Ras oncogene at 85D


EVOLUTIONARY HOMOLOGS

Table of contents

Ras2 and Rop of Drosophila

The product of the ras opposite (rop) gene is an essential component of secretion processes in Drosophila. The ROP gene product is homologous to the Caenorhabditis elegans UNC-18 and the rat munc-18/n-Sec1/rbSec1 proteins, implicated in the final steps of neurotransmitter exocytosis in nerve terminals, and the bovine mSec1 protein implicated in the secretion of catecholamines in chromaffin cells. The mammalian brain protein has been shown to exert its activity in the presynaptic membrane through transient interaction with syntaxin, an integral component of this membrane. rop is highly expressed in the Drosophila nervous system, where it acts as both a positive and negative modulator of neurotransmitter release. It is also expressed in specialized tissues in which intensive exocytic/endocytic cycles take place, including the garland cells, a small group of nephrocytes that take up waste materials from the hemolymph by endocytosis. rop is regulated by a bidirectional promoter shared with Ras2, a member of the R-ras/TC21 branch of the ras supergene family. Ras2 is also highly expressed in the garland cells. These cells are characterized by their labyrinthine channels, long invaginations extending from the cell membrane, and a rich population of a variety of vesicles. Rop is detected in the outer membranes of the labyrinthine channels, and in the outer membranes of many vesicles located nearby the labyrinthine channels, but not in vesicles located in inner parts of the cell. Rop is firmly bound to syntaxin. These findings suggest that similar to its role in the synaptic terminal, Rop is active in the garland cell during the final steps of exocytosis, probably by binding to syntaxin. The distribution of Ras2 (Ras oncogene at 64D) in the cortex of the garland cell is identical to that of Rop, suggesting that Ras2 might be also a component of the exocytotic/endocytic cycle of the cell (Halachmi, 1995).

The Drosophila ras2 promoter region exhibits bidirectional activity, as has been demonstrated for the human c-Ha-ras1 and the mouse c-Ki-ras. Drosophila ras2 provides the only example to date in which the flanking gene (rop) and its product have been isolated. A linking mechanism of control suggests a mutual interaction between the two gene products. The Drosophila ras2 promoter region shares with the human c-Ha-ras1 promoter a CACCC box and an AP-1-like sequence. A 14 bp promoter fragment holding a CACCC element is demonstrated to interact with a specific transcription factor (factor B). This CACCC promoter element represents a stretch of imperfect palindrome. This factor can form a complex with another specific DNA-binding protein (factor A). The binding sites (A + B) for these protein factors are essential for 95% expression of both genes flanking the promoter (ras2 and rop). Region A consists of four overlapping consensus sequences: a TATA-like element, a DSE-like motif (the core sequence of the serum response element), a DRE octamer, which has been shown to play a role in cell proliferation, and a 5 bp direct repeat representing the GATA consensus sequence. Factor A has a very weak affinity to the full promoter region, but when complexed with factor B, binding efficiency is enhanced. Alterations of DNA-protein binding specificities can be achieved by supplementing the growth media with different sera (Lightfoot, 1994).

Rap and Rapgap1 of Drosophila

Mammalian Rap1 is a small, Ras-like GTPase that was first identified as a protein that could suppress the oncogenic transformation of cells by Ras. Rap1 is activated by several extracellular stimuli and may be involved in cellular processes such as cell proliferation, cell differentiation, T-cell anergy and platelet activation. At least three different second messengers, namely diacylglycerol, calcium and cyclic AMP, are able to activate Rap1 by promoting its release of the guanine nucleotide GDP and its binding to GTP. Activation of Rap1 by forskolin and cAMP occurs independent of protein kinase A (also known as cAMP-activated protein kinase). The gene encoding a guanine-nucleotide-exchange factor (GEF) has been cloned and named Epac (exchange protein directly activated by cAMP). This protein contains a cAMP-binding site and a domain that is homologous to domains of known GEFs for Ras and Rap1. Epac binds cAMP in vitro and exhibits in vivo and in vitro GEF activity towards Rap1. cAMP strongly induces the GEF activity of Epac towards Rap1 both in vivo and in vitro. It is concluded that Epac is a GEF for Rap1 that is regulated directly by cAMP and that Epac is a new target protein for cAMP (de Rooij, 1998).

The Rap proteins are highly related to Ras and extremely conserved among diverse species. Indeed, the human and Drosophila Rap proteins are more closely related than are human and Drosophila Ras. Drosophila Rap1 mutants are lethal at the larval stage; embryos that lack maternally provided Rap1 develop abnormally, largely because of defects in morphogenesis, suggesting a role for Rap1 in the regulation of cell shape and cell adhesion. The activity of Ras family proteins is modulated in vivo by the function of GTPase activating proteins, which increase their intrinsic rate of GTP hydrolysis. cDNAs encoding a GAP have been isolated for the Drosophila Rap1 GTPase. Drosophila Rapgap1 encodes an 850-amino acid protein with a central region that displays substantial sequence similarity to human RapGAP. This domain, when expressed in Escherichia coli, potently stimulates Rap1 GTPase activity in vitro. Unlike Rap1, which is ubiquitously expressed, Rapgap1 expression is highly restricted. Rapgap1 is expressed at high levels in the developing photoreceptor cells and in the optic lobe. Rapgap1 mRNA is also localized in the pole plasm in an oskar-dependent manner. Although mutations that completely abolish Rapgap1 function display no obvious phenotypic abnormalities, overexpression of Rapgap1 induces a rough eye phenotype that is exacerbated by reducing Rap1 gene dosage. Thus, Rapgap1 can function as a negative regulator of Rap1-mediated signaling in vivo (Chen, 1997).

The Ras-related Rap GTPases are highly conserved across diverse species but their normal biological function is not well understood. Initial studies in mammalian cells have suggested a role for Rap as a Ras antagonist. More recent experiments indicate functions in calcium- and cAMP-mediated signaling and it has been proposed that protein kinase A-mediated phosphorylation activates Rap in vivo. Ras1-mediated signaling pathways in Drosophila are shown to not be influenced by Rap1 levels, suggesting that Ras1 and Rap1 function via distinct pathways. Moreover, a mutation that abolishes the putative cAMP-dependent kinase phosphorylation site of Drosophila Rap1 can still rescue the Rap1 mutant phenotype. These experiments show that Rap1 is not needed for cell proliferation and cell-fate specification but demonstrate a critical function for Rap1 in regulating normal morphogenesis in the eye disk, the ovary and the embryo. Rap1 mutations also disrupt cell migrations and cause abnormalities in cell shape. These findings indicate a role for Rap proteins as regulators of morphogenesis in vivo (Asha, 1999).

Ras and the release (exchange) factor (GEF) SOS

Son-of-sevenless is a guanine nucleotide-releasing factor that activates Ras1 by promoting the exchange of GDP for GTP.

Growth factor receptors activate Ras by recruiting the nucleotide exchange factor son of sevenless (SOS) to the cell membrane, thereby triggering the production of GTP-loaded Ras. Crystallographic analyses of Ras bound to the catalytic module of SOS have led to the unexpected discovery of a highly conserved Ras binding site on SOS that is located distal to the active site and is specific for Ras-GTP. The crystal structures suggest that Ras-GTP stabilizes the active site of SOS allosterically, Ras-GTP is shown to form ternary complexes with SOScat in solution and increases significantly the rate of SOScat-stimulated nucleotide release from Ras. These results demonstrate the existence of a positive feedback mechanism for the spatial and temporal regulation of Ras (Margarit, 2003).

