Ras oncogene at 85D


Table of contents

Kinase repressor of Ras (Ksr)

Two genes in Drosophila have been identified and characterized whose products are required for activated RAS to signal with normal efficiency, but they do not appear to effect signaling by activated RAF. One encodes the beta subunit of type I geranylgeranyl transferase, a prenylation enzyme essential for targeting RAS to the plasma membrane. The other encodes a protein kinase that has been named kinase suppressor of ras (ksr). KSR functions in multiple receptor tyrosine kinase pathways. Mammalian homologs of KSR have been identified that, together with the Drosophila gene, define a novel class of kinases. These results suggest that KSR is a general and evolutionarily conserved component of the RAS signaling pathway that acts between RAS and RAF (Therrien, 1995).

kinase suppressor of ras (ksr) encodes a putative protein kinase that by genetic criteria appears to function downstream of RAS in multiple receptor tyrosine kinase (RTK) pathways. While biochemical evidence suggests that the role of Ksr is closely linked to the signal transduction mechanism of the MAPK cascade, the precise molecular function of Ksr remains unresolved. To further elucidate the role of Ksr and to identify proteins that may be required for Ksr function, a dominant modifier screen was conducted in Drosophila based on a Ksr-dependent phenotype. Overexpression of the Ksr kinase domain in a subset of cells during Drosophila eye development blocks photoreceptor cell differentiation and results in the external roughening of the adult eye. Therefore, mutations in genes functioning with Ksr might modify the Ksr-dependent phenotype. Approximately 185,000 mutagenized progeny were screened for dominant modifiers of the Ksr-dependent rough eye phenotype. A total of 15 complementation groups of Enhancers and four complementation groups of Suppressors were derived. Ten of these complementation groups correspond to mutations in known components of the Ras1 pathway, demonstrating the ability of the screen to specifically identify loci critical for Ras1 signaling and further confirming a role for Ksr in Ras1 signaling. Mutations in genes encoding known components of the Ras pathway were isolated in a screen for the 14-3-3epsilon, Dsor1/mek, rolled/mapk, pointed, yan, and ksr loci. Furthermore, due to the ability of dominant-negative KSR (KDN) to block RAS/MAPK-mediated signaling, mutations in genes expected to function upstream of ksr were also isolated. These included mutations in the Egfr, Star, Sos, and Ras1 loci. In addition, 4 additional complementation groups were identfied. One of them corresponds to the kismet locus, which encodes a putative chromatin remodeling factor (Therrien, 2000).

Complementation test results reveal that SK2-4 is allelic to Src42A, the closest Drosophila homolog of the Src kinase family. Intriguingly, genetic data (suppression of sE-KDN, enhancement of sev-Ras1V12, and suppression of RafHM7) suggest an inhibitory role for Src42A in Ras1 signaling, which is the opposite of the described function of Src-like kinases in vertebrates. During the course of this work, Src42A was also identified by another laboratory as being a dominant suppressor of a hypomorphic allele of Raf (RafC110). Genetic and molecular characterization of different Src42A alleles clearly supports an inhibitory function for Src42A in different RTK-dependent signaling pathways. Therefore, the further elucidation of the molecular function of Src42A in Drosophila may unveil a novel mechanism of action for this family of nonreceptor tyrosine kinases (Therrien, 2000).

Since alleles of EK2-9 failed to complement two independent P elements recently shown to disrupt the RRM-motif protein locus split ends (spen), it is concluded that EK2-9 is allelic to spen. This gene encodes at least three large (~5500 amino acids) and closely related protein isoforms. The Spen proteins contain three RRMs at the N terminus and a conserved region of unknown function at the C terminus. The presence of three RRMs suggests that the Spen proteins mediate their effect via a RNA binding-dependent mechanism such as RNA processing or transport. Interestingly, mutant alleles of spen have been isolated in several independent genetic screens in Drosophila. They were initially recovered in a screen for mutations affecting peripheral nervous system development. Subsequently, they were isolated in two related screens that, like the KDN screen, were designed to identify novel components of the Ras1 pathway. Mutations in spen were isolated as dominant enhancers of a Raf-induced [E(Raf) 2A complementation group] and a Yan-induced rough eye phenotype. The fact that three separate screens targeting Ras1 signaling (KSR-, Raf-, and Yan-dependent) recovered mutant alleles of spen suggests that this locus is relevant for Ras1-mediated signal transduction. Examination of the genetic interactions, however, indicates that the role of spen in Ras1 signaling may not be straightforward. For example, the ability of the E(Raf)2A/spen alleles to dominantly enhance an activated Raf-dependent phenotype suggests that spen is a negative regulator of the pathway. However, alleles of spen also enhance RafHM7 lethality at 18°, and homozygous mutant clones in the eye are often missing R7 and outer photoreceptor cells, although extra photoreceptor cells are occasionally found. These results are more consistent with a positive role for spen during Ras1 signaling and would agree with the fact that spen mutations have been recovered as enhancers of sE-KDN and gmr-yanact. Alleles of spen were also recovered as enhancers of a loss-of-function phenotype for the Hox gene Deformed (Dfd). In that context, the genetic analysis of spen suggests that it functions in parallel to Dfd for the specification of head cuticular structures. Finally, mutations in spen have been identified as dominant enhancers of an E2F/Dp-induced rough eye phenotype and as dominant suppressors of p21CIP1-induced phenotypes. The fact that S-phase entry is stimulated by the overexpression of E2F and Dp proteins in the eye but inhibited by p21CIP1 overexpression suggests that spen may be involved in the negative regulation of cell cycle progression (Therrien, 2000).

