Ras oncogene at 85D


PROTEIN INTERACTIONS

Table of contents

Drk, an adaptor protein homologous to vertebrate Grb-2

Four tyrosine residues (Y644, Y698, Y767, and Y772) are identifed that become phosphorylated after activation of the Torso (Tor) receptor tyrosine kinase. Phosphotyrosine sites P-Y630 and P-Y918 have also been identifed. Of the six P-Y sites identified, three (Y630, Y644, and Y698) are located in the kinase domain insert region; one (Y918) is located in the C-terminal tail region, and two (Y767 and Y772) are located in the activation loop of the kinase domain. To investigate the function of each P-Y residue in Tor signaling, transgenic Drosophila embryos expressing mutant Tor receptors containing either single or multiple tyrosine to phenylalanine substitutions were generated. Single P-Y mutations have either positive, negative, or no effect on the signaling activity of the receptor. Elimination of all P-Y sites within the kinase insert region results in the complete loss of receptor function, indicating that some combination of these sites is necessary for Tor signaling. Mutation of the C-terminal P-Y918 site reveals that this site is responsible for negative signaling or down-regulation of receptor activity. Mutation of the P-Y sites in the kinase domain activation loop demonstrates that these sites are essential for enzymatic activity. This analysis provides a detailed in vivo example of the extent of cooperativity between P-Y residues in transducing the signal received by a receptor tyrosine kinase and in vivo data demonstrating the function of P-Y residues in the activation loop of the kinase domain (Gayko,1999).

To date, the only members of the Tor pathway that contain SH2 domains are Corkscrew and Drk. Csw, the Drosophila homolog of SHP-2, encodes a nonreceptor tyrosine phosphatase with two N-terminal SH2 motifs. Drk, the Drosophila homolog of Grb2, encodes an adapter protein containing one SH2 and two SH3 motifs and functions to recruit the exchange factor Sos to the activated RTK. Csw associates directly with P-Y630 and there are no direct binding sites for Drk on Tor. Csw has at least two distinct functions in Tor signaling: (1) it regulates positive signaling through Drk because P-Y666 on Csw is a Drk-binding site; (2) it blocks the activity of a negative regulator of Tor signaling that binds to the P-Y918 site. Mutation of Y918 leads to an increase in Tor activity, and P-Y918 is a binding site for a Drosophila Ras-GAP protein that contains two SH2 motifs. Csw dephosphorylates P-Y918 and thus prevents the negative regulator RasGAP from associating with Tor (Gayko, 1999 and references).

Three models by which P-Y644 and P-Y698 residues transduce the Tor signal are envisioned. In the first model, these P-Ys may bind an adapter molecule(s), which would recruit either Csw and/or Drk to Tor. This model predicts that activation of all downstream signaling events is mediated solely by Csw and Drk, a hypothesis that can be tested by examining the phenotype of embryos derived from germlines missing both Csw and Drk activities. Possible candidates for such an adapter include SHC and NCK/DOCK, although it is not known whether these proteins bind Csw/SHP-2 or Drk/Grb2. In a second model, Tor could transduce a signal through activation of Csw and Drk, as well as through Dos. Dos encodes a protein with an amino-terminal pleckstrin homology domain, a polyproline motif that may bind an SH3 domain, and 10 potential P-Y sites with consensus sequences for binding SH2 domains. Previous studies have implicated Dos as a component of the Tor signaling pathway because embryos derived from females that lack maternal Dos activity show a partial loss of function Tor phenotype, and other studies have demonstrated that Dos can bind Grb2 and Csw. In the third model, P-Y644 and P-Y698 could mediate activation of the Raf kinase in a pathway that does not require Ras1 activity. Previous work has suggested the existence of a Ras1-independent pathway of Raf activation. In this scenario, a novel, yet unidentified protein could bind to Y644 and Y698, through either SH2 domain or PTB domains, and could lead to Raf activation in a Ras1-independent manner (Gayko, 1999 and references).

Activation of the Sevenless protein-tyrosine kinase is required for the proper specification of R7 photoreceptors in the Drosophila eye. The activation of a Ras protein, p21Ras1, is a crucial early event in the signaling pathway, and constitutive activation of p21Ras1 is sufficient to induce all of the effects of sevenless action. E(sev)2B, another gene required for proper signaling by Sevenless, encodes a protein of the structure SH3-SH2-SH3. The E(sev)2B protein is required for activation of p21Ras1 but not for any subsequent events; this protein can bind in vitro to Sevenless and to Son of sevenless (Sos), a putative guanine nucleotide exchange factor for p21Ras1. These results suggest that the E(sev)2B protein may act to stimulate the ability of Sos to catalyze p21Ras1 activation by linking sevenless and Sos in a signaling complex. The E(sev)2B locus is therefore renamed Downstream of receptor kinases (DRK) (Simon, 1993).