The sequence of human SOS1 was compared to the sequences of SOS from two insects, Drosophila melanogaster, and Anopheles gambiae, and to that of the worm Caenorhabditis elegans. The SOS protein is highly conserved throughout the REM and cdc25 domains. The REM (Ras exchanger motif) domain and cdc25 domain (so named because of sequence similarity to cdc25, the Ras-specific nucleotide exchange factor in Saccharomyces cerevisiae) of human SOS1 are 52% and 61% identical in sequence, respectively, to the corresponding domains in the D. melanogaster SOS protein. For the human-A. gambiae and human-C. elegans comparisons, the levels of sequence identity are 54% and 63% (A. gamabiae) and 34% and 42% (C. elegans), respectively, for the two domains. Examination of sequence variation on the surface of SOScat reveals that in addition to a patch of highly conserved residues at the active site of SOScat, there is also a patch of conserved residues on the surface extending out of the active site and into the REM domain, surrounding the distal surface of SOScat. At the levels of sequence identity which pertain here (35%-55% overall identity in the pairwise comparisons), it is expected that residues in the hydrophobic core and the active site of SOScat will be highly conserved but that residues on the surface will not be conserved unless they have functional importance. In particular, it is significant that there is a striking conservation in the surface-exposed residues of SOScat that make contact with the distal Ras-GTP (Margarit, 2003).

Several of the residues in SOS that interact with the distal Ras are invariant across all four species. Arg 688, which forms hydrogen bonds with Glu-37 in the Switch 1 region of Ras, is invariant. The two cis-prolines in the hairpin base that forms the core of the cdc25 interface with distal Ras are part of a sequence motif (921SINPPC926 in human SOS1) that is invariant across all four sequences, suggesting that the unusual turn structure is conserved. The conservation of these features of the distal binding site raises the possibility that the distal interaction with Ras-GTP may also be conserved (Margarit, 2003).

A striking feature of the ternary Ras-GTP:SOScat:Ras complex is a network of tightly linked interactions, which span the REM and cdc25 domains and Ras-GTP and are suggestive of an allosteric mechanism whereby Ras-GTP stabilizes SOScat and stimulates its exchange factor activity. The rate of release of fluorescent nucleotide derivatives bound to Ras was measured in the presence of SOScat and if was found that the addition of Ras-GTP significantly accelerates the rate of SOScat-stimulated GDP release from Ras, whereas the addition of Ras-GDP does not. Taken together, these results point to the presence of a hitherto unsuspected positive feedback mechanism in the activation of Ras by SOS (Margarit, 2003).

Signaling from receptor tyrosine kinases (RTKs) requires the sequential activation of the small GTPases Ras and Rac. Son of sevenless (Sos-1), a bifunctional guanine nucleotide exchange factor (GEF), activates Ras in vivo and displays Rac-GEF activity in vitro, when engaged in a tricomplex with Eps8 and E3b1-Abi-1, a RTK substrate and an adaptor protein, respectively. A mechanistic understanding of how Sos-1 coordinates Ras and Rac activity is, however, still missing. This study demonstrate that (a) Sos-1, E3b1, and Eps8 assemble into a tricomplex in vivo under physiological conditions; (b) Grb2 and E3b1 bind through their SH3 domains to the same binding site on Sos-1, thus determining the formation of either a Sos-1-Grb2 (S/G) or a Sos-1-E3b1-Eps8 (S/E/E8) complex, endowed with Ras- and Rac-specific GEF activities, respectively; (c) the Sos-1-Grb2 complex is disrupted upon RTKs activation, whereas the S/E/E8 complex is not; and (d) in keeping with the previous result, the activation of Ras by growth factors is short-lived, whereas the activation of Rac is sustained. Thus, the involvement of Sos-1 at two distinct and differentially regulated steps of the signaling cascade allows for coordinated activation of Ras and Rac and different duration of their signaling within the cell (Innocenti, 2002).

Vulval induction in C. elegans has helped define an evolutionarily conserved signal transduction pathway from receptor tyrosine kinases (RTKs) through the adaptor protein SEM-5 to RAS. One component present in other organisms, a guanine nucleotide exchange factor for Ras, has been missing in C. elegans. To understand the regulation of this pathway it is crucial to have all positive-acting components in hand. The identification, cloning and genetic characterization of C. elegans SOS-1, a putative guanine nucleotide exchanger for LET-60 RAS, is described. RNA interference experiments suggest that SOS-1 participates in RAS-dependent signaling events downstream of LET-23 EGFR, EGL-15 FGFR and an unknown RTK. The previously identified let-341 gene encodes SOS-1. Analyzing vulval development in a let-341 null mutant, an SOS-1-independent pathway has been found that is involved in the activation of RAS signaling. This SOS-1-independent signaling is not inhibited by SLI-1/Cbl and is not mediated by PTP-2/SHP, raising the possibility that there could be another RasGEF (Chang, 2000).

The three proteins Grb2-Sem-5, Shc and Sos have been implicated in the signaling pathway that extends from tyrosine kinase receptors to Ras. Grb2-Sem-5 binds directly to murine Sos1, a Ras exchange factor, through two SH3 domains. Sos is also associated with ligand-activated tyrosine kinase receptors that bind Grb2-Sem-5, and with the Grb2-Sem-5 binding protein, Shc. Ectopic expression of Drosophila Sos stimulates morphological transformation of rodent fibroblasts. These data define a pathway by which tyrosine kinases act through Ras to control cell growth and differentiation (Egan, 1993).

In response to stimulation with epidermal growth factor (EGF), the guanine nucleotide exchange factor human SOS1 (hSOS1) promotes the activation of Ras by forming a complex with Grb2 and the human EGF receptor (hEGFR). hSOS1 is phosphorylated in cells stimulated with EGF or phorbol 12-myristate 13-acetate or following co-transfection with activated Ras or Raf. Co-transfection with dominant negative Ras results in a decrease of EGF-induced hSOS1 phosphorylation. The mitogen-activated protein kinase (MAPK) phosphorylates hSOS1 in vitro within the carboxyl-terminal proline-rich domain. The same region of hSOS1 is phosphorylated in vivo, in cells stimulated with EGF. Tryptic phosphopeptide mapping shows that MAPK phosphorylates hSOS1 in vitro on sites that were also phosphorylated in vivo. Phosphorylation by MAPK does not affect hSOS1 binding to Grb2 in vitro. However, reconstitution of the hSOS1-Grb2-hEGFR complex shows that phosphorylation by MAPK markedly reduced the ability of hSOS1 to associate with the hEGFR through Grb2. Similarly, phosphorylated hSOS1 is unable to form a complex with Shc through Grb2. Thus phosphorylation of hSOS1 down-regulates signal transduction from the hEGFR to the Ras pathway, by affecting hSOS1's interaction with the hEGFR or Shc (Porfiri, 1996).

The human protein Grb2 binds to ligand-activated growth factor receptors and downstream effector proteins through its respective Src-homology (SH) domains SH2 and SH3. Like its homolog from Caenorhabditis elegans, Sem-5, Grb2 apparently forms part of a highly conserved pathway by which these receptors can control Ras activity. The SH3 domains of Grb2 bind to the carboxy-terminal part of hSos1, the human homolog of the Drosophila guanine-nucleotide-releasing factor for Ras, which is essential for control of Ras activity by epidermal growth factor receptor and sevenless. A synthetic 10-amino-acid peptide containing the sequence PPVPPR specifically blocks the interaction. These results indicate that the Grb2/hSos1 complex couples activated EGF receptor to Ras signaling (Li, 1993).