Currently it is unknown how spen activity links Ras1-dependent cell differentiation, Hox-dependent segment specification, and E2F-dependent cell cycle control. Nonetheless, their common requirement for spen function suggests that they are interconnected. In agreement with this idea, other loci have been found in common in the screens mentioned above, as well as in other related screens. One of these loci corresponds to the kismet (kis) gene. In addition to the EK2-4/kis alleles identified in this KDN screen, mutations in kis were recovered as dominant enhancers in the Dfd screen and as dominant suppressors in a Polycomb (Pc) loss-of-function screen. Alleles of kis have also been identified as dominant suppressors of the synthetic lethality generated by the coexpression of activated Sevenless (SevS11) and Ras1V12 (Therrien, 2000).

The ability of kis mutations to suppress the homeotic transformations observed in a Pc mutant background and to suppress the expression of the Hox gene Sex combs reduced suggests that kis is a member of the trithorax group (trxG) of genes. In Drosophila, trxG genes are essential for Hox gene expression, where they appear to counteract the repressive effect of the Polycomb group (PcG) of genes on Hox gene expression. Interestingly, a number of trxG proteins are similar to components of the large (2 MD) ATPase-dependent SWI/SNF complex that facilitates the access of transcription factors to their DNA binding sites by destabilizing the nucleosomes. It is thus possible that trxG proteins control Hox gene expression by directly altering the chromatin at specific Hox loci. Strikingly, sequence analysis of the Kis proteins reveals strong homologies with the CHD proteins, which represent a novel class of putative ATPase subunits of chromatin remodeling complexes, and with Brahma, a trxG protein that is the closest fly homolog to the yeast SWI2/SNF2 ATPase subunit. These observations strongly suggest that the Kis proteins are also involved in chromatin remodeling. Interestingly, Brahma and Kis proteins do not appear to coexist in a common complex. This raises the possibility that Kis is part of a different chromatin remodeling complex that might regulate the transcription of a distinct group of genes. Consistent with this hypothesis, brahma mutations have not been isolated in the Ras1 pathway-dependent screens mentioned above. On the basis of the presumed function for Kis and the observation that kis loss-of-function mutations suppress sev-Ras1V12, and enhance sE-KDN and RafHM7 rough eye phenotypes, it is tempting to speculate that Kis is part of a complex that, in response to Ras1-dependent signals, directly alters the transcription of a specific group of genes required for Ras1-dependent cell differentiation during Drosophila eye development (Therrien, 2000).

Mechanisms that regulate signal propagation through the ERK/MAPK pathway are still poorly understood. Several proteins are suspected to play critical roles in this process. One of these is Kinase Suppressor of Ras (KSR), a component previously identified in RAS-dependent genetic screens in Drosophila and Caenorhabditis elegans. KSR functions upstream of MEK within the ERK/MAPK module. In agreement with this, KSR facilitates the phosphorylation of MEK (officially termed Downstream of raf1) by RAF (accepted FlyBase name: Pole hole). KSR associates independently with RAF and MEK, and these interactions lead to the formation of a RAF/MEK complex, thereby positioning RAF in close proximity to its substrate MEK. These findings suggest that KSR functions as a scaffold that assembles the RAF/MEK functional pair (Roy, 2002).

The KSR kinase domain has been reported to interact with MEK but not phosphorylate MEK. KSR has also been found to interact with RAF, but the regions involved have not been identified. It was important to examine whether forced association between KSR and RAF catalytic domains (KSRc and RAFc) would affect MEK activity. Advantage was taken of two Drosophila transgenic lines that express, during eye development, either RAFc or KSRc fused to the N-terminal portion of Torso4021, which comprises the extracellular and transmembrane regions of the Torso receptor tyrosine kinase (N-Tor4021). Tor4021 is a gain-of-function allele of torso. The aberrant Torso4021 protein has a Y327C change in the extracellular portion that is thought to promote ligand-independent receptor oligomerization (Roy, 2002).