The Drk SH3-SH2-SH3 adaptor protein has been genetically identified in a screen for rate-limiting components acting downstream of the Sevenless (Sev) receptor tyrosine kinase in the developing eye of Drosophila. It provides a link between the activated Sev receptor and Sos, a guanine nucleotide release factor that activates Ras1. A combined biochemical and genetic approach was used to study the interactions between Sev, Drk and Sos. Tyr2546 in the cytoplasmic tail of Sev is required for Drk binding, probably because it provides a recognition site for the Drk SH2 domain. Interestingly, a mutation at this site does not completely block Sev function in vivo. This may suggest that Sev can signal in a Drk-independent, parallel pathway or that Drk can also bind to an intermediate docking protein. Analysis of the Drk-Sos interaction has identified a high affinity binding site for Drk SH3 domains in the Sos tail. The N-terminal Drk SH3 domain is primarily responsible for binding to the tail of Sos in vitro, and for signalling to Ras in vivo (Raabe, 1995).

A Drosophila gene (drk) encodes a widely expressed protein with a single SH2 domain and two flanking SH3 domains, which is homologous to the Sem-5 protein of C. elegans and mammalian GRB2. Genetic analysis suggests that drk function is essential for signaling by the sevenless receptor tyrosine kinase. DRK biological activity correlates with binding of its SH2 domain to activated receptor tyrosine kinases and concomitant localization of DRK to the plasma membrane. In vitro, DRK also binds directly to the C-terminal tail of SOS, a Ras guanine nucleotide-releasing protein (GNRP), which, like Ras1 and DRK, is required for Sevenless signaling. These results suggest that DRK binds autophosphorylated receptor tyrosine kinases with its SH2 domain and the SOS GNRP through its SH3 domains, thereby coupling receptor tyrosine kinases to Ras activation. The conservation of these signaling proteins during evolution indicates that this is a general mechanism for linking tyrosine kinases to Ras (Olivier, 1993).

Drk, the Drosophila homolog of the SH2-SH3 domain adaptor protein Grb2, is required during signaling by the Sevenless receptor tyrosine kinase (Sev). One role of Drk is to provide a link between activated Sev and the Ras1 activator Sos. The ability of activated Ras1 to bypass the requirement for Sev function during R7 development has suggested that the primary function of Sev is to activate Ras. However, the model suggesting that the sole function of activated Sev is to bind Drk-Sos has been questioned by genetic studies that suggest the existence of multiple intracellular signaling pathways downstream of Sev. For example, although the association of Drk and Sos does not depend on the carboxy (C)-terminal SH3 domain of Drk, mutations that affect this domain partially compromise Sev signaling. Furthermore, a C-terminal SH3 domain-truncated Drk cannot rescue the lethality associated with homozygous drk mutations. These data suggest that Drk-binding proteins other than Sos may play important roles in signaling by Sev and other RTKs. Biochemical studies performed with mammalian systems have provided evidence that such Grb2-binding partners do exist. These include Cbl, a proto-oncogene product, and GAB1, a downstream component of the insulin and epidermal growth factor receptors. The possibility that Drk performs functions other than binding to Sos has been been investigated by identification of additional Drk-binding proteins. The phosphotyrosine-binding (PTB) domain-containing protein Disabled (Dab) binds to the Drk SH3 domains (Le, 1998).

To characterize the nature of the in vitro Dab-Drk interaction, it was necessary to determine which domains of Drk are required for binding to Dab. To answer this question, well-characterized mutations were used that had been shown to inactivate the function of either the SH2 or SH3 domain of Grb2. For example, changing the proline 49 residue to leucine (P49L) inactivates the N-terminal SH3 domain, while the arginine 86-to-lysine (R86K) mutation disrupts the SH2 domain and the glycine 203-to-arginine (G203R) mutation affects the C-terminal SH3 domain. The corresponding mutations were introduced, individually or in combination (P49L, R85K, G199R, P49L/G199R), into the [32P]GTK-DRK fusion protein and the ability of the mutant proteins to interact with the lambda gt11-encoded beta-galactosidase-DAB fusion protein was tested. Mutation of the SH2 domain does not affect binding, indicating that the in vitro Dab-Drk interaction does not require a functional Drk SH2 domain. However, the Dab-Drk interaction is dependent on the function of the SH3 domains because simultaneous mutations of both SH3 domains abolish binding. Moreover, while Dab binds to both SH3 domains, it appears to interact more strongly with the C-terminal domain. In addition, in vitro interaction between Drk and Dab requires the presence of the proline-rich region of Dav and suggests that the SH3 domains of Drk bind directly to sequences within the Dab proline-rich core (Le, 1998).