Tyrosine kinase receptors stimulate the Ras signaling pathway by enhancing the activity of the SOS nucleotide-exchange factor. This occurs, at least in part, by the recruitment of an SOS-GRB2 complex to Ras in the plasma membrane. A different signaling pathway to Ras exists that involves activation of the Ras-GRF exchange factor in response to Ca2+ influx. The ability of Ras-GRF to activate Ras in vivo is markedly enhanced by raised Ca2+ concentrations. Activation is mediated by calmodulin binding to an IQ motif in Ras-GRF, because substitutions in conserved amino acids in this motif prevent both calmodulin binding to Ras-GRF and Ras-GRF activation in vivo. So far, full-length Ras-GRF has been detected only in brain neurons. These findings implicate Ras-GRF in the regulation of neuronal functions that are influenced by Ca2+ signals (Farnsworth, 1995).

Members of the Ras subfamily of small guanine-nucleotide-binding proteins are essential for controlling normal and malignant cell proliferation as well as cell differentiation. The neuronal-specific guanine-nucleotide-exchange factor, Ras-GRF/CDC25Mm, induces Ras signaling in response to Ca2+ influx and activation of G-protein-coupled receptors in vitro, suggesting that it plays a role in neurotransmission and plasticity in vivo. Ras-GRF is exclusively expressed in neurons of the postnatal and adult central nervous system and is mainly localized in the synaptosomal fraction. Following activation of muscarinic M1 and M2 receptors, Ras-GRF becomes phosphorylated; this increases its exchange activity. Instead of presenting a Grb2-binding domain, Ras-GRF contains an ilimaquinone domain. When intracellular calcium is increased, this domain is necessary for binding to Ca2+ calmodulin and for Ras-GRF-dependent activation of the Ras/MAPK pathway. Mice lacking Ras-GRF are impaired in the process of memory consolidation, as revealed by emotional conditioning tasks that require the function of the amygdala; learning and short-term memory are intact. Electrophysiological measurements in the basolateral amygdala reveal that long-term plasticity is abnormal in mutant mice. In contrast, Ras-GRF mutants do not reveal major deficits in spatial learning tasks, such as the Morris water maze, a test that requires hippocampal function. Consistent with apparently normal hippocampal functions, Ras-GRF mutants show normal NMDA (N-methyl-D-aspartate) receptor-dependent long-term potentiation in this structure. These results implicate Ras-GRF signaling via the Ras/MAP kinase pathway in synaptic events leading to formation of long-term memories (Brambilla, 1997).

It has been suggested that a key event in growth factor-induced p21Ras activation by the guanine nucleotide exchange factor Sos, is the recruitment of Sos to the plasma membrane by its interaction with the adaptor protein Grb2. However, other evidence argues that the sub cellular localization of Sos is independent of Grb2, and that the Sos/Grb2 interaction can be dispensed with for p21Ras activation. To clarify the role of the Sos/Grb2 interaction in ligand-stimulated p21Ras activation, the observation that overexpression of the Sos C-terminal domain can effectively inhibit p21Ras-dependent signaling in three different mammalian systems has been utilized. Concurrent expression of Grb2, but not SH2 or SH3 domain mutants of Grb2, or the alternative adaptor protein Nck (Drosophila homolog: Dreadlocks) can rescue this inhibitory effect of the C-terminus. This shows that the Grb2/Sos interaction is required to mediate growth factor-dependent activation of p21Ras, and requires the presence of intact SH2 and SH3 domains of Grb2. This approach was also used for a functional analysis of Sos which reveals that growth factor dependent signals are transmitted through both the N-terminal and C-terminal domains (Byrne, 1996).

The guanine nucleotide exchange factor Sos mediates the coupling of receptor tyrosine kinases to Ras activation. To investigate the mechanisms that control Sos activity, the contribution of various domains to its catalytic activity has been analyzed. Using human Sos1 (hSos1) truncation mutants, it has been shown that Sos proteins lacking either the amino or the carboxyl terminus domain, or both; they also display a guanine nucleotide exchange activity that is significantly higher compared with that of the full-length protein. The amino terminal domain of Sos is approximately 600 amino acids long and contains regions of homology to the Dbl (DH) and pleckstin (PH) domains. PH domains in Sos proteins have been implicated in the regulation of Sos guanine nucleotide exchange activity and ligand-dependent membrane targeting. The function of the DH domain is unknown. The catalytic activity of Sos is mediated by a central domain of approximately 420 amino acids that is highly conserved among different Ras exchange factors. The C-terminus domain of Sos proteins is characterized by the presence of multiple proline-rich SH3 binding sites, which mediate interaction with the adaptor molecule Grb2. Both the amino and the carboxyl terminus domains of Sos are involved in the negative regulation of Sos's catalytic activity. In vitro Ras binding experiments suggest that the amino and carboxyl terminus domains exert negative allosteric control on the interaction of the Sos catalytic domain with Ras. The guanine nucleotide exchange activity of hSos1 is not augmented by growth factor stimulation, indicating that Sos activity is constitutively maintained in a downregulated state. Deletion of both the amino and the carboxyl terminus domains is sufficient to activate the transforming potential of Sos. These findings suggest a novel negative regulatory role for the amino terminus domain of Sos and indicate a cooperation between the amino and the carboxyl terminus domains in the regulation of Sos activity (Corbalan-Garcia, 1998).

The crystal structure of human H-Ras complexed with the Ras guanine-nucleotide-exchange-factor region of the Son of sevenless (Sos) protein has been determined at 2.8 A resolution. The normally tight interaction of nucleotides with Ras is disrupted by Sos in two ways. (1) The insertion into Ras of an alpha-helix from Sos results in the displacement of the Switch 1 region of Ras, opening up the nucleotide-binding site. (2) Side chains presented by this helix and by a distorted conformation of the Switch 2 region of Ras alter the chemical environment of the binding site for the phosphate groups of the nucleotide and the associated magnesium ion, so that their binding is no longer favoured. Sos does not impede the binding sites for the base and the ribose of GTP or GDP, so the Ras-Sos complex adopts a structure that allows nucleotide release and rebinding (Boriack-Sjodin, 1998).

Ras and Rac are membrane-associated GTPases that function as molecular switches activating intracellular mitogen-activated protein kinase (MAPK) cascades and other effector pathways in response to extracellular signals. Activation of Ras and Rac into their GTP-bound conformations is directly controlled by specific guanine-nucleotide exchange factors (GEFs), which catalyze GDP release. Several Ras-specific GEFs that are related to the budding yeast protein Cdc25p have been described, whereas GEFs for Rac-related GTPases contain a region that is homologous to the oncoprotein DbI. The Ras-GRF1 and Ras-GRF2 proteins, which couple Ras activation to serpentine receptors and calcium signals, contain both Cdc25 and DbI homology (DH) regions. Ras-GRF2 is a bifunctional signaling protein that is able to bind and activate Ras and Rac, and thereby coordinate the activation of the extracellular-signal-regulated kinase (ERK) and stress-activated protein kinase (SAPK) pathways (Fan, 1998).