N-Tor4021RAFc behaves as an activated form of RAF and transforms cone cells into additional R7 photoreceptor cells when expressed in the developing Drosophila eye (Dickson, 1992). In contrast, N-Tor4021KSRc is a potent dominant-negative form of KSR that blocks signaling through the MAPK pathway, presumably by sequestering MEK. It strongly antagonizes photoreceptor cell differentiation (Therrien, 1996). It was reasoned that crossing flies expressing N-Tor4021RAFc (BT98) to flies expressing N-Tor4021KSRc (KDN) should lead to the formation of hetero-oligomers, which might result in activation of endogenous MEK and MAPK. BT98 and KDN transgenic flies were crossed to a wild-type strain or to each other to assess the effect on MAPK compared with wild-type flies. Third instar eye discs of the different genotypes were stained with an anti-phospho-MAPK (anti-pMAPK) antibody; compared with wild-type pMAPK levels, BT98/+ flies had slightly elevated pMAPK levels posterior to the morphogenetic furrow at a position consistent with the expected transgene expression pattern. In agreement with elevated pMAPK, BT98/+ adult eyes have additional R7 photoreceptor cells that result in a rough eye phenotype. In contrast, the KDN/+ flies have reduced pMAPK levels, which is also consistent with the roughening of the external adult eye surface due to a block in photoreceptor cell differentiation. Strikingly, the BT98/KDN flies show a massive accumulation of pMAPK posterior to the morphogenetic furrow that correlates with a robust enhancement of activated RAF rough eye phenotype. These findings strongly suggest that N-Tor4021KSRc brings endogenous MEK to N-Tor4021RAFc, thus strongly enhancing endogenous MEK and MAPK activation (Roy, 2002).

To verify that the association between RAFc and KSRc is responsible for MEK phosphorylation, RAFc and KSRc were fused to the FK506-binding protein (FKBP) and FKBP-rapamycin-binding domain (FRB), respectively, to allow their heterodimerization in a rapamycin-dependent manner. A functional or inactivated myristoylation signal was introduced to examine the influence of membrane localization. RAFc and KSRc derivatives were tagged with the polyoma (pyo) and the Flag epitopes, respectively, to allow their detection. The FKBP-RAFc and FRB-KSRc fusion proteins were expressed separately or together along with myc-epitope-tagged MEKDA in the absence or the presence of rapamycin. Expression of KSRc or RAFc variants alone or together does not result in MEK phosphorylation in the absence of rapamycin. As expected, FRB-KSRc physically interacts with MEK as revealed by the associated mycMEKDA in anti-Flag immunoprecipitates, whereas the FKBP-RAFc did not interact with MEK. Rapamycin treatment does not affect the behavior of the KSRc and RAFc constructs when they are expressed alone, but promotes their heterodimerization when expressed together. This results in complex formation between RAF and MEK and leads to MEK phosphorylation. Interestingly, this effect is accompanied by a clear and strong mobility shift of FKBP-RAFc. Permutation of the myristoylation signal or no myristoylation signal on the two types of fusion proteins give identical results, which suggests that no additional membrane components are required in this event. Together, these results show that induced association between RAFc and KSRc results in MEK phosphorylation (Roy, 2002).

To address whether MEK phosphorylation depends on the catalytic function of RAFc or KSRc, the activity of two kinase-inactivated mutants, RAFcK498S and KSRcK705M was tested. As expected, kinase-inactivated RAFc does not support rapamycin-dependent MEK phosphorylation. Interestingly, this mutant does not display a mobility shift. In contrast, KSRcK705M, which interacts normally with MEK, does promote MEK phosphorylation, although not as efficiently as wild-type KSR. These results suggest that the ability of KSR to promote MEK phosphorylation does not absolutely require a catalytically intact function (Roy, 2002).

Although previous genetic and biochemical data on KSR have suggested its involvement in the MAPK module, they have never demonstrated it. The RNAi technique was used to deplete endogenous KSR levels in S2 cells as well as the levels of the other major components and examine the effect on MAPK activation. A stable S2 cell line expressing RASV12 under the control of a heavy metal-inducible promoter was used to activate the MAPK pathway. Addition of copper to cell culture media augmented total RAS1 levels, which resulted in a modest increase in phospho-MEK (pMEK) and a robust elevation of pMAPK (Roy, 2002).