Dab is expressed in the ommatidial clusters, and loss of Dab function disrupts ommatidial development. Intense anti-Dab staining is observed both in the morphogenetic furrow and in developing ommatidial clusters posterior to the furrow. An apical-to-basal cross section revealed that Dab is localized to a small region just below the apical surface of the retinal epithelium. To determine which cells express Dab, the discs were costained with an antibody to ELAV, a neuronal marker present in the nuclei of developing and mature photoreceptors. The results from these experiments showed that Dab accumulates at the apical membrane of the developing photoreceptor cells. However, it was not possible to assign Dab expression to particular photoreceptors due to the apical constriction of these cells. The subcellular localization of Dab is similar to that of Drk, consistent with its role as a Drk-binding partner (Le, 1998).

Numerous abnormalities are observed in dab homozygous mutant clones. The most common defects are the absence of the R7 cell and the lack of one or more outer photoreceptors (R1 to R6) in mosaic ommatidia. In addition, large dab mutant clones show extensive ommatidial disorganization. including regions in which no photoreceptors are present. This phenotype is observed with three different alleles of dab and resembles those observed in clones of cells homozygous for weak alleles of either Sos or Ras1. These results indicate that Dab has an important function during photoreceptor and ommatidial development. Reduction of Dab function attenuates signaling by a constitutively activated Sev. Biochemical analysis suggests that Dab binds Sev directly via its PTB domain, becomes tyrosine phosphorylated upon Sev activation, and then serves as an adaptor protein for SH2 domain-containing proteins. Taken together, these results indicate that Dab is a novel component of the Sev signaling pathway (Le, 1998).

Disabled has been implicated in other RTK signaling pathways. A murine DAB-related protein, mDAB1, has been identified as a tyrosine-phosphorylated protein that binds to the non-receptor protein tyrosine kinase Src. Recently, several reports have shown that mice lacking mDAB1 function have neuronal defects similar to those seen in reeler mice, including abnormal cortical lamination resulting from disruptions of neuronal migration processes. These results suggest that mDAB1 might participate in a signaling pathway triggered by REELIN, a secreted protein released near the targets of migrating neurons. The neuronal defects associated with Drosophila and mouse dab mutations and the identification of DAB as a putative adaptor protein acting downstream of the receptor tyrosine kinase Sev suggest that Dab may function downstream of many RTKs, including ones required for proper development of the Drosophila central nervous system (Le, 1998).

Sos, a guanine nucleotide exchange/release factor

Activation of p21ras by receptor tyrosine kinases is thought to result from recruitment of guanine nucleotide exchange factors such as Son-of-sevenless (Sos) to plasma membrane receptor substrates via adaptor proteins such as Grb2. This hypothesis was tested by evaluating the ability of truncation and deletion mutants of Drosophila (d)Sos to enhance [32P]GTP loading of p21ras when expressed in 32P-labeled COS or 293 cells. The dSos catalytic domain (residues 758-1125), expressed without the dSos NH2-terminal (residues 1-757) or adaptor-binding COOH-terminal (residues 1126-1596) regions, exhibits intrinsic exchange activity as evidenced by its rescue of mutant Saccharomyces cerevisiae deficient in endogenous GTP/GDP exchange activity. This dSos catalytic domain fails to affect GTP p21ras levels when expressed in cultured mammalian cells unless the NH2-terminal domain is also present. Surprisingly, the COOH-terminal adaptor binding domain of dSos is not sufficient to confer p21ras exchange activity to the Sos catalytic domain in these cells in the absence of the NH2-terminal domain. This function of promoting catalytic domain activity could be localized by mutational analysis to the pleckstrin and Dbl homology sequences located just NH2-terminal to the catalytic domain. The results demonstrate a functional role for these pleckstrin and Dbl domains within the dSos protein, and suggest the presence of unidentified cellular elements that interact with these domains and participate in the regulation of p21ras (McCollam, 1995).

