Ras oncogene at 85D


PROTEIN INTERACTIONS

Table of contents

Ras GTPase-activating proteins (GAPs)

Mammalian Ras GTPase-activating protein (GAP), p120 Ras-GAP, has been implicated as both a downregulator and effector of Ras proteins, but its precise role in Ras-mediated signal transduction pathways is unclear. To begin a genetic analysis of the role of p120 Ras-GAP a homolog from the fruit fly was identifed through its ability to complement the sterility of a Schizosaccharomyces pombe (fission yeast) gap1 mutant strain. Drosophila RasGAP and human p120 Ras-GAP share 47% amino acid identity (68% similarity) overall. The p120 Ras-GAP protein identified here is the third GAP for Ras proteins to be identified in Drosophila and shows that the fly has one of each of the types of RasGAPs found in mammals. Like its mammalian homologs, the Drosophila protein can be divided into two regions. The N-terminal part consists of protein-protein (SH2 and SH3) and protein-lipid (PH and C2/C2CalB) interaction domains. The C-terminal part contains the GAP catalytic domain which is conserved in RasGAPs from yeast to mammals. One notable difference between RasGAP and the mammalian p120 Ras-GAP isoform is that the latter has a proline-rich sequence near the amino terminus which is absent in the Drosophila protein. this region appears to be required for in vitro association of P120Ras-GAP with Src faminly tyrosine kinases via their SH2 domain. Drosophila RasGAP also lacks a 70-amino-acid hydrophobic region found at the amino terminus of p120 Ras-Gap. Like its mammalian homolog, Drosophila RasGAP stimulates the intrinsic GTPase activity of normal mammalian H-Ras but not that of the oncogenic Val12 mutant. Drosophila RasGAP is tyrosine phosphorylated in embryos and its Src homology 2 (SH2) domains can bind in vitro to a small number of tyrosine-phosphorylated proteins expressed at various developmental stages. Ectopic expression of RasGAP in the wing imaginal disc reduces the size of the adult wing by up to 45% and suppresses ectopic wing vein formation caused by expression of activated forms of Breathless and Heartless, two Drosophila receptor tyrosine kinases of the fibroblast growth factor receptor family. Both Drosophila FGF receptors have exact matches to the optimal binding site (phospho YxxPxD, where x is any amino acid) for the SH2 domains of mammalian p120 Ras-GAP. The in vivo effects of RasGAP overexpression requires intact SH2 domains, indicating that intracellular localization of RasGAP through SH2-phosphotyrosine interactions is important for its activity. These results show that RasGAP can function as an inhibitor of signaling pathways mediated by Ras and receptor tyrosine kinases in vivo. Genetic interactions, however, suggested a Ras-independent role for RasGAP in the regulation of growth. Drosophila RasGAP appears to be not efficiently recruited to the plasma membrane by the Egfr RTK, and this is consistent with the failure to detect an effect of RasGAP overexpression on photoreceptor development in the eye, a process controlled by the Egfr and Sev RTKs, neither of which contains the RasGAP SH2 binding consensus. The system described here should enable genetic screens to be performed to identify regulators and effectors of p120 RasGAP (Feldmann, 1999).

A Drosophila gene with similarity to the mammalian Ras GTPase activating protein has been isolated in screens for mutations that affect eye development. Inactivation of the locus, GTPase-activating protein 1 (Gap1), mimics constitutive activation of the Sevenless receptor tyrosine kinase and eliminates the need for a functional Sevenless protein in the R7 cell. These results suggest that Gap1 acts as a negative regulator of Sevenless signaling by down-regulating the activity of the Ras1 protein, which has been shown to be a key element in signaling by Sevenless (Gaul, 1992).

The X-linked prune (pn) eye-colour mutation of Drosophila has a highly specific, complementary lethal interaction with the conditional dominant Killer of prune (awdK-pn) mutation. Although awdK-pn flies have no apparent phenotype on their own, pn;awdK-pn double mutants die as second or third larval instars. The awd locus encodes a nucleoside diphosphate kinase, an enzyme that catalyses the transfer of high-energy phosphate bonds between nucleoside diphosphates and nucleoside triphosphates, which is essential for the normal development of Drosophila. Analysis of the pn locus has suggested that the complementary DNA, TcD37, encodes a putative pn+ product. The nucleotide sequence of TcD37 reveals the similarity of its deduced protein product to the catalytic domain of mammalian GTPase-activating proteins (GAPs); GAPs stimulate the GTPase activity of Ras. These results suggest that the Drosophila TcD37 protein participates in a biochemical pathway similar to that of Ras and GAPs in mammals and yeast. It is proposed that the interaction between pn and awd is due to a neomorphic mutation that enhances the ability of AwdK-pn nucleoside diphosphate kinase to induce a regulatory GTPase into a GTP-bound 'on' state, whereas Pn modulates the activity of this GTPase either by switching it to a GDP-bound 'off' state or by interfering with its effector function (Teng, 1991).

