mutant protein, suggesting that SUR-8 may mediate its effects through Ras binding. A structural and functional SUR-8 homolog in humans specifically binds K-Ras and N-Ras but not H-Ras in vitro (Sieburth, 1998).

A Saccharomyces cerevisiae gene encoding adenylate cyclase has been analyzed by deletion and insertion mutagenesis to localize regions required for activation by the S. cerevisiae RAS2 protein. The NH2-terminal 657 amino acids were found to be dispensable for the activation. However, almost all 2-amino acid insertions in the middle 600 residues (comprising leucine-rich repeats) and deletions in the COOH-terminal 66 residues completely abolish activation by the RAS2 protein, whereas insertion mutations in the other regions generally have no effect. Chimeric adenylate cyclases were constructed by swapping the upstream and downstream portions surrounding the catalytic domains between the S. cerevisiae and Schizosaccharomyces pombe adenylate cyclases and examined for activation by the RAS2 protein. The fusion containing both the NH2-terminal 1600 residues and the COOH-terminal 66 residues of the S. cerevisiae cyclase render the catalytic domain of the S. pombe cyclase, which otherwise did not respond to RAS proteins, activatable by the RAS2 protein. Thus the leucine-rich repeats and the COOH terminus of the Sa. cerevisiae adenylate cyclase appear to be required for interaction with RAS proteins (Suzuki, 1998).

Ras and cell junctions

The dynamic rearrangement of cell-cell junctions such as tight junctions and adherens junctions is a critical step in various cellular processes, including establishment of epithelial cell polarity and developmental patterning. Tight junctions are mediated by molecules such as occludin and its associated ZO-1 and ZO-2 (see Drosophila Discs large 1); adherens junctions are mediated by adhesion molecules such as cadherin and its associated catenins. The transformation of epithelial cells by activated Ras results in the perturbation of cell-cell contacts. The ALL-1 fusion partner from chromosome 6 (AF-6) has been identified as a Ras target. AF-6 has the PDZ domain, which is thought to localize AF-6 at the specialized sites of plasma membranes such as cell-cell contact sites. The roles of Ras and AF-6 were investigated in the regulation of cell-cell contacts. AF-6 accumulates at the cell-cell contact sites of polarized MDCKII epithelial cells and has a distribution similar to that of ZO-1 but somewhat different from those of catenins. Immunoelectron microscopy reveals a close association between AF-6 and ZO-1 at the tight junctions of MDCKII cells. Native and recombinant AF-6 interacts with ZO-1 in vitro. ZO-1 interacts with the Ras-binding domain of AF-6; this interaction was inhibited by activated Ras. AF-6 accumulates with ZO-1 at the cell-cell contact sites in cells lacking tight junctions such as Rat1 fibroblasts and PC12 rat pheochromocytoma cells. The overexpression of activated Ras in Rat1 cells results in the perturbation of cell-cell contacts, followed by a decrease of the accumulation of AF-6 and ZO-1 at the cell surface. These results indicate that AF-6 serves as one of the peripheral components of tight junctions in epithelial cells and cell-cell adhesions in nonepithelial cells, and that AF-6 may participate in the regulation of cell-cell contacts, including tight junctions, via direct interaction with ZO-1 downstream of Ras (Yamamoto, 1997).

Hepatocyte growth factor/scatter factor (HGF/SF) stimulates the motility of epithelial cells, initially inducing centrifugal spreading of colonies followed by disruption of cell-cell junctions and subsequent cell scattering. In Madin-Darby canine kidney cells, HGF/SF-induced motility involves actin reorganization mediated by Ras, but whether Ras and downstream signals regulate the breakdown of intercellular adhesions has not been established. Both HGF/SF and V12Ras induce the loss of the adherens junction proteins E-cadherin and beta-catenin from intercellular junctions during cell spreading, and the HGF/SF response is blocked by dominant-negative N17Ras. Desmosomes and tight junctions are regulated separately from adherens junctions, because the adherens junctions are not disrupted by V12Ras. MAP kinase, phosphatidylinositide 3-kinase (PI 3-kinase), and Rac are required downstream of Ras, because loss of adherens junctions is blocked by the inhibitors PD098059 and LY294002 or by dominant-inhibitory mutants of either or both MAP kinase kinase 1 or Rac1. All of these inhibitors also prevent HGF/SF-induced cell scattering. Interestingly, activated Raf or the activated p110alpha subunit of PI 3-kinase alone does not induce disruption of adherens junctions. These results indicate that activation of both MAP kinase and PI 3-kinase by Ras are required for adherens junction disassembly and that the disassembly process is essential for the motile response to HGF/SF (Potempa, 1998).