The Son of sevenless (Sos) protein functions as a guanine nucleotide transfer factor for Ras and interacts with the receptor tyrosine kinase Sevenless through the protein Drk, a homolog of mammalian Grb2. In vivo structure-function analysis reveals that the amino terminus of Sos is essential for its function in flies. A molecule lacking the amino terminus is a potent dominant negative. In contrast, a Sos fragment lacking the Drk binding sites is functional and its activity is dependent on the presence of the Sevenless receptor. Membrane localization of Sos is independent of Drk. This paper discusses a possible role for Drk as an activator of Sos and a Drk-independent interaction between Sos and Sevenless is proposed, one likely to be mediated by the pleckstrin homology domain within the amino terminus (Karlovich, 1995).

The establishment of axon trajectories is ultimately determined by the integration of intracellular signaling pathways. Here, a genetic approach in Drosophila has demonstrated that both Calmodulin and Son of sevenless signaling pathways are used to regulate which axons cross the midline. A loss in either signaling pathway leads to abnormal projection of axons across the midline and these increase with roundabout or slit mutations. When both Calmodulin and Son of sevenless are disrupted, the midline crossing of axons mimics that seen in roundabout mutants, although Roundabout remains expressed on crossing axons. Calmodulin and Son of sevenless also regulate axon crossing in a commissureless mutant. These data suggest that Calmodulin and Son of sevenless signaling pathways function to interpret midline repulsive cues that prevent axons crossing the midline (Fritz, 2000).

A novel CaM inhibitor, called kinesin-antagonist (KA), has been expressed using the neurogenic enhancer element of the fushi tarazu gene (ftzng) in a subset of CNS neurons that normally do not cross the midline. KA expression decreases endogenous CaM activation of target proteins in the growth cone and this leads to specific axon guidance defects including stalls at selected choice points, failure to fasciculate properly and abnormal crossing of the midline. robo and slit mutations and KA interact synergistically to increase the number of axon bundles abnormally crossing the midline. KA also induces axon bundles to cross the midline in the absence of Comm protein. Sos-dependent crossovers are enhanced by KA or by slit mutation. KA and Sos also interact to increase the number of axon bundles crossing in a comm mutant. Thus, the data demonstrate that both CaM and Sos signaling pathways are required to prevent certain axons crossing the midline (Fritz, 2000).

Whether CaM and Sos-mediated signaling is working directly downstream of Robo or in closely associated, but parallel signaling pathways to prevent axons from crossing is difficult to ascertain from this genetic data alone. If these signaling pathways lie downstream of Robo, the data suggest that both CaM and Sos are activated upon Slit binding to Robo, and result in growth cone repulsion. Interestingly, increased levels of calcium have been implicated in growth cone retraction and growth cone collapse, two ways in which a growth cone may respond to a repulsive agent. In addition, retrograde actin flow, which leads to filopodial retraction, is stimulated by CaM activation of myosin light chain kinase. Two other CaM target proteins, cAMP adenylyl cyclase and phosphodiesterase, regulate cAMP cellular concentrations thus altering neuronal response to Netrin 1 and other guidance cues. Activation of a Sos signaling pathway can affect cytoskeletal dynamics by activating various GTPases known to regulate growth cone behavior and axon guidance. Moreover, the cytoplasmic tail of Robo, known to be essential for signaling function, has a tyrosine residue that could recruit Sos via Drk or dreadlocks (dock), another SH2-SH3 adapter protein that affects axon guidance. Alternatively, Robo may bind Enabled, a known substrate for Abelson tyrosine kinase (Abl), which has been implicated in commissure formation. If Sos binds to phosphotyrosine residues on Ena (also via an adapter protein) it could be indirectly recruited to Robo (Fritz, 2000 and references therein).

Another possibility is that a disruption in both the CaM and Sos signaling pathways indirectly causes abnormal crossovers. CaM has been identified as a player downstream of several guidance molecules. Indeed, the gaps in the longitudinal connectives observed with increasing copies of KA in a comm mutant or in KA robo mutants, which are not seen in robo mutants alone, suggest CaM may function downstream of other guidance cue receptors to allow extension through the connective. Once these signals are attenuated by expression of KA, axons may inadvertently cross the midline. However, if CaM only functions in cell adhesive mechanisms within the connectives, it is difficult to explain why axons cross the midline in comm mutants when no other axons cross and the presence of Slit is still being read by Robo (Fritz, 2000 and references therein).