Btl (Breathless) and Htl (Heartless), the two FGFRs (fibroblast growth factor receptors) in Drosophila melanogaster, control cell migration and differentiation in the developing embryo. These receptors signal through the conserved Ras/mitogen-activated protein kinase pathway, but how they regulate Ras activity is not known. The present study shows that there is a direct interaction between p120 RasGAP (Ras GTPase-activating protein), a negative regulator of Ras, and activated FGFRs in Drosophila. The interaction is dependent on the SH2 (Src homology 2) domains of RasGAP, which have been shown to interact with a phosphotyrosine residue within the consensus sequence (phospho)YXXPXD. A potential binding site that matches this consensus is found in both Btl and Htl, located between the transmembrane and kinase domains of each receptor. A peptide corresponding to this region is capable of binding RasGAP only when the tyrosine residue is phosphorylated. This tyrosine residue appears to be conserved in human FGFR-1 and mediates the association with the adapter protein CrkII, but no association between dCrk (Drosophila homologue of CrkII) and the activated FGFRs was detected. RasGAP is a substrate of the activated FGFR kinase domain, and mutation of the tyrosine residue within the potential binding site on the receptor prevents tyrosine phosphorylation of RasGAP. RasGAP attenuates FGFR signalling in vivo and this ability is dependent on both its SH2 domains and its GAP activity. On the basis of these results, it is proposed that RasGAP is directly recruited into activated FGFRs in Drosophila and plays a role in regulating the strength of signalling through Ras and the mitogen-activated protein kinase pathway (Woodcock, 2004).

Alignments of the mammalian FGFR-1 to the Drosophila FGFRs revealed a potential binding site for the RasGAP SH2 domains, conserved between species, in the form of a tyrosine residue within the consensus sequence, (phospho)YXXPXD, lying between the transmembrane domain and the cytoplasmic tyrosine kinase domain of the receptors. The fact that the corresponding tyrosine residue was shown to be both an autophosphorylation site and a docking site for SH2 domains in mammalian FGFR-1 suggested that this juxtamembrane tyrosine is a good candidate for a RasGAP-binding site on Btl and Htl. This was shown to be the case in three separate experiments. (1) A peptide corresponding to this region of Btl and Htl can precipitate RasGAP expressed in Drosophila only when the peptide is phosphorylated on the tyrosine. (2) This phosphorylated peptide readily competes with Tor-Htl for binding to immobilized RasGAP SH2 domains, whereas the non-phosphorylated peptide can not compete with this binding. (3) If the juxtamembrane Tyr402 of Htl is mutated, RasGAP is no longer tyrosine-phosphorylated when expressed in S. pombe. This suggests that RasGAP can not be recruited to the mutated receptor and therefore is not in a position to be phosphorylated by the active receptor kinase domain. The evidence provided indicates that the juxtamembrane tyrosine residue of Btl and Htl is both an autophosphorylation site and a binding site for the SH2 domains of RasGAP (Woodcock, 2004).

Why does RasGAP have tandem SH2 domains? Two possible reasons for the existence of tandem SH2 domains in the adapter region of RasGAP are (1) they may be required to bind two separate docking sites simultaneously and (2) they may stabilize the interaction with one docking site by increasing the avidity. The individual requirement of the SH2 domains in the interaction with Btl and Htl was tested in two experiments. One of them showed that the immobilized pTyr peptide of Btl/Htl was unable to precipitate RasGAP with single N- and C-terminal mutant SH2 domains to the same extent as wild-type RasGAP. This shows that the two SH2 domains in RasGAP work together to increase the strength of the interaction with the juxtamembrane phosphotyrosine of FGFRs. The second experiment showed that both SH2 domains of RasGAP were required for its maximal tyrosine phosphorylation in response to FGFR signalling in Drosophila. This suggests that when RasGAP has only one intact SH2 domain, it cannot form a stable enough complex with an activated tyrosine kinase, probably the FGFR in this case, to be phosphorylated efficiently. These results indicate that the tandem SH2 domains of the RasGAP adapter region are essential for both the initial interaction with FGFRs and its subsequent phosphorylation on tyrosine. The fact that the activated FGFRs will be dimerized means that there will be two juxtamembrane phosphotyrosine-binding sites in the receptor complex, both of which may be bound to the tandem SH2 domains of RasGAP, adding to the avidity of the association and stabilizing the interaction. The requirement for both SH2 domains in the binding of RasGAP to FGFRs is consistent with the requirement of both SH2 domains for the attenuation of ectopic FGFR signalling by RasGAP in the wing (Woodcock, 2004).

There is a corresponding juxtamembrane tyrosine residue equivalent to that of Btl and Htl in mammalian FGFR-1 that is autophosphorylated and binds the SH2 domain of mammalian CrkII. However, when dCrk, the Drosophila homologue of CrkII, was tested for its ability to bind the FGFR homologues in Drosophila, no association could be detected. This suggests that the interaction between Crk and FGFRs is not conserved between mammals and Drosophila. Therefore it is unlikely that in Drosophila, RasGAP and dCrk compete in vivo for binding to the juxtamembrane tyrosine residues of Btl and Htl. In mammals, it is possible that CrkII and p120 RasGAP may both compete with each other for binding to the juxtamembrane tyrosine residue of FGFR-1 (Woodcock, 2004).

Drk, the Drosophila homologue of mammalian Grb2, has been shown to act upstream of Ras in RTK signalling pathways and acts by recruiting the Ras activator, Sos, to the site of receptor activation. Drk binds directly to activated RTKs or to substrates of the activated RTKs. Since activation of Ras has been shown to partially rescue both btl and htl mutant phenotypes, the ability of Drk to interact directly with these FGFRs was tested. However, a GST fusion protein of wild-type Drk was not capable of associating with activated Btl or Htl expressed in adult Drosophila. This result suggests that the recruitment of Drk to the site of FGFR activation in Drosophila is not directly to the receptor, but requires a receptor substrate, as in the case of mammals that utilize the adapter-like protein FRS2. The lack of an FRS2 homologue in Drosophila suggests the involvement of a novel adapter in FGFR signalling; one candidate is the cytoplasmic protein Dof, which is essential for FGFR signalling in Drosophila. It acts between FGFRs and Ras, and it contains a number of tyrosine residues that lie within a consensus binding sequence for the Drk SH2 domain (Woodcock, 2004).