To further unravel the molecular function of KSR, a KSR-dependent MEK phosphorylation assay was reconstituted using native KSR and RAF proteins. However, mouse KSR1 has been reported to block signaling through the ERK/MAPK pathway when overexpressed in various cell lines. This effect is likely owing to sequestration of specific components of the pathway that are in limiting amounts. RAF and MEK are prime candidates, as they have been reported to interact with mKSR1. To circumvent this problem, KSR was coexpressed together with RAF and MEK. RASV12 was also used to activate the MAPK pathway. To prevent possible MAPK-dependent negative-feedback effects, a kinase-inactivated version of MEK (MEKDA) was used throughout this work. Compared with myc-epitope-tagged MEKDA expressed alone, RASV12 coexpression slightly increases pMEK levels. Coexpression of KSR or pyo-epitope-tagged RAF in those two conditions (absence or presence of RASV12) does not significantly alter the pMEK profiles. Strikingly, coexpression of MEK with KSR and RAF without RASV12 induces MEK phosphorylation, which strongly increases upon addition of RASV12. These results suggest that native KSR promotes the ability of RAF to phosphorylate MEK. Although RAS activity strongly increases the effect of KSR, the fact that an effect of KSR is detected in the absence of RASV12 suggests among different possibilities that either endogenous RAS activity contributes to that effect or that the higher levels of the transiently expressed proteins somewhat bypassed the normal RAS-dependency of MEK phosphorylation by RAF (Roy, 2002).

Next, tests were performed to see whether the kinase function of RAF and the putative kinase function of KSR were required for MEK phosphorylation in this assay. Kinase-inactivated mutants for pyoRAF (K498M) and KSR (K705M) were generated, and their respective effect was tested. As expected, RAF kinase function is absolutely required for MEK phosphorylation. In contrast, KSRK705M still promotes MEK phosphorylation, although slightly less efficiently than wild-type KSR. The ability of KSR to stimulate MEK phosphorylation does not absolutely depend on its putative kinase function (Roy, 2002).

Advantage was taken of the simple cotransfection assay in S2 cells to examine the effect of five additional mutations in KSR. Evidence had been presented that mKSR1 can phosphorylate RAF. As a kinase-inactive mutant, two conserved aspartic residues were changed in subdomains VI and VII to alanine residues. Because the KSRK705M mutant is still active, a KSR kinase-defective mutant [similar to the reported mutants (D800A-D817A)] was generated to test its effect. The four other mutants affected independent regions of KSR. KSRL50S-R51G lies within the CA1 domain. This mutation has been recovered as a hypomorphic loss-of-function allele in a RAS-dependent genetic screen in Drosophila (Therrien, 1995). KSRC398S-C401S disrupts the integrity of the cysteine-rich motif. A similar mutation in mKSR1 has been shown to abrogate KSR's ability to promote RAS-dependent Xenopus oocyte maturation. KSR contains an FXFP motif that has been proposed to function as a MAPK docking site. The function of this motif in KSR is unknown. The first phenylalanine residue of the motif, KSRF518W, was changed to verify its functional relevance. Finally, a mutation in KSR isolated in a RAS-dependent genetic screen in C. elegans (Sundaram, 1995) has been reported to prevent the association between KSR and MEK (Stewart, 1999). A similar mutation, KSRC922Y, was generated to examine the effect of impairing the KSR/MEK interaction. Interestingly, in contrast to KSRK705M, which still supports MEK phosphorylation, KSRD800A-D817A and KSRC922Y completely abrogates KSR activity. In addition, KSRL50S-R51G shows a severe reduction of activity, whereas KSRC398S-C401S shows only a slight, but reproducible reduction in KSR activity, and KSRF518W is as active as wild-type KSR. Given that the two KSR mutations (L50S-R51G and C922Y) corresponding to loss-of-function mutations isolated in genetic screens also disrupts the ability of KSR to induce MEK phosphorylation by RAF, it strongly suggests that the assay recapitulates the normal function of KSR (Roy, 2002).

It was then investigated whether the effect of the mutations could be caused by defects in KSR's ability to associate with MEK and/or RAF. To verify this, KSR or the mutant variants were immunoprecipitated from cell lysates used to analyze pMEK levels and the amount of coimmunoprecipitated mycMEKDA and pyoRAF was examined. As reported for mKSR1, Drosophila KSR also associates with MEK and RAF. Two mutants, KSRD800A-D817A and KSRC922Y, do not interact with MEK. Interestingly, they also fail to stimulate MEK phosphorylation. In contrast, KSRK705M, which is almost as active as wild-type KSR, is not significantly affected by its association with MEK. These results suggest that the ability of KSR to interact with MEK is critical to stimulate MEK phosphorylation. Intriguingly, KSRL50S-R51G associates normally with MEK, yet is severely diminished in its capacity to promote MEK phosphorylation. Although no drastic effect regarding the binding properties of this mutant was observed, there is a very reproducible demonstration of an approximately twofold decrease in KSR's capacity to associate with RAF. This observation suggests that the CA1 domain mediates an association with RAF. KSRD800A-D817A and KSRC922Y also show a similar RAF-association defect. Their reduced interaction with RAF might be caused by their inability to bind MEK, which normally might stabilize the KSR/RAF interaction. Alternatively, these mutations might induce structural perturbations that reduce the KSR/RAF association independent of the inability of MEK to bind KSR. Taken together, these data indicate that KSR associates independently with MEK and RAF, and that these interactions appear to be critical for KSR activity (Roy, 2002).