Since CaM and Sos appear to interpret a midline repulsive cue, the existence of an additional midline repulsion system working in parallel to Robo represents an interesting possibility. In robo mutants, axons cross the midline but then move to the longitudinal connective, instead of collapsing at the midline as observed in slit mutants. It has been suggested that this occurs because the continued presence of Slit at the midline is detected by a second receptor system, and candidate genes include a second robo gene or karussel. As the data shows, heterozygous slit mutations interact very strongly with single copies of KA, Sos or KA Sos together, to force axons across the midline. The interaction between Sos and slit mutations, especially when compared to the lack of Sos and robo interaction, is particularly striking. It seems that if the activity of both repulsion systems is decreased due to the reduction of a common ligand (Slit), a disruption in CaM and/or Sos signaling dramatically increases midline crossing errors. Most of these results, including the synergistic effects of KA and Sos, robo and slit mutations, the robo-like phenotype of KA Sos mutants, and the enhancement of crossovers in comm mutants can be explained by a parallel decrease in both midline repulsive systems upon disruption of the CaM and Sos signaling pathways. Thus while the mechanisms by which CaM and Sos contribute to an axon guidance decision at the midline remain unclear, the data clearly indicate that CaM and Sos signaling pathways are critical to the transduction of repulsive information at the midline (Fritz, 2000 and references therein).

Rho family GTPases are ideal candidates to regulate aspects of cytoskeletal dynamics downstream of axon guidance receptors. To examine the in vivo role of Rho GTPases in midline guidance, dominant negative (dn) and constitutively active (ct) forms of Rho, Drac1, and Dcdc42 are expressed in the Drosophila CNS. When expressed alone, only ctDrac and ctDcdc42 cause axons in the pCC/MP2 pathway to cross the midline inappropriately. Heterozygous loss of Roundabout enhances the ctDrac phenotype and causes errors in embryos expressing dnRho or ctRho. Homozygous loss of Son-of-Sevenless (Sos) also enhances the ctDrac phenotype and causes errors in embryos expressing either dnRho or dnDrac. CtRho suppresses the midline crossing errors caused by loss of Sos. CtDrac and ctDcdc42 phenotypes are suppressed by heterozygous loss of Profilin, but strongly enhanced by coexpression of constitutively active myosin light chain kinase (ctMLCK), which increases myosin II activity. Expression of ctMLCK also causes errors in embryos expressing either dnRho or ctRho. These data confirm that Rho family GTPases are required for regulation of actin polymerization and/or myosin activity and that this is critical for the response of growth cones to midline repulsive signals. Midline repulsion appears to require down-regulation of Drac1 and Dcdc42 and activation of Rho (Fritz, 2002).

Thus, when expressed alone, only ctDrac and ctDcdc42 cause midline crossing errors. However, the mutant GTPases interact genetically with mutations in robo, Sos, and chic and with overexpression of ctMLCK. The interactions are surprisingly specific. Midline crossing errors caused by expression of ctDrac or ctDcdc42 are suppressed by heterozygous loss of Profilin and enhanced by expression of ctMLCK. These results indicate that Drac1 and Dcdc42 encourage axons to cross the midline by regulating actin polymerization and/or myosin activity. CtRho and dnRho interact strongly with expression of ctMLCK or heterozygous loss of Robo, which suggests that regulation of myosin activity by Rho is crucial for midline repulsion. This work demonstrates that Rho, Drac1, and Dcdc42 are involved in dictating which axon may cross the midline, presumably by aiding in the transduction of attractive and/or repulsive cues operating at the midline. By using mutations in signaling molecules known to prevent pCC/MP2 axons from crossing the midline, this analysis concentrates on how Rho, Drac1, and Dcdc42 may regulate cytoskeletal dynamics in response to midline repulsive cues (Fritz, 2002).

Expression of dnRho may specifically interfere with retraction of filopodia in response to repulsive cues, leading to increased midline crossing errors. A global increase in myosin activity caused by expression of either ctRho or ctMLCK, or even a Rho GEF, may cause axon guidance errors by increasing the forward movement of the growth cone. Midline attractive activity (e.g., Netrins) probably also influences how much myosin activity is available to move a growth cone over the midline. The literature and these experiments are most consistent with a model in which Rho is activated by repulsive guidance signals. Activation of ephrinA5 receptors causes an increase in Rho activity resulting in a growth cone collapse. Plexin B, the receptor for repulsive semaphorins, binds to and seems to activate Rho. Activation of Robo by Slit recruits srGAP1, which appears to prevent it from binding to and inactivating Rho. The genetic interactions seen between Sose49 mutations and expression of ctRho or dnRho are consistent with Sos acting as a GEF for Rho in pCC/MP2 neurons. DnRho strongly enhances the midline crossing errors caused by loss of Sos, while ctRho almost completely suppresses them. Since Sos-dependent signaling pathways are required for response to midline repulsive cues, this is further evidence that Rho is activated downstream of repulsive guidance signals, although a role downstream of selected attractants cannot be ruled out (Fritz, 2002).