In conclusion, a model is proposed in which, after ligand binding, the FGFRs Btl and Htl undergo autophosphorylation on the juxtamembrane tyrosine residue, thereby providing a docking site for RasGAP. This association is stabilized by the fact that RasGAP possesses two SH2 domains, both of which are required for maximal binding to the FGFRs. Once recruited, RasGAP becomes a substrate for the active kinase domain of the receptor, potentially providing further docking sites at the location of receptor activation. Recruitment of RasGAP to the activated FGFRs would allow it to act on its substrate, namely RasGTP, thus negatively regulating the downstream signal through Ras effector pathways, such as the MAPK pathway. This model is consistent with the observations that the ability of RasGAP to attenuate FGFR signalling in vivo requires its GAP activity and both its SH2 domains, but not its SH3 domain, which is dispensable for FGFR binding. However, the physiological relevance of the association of RasGAP with FGFRs remains to be established. The recent identification of mutants defective in the gene encoding RasGAP, vacuolar peduncle (vap), will make it possible to examine the effects of loss of RasGAP activity on FGFR signalling pathways regulating morphogenesis and differentiation in the Drosophila embryo (Woodcock, 2004).

RalGDS, an effector protein for Ras

The Ral GTPase is activated by RalGDS, which is one of the effector proteins for Ras. Previous studies have suggested that Ral might function to regulate the cytoskeleton; however, its in vivo function is unknown. A Drosophila homolog of Ral has been identified that is widely expressed during embryogenesis and imaginal disc development. Two mutant Drosophila Ral (DRal) proteins, DRal(G20V) and DRal(S25N), were generated and analyzed for nucleotide binding and GTPase activity. The biochemical analyses demonstrated that DRal(G20V) and DRal(S25N) act as constitutively active and dominant negative mutants, respectively. Overexpression of the wild-type DRal does not cause any visible phenotype, whereas DRal(G20V) and DRal(S25N) mutants cause defects in the development of various tissues, including the cuticular surface, which is covered by parallel arrays of polarized structures such as hairs and sensory bristles. The dominant negative DRal protein causes defects in the development of hairs and bristles. These phenotypes are genetically suppressed by loss of function mutations of hemipterous and basket, encoding Drosophila Jun NH(2)-terminal kinase kinase (JNKK) and Jun NH(2)-terminal kinase (JNK), respectively. Expression of the constitutively active DRal protein causes defects in the process of dorsal closure during embryogenesis and inhibits the phosphorylation of JNK in cultured S2 cells. These results indicate that DRal regulates developmental cell shape changes through the JNK pathway (Sawamoto, 1999a).

The small GTP-binding protein Ral is activated by RalGDS, one of the effector molecules for Ras. Active Ral binds to a GTPase activating protein for CDC42 and Rac. Although previous studies have suggested a role for Ral in the regulation of CDC42 and Rac (two proteins involved in arranging the cytoskeleton), Ral's in vivo function is largely unknown. To examine the effect on development of overexpressing Ral, transgenic Drosophila were generated that overexpress wild-type or mutated Ral during eye development. While wild-type Ral causes no developmental defects, expression of a constitutively activated protein results in a rough eye phenotype. Activated Ral does not affect cell fate determination in the larval eye discs but causes severe disruption of the ommatidial organization later in pupal development. Phalloidin staining shows that activated Ral perturbs the cytoskeletal structure and cell shape changes during pupal development. This phenotype is similar to that caused by RhoA overexpression. In addition, the phenotype is synergistically enhanced by the coexpression of RhoA. These results suggest that Ral functions to control the cytoskeletal structure required for cell shape changes during Drosophila development (Sawamoto, 1999b).

A Ral guanine exchange factor-Ral pathway is conserved in Drosophila melanogaster and sheds new light on the connectivity of the Ral, Ras, and Rap pathways

Ras GTPases are central to many physiological and pathological signaling pathways and act via a combination of effectors. In mammals, at least three Ral exchange factors (RalGEFs) contain a Ras association domain and constitute a discrete subgroup of Ras effectors. Despite their ability to bind activated Rap as well as activated Ras, they seem to act downstream of Ras but not downstream of Rap. This study revisited the Ras/Rap-Ral connections in Drosophila by using iterative two-hybrid screens with these three GTPases as primary baits and a subsequent genetic approach. It was shown that (1) the Ral-centered protein network appears to be extremely conserved in human and flies, (2) in this network, Ral guanine nucleotide exchange factor 2 (RGL) is a functional Drosophila orthologue of RalGEFs, and (3) the RGL-Ral pathway functionally interacts with both the Ras and Rap pathways. These data do not support the paradigmatic model where Ral is in the effector pathway of Ras. They reveal a signaling circuitry where Ral is functionally downstream of the Rap GTPase, at odds with the pathways described for mammalian cell lines. Thus, in vivo data show variations in the connectivity of pathways described for cell lines which might display only a subset of the biological possibilities (Mirey, 2003).

The functions of Ral proteins remain unclear. They are not oncogenic per se, but they facilitate Ras transformation, participate in cell motility, and are required for metastatic evolution of Ras-transformed cells as well as for Ras-induced stimulation of cyclin D1 expression. They are involved in phospholipase D activation, endocytosis, and exocytosis. There is not yet a unifying theory that relates these latter functions to the former cancer-connected phenotypes (Mirey, 2003 and references therein).