Intersectin, a protein containing two EH and five SH3 domains, has been idnetified as a component of the endocytic machinery. The N-terminal SH3 domain (SH3A), unlike other SH3 domains from intersectin or various endocytic proteins, specifically inhibits intermediate events leading to the formation of clathrin-coated pits. A brain-enriched, 170 kDa protein (p170) interacts specifically with SH3A. Screening of combinatorial peptides reveals the optimal ligand for SH3A as Pp(V/I)PPR. The 170 kDa mammalian son-of-sevenless (mSos1) protein, a guanine-nucleotide exchange factor for Ras, contains two copies of the matching sequence, PPVPPR. Immunodepletion studies confirm that p170 is mSos1. Intersectin and mSos1 are co-enriched in nerve terminals and are co-immunoprecipitated from brain extracts. SH3A competes with the SH3 domains of Grb2 in binding to mSos1, and the intersectin-mSos1 complex can be separated from Grb2 by sucrose gradient centrifugation. Overexpression of the SH3 domains of intersectin blocks epidermal growth factor-mediated Ras activation. These results suggest that intersectin functions in cell signaling in addition to its role in endocytosis and may link these cellular processes (Tong, 2000).

Given that intersectin is involved in the formation of clathrin-coated pits, and that the intersectin SH3A domain interacts specifically with cellular targets that function early in the formation of a clathrin-coated bud, it is interesting to speculate that mSos1 may also play a role in clathrin-coated pit formation, possibly through activation of Ras. Many vesicular budding events that are mediated by coat proteins are initiated by the activation of small GTP-binding proteins through the actions of guanine-nucleotide exchange factors. Overexpression of mutant forms of Ras, as well as the small GTP-binding protein Ral, which plays a major role in mediating downstream Ras function, blocks the internalization of the EGF receptor. Furthermore, mSos1 can activate Rac, which has been implicated in transferrin receptor endocytosis. Finally, it should be noted that the long form of intersectin, which is generated by alternative splicing in neuronal tissues, contains DH, PH and C2 domains. Comparison of the primary structure of the DH and PH domains with other proteins suggests that the long form of intersectin may be a guanine-nucleotide exchange factor for Rho. Further work is necessary to clarify the involvement of GTP-binding proteins in clathrin-mediated endocytosis (Tong, 2000 and references therein).

Another possible function of the intersectin-mSos1 complex is to couple the molecular machineries for endocytosis and signal transduction. For example, it has been demonstrated that dynamin-dependent endocytosis of the EGF receptor is necessary for EGF-dependent activation of the MAP-kinase pathway. The ability of insulin-like growth factor-1 (IGF-1) to activate the SHC/MAP-kinase pathway, but not the insulin receptor substrate 1 pathway, is also dependent on clathrin-mediated endocytosis of the IGF receptor. Furthermore, endocytosis of the beta2-adrenergic receptor is necessary for coupling to MAP-kinase activation. Specifically, overexpression of a mutant form of beta-arrestin, which prevents the beta2-adrenergic receptor from targeting to clathrin-coated pits, blocks agonist activation of MAP kinase. Thus, it is possible that the clathrin-coated pit can function as a membrane microdomain, directing the assembly of signaling complexes, much as has been proposed for caveoli. In fact, activation of the EGF receptor can lead to the formation of signaling complexes that include mSos1. Such signaling complexes are localized largely in endosomes (Tong, 2000 and references therein).

Given the evidence for a link between endocytosis and signaling, it is interesting to speculate that intersectin could play an important role in bringing together endocytic proteins such as dynamin, with signaling molecules such as mSos1. In fact, the data demonstrating that overexpression of the SH3 domains of intersectin functions in a dominant-negative manner to block EGF-dependent Ras activation strongly support a role for intersectin in cell signaling. Moreover, human intersectin has been found to interact by yeast two-hybrid screening with the proto-oncogene product, c-Cbl, a tyrosine kinase substrate with ubiquitin ligase activity, and transfection experiments have revealed that full-length intersectin functions in cell signaling pathways leading to activation of the Elk-1 transcription factor. Finally, in a genetic screen in Drosophila, Dap160, the Drosophila homolog of intersectin (Roos, 1998), was selected as a negatively regulating component of the Sevenless receptor tyrosine kinase/MAP-kinase pathway (Rintelen and Hafen, personal communication to Tong, 2000). Thus, intersectin appears to have a dual function in both endocytosis as well as signal transduction pathways, and it may play a role as an interface between these two important cellular processes (Tong, 2000 and references therein).

The PDZ domain-containing proteins, such as PSD-95 and GRIP, have been suggested to be involved in the targeting of glutamate receptors, a process that plays a critical role in the efficiency of synaptic transmission and plasticity. To address the molecular mechanisms underlying AMPA receptor synaptic localization, several GRIP-associated proteins (GRASPs) have been identified that bind to distinct PDZ domains within GRIP. GRASP-1 is a neuronal rasGEF associated with GRIP and AMPA receptors in vivo. Overexpression of GRASP-1 in cultured neurons specifically reduces the synaptic targeting of AMPA receptors. In addition, the subcellular distribution of both AMPA receptors and GRASP-1 is rapidly regulated by the activation of NMDA receptors. These results suggest that GRASP-1 may regulate neuronal ras signaling and contribute to the regulation of AMPA receptor distribution by NMDA receptor activity (Ye, 2000).

LTP and LTD have been proposed to be mediated, in part, by changes in AMPA receptor function. Increases in AMPA receptor responses have been observed during the expression of LTP. Recently, it has been shown that a high proportion of synapses in hippocampal CA1 region contains only NMDA receptors and acquires AMPA receptors only after the induction of LTP. This emergence of AMPA receptor current seems due to the appearance of synaptic AMPA receptors. Moreover, NMDA receptor-dependent LTD in cultured neurons has recently been observed to correlate with a decrease in the levels of synaptic AMPA receptors. Previous studies have suggested that AMPA receptor-associated proteins, such as GRIP, are involved in the synaptic targeting of AMPA receptors. In this study, GRASP-1 has been added to this complex and evidence is provided that GRASP-1 may also be important in regulation of AMPA receptor function and may play a role in AMPA receptor synaptic targeting. Overexpression of GRASP-1 in neurons downregulates synaptic AMPA receptor clusters, while it has no effect on synaptic NMDA receptor synaptic targeting. Both the rasGEF catalytic domain and the C-terminal 'regulatory' domain were required for this activity. Activation of NMDA receptors dramatically induces the redistribution of both GRASP-1 and AMPA receptors from punctate membrane structures to a more diffuse pattern. Together with the GRASP-1 overexpression data, these results suggest that the overall spatial distribution of GRASP-1, as well as the absolute levels, may be important for AMPA receptor targeting. These results suggest that GRASP-1 and possibly ras signaling may play a role in the regulation of AMPA receptor synaptic targeting and its regulation by NMDA receptor activity (Ye, 2000).

Phospholipase C-gamma1 (PLC-gamma1: see Drosophila Small wing) hydrolyzes phosphatidylinositol 4,5-bisphosphate to the second messengers inositol 1,4,5-trisphosphate and diacylglycerol (DAG). PLC-gamma1 is implicated in a variety of cellular signalings and processes including mitogenesis and calcium entry. However, numerous studies demonstrate that the lipase activity is not required for PLC-gamma1 to mediate these events. The phospholipase activity of PLC-gamma1 plays an essential role in nerve growth factor (NGF)-triggered Raf/MEK/MAPK pathway activation in PC12 cells. Employing PC12 cells stably transfected with an inducible form of wild-type PLC-gamma1 or lipase inactive PLC-gamma1 with histidine 335 mutated into glutamine in the catalytic domain, it is shown that NGF provokes robust activation of MAP kinase in wild-type but not in lipase inactive cells. Both Ras/C-Raf/MEK1 and Rap1/B-Raf/MEK1 pathways are intact in the wild-type cells. By contrast, these signaling cascades are diminished in the mutant cells. Pretreatment with cell permeable DAG analog 1-oleyl-2-acetylglycerol rescues the MAP kinase pathway activation in the mutant cells. These observations indicate that the lipase activity of PLC-gamma1 mediates NGF-regulated MAPK signaling upstream of Ras/Rap1 activation probably through second messenger DAG-activated Ras and Rap-GEFs (Rong, 2004).