Clearly, regulation of Rho family GTPase activity is necessary to prevent axons from crossing the midline inappropriately. Midline repulsive signaling involves regulation of all three GTPases; Drac1 and Dcdc42 are likely downregulated, while Rho seems to be activated downstream of repulsive signals. The Rho family GTPases influence actin polymerization and/or myosin force generation to regulate the processes of growth cone motility that are required for proper response to axon guidance signals (Fritz, 2002).

C3g, a potential guanine nucleotide exchange/release factor for Ras

The cellular signal transduction pathways by which C3G, a RAS family guanine nucleotide exchange factor, mediates v-crk transformation are not well understood. v-crk transformation leads to an elevation of tyrosine phosphorylation on characteristic proteins including the focal adhesion molecules p130cas and Paxillin, and an increase in the activity of JUN kinase (JNK) and MAPK. The v-crk oncogene is unusual in that it encodes an adaptor protein, which is comprised of viral Gag sequences fused to the SH2 and the N-terminal SH3 domain of c-CRK. Consistent with its identification as an adaptor protein, v-CRK has been shown to bind a number of cellular proteins via its SH2 domain, including the adaptor molecules c-CBL, p130cas and Paxillin. In addition, the v-CRK SH3 domain is capable of binding the tyrosine kinases ABL and ARG, the RAS guanine nucleotide exchange factor (GEF) SOS and the putative RAP1 GEF C3G. Due to the identification of these v-CRK-binding partners, progress has been made in the elucidation of the signal transduction mechanisms utilized in v-crk transformation. The ability of v-CRK to bind to the RAS family GEF SOS suggests an involvement of the RAS pathway. Expression of dominant-negative H-RASN17 causes morphological reversion of v-crk-transformed NIH 3T3 cells, suggesting that RAS activation is involved in v-crk transformation (Ishimaru, 1999).

Reported here is the identification of Drosophila C3G, which, like its human cognate, specifically binds to CRK but not DRK/GRB2 adaptor molecules. During Drosophila development, constitutive membrane binding of C3G, which also occurs during v-crk transformation, results in cell fate changes and overproliferation, mimicking overactivity of the RAS-MAPK pathway. To determine whether DC3G can influence cell proliferation, the wild-type, activated and dominant-negative versions were expressed under the control of the decapentaplegic disc enhancer fragment (dppGAL4). This enhancer fragment drives gene expression in all imaginal discs in a spatially restricted pattern closely resembling that of endogenous dpp. In the wing imaginal disc, dppGAL4 is expressed in a broad stripe just anterior to the antero-posterior compartment border, which will run between the third and fourth vein in the adult wing. The expression patterns of the DC3G transgenes were visualized directly using anti-MYC antibodies. Expression of wild-type DC3G under dppGAL4 control results in a pattern indistinguishable from that of beta-galactosidase under dppGAL4 control, showing that overexpression of wild-type DC3G does not affect cell proliferation. In contrast, expression of activated DC3G under dppGAL4 control results in a gross distortion of the dppGAL4 pattern. Instead of forming a well-defined stripe, the staining is diffuse and broadened considerably, often to encompass large portions of the wing pouch. To determine whether activated DC3G induces cell proliferation, third instar larval discs were labeled with bromodeoxyuridine (BrdU), which is incorporated by cells during S phase. dppGAL4/UASMyDC3G wing discs show an increase in BrdU incorporation in a band corresponding to the domain of dppGAL4 expression. These findings indicate that activated DC3G drives the cells in which it is expressed to overproliferate (Ishimaru, 1999).