In mammalian cell lines, Ral proteins were shown to be involved in not only H-Ras and K-Ras but also TC21 signaling via a family of Ras effectors, the RalGEFs. Once Ras is bound to GTP, it binds and activates these RalGEFs, which in turn activate Ral proteins. There are also Ras-independent pathways that activate Ral (Mirey, 2003 and references therein).

Rap proteins are GTPases once described as antagonistic to Ras oncoproteins. Their function remains elusive. They were reported to be functionally connected to integrin signaling, and they are able to bind RalGDS, one of the mammalian RalGEFs, with a higher affinity than Ras, yet this interaction does not lead to the activation of Ral in cell lines (Mirey, 2003 and references therein).

This study addressed the following questions: (1) The contribution of the Ral pathway to cellular functioning was examined by using approaches with different methodological biases; (2) Within the frame of a whole organism it was asked where cells have to communicate with neighboring cells of different types and integrate various signals; (3) It was desirable to have several readouts, assuming that signaling pathways might be using signaling modules following different architectures in different situations; and (4) The power of genetics was used to establish signaling cascades as well as functional interactions between distinct signaling pathways (Mirey, 2003).

First, it was shown that an exchange factor for Ral of the RalGEF family, which is an orthologue of mammalian RGL, exists in Drosophila. In fact, flies express two orthologues, RGL1 and RGL2, probably generated by the use of two promoters and alternative splicing. RGL1 and RGL2 share the same RalGEF domain as well as the C-terminal domain that binds Ras and Rap, but they differ in their N termini. Combined data from several two-hybrid screens, including the present one, suggest that the Ras/Rap-Ral network is very similar in mammals and in Drosophila. Physical interactions connect RGL to RAS1 (Ras in humans), RAS2 (R-Ras and/or TC21 in humans), and RAS3 (Rap1 in humans) as well as RAL to RLIP (RLIP76 in humans) and SEC5 (the same in humans). RLIP is connected to the orthologous µ2 chains of the AP2 complexes as well as to REPS (the same in humans). The conservation of such a large network confirms that Drosophila is a suitable model to study the Ral pathway in a physiological context. It is noteworthy that in Caenorhabditis elegans, all the proteins of this network exist and certain interactions have been shown, as opposed to what is seen in S. cerevisiae, suggesting that they are important for metazoans (Mirey, 2003).

Several lines of transgenic flies were generated to decipher the functional relationships between the different actors. Phenotypes of the transgenic flies suggest that, like in mammals, the function of Rgl is not totally accounted for by the activation of Ral, since an activated allele of Ral does not mimic the activated alleles of Rgl. Could activated Rgl phenotypes be due to the titration of endogenous RAS1 or RAP1 by the RA domain of the RGL transgenes? If so, coexpression of activated RGL with either wild-type Ras1 or wild-type Rap1 should attenuate the Rgl phenotypes. This is not the case. Flies coexpressing activated RGL and RAS1 display some new phenotypes which are not seen when each transgene is individually expressed (extra veins under en-GAL4; heterogeneity of ommatidia size under GMR-GAL4) or keep displaying the Rgl phenotype (bristle morphology under sca-GAL4). Flies coexpressing activated RGL and RAP1 even display an enhanced Rgl phenotype (in eyes and on wings) or keep displaying the bristle morphology phenotype due to activated RGL. Ral-independent functions of RGL might be mediated by protein-protein interactions with domains other than the Ras/Rap and Ral interacting domains, and recently, mammalian RalGDS was shown to interact with ß-arrestin. However the titration hypothesis cannot be ruled out totally. An alternative explanation might be that Ral has to cycle between a GDP state and a GTP state, which would be accelerated by activated RalGEF and not mimicked by activated Ral that is blocked in a GTP-bound state. Although the existence of Ral-independent functions of RalGEFs is suggested both in mammals and in flies, the clarification of this question requires further investigation (Mirey, 2003).

But is RGL an actual exchange factor for RAL? The effects in bristle development of a dominant-negative Ral are suppressed by the increased expression of Rgl. The simplest explanation is that RGL is a bona fide exchange factor for Ral (Mirey, 2003).

Interactions were investigated between the Ral pathway and two of its interlocutors, the Ras and Rap GTPases. In mammalian cell lines, the RasG12V E37G, RasG12V Y40C, and RasG12V T35S alleles activate the Ral pathway via interaction with RalGEFs, the PI3K pathway, and the Raf pathway, respectively, although things might be more complicated since RasG12V Y40C might be acting together with RasG12V E37G to activate RalGEFs upon epidermal growth factor stimulation. RasG12V E37G does not activate the Raf nor the PI3K pathway. In Drosophila also, these different Ras alleles drive different pathways; however, nothing is known about the connection between the Ras and Ral pathways (Mirey, 2003).