Two important Ras guanine nucleotide exchange factors, Son of sevenless (Sos) and Ras guanine nucleotide releasing protein (RasGRP), have been implicated in controlling Ras activation when cell surface receptors are stimulated. To address the specificity or redundancy of these exchange factors, Sos1/Sos2 double- or RasGRP3-deficient B cell lines were generated and their ability to mediate Ras activation upon B cell receptor (BCR) stimulation was determined. The BCR requires RasGRP3; in contrast, epidermal growth factor receptor is dependent on Sos1 and Sos2. Furthermore, BCR-induced recruitment of RasGRP3 to the membrane and the subsequent Ras activation are significantly attenuated in phospholipase C-gamma2-deficient B cells. This defective Ras activation is suppressed by the expression of RasGRP3 as a membrane-attached form, suggesting that phospholipase C-gamma2 regulates RasGRP3 localization and thereby Ras activation (Oh-hora, 2003).

The Ras-specific guanine nucleotide-exchange factors Son of sevenless (Sos) and Ras guanine nucleotide-releasing factor 1 (RasGRF1) transduce extracellular stimuli into Ras activation by catalyzing the exchange of Ras-bound GDP for GTP. A truncated form of RasGRF1 containing only the core catalytic Cdc25 domain is sufficient for stimulating Ras nucleotide exchange, whereas the isolated Cdc25 domain of Sos is inactive. At a site distal to the catalytic site, nucleotide-bound Ras binds to Sos, making contacts with the Cdc25 domain and with a Ras exchanger motif (Rem) domain. This allosteric Ras binding stimulates nucleotide exchange by Sos, but the mechanism by which this stimulation occurs has not been defined. A crystal structure of the Rem and Cdc25 domains of Sos is presented, determined at 2.0-Å resolution in the absence of Ras. Differences between this structure and that of Sos bound to two Ras molecules show that allosteric activation of Sos by Ras occurs through a rotation of the Rem domain that is coupled to a rotation of a helical hairpin at the Sos catalytic site. This motion relieves steric occlusion of the catalytic site, allowing substrate Ras binding and nucleotide exchange. A structure of the isolated RasGRF1 Cdc25 domain determined at 2.2-Å resolution, combined with computational analyses, suggests that the Cdc25 domain of RasGRF1 is able to maintain an active conformation in isolation because the helical hairpin has strengthened interactions with the Cdc25 domain core. These results indicate that RasGRF1 lacks the allosteric activation switch that is crucial for Sos activity (Freedman, 2006).

Ras is a critical signaling molecule that cycles between inactive GDP-bound and active GTP-bound states. The activation of Ras by receptor tyrosine kinases proceeds through the recruitment of the nucleotide-exchange factor Son of sevenless (Sos) to the plasma membrane, where it encounters Ras and stimulates release of GDP, allowing its replacement by GTP. In some cells, G protein-coupled receptors rely on relatives of Sos, such as Ras guanine nucleotide-releasing factor 1 (RasGRF1), also known as p140Ras-GRF or Cdc25, for initiating Ras signaling (Mattingly, 1996; Wei, 1993; Tian 2004; Freedman, 2006 and references therein).

The region of Sos that is required for Ras-specific nucleotide-exchange activity, Soscat, contains a Ras exchanger motif (Rem) domain and a Cdc25 homology domain (Kim, 1998; Boriack-Sjodin, 1998). In addition, Sos requires allosteric activation through a second Ras-binding site that bridges the Rem and Cdc25 domains (Margarit, 2003; Sondermann, 2004). When Sos is activated, the Cdc25 domain of Sos inserts a helical hairpin between two flexible regions of Ras, switch 1 and switch 2, opening the nucleotide-binding site of Ras for GDP release (Boriack-Sjodin, 1998). Ras·GTP binds more tightly to the allosteric site than does Ras·GDP, leading to positive feedback on the initiation of nucleotide exchange (Margarit, 2003; Sondermann, 2004). Ras binding at the allosteric site has been shown to increase the affinity of Ras for the Sos catalytic site (Sondermann, 2004), but the structural basis for this allosteric activation has not been clear. In contrast to Sos, which requires Ras binding to the allosteric site for activity, the Cdc25 domain of RasGRF1 is active on its own (Lenzen, 1998; Freedman, 2006 and references therein).

To identify the conformational changes that accompany Sos activation, the crystal structure of Soscat, containing the Rem and Cdc25 domains in the absence of Ras was determined at 2.0-Å resolution and the structure of the Cdc25 domain of RasGRF1, also without Ras bound, was determined at 2.2-Å resolution. Comparison of these structures with that of Soscat bound to Ras (Boriack-Sjodin, 1998; Margarit, 2003) reveals the switch by which allosteric Ras binding conveys an activating signal to the Sos catalytic site and the structural basis for RasGRF1 activity in the absence of allosteric activation (Freedman, 2006).

Nucleotide-exchange assays were performed in which the release rate of fluorescently labeled GDP from Ras was monitored in the presence and absence of nucleotide-exchange factor. Guided by secondary structure prediction and sequence alignment to Sos, a construct was created of RasGRF1 that spans residues 1,028 to 1,262, RasGRF1Cdc25, which is 51 residues shorter than that used in earlier biochemical studies. The rate of nucleotide release from Ras in the presence of RasGRF1Cdc25 was comparable to the value for 1 µM exchange factor reported previously and is significantly higher than the intrinsic rate of nucleotide release by isolated Ras (1.8 ± 0.2 x 10–4 s–1) (Freedman, 2006).

Soscat (Rem-Cdc25) displays a basal level of nucleotide-exchange activity attributable to allosteric activation by Ras·GDP, normally present as the substrate in nucleotide-exchange assays (Sondermann, 2004, Guo, 2005). To minimize this interference, 0.1 µM substrate Ras·GDP was used, a concentration 10-fold lower than the substrate concentrations used in previous studies and >100-fold lower than the value estimated for the dissociation constant (>25 µM) for Ras·GDP binding at the allosteric site of Sos. Under these conditions, the rate of nucleotide release from Ras in the presence of Sos (5 ± 2 x 10–4 s–1 for 1 µM exchange factor) is comparable to the intrinsic rate of nucleotide release by isolated Ras and also to the observed nucleotide-release rate in the presence of Soscat W729E, a mutant that is impaired in binding allosteric Ras (4.7 ± 0.5 x 10–4 s–1 for 1 µM exchange factor. In the presence of saturating concentrations of RasY64A, a mutant of Ras that binds to the allosteric site of Sos but not to the active site, the nucleotide-release rate is increased over that of unstimulated Soscat (i.e., Sos in which the allosteric site is predominantly unoccupied) by a factor of 75 (380 ± 20 x 10–4 s–1 for 1 µM exchange factor and 40 µM RasY64A·GMPPNP). Given these results, uncomplexed Soscat is referred to as "inactive" and Ras-bound Soscat is referred to as "active" (Freedman, 2006).