Ras and Channels

Recent studies show that AMPA receptor trafficking is important in synaptic plasticity. However, the signaling controlling this trafficking is poorly understood. Small GTPases have diverse neuronal functions and their perturbation is responsible for several mental disorders. The roles of small GTPases Ras and Rap in the postsynaptic signaling underlying synaptic plasticity were examined. Ras relays the NMDA receptor and CaMKII signaling that drives synaptic delivery of AMPA receptors during long-term potentiation. In contrast, Rap mediates NMDA-receptro-dependent removal of synaptic AMPA receptors that occurs during long-term depression. Ras and Rap exert their effects on AMPA receptors that contain different subunit composition. Thus, Ras and Rap, whose activity can be controlled by postsynaptic enzymes, serve as independent regulators for potentiating and depressing central synapses (Zhu. 2002).

The cytoplasmic carboxyl tails of AMPA receptor constituent subunits, which show either long or short forms, control the trafficking characteristics of AMPA receptors. AMPA receptors with long cytoplasmic tails (e.g., GluR1 or GluR4) are restricted from synapses and delivered to synapses during activity-induced synaptic enhancement. AMPA-Rs with only short cytoplasmic tails (e.g., GluR2 or GluR3) cycle continuously from nonsynaptic to synaptic sites in an activity independent manner; their number at synapses can be reduced after activity-induced synaptic depression. The results indicate that spontaneous neural activity continuously adds into the synapses AMPA receptors containing long cytoplasmic tails via Ras activity and continuously removes from synapses AMPA receptors containing only short cytoplasmic tails via Rap activity. Similarly, this study argues that LTP adds to synapses AMPA receptors containing long cytoplasmic tails while LTD removes receptors containing only short cytoplasmic tails. These results indicate the existence of a replacement mechanism at synapses that can exchange AMPA receptors containing long cytoplasmic tails with those containing only short cytoplasmic tails, which explains the observation that LTP and LTD can reverse each other. In fact, this replacement has previously been detected and may itself be under some form of regulation. For example, a more robust replacement appears to occur in dissociated neuronal preparations where LTD stimuli lead to rapid removal of AMPA receptors with long cytoplasmic tails. Thus, the rate of receptor replacement and relative number of receptors with long or short cytoplasmic tails at a synapse may control the amount of LTP or LTD available at that synapse (Zhu, 2002).

Synaptic trafficking of AMPA-Rs, controlled by small GTPase Ras signaling, plays a key role in synaptic plasticity. However, how Ras signals synaptic AMPA-R trafficking is unknown. This study shows that low levels of Ras activity stimulate extracellular signal-regulated kinase kinase (MEK)-p42/44 MAPK (extracellular signal-regulated kinase [ERK]) signaling, whereas high levels of Ras activity stimulate additional Pi3 kinase (Pi3K)-protein kinase B (PKB) signaling, each accounting for ~50% of the potentiation during long-term potentiation (LTP). Spontaneous neural activity stimulates the Ras-MEK-ERK pathway that drives GluR2L into synapses. In the presence of neuromodulator agonists, neural activity also stimulates the Ras-Pi3K-PKB pathway that drives GluR1 into synapses. Neuromodulator release increases with increases of vigilance. Correspondingly, Ras-MEK-ERK activity in sleeping animals is sufficient to deliver GluR2L into synapses, while additional increased Ras-Pi3K-PKB activity in awake animals delivers GluR1 into synapses. Thus, state-dependent Ras signaling, which specifies downstream MEK-ERK and Pi3K-PKB pathways, differentially control GluR2L- and GluR1-dependent synaptic plasticity (Qin, 2005).