If a Ras-Ral pathway exists, a dominant-negative allele of Ral should attenuate effects due to RasG12V E37G but not phenotypes due to RasG12V Y40C or RasG12V T35S. Indeed, in HeLa cells, dominant-negative alleles of Ral do block a RasG12V E37G phenotype (Ikeda, 1998). Reciprocally, RasG12V E37G, but not the two other alleles, might attenuate a Ral dominant-negative phenotype. The two-hybrid results show that, as in mammals, fly RGL behaves as an effector of fly RAS1, and this interaction is mediated by the RA domain of RGL. When searching for genetic interactions between Ras1 and Ral, it was found that all three Ras alleles enhanced the RalS25N loss-of-bristle phenotype. These results show an actual genetic interaction between the Ras and Ral pathways but do not support the classical model of a linear pathway from Ras to Ral, a conclusion strengthened by the absence of the Ras allele specificity of the observed interactions. An alternative model would be an intersection of the Ral and Ras pathways and would involve a yet undefined Ras effector whose interaction with Ras would not be selective for the three effector loop mutations tested in this study (Mirey, 2003).

Rap1 is another GTPase of the Ras family that can interact with most Ras effectors, including RalGEFs. No functional Rap1-RalGEF, Rap1-PI3K, or Rap1-Raf interactions have been documented, except for an isoform of B-Raf, described as activated by Rap1. The originally suggested antagonism between Ras and Rap remains a murky issue, and Rap1 and Ras seem to participate in rather independent pathways, although recent data challenge this idea, at least in vesicle trafficking at synapses. In Drosophila, where Rap1 is required for morphogenesis, Ras1 and Rap1 act in distinct pathways (Asha, 1999). No functional effector of Rap has been identified. Two-hybrid data show that Drosophila RGL behaves as a Rap1 effector. Genetic data support the idea that this interaction is functional: a dominant-negative allele of Ral is able to rescue lethality caused by an activated allele of Rap1, and reciprocally, in the surviving flies, activated Rap1 rescues the bristle development phenotype of a dominant-negative Ral. Similarly, in eyes, Rgl and Ral seem to act downstream of Rap1. Although an alternative model where Rap and Ral signals converge towards a common downstream target cannot be ruled out, the results rather argue in favor of a linear Rap-Rgl-Ral pathway. Consistent with this model, preliminary data with an engrailed-GAL4 driver show that the phenotype displayed by RalS25N in wings mimics the one obtained by overexpression of a negative regulator of Rap, RapGAP. Thus, titrating RGL proteins by the expression of a dominant-negative Ral mimics the inactivation of Rap1 by an excess of its GAP (Mirey, 2003).

Taken together, the genetic data from Drosophila shed a different light on signaling networks as they were established in mammalian cell lines. In both developmental systems used in this study (eye and notum), Ras1 and Ral do not seem to be linearly connected. In contrast, Rap1 and Ral are shown to act as if they were participating in a common transduction pathway. These data do not rule out that in some other tissues, a Ras-Ral pathway might indeed exist, but they suggest that a molecular Lego might assemble signaling modules following various architectures in different tissues. It is speculated that this should also be the case in mammals. An alternative model would be that a Ras-Ral pathway cannot be revealed in this experimental system, just as the Rap-Ral pathway couldn't be revealed in mammalian cell lines, and that, in the same tissue, Ras-Ral and Rap-Ral pathways are functional (Mirey, 2003).

A mouse homologue of the Drosophila tumor suppressor l(2)tid gene defines a novel Ras GTPase-activating protein (RasGAP)-binding protein

p120 GTPase-activating protein (GAP) down-regulates Ras by stimulating GTP hydrolysis of active Ras. In addition to its association with Ras, GAP has been shown to bind to several tyrosine-phosphorylated proteins in cells stimulated by growth factors or expressing transforming tyrosine kinase variants. A novel GAP-binding protein, mTid-1, a DnaJ chaperone protein that represents the murine homolog of the Drosophila tumor suppressor l(2)tid gene (Kurzik-Dumke, 1995), has been cloned and characterized. Three alternatively spliced variants of mTid-1 were isolated, two of which correspond to the recently identified hTid-1L and hTid-1S forms of the human TID1 gene that exhibit opposing effects on apoptosis. Both cytoplasmic precursor and mitochondrial mature forms of mTid-1 associate with GAP in vivo. Interestingly, although mTid-1 is found tyrosine-phosphorylated in v-src-transformed fibroblast cells, GAP selectively binds to the unphosphorylated form of mTid-1. In immunofluorescence experiments, GAP and Tid-1 were shown to colocalize at perinuclear mitochondrial membranes in response to epidermal growth factor stimulation. These findings raise the possibility that Tid chaperone proteins may play a role in governing the conformation, activity, and/or subcellular distribution of GAP, thereby influencing its biochemical and biological activity within cells (Trentin, 2001).

The Drosophila l(2)tid gene is the first member of a DnaJ chaperone family to be classified as a tumor suppressor. Recessive mutations at the l(2)tid locus cause defects in differentiation and morphogenesis of larval imaginal discs leading to neoplastic growth of these cells into lethal tumors (Kurzik-Dumke, 1995). The ubiquitously expressed DnaJ family of proteins serve as regulatory factors to the evolutionary conserved heat shock 70 (Hsp70) superfamily of molecular chaperones. This protein family is defined by a highly conserved J-domain, which functions as the binding region for Hsp70 chaperones and orchestrates their interaction with specific substrates. Hsp70 proteins and their associated DnaJ co-chaperone mediate a variety of cellular activities including the folding of newly synthesized polypeptides, the translocation of proteins across membranes, and assembly of multimeric protein complexes. More recently, genetic and biochemical studies have implicated DnaJ and Hsp70 proteins as important components of intracellular signaling pathways linked to cell survival and growth regulation. In this context, they regulate many facets of the signaling process that have been described for protein modules such as pleckstrin homology SH2 and SH3 domains, namely subcellular localization, regulation of enzymatic activity, and enzyme/substrate recognition. For example, genetic studies of v-src toxicity in yeast indicate that the DnaJ protein, Ydj1, is necessary for the correct subcellular targeting and kinase activation of v-src. Ydj1 has also been implicated as a positive regulator of cell cycle progression essential for efficient recognition and phosphorylation of cyclin CLN3 by cdc28, events that signal CLN3 degradation. Additional biochemical evidence suggests that members of the DnaJ and Hsp70 family interact with and modulate the growth-suppressive properties of several tumor suppressor proteins, including p53, Wilms' tumor suppressor (WT1), retinoblastoma (Rb) and the double-stranded RNA-activated protein kinase PKR. The findings presented in this paper suggest a possible novel role for GAP in collaboration with Tid-1·Hsp 70 chaperone complexes in the integration of mitogenic-signaling pathways at the plasma membrane and control of apoptotic signal transduction at mitochondrial membranes (Trentin, 2001).