The effects of C3G overactivity can be suppressed by reducing the gene dose of components of the RAS-MAPK pathway and of RAP1. A 50% reduction in gene activity of Ras1, ksr and rolled (MAPK) leads to a remarkable phenotypic rescue, while reduction in the gene dose of Sos or drk has no effect on the phenotype. The phenotypic defects resulting from expression of dominant-negative DC3G mimic the defects resulting from interference with the RAS1-MAPK pathway. Taken together, these findings show that DC3G can activate the RAS1-MAPK pathway. This activation could be achieved either by direct stimulation of RAS1 or, indirectly, by stimulating another RAS family GTPase, which in turn activates RAS1. Several independent biochemical studies have shown that hC3G acts as a GEF for RAS family members in vitro. It is most efficient against mammalian RAP1, less so toward R-RAS, and only weakly active against H-RAS. Nevertheless, the possibility that in vivo DC3G directly activates RAS1, the Drosophila H-RAS homolog, cannot be discounted. The transgenes provide high levels of membrane-tagged DC3G, such that its local concentration at the membrane may be high enough to drive guanine nucleotide exchange on the less favored RAS1 substrate. Alternatively, DC3G may activate the RAS1-MAPK pathway indirectly by activating R-RAS or RAP1. However, during eye development, expression of activated R-RAS leads to a loss of photoreceptor cells, the phenotypic opposite of activated DC3G and activated RAS1. This excludes the possibility that R-RAS acts as the relevant intermediary between DC3G and RAS1. Currently, the effects of activated RAP1 on eye development are unknown. The function of Rap1 has usually been studied using viable alleles, such as the Roughened mutation, which cause the loss, predominantly, of the R7 photoreceptor in the eye. The genetic classification of these alleles is somewhat unclear, making it difficult to extrapolate what the phenotypic consequences of RAP1 overactivity might be. To resolve this, a transgene was generated containing wild-type Rap1 under UAS promoter control (UASRap1) and it was expressed in the developing eye using GMRGAL4. The phenotype of Rap1 overexpression is very similar to activated DC3G and is characterized by morphogenetic defects and, most importantly, by the presence of supernumerary photoreceptor cells. This finding, together with the fact that the activated DC3G phenotype is strongly suppressed by a 50% reduction in Rap1 gene dose using complete loss-of-function alleles, strongly argues that RAP1 activation is involved in generating the DC3G overactivity phenotype. These findings provide the first in vivo evidence that membrane localization of C3G can trigger activation of RAP1 and RAS resulting in the activation of MAPK, one of the hallmarks of v-crk transformation previously thought to be mediated through activation of SOS. It seems possible that the recruitment of CRK-bound C3G to the plasma membrane, leading to RAP1 stimulation, is triggered by EGFR activity. Since the cytoplasmic domain of EGFR contains two putative CRK SH2-binding sites, the activated receptor may also interact directly with the CRK adaptor. Thus, CRK, C3G and RAP1 may provide an alternate route by which EGFR can stimulate the MAPK pathway (Ishimaru, 1999).

The guanine nucleotide exchange factor (GEF) Son-of-sevenless (Sos) encodes a complex multidomain protein best known for its role in activating the small GTPase RAS in response to receptor tyrosine kinase (RTK) stimulation. Much less well understood is SOS's role in modulating RAC activity via a separate GEF domain. In the course of a genetic modifier screen designed to investigate the complexities of RTK/RAS signal transduction, a complementation group of 11 alleles was isolated and mapped to the Sos locus. Molecular characterization of these alleles indicates that they specifically affect individual domains of the protein. One of these alleles, SosM98, which contains a single amino acid substitution in the RacGEF motif, functions as a dominant negative in vivo to downregulate RTK signaling. These alleles provide new tools for future investigations of SOS-mediated activation of both RAS and RAC and how these dual roles are coordinated and coregulated during development (Silver, 2004).

Ras is ubiquitinated: Impairment of ubiquitylation by mutation in Drosophila E1 promotes both cell-autonomous and non-cell-autonomous Ras-ERK activation in vivo

Ras signaling can promote proliferation, cell survival and differentiation. Mutations in components of the Ras pathway are found in many solid tumors and are associated with developmental disorders. This study demonstrates that Drosophila tissues containing hypomorphic mutations in E1 (Ubiquitin activating enzyme 1), the most upstream enzyme in the ubiquitin pathway, display cell-autonomous upregulation of Ras-ERK activity and Ras-dependent ectopic proliferation. Ubiquitylation is widely accepted to regulate receptor tyrosine kinase (RTK) endocytosis upstream of Ras. However, although the ectopic proliferation of E1 hypomorphs is dramatically suppressed by removing one copy of Ras, removal of the more upstream components Egfr, Grb2 or sos shows no suppression. Thus, decreased ubiquitylation may lead to growth-relevant Ras-ERK activation by failing to regulate a step downstream of RTK endocytosis. This study further demonstrates that Drosophila Ras is ubiquitylated. These findings suggest that Ras ubiquitylation restricts growth and proliferation in vivo. An intriguing observation is that complete inactivation of E1 causes non-autonomous activation of Ras-ERK in adjacent tissue, mimicking oncogenic Ras overexpression. Maintaining sufficient E1 function is required both cell autonomously and non-cell autonomously to prevent inappropriate Ras-ERK-dependent growth and proliferation in vivo and may implicate loss of Ras ubiquitylation in developmental disorders and cancer (Yan, 2009).