The results suggest that Ras signals synaptic insertion of AMPA-Rs via stimulating phosphorylation of S845 and S831 of GluR1 and S842 of GluR2L. Because Ras downstream signaling molecules ERK and PKB are unlikely to directly phosphorylate GluR1 and GluR2L, other molecules probably exist at synapses to relay the signaling. Two likely candidates are cAMP-dependent protein kinase (PKA) and calcium/calmodulin-dependent protein kinase II (CaMKII), since they can phosphorylate S845 and S831 of GluR1, respectively. Protein kinase C (PKC) is another putative candidate because it can phosphorylate S831, as well as S845, albeit to a lesser extent. However, whether ERK and PKB stimulate PKA, CaMKII, and/or PKC remains to be examined. In contrast, serine/threonine kinases Rsk (see Drosophila S6kII) and mTOR-S6K, which relay downstream Ras signaling in nonneuronal cells, may also serve as the relays. In particular, both Rsk and mTOR are expressed at synapses, and disruption of Rsk and mTOR signaling leads to mental retardation. Thus, determining the precise functional relationships (i.e., sequential or parallel, and downstream or upstream) of the signaling molecules involved in Ras pathways during LTP is central to answer many important questions related to the mechanisms of synaptic plasticity (Qin, 2005).

Though NMDA-R-dependent forms of synaptic plasticity have been extensively examined in vitro, little is known about their properties in the intact brain. Previous studies have shown that both the occurrence and magnitude of LTP induced by electric tetanization stimuli are higher in awake than sleeping animals. However, the mechanisms of this state-dependent LTP are unclear, because the LTP-inducing stimuli do not mimic physiological activity in these states. Both GluR2L and GluR1 mediate LTP in juvenile and adult animals. This study reports that synaptic activity in sleeping animals is sufficient for driving GluR2L but not GluR1 into synapses, whereas synaptic activity in awake animals drives more GluR2L as well as GluR1 into synapses, suggesting more synaptic plasticity in awake animals. Based on these findings, it is proposed that state-dependent physiological factors, such as neuromodulators, may control the state-dependent plasticity. Indeed, neuromodulator agonists (for example, histamine, a monoamine neuromodulator) can drive more GluR2L as well as GluR1 into synapses, by stimulating Ras signaling. These results are consistent with the previous findings that neuromodulators, whose release increases in general during the awake behavioral state, stimulate ERK and Pi3K signaling and potentiate LTP. It remains to be determined whether other state-dependent factors (i.e., neuronal firing patterns, hormones, and neurotrophic factors) regulate synaptic plasticity and how these factors interact in the intact brain (Qin, 2005).

Memory consolidation seems to occur during sleep and waking, while learning occurs in the conscious state. It is believed that the learning and memory processes require synaptic plasticity. This study shows that synaptic potentiation is present in both sleeping and awake states. Interestingly, synaptic plasticity in sleeping and awake states is controlled by different levels of Ras signaling and mediated by trafficking of distinct AMPA-Rs. The obvious puzzles are whether and how Ras-regulated, subunit-specific AMPA-R trafficking correlates with the different forms of memory consolidation and learning (e.g., declarative vs. procedural or explicit vs. implicit). Manipulating Ras signaling and trafficking of AMPA-Rs in intact animals (e.g., in vivo expression of Ras mutants and GluRct-GFP) during different behavioral states (e.g., slow-wave sleep, REM sleep, quiescent alert, and active exploring) and monitoring changes in learning and memory behavior promise to reveal new insights into these pivotal questions (Qin, 2005).

NMDA receptors (NMDARs) control bidirectional synaptic plasticity by regulating postsynaptic AMPA receptors (AMPARs). NMDAR activation can have differential effects on AMPAR trafficking, depending on the subunit composition of NMDARs. In mature cultured neurons, NR2A-NMDARs promote, whereas NR2B-NMDARs inhibit, the surface expression of GluR1, primarily by regulating its surface insertion. In mature neurons, NR2B is coupled to inhibition rather than activation of the Ras-ERK pathway, which drives surface delivery of GluR1. Moreover, the synaptic Ras GTPase activating protein (GAP) SynGAP is selectively associated with NR2B-NMDARs in brain and is required for inhibition of NMDAR-dependent ERK activation. Preferential coupling of NR2B to SynGAP could explain the subtype-specific function of NR2B-NMDARs in inhibition of Ras-ERK, removal of synaptic AMPARs, and weakening of synaptic transmission (Kim, 2005 ).

The ERK1/2 signaling pathway is activated by calcium influx through NMDARs and plays an important role in synaptic plasticity and cell survival. NMDAR-dependent ERK activation involves the small GTPase Ras, which is stimulated by specific guanine nucleotide exchange factors (GEFs) and inhibited by GTPase activating proteins (GAPs). The RasGEF RasGRF1 is reported to bind directly to the NR2B subunit of NMDARs. SynGAP, a RasGAP highly enriched in the postsynaptic density (PSD), can associate with NMDARs through binding to PSD-95 family proteins. The exact function of these Ras regulatory proteins in synaptic plasticity has not been established, and how they are functionally coupled to NMDARs remains unclear (Kim, 2005).