The l(2)tid gene was originally classified as a tumor suppressor in Drosophila in which recessive mutations lead to malignant transformation of the imaginal discs of the larva (Kurzik-Dumke, 1992). Although TID1 has not previously been recognized as a tumor suppressor in humans, it is tempting to speculate on its tumor suppressor function in a mammalian setting as well. Importantly, the data point to a potential role for mTid-1 in GAP-mediated regulation of cell growth. In this capacity, mTid-1-Hsp/Hsc70 heterocomplexes may govern the conformational maturation and/or activity of GAP required for its role as a negative regulator of Ras or as a regulator of cytoskeletal organization. mTid-1 may also assist in the assembly of complexes consisting of GAP and other signaling proteins such as p62dok and p190 involved in GAP-directed activities. Alternatively, the association of mTid-1 with GAP may function to sequester GAP from the cytosol to the mitochondria, thereby modulating its interaction with and GTPase-promoting activity towards Ras in response to growth factor receptor activation. At any rate, one may envision that in the absence of functional Tid proteins, GAP may escape the regulation imposed by Hsp70 association and consequently affect the ability of GAP to effectively down-regulate Ras, which may contribute to a hyperproliferative phenotype. Significantly, it has been shown that loss of expression of another Ras GTPase-activating protein, neurofibromatosis-1, in neurofibroma tumors correlates with increased levels of activated Ras. Additional investigations of the biochemical function of Tid-1 and GAP complexes may improve understanding of how Tid-1 DnaJ proteins may exert their effects on cell survival and cell growth (Trentin, 2001).

Sprouty: a Ras pathway antagonist that binds Drk and Gap1

Sprouty was identified in a genetic screen as an inhibitor of Drosophila EGF receptor signaling. The Egfr triggers cell recruitment in the eye, and sprouty minus eyes have excess photoreceptors, cone cells, and pigment cells. Tests provide evidence that Sprouty interacts specifically with the Egfr pathway. Halving the dose of sprouty (1) strongly enhances the rough eye caused by the misexpression of rhomboid, a specific activator of Egfr signaling; suppresses the rough eye caused by underrecruitment of photoreceptors in a hypomorphic allele of spitz, the TGF-like ligand of the Egfr; (3) suppresses the phenotypes of Egfr hypomorphic mutations both in the eye and the wing and (4) flies heterozygous for both sprouty and argos have mildly rough eyes, caused by a slight overrecruitment of all types of cell, although heterozygosity for either mutation alone causes no phenotype. Other genetic interactions between sprouty and the Egfr pathway are also detailed. All point to the same conclusion: Sprouty inhibits Egfr signaling (Casci, 1999).

The expression of sprouty in the eye imaginal disc was documented. In eye imaginal discs, sprouty is detected only behind the morphogenetic furrow, in the region where Egfr-mediated recruitment occurs. It appears successively in each of the newly recruited cells, both in photoreceptors and cone cells, a pattern that strongly suggests that sprouty expression is dependent on Egfr signaling itself. This is confirmed by loss of sprouty staining in many cells when Egfr signaling is blocked (with a dominant-negative form of the receptor) and an increased number of cells expressing sprouty in the presence of an activated form of the receptor. These results in the eye are consistent with observations that sprouty expression also follows, and is dependent upon, Egfr activation in the follicle cells of the egg. Similarly, sprouty expression has been shown to be dependent on Fgfr signaling in the trachea. Although the widespread dependence of sprouty expression on RTK signaling could be indirect, it has important developmental consequences, as it implies that Sprouty participates in negative feedback control of signaling (Casci, 1999)

Hacohen (1998) found sprouty to behave nonautonomously in the trachea. In contrast, in the current study sprouty is shown to act strictly cell autonomously in R7 cells. In sevenless mutant ommatidia mosaic for sprouty, all R7s are sprouty minus. Ommatidia are found where the only sprouty minus cell is the R7, implying that absence of Sprouty in a cell can be sufficient to transform it into an R7, even when all its neighbors are sprouty plus (Casci, 1999).

To determine where in the Ras signal transduction pathway Sprouty acts, it was asked whether altering its amount could modify the phenotypes caused by expressing constitutive forms of the Egfr, Ras1, and Raf in the eye. Halving the dose of sprouty enhances the phenotype of constitutive Egfr but has no effect on the constitutive Ras1 or Raf phenotypes. Overexpression of sprouty is able to rescue the phenotype caused by expression of the constitutive Egfr. These interactions indicate that Sprouty acts downstream of (or parallel to) the Egfr, but upstream of Ras1 and Raf (Casci, 1999).