It was also found that the isolated Sos Cdc25 domain (SosCdc25, residues 750–1,049) does not stimulate nucleotide release from Ras (the release rate is 1.5 ± 0.2 x 10–4 s–1 for 1 µM exchange factor. A longer construct (residues 731–1,049, containing the Cdc25 domain plus 19 residues that link the Rem and Cdc25 domains) also is inactive, as are both constructs in the presence of RasY64A. Circular dichroism spectroscopy was used to confirm that the inactivity of SosCdc25 does not result simply from lack of folding. Like Soscat, SosCdc25 is well folded, displaying a predominantly helical spectrum and a cooperative unfolding transition upon titration with chemical denaturant (Freedman, 2006).

Soscat (Rem-Cdc25) was crystallized in the absence of Ras and determined its structure was determined at 2.0-Å resolution. The overall structure of the Cdc25 domain of uncomplexed, inactive Soscat is similar to that of Ras-bound, active Soscat. There are, however, localized conformational changes in the Cdc25 and Rem domains in the absence of Ras. In the Cdc25 domain, the helical hairpin, a critical Ras-binding element, is rotated inward by ~10° in the structure of the uncomplexed Cdc25 domain of Soscat compared with its orientation in the Ras-bound structure. This conformational change is an en bloc movement of the helical hairpin with respect to the rest of the Cdc25 domain, as indicated by a distance difference matrix. A similar rotation of the Rem domain also is observed (Freedman, 2006).

The crystal structure of RasGRF1Cdc25 at 2.2-Å resolution was also determined. The structure of the Cdc25 domain of RasGRF1 is very similar to that of Sos, consistent with the 30% sequence identity within the two Cdc25 domains. The orientation of the helical hairpin of RasGRF1 resembles that of active Soscat and is rotated outward relative to that of uncomplexed Soscat. Distance difference matrices confirm that the differences between the Cdc25 domains of RasGRF1 and inactive Soscat are localized to the helical hairpin position relative to the rest of the Cdc25 domain, and that the conformation of RasGRF1Cdc25 is more similar to that of active Soscat (Freedman, 2006).

Sos engages Ras at the catalytic site by binding Tyr-64 from the switch 2 region of Ras in a deep pocket abutting the helical hairpin (Boriack-Sjodin, 1998), and the inability of Sos to release nucleotide from the RasY64A mutant shows that this interaction is essential for Sos-catalyzed nucleotide exchange (Hall, 2001). In the structure of uncomplexed Soscat, the inward-rotated helical hairpin generates extensive steric clashes with Ras modeled at the active site, effectively blocking access to the Tyr-64 binding pocket and rendering uncomplexed Sos inactive (Freedman, 2006).

Upon binding to the Sos allosteric site, nucleotide-bound Ras pulls the Rem domain downward by ~10°. The Rem and Cdc25 domains of Sos share an extensive interface, including a four-stranded Δ-sheet that incorporates two strands from the turn of the helical hairpin and two strands from the Rem domain. The structure of this Δ-sheet is unaltered in the uncomplexed and Ras-bound Sos structures, and so the position of the helical hairpin appears to be coupled strongly to the orientation of the Rem domain. When Ras binding to the allosteric site rotates the Rem domain, the helical hairpin is pulled along, opening the catalytic site for Ras. Another possible link between the Rem and Cdc25 domains of Sos is the hydrophobic interface between the Rem and Cdc25 domains, which has been shown by mutagenesis to be essential for Sos activity (Hall, 2001). A complex of Soscat bound to Ras at the allosteric site alone has not been crystallized. Because Soscat (Rem-Cdc25) requires occupation of the allosteric site for Ras interaction at the catalytic site (Sondermann, 2004), it is believed that the activating conformational change is attributable to the Ras molecule at the allosteric site and not the one at the catalytic site. The rotation of the Rem domain when Ras is not bound to the allosteric site has been seen previously in a crystal structure of autoinhibited Sos in which the allosteric site is blocked by the DH and PH domains (Sondermann, 2004), but low resolution of the data (3.6 Å) precluded a definitive analysis (Freedman, 2006).

Epac2, a Sos homolog that activates the Ras-related protein Rap1, is autoinhibited by regulatory domains that prevent Rap1 binding to the active site (Rehmann, 2006). Interestingly, the helical hairpin in the Cdc25 domain of inactive Epac2 is pivoted inward relative to that of active Sos, blocking the active site. As in Sos, the interaction between the helical hairpin of Epac2 and the Rem domain includes an interdomain β-sheet. A conformational switch driven by movements of the Rem domain and the helical hairpin thus appears to be a conserved feature among a subset of nucleotide-exchange factors for the Ras superfamily (Freedman, 2006).

The results discussed so far indicate that the helical hairpin of RasGRF1 is stable in the active conformation, whereas that of Sos is not. Strikingly, the helical hairpin of RasGRF1 is buttressed on either side by projections extending from the Cdc25 domain core, that are called flap1 and flap2, whereas the helical hairpin of Sos interacts less closely with the corresponding flaps. The bulky side chains of Tyr-1048, Phe-1051, and Phe-1052 from flap1 of RasGRF1 interact with Ile-1210 and Ile-1214 from the helical hairpin. Sos contains smaller residues at this interface, including Pro-801, Leu-804, and Val-805 in flap1 and Val-964 and Thr-968 in the helical hairpin. When activated by allosteric Ras binding, the helical hairpin of Soscat is rotated away from flap1. However, in the absence of allosteric Ras, the helical hairpin forms a tighter interface with flap1. A similar collapse of the RasGRF1 helical hairpin to a Sos-like inactive conformation appears to be prevented by the bulky residues in the flap1-helical hairpin interface (Freedman, 2006).

The link between flap2 and the helical hairpin of RasGRF1 is maintained by Arg-1160 and Arg-1165 in flap2 that bridge to Asp-1185 in the helical hairpin. Phe-1188 and Met-1181 from the helical hairpin enclose the arginine residues in flap2. In contrast, flap2 of Sos does not interact with the helical hairpin. The residues that anchor flap1 and flap2 of RasGRF1 to the helical hairpin are conserved in RasGRF1 sequences but not in Sos sequences (Freedman, 2006).

To further analyze the significance of these structural features, the effects were tested computationally of swapping residues from the Cdc25 domain of RasGRF1 into the Cdc25 domain of Sos and vice versa. Residues differing in the two polypeptide chains were allowed either to retain their original identity or to 'mutate' to the corresponding amino acid residue from the other protein. Monte Carlo-simulated annealing then was used to allow side chains to move while the backbone remained fixed. The energetic consequence of each substitution was calculated and used to determine whether a substitution move during the simulation was kept or discarded. In this way, the Sos or RasGRF1 sequence could accumulate substitutions that stabilize the observed backbone conformation in each simulation. Repetition of these simulations allowed the calculation of a substitution frequency for each residue, reflecting the number of times the wild-type residue swapped with the corresponding residue from the other protein in the low-energy sequences (Freedman, 2006).

These computational experiments yield the striking result that the Sos structure acquires several buried residues from the RasGRF1 sequence with high frequency, whereas relatively few buried residues in RasGRF1 are replaced by their counterparts in Sos. In RasGRF1, the positions that switch to the Sos sequence are located in the Cdc25 domain core, remote from the helical hairpin. This outcome differs from the results for Sos, in which a large number of sequence swaps occur in the helical hairpin or in abutting regions of the Cdc25 domain (Freedman, 2006).