Previous work has reported that the association between mKSR1 and RAF is RAS-dependent (Therrien, 1996; Xing, 1997). Interestingly, Drosophila KSR can associate with RAF without coexpressing an activated form of RAS. It was therefore of interest to determine whether endogenous RAS1 activity might contribute to this interaction. To verify this, pyoRAF and KSR were coexpressed in the presence of dsRNA for GFP, RAS1, MEK, or MAPK; then the levels of immunoprecipitated RAF and the levels of associated KSR were measured. As for the negative controls, addition of dsRAS1 RNA did not perturb the KSR/RAF association, which suggests that the Drosophila KSR/RAF interaction can occur in the absence of RAS-mediated signals. The inability of dsMEK RNA to alter the KSR/RAF association is another indication that KSR interacts with RAF independently of MEK (Roy, 2002).

Given that KSR appears to associate independently with RAF and MEK, tests were performed to see whether KSR might physically link RAF and MEK together. Fixed amounts of RAF and MEK were coexpressed alone or in the presence of increasing amounts of KSR, and the levels of associated MEK were evaluated by probing the anti-pyo immunoprecipitates with anti-myc. Coexpression of RAF and MEK alone does not result in the formation of a stable RAF/MEK association. In sharp contrast, expression of KSR allows the formation of a RAF/MEK complex. The levels of MEK associated with RAF increase upon augmenting the expression levels of KSR as predicted if KSR physically connects RAF and MEK. If this model is correct, KSRC922Y should not induce the formation of a RAF/MEK complex because it no longer binds to MEK. Interestingly, when coexpressed with RAF and MEK, wild-type KSR shows greater protein stability compared with KSRC922Y. Therefore more DNA for the mutant construct was transfected to compare the effect of equal amounts of proteins. As predicted, KSRC922Y can not promote the formation of a RAF/MEK complex (Roy, 2002).

The fact that KSR induces the formation of a RAF/MEK complex in the absence of cotransfected RASV12 is intriguing. Whether endogenous RAS1 activity is involved in the RAF/MEK complex formation was therefore verified by eliminating RAS's contribution using dsRAS1 RNA. Surprisingly, dsRAS1 RNA does not affect the complex. Similarly, coexpression of RASV12 does not significantly enhance its formation. Together, these findings strongly suggest that KSR connects RAF and MEK and that the assembly does not require RAS activity (Roy, 2002).

CNK, a RAF-binding multidomain protein required for RAS signaling

kinase suppressor of ras (ksr) is required for efficient signal transmission within the Ras/MapK cascade. A screen for mutations that modify a ksr-dependent phenotype has identified a novel gene, Connector enhancer of KSR (cnk), which functions either upstream or in parallel to Raf in the Ras pathway. cnk encodes a protein of 1557 amino acids containing several protein-protein interaction domains, suggesting that Cnk brings different signaling molecules together. Searches of protein and translated nucleotide databases reveal a multidomain organization for the Cnk protein. These domains include a sterile alpha motif (SAM) domain, a PSD-95/DLG-1/ZO-1 (PDZ) domain, and a pleckstrin homology (PH) domain. In addition, protein sequence analysis has identified two proline-rich stretches containing SH3 domain consensus binding sites and several potential tyrosine phosphorylation sites corresponding to different SH2 domain consensus binding sites. This structural organization is reminiscent of multiadaptor proteins and suggests that the function of Cnk is to bring together different molecules. A search of the expressed sequence tags (EST) database identified several sequences encoding SAM, PDZ, or PH domains similar to those in CNK. Full-length cDNA clones were isolated corresponding to a few of these ESTs and their sequences were determined. One of them encodes a protein of 751 amino acids that, although smaller than CNK, has a similar modular arrangement: a SAM domain at the extreme N terminus, followed by a PDZ domain and then a PH domain. A putative C. elegans ORF contains the same organization of protein modules. A closer inspection of sequence similarities between these three proteins identified another conserved region of about 80 amino acids between the SAM and PDZ domains that might represent a novel protein module. This putative domain was named "CRIC" for conserved region in CNK (Therrien, 1998).

To determine whether cnk has a role during Ras signaling, the ability of cnk to modify the rough eye phenotype caused by overexpression of Ras1V12 (constitutively active Ras), sevS11 (activated Sevenless RTK, and RafTor4021 (activated RAF) was tested. cnk dominantly suppresses both Ras1V12 and sevS11 rough eye phenotypes but is unable to modify the RafTor4021 rough eye phenotype. Analysis of apical sections of adult fly retinae support this conclusion, and multiple cnk alleles have the same effect. The ability of cnk to enhance KDN (dominant negative Kinase suppressor of Ras) and to suppress both Ras1V12 and sevS11 phenotypes suggests that the EK2-3 locus encodes a positive component of RAS signaling. In addition, the finding that cnk does not modify the RafTor4021 phenotype suggests that cnk is required upstream or in parallel to Raf. The finding that cnk loss-of-function alleles modify sensitized genetic backgrounds like those produced by KDN or sev-Ras1V12 suggests that this gene is normally required for photoreceptor differentiation. This conclusion is supported by examination of the mild rough eyes of a hypomorphic allele. Analysis of apical eye sections of hypomorphic allele homozygotes reveals the absence of R7 photoreceptors in more than 80% of the ommatidia, with roughly 15% of the ommatidia also missing an outer photoreceptor. This phenotype is similar to that observed for hypomorphic alleles of several components of the Ras pathway. Because R7 and outer photoreceptor cell differentiation depend on Sevenless and Epidermal growth factor, respectively, this observation suggests that cnk functions downstream of both receptors (Therrien, 1998).