Both full-length Sprouty and a truncated Sprouty containing residues 1-369 (i.e., without the cys-rich domain and C-terminal residues) were assayed for their ability to bind in vitro translated members of the Ras pathway. Strong interactions are detected between Sprouty and Drk, an SH2-SH3 containing adaptor protein homologous to mammalian Grb2, and between Sprouty and Gap1, a Ras GTPase-activating protein. No interactions were seen between Sprouty and several other proteins involved in the Ras pathway: Sos, Dos, Csw, Ras1, Raf, and Leo (14-3-3). The interactions with Drk and Gap1 did not require the presence of the C-terminal cysteine-rich domain, the region of Sprouty most conserved between flies and humans. Since the well-conserved cysteine-rich domain of Sprouty is not required for binding to Drk or Gap1, it might instead target the protein to the plasma membrane. To test this, two truncated forms of Sprouty were expressed in cultured cells. One form lacks the conserved cysteine-rich domain, whereas a second exclusively comprises the cysteine-rich domain. The form with the cysteine-rich domain is membrane associated and is indistinguishable from the wild-type protein. In sharp contrast, the form lacking the cysteine-rich domain is distributed uniformly throughout the cell, with no specific localization to membranes. Cell fractionation confirms these results. It is concluded that the 147-residue cysteine-rich domain in Sprouty, which corresponds to the most conserved region in the published human ESTs, is responsible for the specific localization of Sprouty to the plasma membrane (Casci, 1999).

Sprouty's function is, however, more widespread. It also interacts genetically with the receptor tyrosine kinases Torso and Sevenless, and it was first discovered through its effect on FGF receptor signaling. In contrast to an earlier proposal that Sprouty is extracellular, biochemical analysis suggests that Sprouty is an intracellular protein, associated with the inner surface of the plasma membrane. Sprouty binds to two intracellular components of the Ras pathway, Drk and Gap1. These indicate that Sprouty is a widespread inhibitor of Ras pathway signal transduction (Casci, 1999).

DOS, a presumptive adaptor protein

The SH2 domain-containing phosphotyrosine phosphatase Corkscrew (Csw) is an essential component of the signaling pathway initiated by the activation of the sevenless receptor tyrosine kinase (Sev) during Drosophila eye development. Genetic and biochemical approaches have been used to identify a substrate for Csw. Expression of a catalytically inactive Csw was used to trap Csw in a complex with a 115 kDa tyrosine-phosphorylated substrate. This substrate was purified and identified as the product of the daughter of sevenless (dos) gene. Mutations of dos were identified in a screen for dominant mutations that enhance the phenotype caused by overexpression of inactive Csw during photoreceptor development. Analysis of dos mutations indicates that Dos is a positive component of the Sev signaling pathway and suggests that Dos dephosphorylation by Csw may be a key event during signaling by Sev (Herbst, 1996).

The specification of the R7 photoreceptor cell in the developing eye of Drosophila is dependent upon activation of the Sevenless (SEV) receptor tyrosine kinase. By screening for mutations that suppress signaling via a constitutively activated SEV protein, a novel gene, daughter of sevenless (dos) has been identified. DOS is required not only for signal transduction via SEV but also in other receptor tyrosine kinase signaling pathways throughout development. The presence of an amino-terminally located pleckstrin homology domain and many potential tyrosine phosphorylation sites suggests that DOS functions as an adaptor protein able to interact with multiple signaling molecules. The genetic analysis demonstrates that DOS functions upstream of Ras1 and defines a signaling pathway that is independent of direct binding of the DRK SH2/SH3 adaptor protein to the SEV receptor tyrosine kinase (Raabe, 1996).

The pleckstrin homology (PH) domain-containing protein Daughter of Sevenless (Dos) is an essential component of the Sevenless receptor tyrosine kinase (SEV) signaling cascade, which specifies R7 photoreceptor development in the Drosophila eye. Previous results have suggested that Dos becomes tyrosine phosphorylated during Sev signaling and collaborates with the protein tyrosine phosphatase Csw. This possibility was investigated by identifying tyrosine residues 801 and 854 of Dos as the phosphorylated binding sites for the Csw SH2 domains. These sites become phosphorylated in response to Sev activation and phosphorylation of both sites is required to allow Csw to bind Dos. Mutant Dos proteins in which either Y801 or Y854 of Dos has been changed to phenylalanine are unable to function during signaling by Sev and other receptor tyrosine kinases. In contrast, a mutant Dos protein in which all tyrosine phosphorylation sites except Y801 and Y854 have been removed is able effectively to provide Dos function during Sev signaling and to rescue the lethality associated with dos loss-of-function mutations. These results indicate that a primary role for Dos during signaling by Sev and other receptor tyrosine kinases is to become phosphorylated at Y801 and Y854 and then recruit Csw (Herbst, 1999).