These results indicate that RasGRF1 residues may be better than Sos residues at stabilizing the helical hairpin in the active backbone conformation. For example, active Sos acquires some high-frequency substitutions to RasGRF1 residues in the interface between flap1 and the helical hairpin. Sos residues Val-964, Thr-968, and Val-805 are replaced with the corresponding residues in RasGRF1, Ile, Ile, and Phe, respectively. Presumably, the larger side chains more effectively fill the gap between the helical hairpin and flap1 in the active Sos conformation imposed during the simulations in the absence of the Rem domain and allosteric Ras. Interestingly, in a similar simulation with the inactive Sos backbone structure, this interface does not acquire RasGRF1 residues. This finding is consistent with the observation that the flap1-helical hairpin interface is more tightly packed in inactive Sos, and so bulky RasGRF1 residues would destabilize this conformation and be rejected as higher-energy changes. In other respects, the inactive Sos simulation is similar to that of active Sos (Freedman, 2006).

The conformational switch used by Sos seems to occur at the expense of the conformational stability apparent in RasGRF1. For example Tyr-915 in Sos, Phe-930, Tyr-796, Met-824, Glu-792, Tyr-974, and Asn-866, which interact with the base of the Sos helical hairpin and accommodate the conformational switch, mutate to RasGRF1 residues in almost every Sos simulation, whereas the corresponding residues in RasGRF1 remain unchanged (Freedman, 2006).

Sos and RasGRF1 are homologous exchange factors that contain Rem and Cdc25 domains. Biochemical characterization of RasGRF1 (Lenzen, 1998) had established that only the Cdc25 domain is required for Ras-specific nucleotide-exchange activity, and so the subsequent discovery that Sos is inactive without allosteric Ras binding to the Rem and Cdc25 domains was surprising. This study now shows that this functional distinction between Sos and RasGRF1 is reflected in the structures of the Cdc25 domains of the two proteins. The helical hairpin jutting out from the Cdc25 domain of Sos switches between conformations that either block or support Ras binding to the catalytic site. The open and active conformation of the Sos Cdc25 domain is induced allosterically by the binding of Ras to the distal site formed by the Rem and Cdc25 domains, causing a pivoting of the Rem domain upon allosteric Ras binding that is coupled to the pivoting of the helical hairpin relative to the Cdc25 domain core (Freedman, 2006).

There is no structural information about the Rem domain of RasGRF1, and its function has not been fully explored. Previous studies have suggested that the Rem domain of RasGRF1 is regulatory in function, containing phosphorylation sites and PEST motifs. The level of activity observed for the isolated Cdc25 domain of RasGRF1, although significantly greater than the intrinsic rate of nucleotide release from Ras, is much lower than the maximum rate observed for allosterically activated Soscat. At this time it is unknown whether RasGRF1 is truly less active than Sos or whether a binding partner will be found that triggers enhanced RasGRF1 activity (Freedman, 2006).

Activation of the small guanosine triphosphatase H-Ras by the exchange factor Son of Sevenless (SOS) is an important hub for signal transduction. Multiple layers of regulation, through protein and membrane interactions, govern activity of SOS. This study characterized the specific activity of individual SOS molecules catalyzing nucleotide exchange in H-Ras. Single-molecule kinetic traces revealed that SOS samples a broad distribution of turnover rates through stochastic fluctuations between distinct, long-lived (more than 100 seconds), functional states. The expected allosteric activation of SOS by Ras-guanosine triphosphate (GTP) was conspicuously absent in the mean rate. However, fluctuations into highly active states were modulated by Ras-GTP. This reveals a mechanism in which functional output may be determined by the dynamical spectrum of rates sampled by a small number of enzymes, rather than the ensemble average (Iversen, 2014).

Intersectin: A SOS interacting protein that provides a link between the endocytic and mitogenic machinery of the cell

Endocytosis is a regulated physiological process by which cell surface proteins are internalized along with extracellular factors such as nutrients, pathogens, peptides, toxins, etc. The process begins with the invagination of small regions of the plasma membrane that ultimately form intracellullar vesicles. These internalized vesicles may shuttle back to the plasma membrane to recycle the membrane components or they may be targeted for degradation. One role for endocytosis is in the attenuation of receptor signaling. For example, desensitization of activated membrane bound receptors such as G-protein coupled receptors (GPCRs) or receptor tyrosine kinases (RTKs) occurs, in part, through endocytosis of the activated receptor. However, accumulating evidence suggests that endocytosis also mediates intracellular signaling. Endocytosis is a critical component in cellular signal transduction, both in the initiation of a signal as well as in the termination of a signal. The adaptor protein, intersectin (ITSN) provides a link to both the endocytic and the mitogenic machinery of a cell. ITSN functions at a crossroad in the biochemical regulation of cell function (O'Bryan, 2001).

ITSN (also known as Ese-1, EHSH-1, Dap-160) has been isolated by a number of groups based on its ability to bind proline-rich peptides (ITSN), to form a complex with dynamin. ITSN is a 145 kDa adaptor protein consisting of two amino-terminal Eps15 homology domains (EH), a central coiled-coil domain (CC) and has five tandem Src homology 3 domains (SH3). EH domains promote the interaction with Asp-Pro-Phe sequences and are present in numerous endocytic accessory proteins. CC domains promote both homo- and hetero-typic interactions with other CC-containing proteins and are also widely distributed. SH3 domains recognize Pro-rich sequences within target proteins and are present in a variety of cytoskeletal and signaling proteins. ITSN is conserved throughout evolution with homologues present in humans, rodents, Xenopus, Drosophila and likely C. elegans. ITSN proteins are present predominantly in the nervous system with lower expression elsewhere. In addition, there is a larger (~200 kDa) isoform of ITSN, termed intersectin-long (ITSN-L), that is predominantly expressed in the nervous system. This iso-form is derived by alternative RNA splicing. ITSN-L possesses a carboxy-terminal extension encoding a Dbl homology domain (DH), a pleckstrin homology domain (PH) and a C2 domain. DH domains function as guanine nucleotide exchange factors (GEFs) for the Rho subfamily of Ras-like GTPases, whose members include Rho, Rac and Cdc42. These domains function in concert with PH domains which direct interaction with lipids and membrane. Thus, ITSN-L may serve to regulate Rho family activation within the nervous system. C2 domains bind phospholipid membranes, proteins or soluble inositol polyphosphates using both Ca 2+ -dependent and -independent mechanisms. Interestingly, EH domains also bind Ca2+ although an importance for this activity in the function of this domain has not been demonstrated. An ITSN-related protein, termed ITSN-2/Ese-2, has also been identified. ITSN-2 shares a similar structural architecture with ITSN-1 possessing both long and short isoforms. In contrast to ITSN-1L, ITSN-2L appears to be more widely expressed suggesting that this isoform is a more general regulator of Rho family members (O'Bryan, 2001).