In Drosophila, components of the RAS pathway are generally required for cell proliferation and/or survival. To test whether cnk is similarly required, large clones of cnk homozygous mutant tissue were generated in the eye. cnk appears to be required for cell proliferation and/or survival, since mutation of this gene prevents normal eye development in a similar manner as is observed when loss-of-function mutations in Ras1 or ksr are tested. Evidence is found indicating that cnk is required for RAS-dependent signaling pathways outside the eye. For example, like other components of this pathway, cnk loss-of-function alleles are synthetic lethal with a hypomorphic allele of Raf, RafHM7. Induction of cnk mutant clones in the wing prevents wing vein formation. Wing vein formation is controlled by Egfr, indicating a role for cnk in other RAS-dependent pathways (Therrien, 1998).

To identify a putative site of action for Cnk, its localization within the cell was determined. The protein is mainly confined to the apical portions of cells where adherens junctions are found. In addition, analysis of several focal planes reveals a significant membrane localization throughout the cells, but no cytoplasmic or nuclear staining. The apical localization of Cnk is similar to that of Sevenless and the two adaptor molecules Drk and Dos. In cultured cells, Cnk is found in the cytoplasm, but, interestingly, it also accumulates in the membrane at cell-cell contact points. Since Cnk and Sevenless appear to colocalize in the eye disc and the Cnk amino acid sequence contains several SH2 domain consensus binding sites, it was asked whether Cnk could be tyrosine phosphorylated in a Sevenless-dependent manner. Constitutively active Sevenless induces tyrosine phosphorylation of Cnk. Cnk is also a target for Egf-dependent tyrosine phosphorylation in cultured cells. These results do not distinguish whether these RTKs directly phosphorylate Cnk or induce another tyrosine kinase, which in turn phosphorylates Cnk (Therrien, 1998).

To determine if an increase in wild-type Cnk level could transform cone cells into R7 cells, the effect of overexpressing wild-type Cnk was examined in the developing eye. Adult flies overexpressing Cnk have a mild rough eye phenotype. Apical sections of these eyes reveal missing R7 and outer photoreceptor cells, indicating that when overexpressed in the eye, wild-type CNK has a mild dominant-negative effect. This effect might be explained by the titration of one or more CNK-interacting proteins that are normally required for endogenous Ras signaling. The absence of extra R7 cells indicates that overexpression of Cnk is not sufficient to activate the Ras pathway. Cnk overexpression massively enhances an activated Ras1 phenotype. This result indicates that CNK cooperates with activated RAS1 and suggests that CNK function is RAS dependent. Cnk is shown to physically interact with the C-terminal kinase domain of Raf and appears to localize to cell-cell contact regions. Together, these findings suggest that Cnk is a novel component of a Ras-dependent signaling pathway that regulates Raf function and/or targets Raf to a specific subcellular compartment upon Ras activation (Therrien, 1998).

A misexpression screen identifies genes that can modulate RAS1 pathway signaling in Drosophila melanogaster

Differentiation of the R7 photoreceptor cell is dependent on the Sevenless receptor tyrosine kinase, which activates the Ras1/mitogen-activated protein kinase signaling cascade. Kinase suppressor of Ras (Ksr) functions genetically downstream of Ras1 in this signal transduction cascade. Expression of dominant-negative Ksr (KDN) in the developing eye blocks Ras pathway signaling, prevents R7 cell differentiation, and causes a rough eye phenotype. To identify genes that modulate Ras signaling, a screen was carried out for genes that alter Ras1/Ksr signaling efficiency when misexpressed. In this screen, three known genes, Lk6, misshapen, and Akap200, were recovered. Seven previously undescribed genes were also recovered; one encodes a novel rel domain member of the NFAT family, and six encode novel proteins. These genes may represent new components of the RAS pathway or components of other signaling pathways that can modulate signaling by RAS (Huang, 2000).