Structural and enzymatic studies of SHP-2, the mammalian homolog of Csw, have shown that binding of the N-terminal SH2 domain to a phosphorylated tyrosine-containing peptide leads to a marked increase in SHP-2 catalytic activity. Thus, an expected consequence of the binding of Csw to Dos is a substantial increase in Csw catalytic activity. Given the requirement for the Csw catalytic activity during Sev signaling, a key question is the identity of the key Csw target(s) whose dephosphorylation is required for R7 photoreceptor development. Dos itself might be a crucial target of Csw. In particular, Dos might contain sites of tyrosine phosphorylation that serve as docking sites for proteins inhibitory to Sev signaling and these sites might be dephosphorylated by Csw. This idea is based both on studies showing that Dos is a potential Csw substrate and on genetic studies indicating that reduced Csw or Dos function decreases the strength of Sev signaling. Given this model, an expectation is that mapping the sites of tyrosine phosphorylation in catalytically inactive CswCS-bound Dos might identify both the Csw SH2 domain-binding sites and additional Csw target sites. Instead, this analysis reveals phosphorylation of only the two Csw SH2 domain-binding sites, Y801 and Y854. Together, the (1) failure to find additional sites of Dos tyrosine phosphorylation in CswCS-bound Dos and (2) the ability of DosYY (in which all tyrosines outside of the Dos PH domain except Y801 and Y854 are either removed by deletion or changed to phenylalanine) to provide Dos function during Sev signaling, suggest that the primary role of Dos may be to activate the catalytic activity of Csw towards other, as yet unidentified, proteins. In this model, the role of Csw's dephosphorylation of Dos would be to down-regulate signaling by eliminating its own binding sites (Herbst, 1999).

The Drosophila nonreceptor protein tyrosine phosphatase, Corkscrew (Csw), functions positively in multiple receptor tyrosine kinase (RTK) pathways, including signaling by the Epidermal growth factor receptor (Egfr). Detailed phenotypic analyses of csw mutations have revealed that Csw activity is required in many of the same developmental processes that require Egfr function. However, it is still unclear where in the signaling hierarchy Csw functions relative to other proteins whose activities are also required downstream of the receptor. To address this issue, genetic interaction experiments were performed to place csw gene activity relative to the Egfr, spitz (spi), rhomboid (rho), daughter of sevenless (Dos), kinase-suppressor of ras (ksr), ras1, D-raf, pointed (pnt), and moleskin. The Egfr-dependent formation of VA2 muscle precursor cells was followed as a sensitive assay for these genetic interaction studies. Csw is shown to have a positive function during mesoderm development. Tissue-specific expression of a gain-of-function csw construct rescues loss-of-function mutations in other positive signaling genes upstream of rolled (rl)/MAPK in the EGFR pathway. Levels of Egfr signaling in various mutant backgrounds during myogenesis could be inferred. This work extends previous studies of Csw during Torso and Sevenless RTK signaling to include an in-depth analysis of the role of Csw in the EGFR signaling pathway (Hamlet, 2001).

A variety of genetic interaction experiments between gain- and loss-of-function mutations and/or constructs in genes involved in Egfr signaling has resulted in three principal findings. (1) Consistent with findings in the developing retina, Cswsrc90 functions like a bona fide gain-of-function protein in several Egfr-initiated developmental processes during oogenesis, embryogenesis, and metamorphosis. (2) Csw plays a positive role in Egfr signaling during myogenesis. (3) Tracking the formation of VA2 precursor cells serves as a sensitive assay to infer levels of Egfr signaling in various mutant genetic backgrounds (Hamlet, 2001).

Genetic interaction data between csw and Dos are consistent with a model whereby a direct interaction between Csw and Dos is essential for Drosophila Egfr signaling. A Dos protein containing only the pTyr sites that bind to the Csw SH2 domains is sufficient to provide wild-type Dos function. A vertebrate Dos homolog, Gab1, and SHP-2 associate upon activation of the vertebrate Egfr, results in an increase in MAPK signaling (Hamlet, 2001 and references therein).

The readout from the putative Dos dominant-negative mutant embryos is in the same range as that of dominant-negative csw mutant embryos. The identical genetic interaction of csw and Dos with cswsrc90 places their function in a category separate from that of the other signaling genes analyzed and suggests that they both function at the same level in the Egfr pathway (Hamlet, 2001).

Interestingly, Dos mutant embryos phenocopy the putative dominant-negative csw mutant embryos but not the protein null csw mutant embryos. These results suggest that the dominant-negative csw mutant phenotype reflects loss of Dos function. Since the cswVA199 mutation generates a truncated Csw protein where only the SH2 domains are expressed, perhaps the SH2 domains still bind to and sequester Dos function away from the signaling pathway (Hamlet, 2001).

Ras interaction with PI3K

Ras-dependent and Ras-independent effects of PI3K in Drosophila motor neurons

The lipid kinase PI3K plays key roles in cellular responses to activation of receptor tyrosine kinases or G protein coupled receptors such as the metabotropic glutamate receptor (mGluR). Activation of the PI3K catalytic subunit p110 occurs when the PI3K regulatory subunit p85 binds to phosphotyrosine residues present in upstream activating proteins. In addition, Ras is uniquely capable of activating PI3K in a p85-independent manner by binding to p110 at amino acids distinct from those recognized by p85. Because Ras, like p85, is activated by phosphotyrosines in upstream activators, it can be difficult to determine if particular PI3K-dependent processes require p85 or Ras. This study asked if PI3K requires Ras activity for either of two different PI3K-regulated processes within Drosophila larval motor neurons. To address this question, the effects on each process were determined of transgenes and chromosomal mutations that decrease Ras activity, or mutations that eliminate the ability of PI3K to respond to activated Ras. It was found that PI3K requires Ras activity to decrease motor neuron excitability, an effect mediated by ligand activation of the single Drosophila mGluR DmGluRA. In contrast, the ability of PI3K to increase nerve terminal growth is Ras-independent. These results suggest that distinct regulatory mechanisms underlie the effects of PI3K on distinct phenotypic outputs (Johnson, 2012).

Table of contents


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

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