The domain structure of ITSN-1 suggests that this protein may act as a scaffolding or adaptor protein that regulates various biochemical pathways. ITSN localizes to clathrin coated pits (CCPs) via the EH region, suggesting that it may serve to assemble multiprotein complexes at sites of CCP formation. Indeed, ITSN associates directly with a number of proteins including epsin, secretory carrier membrane protein 1 (SCAMP1), HIV Rev binding protein, Eps15, SNAP-23/25, dynamin, synaptojanin and Sos, a Ras GEF. Several lines of evidence suggest that ITSN does indeed regulate cellular signaling pathways. (1) ITSN, through its SH3 domains, forms a stable complex with Sos both in vitro and in vivo. (2) ITSN directly activates mitogenic signaling pathways. (3) Overexpression of ITSN is sufficient to induce morphological transformation of rodent fibroblast as well as accelerate hormone-induced Xenopus oocyte maturation in culture. (4) ITSN-L possesses an exchange factor domain for the Rho family of GTPases suggesting that ITSN may regulate Rho, Rac or Cdc42 as well as Ras. Together these data provide compelling evidence that ITSN directly participates in cellular signaling (O'Bryan, 2001).

Intersectin is a member of a growing family of adaptor proteins that possess conserved Eps15 homology (EH) domains as well as additional protein recognition motifs. In general, EH domain-containing proteins play an integral role in clathrin-mediated endocytosis. Indeed, intersectin functions in the intermediate stages of clathrin-coated vesicle assembly. However, recent evidence suggests that components of the endocytic machinery also regulate mitogenic signaling pathways. Evidence is provided that intersectin has the capacity to activate mitogenic signaling pathways. (1) Intersectin overexpression activates the Elk-1 transcription factor in an MAPK-independent manner. This ability resides within the EH domains, since expression of the tandem EH domains is sufficient to activate Elk-1. (2) Intersectin cooperates with epidermal growth factor to potentiate Elk-1 activation; however, a similar level of Elk-1 activation is obtained by expression of the tandem EH domains suggesting that the coiled-coil region and SH3 domains act to regulate the EH domains. (3) Intersectin expression is sufficient to induce oncogenic transformation of rodent fibroblasts. And (4), intersectin cooperates with progesterone to accelerate maturation of Xenopus laevis oocytes. Together, these data suggest that intersectin links endocytosis with regulation of pathways important for cell growth and differentiation (Adams, 2000).

Intersectin is a multiple EH and SH3 domain-containing protein, that serves as a component of the endocytic machinery. Overexpression of the SH3 domains of intersectin blocks transferrin receptor endocytosis, possibly by disrupting targeting of accessory proteins of clathrin-coated pit formation. Mammalian Sos, a guanine-nucleotide exchange factor for Ras, is an intersectin SH3 domain-binding partner. Overexpression of intersectin's SH3 domains blocks activation of Ras and MAP kinase in various cell lines. Several studies suggest that activation of MAP kinase downstream of multiple receptor types is dependent on endocytosis. Thus, the dominant-negative effect of the SH3 domains on Ras/MAP kinase activation may be indirectly mediated through a block in endocytosis. Consistent with this idea, incubating cells at 4°C or with phenylarsine oxide, a treatment that inhibits EGF receptor endocytosis, blocks EGF-dependent activation of MAP kinase. However, under these conditions, Ras activity is unaffected and overexpression of the SH3 domains of intersectin is still able to block Ras activation. Thus, intersectin SH3 domain overexpression can effect EGF-mediated MAP kinase activation directly through a block in Ras, consistent with a functional role for intersectin in Ras activation (Tong, 2000).

The classical model for the activation of the nucleotide exchange factor Son of sevenless (SOS) involves its recruitment to the membrane, where it engages Ras. The recent discovery that RasGTP is an allosteric activator of SOS indicates that the regulation of SOS is more complex than originally envisaged. Crystallographic and biochemical analyses are presented of a construct of SOS that contains the Dbl homology-pleckstrin homology (DH-PH) and catalytic domains; the DH-PH unit blocks the allosteric binding site for Ras and suppresses the activity of SOS. SOS is dependent on Ras binding to the allosteric site for both a lower level of activity, which is a result of RasGDP binding, and maximal activity, which requires RasGTP. The action of the DH-PH unit gates a reciprocal interaction between Ras and SOS, in which Ras converts SOS from low activity form to high activity form as RasGDP is converted to RasGTP by SOS (Sondermann, 2004).

The signaling protein Ras is a molecular switch that cycles between inactive GDP bound and active GTP bound states. Receptors that signal through tyrosine kinases activate Ras by recruiting the Ras-specific nucleotide exchange factor Son of sevenless (SOS) to the plasma membrane, where SOS and Ras form a complex that results in the expulsion of otherwise tightly bound nucleotides from Ras. Ras is kept under strict control in the cell, and the unregulated activation of Ras is a consistent hallmark of many cancers (Sondermann, 2004 and references therein).

SOS is a complex multidomain protein of about 1330 residues. The N-terminal domain (200 residues) contains two tandem histone folds of unknown function and is followed by a Dbl homology (DH) domain (200 residues) and a pleckstrin homology (PH) domain (150 residues) that together are implicated in the activation of the small GTPase Rac1. The next two domains are both required for the Ras-specific nucleotide exchange activity of SOS and are always found together in other Ras-specific nucleotide exchange factors. The first of these is the Ras exchanger motif (Rem) domain (200 residues), which is followed by the Cdc25 domain (300 residues; named for homology to Cdc25, the Ras activator protein in yeast). These two domains together are referred to as SOScat. Finally, the 250 residues in the C-terminal region provide docking sites for adaptor proteins such as Grb2 (Sondermann, 2004 and references therein).

The structure of nucleotide-free Ras in complex with SOScat shows that Ras is bound in such a way that its nucleotide binding site is almost completely disrupted. The interaction between Ras and SOS is localized entirely to the Cdc25 domain, and the position and function of the Rem domain, which interacts with a surface of the Cdc25 domain that is distal to the active site, was puzzling at first. A recent crystallographic study uncovered a role for the Rem domain in a previously unsuspected allosteric mechanism in SOS. RasGTP, the product of the exchange reaction, interacts with a distal binding site on SOScat that is between the Rem and Cdc25 domains, thereby forming a bridge between these two domains. Binding of RasGTP to this distal allosteric site results in increased Ras exchange activity, indicating the presence of a positive feedback loop in the activation of Ras by SOS (Sondermann, 2004).

Initial models for the regulation of SOS emphasized its recruitment to the membrane as the key step of activation, since Ras is membrane bound. The regulation of SOS is likely to be more complex than simple membrane recruitment. In addition to the Grb2-mediated recruitment of SOS to the plasma membrane, early experiments have suggested a role for the N-terminal segment of SOS in its activation. Deletion of the C-terminal docking segment or the N-terminal 550 amino acids (including the histone domain and the DH-PH unit) increases SOS activity in cellular assays. These results, as well as a recent genetic study of Drosophila SOS (Silver, 2004), suggest that there is a complex but poorly characterized interplay between the domains of SOS that results in modulation of the ability of SOS to activate Ras (Sondermann, 2004).

The present study investigates a construct of SOS (SOSDH-PH-cat) that contains the DH-PH unit in addition to the catalytic unit (SOScat). By determining the structure of SOSDH-PH-cat, it has been shown that the DH-PH unit inhibits SOS by blocking the distal allosteric RasGTP binding site of SOS. Surprisingly, blockage of the allosteric Ras binding site suppresses both the unstimulated (by RasGTP) and the allosterically stimulated levels of activity of SOS, leading to the discovery that the basal level of SOS activity is dependent on the binding of RasGDP to the distal site. It appears that the SOS protein has evolved to have its nucleotide exchange activity be masked until as yet undiscovered signals trigger the displacement of the DH-PH unit and the opening of the allosteric site, allowing Ras itself to stimulate SOS to first low and then high levels of activity (Sondermann, 2004).

Evolutionary homologs: Table of contents

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

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