Altered AMPAR trafficking has emerged as a major postsynaptic mechanism for the expression of synaptic plasticity. A prevailing model is that NMDAR-dependent LTP is mediated by the surface insertion and synaptic delivery of GluR1, that is driven by CaM kinase II and the Ras-ERK pathway. In contrast, LTD is supposed to result, at least in part, from the removal of synaptic AMPARs by the increased endocytosis and/or reduced recycling of GluR2/3 subunits (Kim, 2005).

This study investigates the links between NMDAR subtypes, Ras-ERK signaling, and AMPAR trafficking. NR2A and NR2B are found to have antagonistic actions on Ras-ERK activation and AMPAR distribution in mature neurons. NR2A-NMDARs promote, whereas NR2B-NMDARs inhibit, the surface expression of GluR1 -- primarily by regulating GluR1 surface insertion. Potentially accounting for this difference is that NR2B is coupled to the inhibition rather than the activation of the Ras-ERK pathway. This functional coupling is correlated with the specific biochemical association of SynGAP with NR2B-NMDARs (Kim, 2005).

Ras interaction with Minibrain homologs

Dual-specificity tyrosine-phosphorylated and regulated kinase 1A (Dyrk1A) is the human homologue of Drosophila Minibrain. In Drosophila, mnb is involved in postembryonic neurogenesis. In human, DYRK1A maps within the Down syndrome critical region of chromosome 21 and is overexpressed in Down syndrome embryonic brain. Despite its potential involvement in the neurobiological alterations observed in Down syndrome patients, the biological functions of the serine/threonine kinase DYRK1A have not yet been identified. DYRK1A overexpression potentiates nerve growth factor (NGF)-mediated PC12 neuronal differentiation by up-regulating the Ras/MAP kinase signaling pathway independently of its kinase activity. Furthermore, DYRK1A prolongs the kinetics of ERK activation by interacting with Ras, B-Raf, and MEK1 to facilitate the formation of a Ras/B-Raf/MEK1 multiprotein complex. These data indicate that DYRK1A may play a critical role in Ras-dependent transducing signals that are required for promoting or maintaining neuronal differentiation and suggest that overexpression of DYRK1A may contribute to the neurological abnormalities observed in Down syndrome patients (Kelly, 2005).

Shp2 signaling upstream of Ras

SHP2 down-regulates PI3K activation by dephosphorylating; however, the mechanisms explaining the positive role of the Gab1/SHP2 pathway in EGF-induced Ras activation remain ill defined. Substrate trapping experiments suggest that SHP2 dephosphorylates other Gab1 phosphotyrosines located within a central region displaying four YXXP motifs. Because these sites are potential docking motifs for Ras-GAP, whether SHP2 dephosphorylates them to facilitate Ras activation was tested. A Gab1 construct preventing SHP2 recruitment promotes membrane relocation of RasGAP. Moreover, a RasGAP-inactive mutant restores the activation of Ras in cells transfected with SHP2-inactivating Gab1 mutant or in SHP2-deficient fibroblasts, supporting the hypothesis that RasGAP is a downstream target of SHP2. To determine whether Gab1 is a RasGAP-binding partner, a Gab1 mutant deleted of four YXXP motifs was produced. The deletion suppresses RasGAP redistribution and restores the defective Ras activation caused by SHP2-inactivating mutations. Moreover, Gab1 interacts with RasGAP SH2 domains, only under conditions where SHP2 is not activated. To identify Ras-GAP-binding sites, Tyr to Phe mutants of Gab1 YXXP motifs were produced. Gab1 constructs mutated on Tyr(317) are severely affected in RasGAP binding and are the most active in compensating for Ras-defective activation and blocking RasGAP redistribution induced by SHP2 inactivation. Thus a Ras-negative regulatory tyrosine phosphorylation site involved in RasGAP binding has been identified and an important SHP2 function has been demonstrated to down-regulate this site's phosphorylation to disengage RasGAP and sustain Ras activation (Montigner, 2005).

Evolutionary homologs: Table of contents

Ras85D: Biological Overview | Regulation | Protein Interactions | Effects of Mutation | Ras as Oncogene | References

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