One of the misexpression interactors, MESR2, was an insertion upstream of the Akap200 locus. DAKAP200 refers to Drosophila A kinase anchor protein of molecular weight 200 kd, and binds the regulatory II (rII) subunit of cyclic AMP-dependent protein kinase (PKA). The Akap200 gene produces two different transcripts, one that contains the binding site for RII and one where the exon encoding for the RII binding site is spliced out to generate a protein that does not interact directly with PKA. Both isoforms of AKAP200 are expressed at relatively similar levels throughout development as well as in adult heads (Huang, 2000).

PKA is the principal mediator of signals that activate adenylate cyclase. cAMP signals are often targeted to effectors that accumulate to discrete intracellular locations. This targeting is due to a nonuniform distribution of PKA molecules within cells. In Drosophila, PKA has been implicated in normal developmental events in all imaginal tissues through the Hedgehog signaling pathway and is involved in signaling pathways that generate cell polarity: this requires that Hh be localized to distinct intracellular locations. Subcellular localization of PKA occurs through association with AKAPs. AKAPs are a functionally related family of proteins, defined by their ability to associate with PKA. Each AKAP contains a unique targeting domain that directs the complex to a defined intracellular location where PKA is placed proximal to both a signal generator (adenylate cyclase) as well as to potential PKA effector molecules. Coordinate binding of specific combinations of enzymes can allow such complexes to respond to distinct second messenger-mediated signals (Huang, 2000).

Studies in mammalian cells have suggested that PKA signaling via Rap1, another small molecular weight GTP-binding protein, antagonizes RAS1 signaling by competing for RAS pathway components such as B-Raf and MAPK. However, more recent studies suggest no genetic interaction between Drosophila Rap1 and RAS1. In Drosophila, overexpression of Rap1 in a heterozygous RAS1 mutant background has no effect on photoreceptor determination, suggesting no interaction between the two gene functions. A heterozygous Rap1 mutation does not reduce the number of R7 cells in a sev-RAS1V12 rough eye, also suggesting that the two pathways do not interact. Although there is no direct evidence linking PKA activation to MAPK activation via Rap1, there may be a still unknown pathway by which these molecules can signal (Huang, 2000).

The screen isolated Akap200 as a misexpression enhancer of KDN and suppressor of RAS1V12. This suggests that overexpression of this AKAP decreases signaling through RAS1. Overexpression of an AKAP might cause mislocalization of PKA molecules to the plasma membrane. This could activate a signaling pathway that normally is not utilized in this cell or at this time in development. If PKA and Rap1 are involved in RAS signaling, why were they not uncovered in previous loss-of-function screens? One possibility is that mutations in either gene may not be dose sensitive and therefore be unable to dominantly modify a rough eye phenotype. Another is the possibility that overexpression of an AKAP causes abnormal targets of PKA to become activated. Whether PKA signals through Rap1 is still unclear; however, the reported effects of attenuating RAS1/MAPK signaling are supported by this study. The enhancement of the KDN rough eye phenotype could be due to the additive effects of inefficient signaling due to KDN as well as the attenuation of MAPK by mislocalized PKA. In the activated RAS1V12 background, the attenuating effects of activated PKA due to mislocalization to the plasma membrane might reduce the amount of signaling through the pathway to suppress the RAS1-dependent rough eye phenotype (Huang, 2000).

Overexpression of msn in an sE-KDN background enhances the rough eye phenotype. msn encodes the Drosophila homolog of Nck interacting kinase (NIK), a member of the mammalian SPS1 subfamily of the STE20 kinase family. msn is an essential gene involved in dorsal closure during embryogenesis in Drosophila and mutant clones result in misshapen rhabdomeres (due to defects in polarity, malformed and missing photoreceptors) in the adult eye. STE20, the founding member of the family in yeast, acts in the pheromone signaling pathway and activates the yeast MAPK module. However, the upstream activators of the STE20 pathway are not known. msn, the STE20 homolog in Drosophila, acts upstream of the c-Jun amino-terminal kinase (JNK) MAPK cascade required for dorsal closure and has recently been implicated to act downstream of the Frizzled receptor in the epithelial planar polarity pathway. Published results have suggested that msn, when overexpressed in the eye in an otherwise wild-type background, can generate a rough eye. However, in the experiments described here, no effect on eye morphology was found using sE-GAL4 to drive either UAS-msn or EP(3)0609 and EP(3)0549, the two EP lines upstream of the msn gene. Drosophila Jun has been implicated as a downstream target of both the JNK MAPK and RAS1/MAPK signal transduction pathways by overexpression analysis of dominant-negative mutations; however, no other components of either pathway are shared. If the JNK pathway can partially compensate for the RAS1 pathway in eye development, one would expect that msn overexpression would suppress sE-KDN. The overexpression results from this screen suggest that as a misexpression suppressor of RAS1, msn decreases signaling in the pathway. msn may independently inhibit neuronal cell fate, although there is no previous evidence for this. It is possible that the JNK signaling pathway may compete with the RAS pathway for common components. Alternatively, this interaction may be tissue specific and only uncovered in the eye with overexpression. There is also the possibility that misexpression of msn causes promiscuous signaling through an independent pathway that also affects eye morphology. Although no phenotype is seen when msn is overexpressed in the eye, this situation may sensitize the eye and nonspecifically enhance the sE-KDN phenotype (Huang, 2000 and references therein).