Therefore, impaired ubiquitin pathway function due to mutation in E1 results in a growth-relevant, cell-autonomous increase in Ras-ERK activity. It is widely accepted that RTK endocytosis is regulated by ubiquitylation and that a failure of RTK ubiquitylation promotes increased signaling through Ras. Contributions from upstream regulators of Ras to the phenotypes of E1 mutants in the current system cannot be ruled out; however, mutation in Ras dominantly suppress the increased proliferation and pupal lethality of E1 hypomorphs strongly, whereas mutations in Egfr, drk and sos did not. One possible explanation is that multiple upstream steps that converge on Ras are regulated by ubiquitylation. Alternatively, it is possible that an as-yet-unidentified regulator of Ras is regulated by ubiquitylation. However, the simplest model to explain the current findings is that the cell-autonomous increase in Ras activity may be independent of Egfr and Grb2/sos and occurs at the step of Ras. Indeed, this study has demonstrated that Drosophila Ras is ubiquitylated. These findings suggest the exciting model that decreased ubiquitylation of Ras itself causes increased activation of ERK. This may be a mechanism highly conserved between Drosophila and mammals, because a recent study reports di-ubiquitylation of H-Ras and N-Ras in vitro (Jura, 2006). Whereas Jura established ubiquitylation of H-Ras and N-Ras in a tissue-culture context, the physiological relevance of Ras ubiquitylation has not been investigated. These Drosophila studies demonstrate that in vivo, the activation of Ras is highly sensitive to ubiquitylation. Impairing ubiquitylation leads to increased Ras-ERK activation that promotes ectopic cell proliferation and confers increased resistance to cell death in vivo in a developmental context (Yan, 2009).

How does Ras ubiquitylation restrict signaling through downstream effectors? It is possible that ubiquitylated Ras adopts a conformation that no longer interacts with Raf. Alternatively, ubiquitylation may alter Ras localization, thus isolating it from downstream effectors. Indeed, Jura (2006) showed that a construct of H-Ras fused to ubiquitin (to mimic constitutively ubiquitylated Ras) preferentially localizes to the endosomes (Yan, 2009).

It is generally assumed that E1 activity is not limiting; decreasing E1 activity so it becomes limiting could amplify substrate specificities such that some ubiquitin-mediated processes are affected early and dramatically whereas others are affected to a lesser extent or at a later time. The extreme sensitivity of E1 phenotypes to Ras gene dosage strongly supports the argument that Ras regulation is affected early and/or dramatically upon a decrease in E1 function and implies that maintaining sufficient activity of the ubiquitin pathway is crucial to prevent inappropriate Ras-ERK activation in vivo (Yan, 2009).

This paper also presents intriguing observation that that there is growth-relevant Ras-ERK activation in cells adjacent to E1 null clones, and this Ras activation mimics oncogenic Ras. What is the mechanism underlying non-autonomous Ras activation? Given the pleiotropic effects caused by the global loss of ubiquitylation, elucidating this experimentally is difficult. Previous work in mammalian systems reports that Ras activation increases the release of EGF-like ligands, and this study has demonstrated that a cell-autonomous increase in Ras signaling through ERK is sufficient to promote activation of Ras in neighboring cells. Thus, it is possible that the cell-autonomous increase in Ras activation in E1 null cells promotes the non-autonomous Ras activation. Investigating the role of ubiquitylation in preventing non-autonomous Ras activation in the future will be exciting and may elucidate the ability of stromal cells to promote growth and invasiveness of adjacent tumor cells (Yan, 2009).

By demonstrating that maintaining sufficient ubiquitin pathway activity is crucial for Ras regulation both cell-autonomously and non-autonomously, this study provides further support for the previous suggestion that E1 may be a tumor suppressor gene. In fact, one study using comparative genomic hybridization reports a loss in DNA copy number of the human E1 chromosomal region in breast cancer lines and tumors. Microarray and/or serial analysis of gene expression (SAGE) methods reveal significantly decreased E1 RNA levels in many cancer cell lines and tumors. Previous SAGE studies have shown that E1 levels drop dramatically in the leukocytes and luminal epithelial cells of invasive ductal carcinomas compared to those of normal breast tissue and ductal carcinomas in situ, potentially implicating E1 loss in breast cancer progression. Given these reports and the findings of Ras-dependent overgrowth due to mutation of E1 in vivo, it is proposed that downregulating E1, either by mutation or other means, could be a mechanism employed by tumor cells to achieve cell death resistance and Ras activation. Identification of the ubiquitin ligase or ligases targeting Ras is of high importance, as such ligase(s) may play a crucial role in normal proliferation and may be dysregulated in developmental disorders and in cancer (Yan, 2009).

Table of contents


Ras85D: Biological Overview | Evolutionary Homologs | Regulation | Effects of Mutation | References

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