Another misexpression interactor, MESR8, corresponds to two independent insertions upstream of the Lk6 gene. The Lk6 gene was originally identified biochemically as a microtubule-binding protein. LK6 localizes to centrosomes in the early blastoderm embryo and appears to be expressed ubiquitously throughout development. Loss-of-function mutations in Lk6 are viable and have no visible adult phenotype. Constitutive overexpression of Lk6 under the ubiquitin promoter causes microtubule defects in both eggs and embryos; these defects include fragmented mitotic spindles, abnormal asters, and abnormal metaphases, suggesting that LK6 may stabilize microtubules in vivo. Overexpression of phenotypes in the embryo suggests the same. Overexpression of Lk6 does not appear to affect imaginal tissues. The identification of a cytoskeleton-associated kinase that can genetically interact with the RAS pathway is consistent with studies in mammalian cells that indicate that the activation of the RAS/MAPK pathway can lead to spindle instability. Activated RAS induces chromosome aberrations such as dicentrics, acentrics, and double minutes. Genomic instability as measured by micronucleus formation increases tenfold in cells expressing activated RAS. Micronucleus formation induced by activated MAPK is due largely to disruption of the mitotic spindle rather than double-strand chromosome breaks (Huang, 2000 and references therein).

In the experiments described here, the overexpression of Lk6 in the developing eye has no visible phenotype. However, when overexpressed in the sev-RAS1V12 background, Lk6 is able to partially suppress the associated rough eye by reducing the number of supernumerary R7 cells. Since the supernumerary R7 cells in the sev-RASV12 background are generated postmitotically by cone cell to R7 transformations, in this instance LK6 may be playing a role in differentiation rather than proliferation. LK6 may be redundant with other kinases expressed in the eye, which normally act at other stages of development, or activate normally inactive signaling pathways when overexpressed. It is possible that expressing LK6 in the sev-RASV12 background could have a deleterious effect on cell differentiation and cause the improperly fated photoreceptors to die (Huang, 2000).

The MESR1 interactor identified in this screen is an insertion upstream of a novel gene that encodes a protein containing a rel domain. Sequence comparison of the rel domain with other known proteins of this class identifies it as a distant member of the NFAT family of transcription factors with mammalian NFAT5 its closest relative. NFAT1-4 are highly regulatable transcription factors that are sequestered in the cytoplasm of resting cells of the mammalian immune system. Upon activation of Calcineurin by intracellular calcium release, NFAT1-4 translocate to the nucleus where they bind cooperatively to DNA with the AP-1 heterodimer, FOS and JUN. The mammalian NFAT5 protein is distantly related to the others in sequence homology as well as function. NFAT5 appears to be constitutively nuclear, does not seem to interact with FOS and JUN, and does not transactivate well-characterized NFAT1-4 target promoters; its function and specificity remain unclear. Overexpression of this gene in the eye suppresses an activated RAS1 rough eye, implying that it acts to decrease RAS signaling efficiency. This protein may act to promote a nonneuronal cell fate or actively repress neuronal differentiation. There are four putative MAPK phosphorylation sites in the Drosophila NFAT5 homolog but it is unknown whether any of these sites are phosphorylated in vivo. No loss-of-function mutations have been identified to date. This protein may bind to RAS-dependent promoters; identification of target genes will help elucidate the wild-type function of this gene in flies (Huang, 2000).

Bimodal regulation of RAF by CNK in Drosophila

Connector enhancer of KSR (CNK) is a multidomain-containing protein previously identified as a positive regulator of the RAS/MAPK pathway in Drosophila. Using transfection experiments and an RNAi-based rescue assay in Drosophila S2 cells, it has been demonstrated that CNK has antagonistic properties with respect to RAF activity. CNK's N-terminal region contains two domains (SAM and CRIC) that are essential for RAF function. Unexpectedly, the C-terminal region of CNK contains a short bipartite element that strongly inhibits RAF catalytic function. Interestingly, CNK's opposite properties appear to prevent signaling leakage from RAF to MEK in the absence of upstream signals, but then transforms into a potent RAF activator upon signal activation. Together, these findings suggest that CNK not only participates in the elusive RAF activation process, but might also contribute to the switch-like behavior of the MAPK module (Douziech, 2003).

Three lines of evidence support the claim that CNK is essential for RAF activity. (1) Depletion of endogenous CNK prevents MAPK activation by RASV12, but not by activated RAF. (2) NT-CNK (deleted of the C-terminal repressive function) cooperates with RASV12 to activate MAPK (or MEK), but not with activated RAF. This result not only places CNK's positive effect between RAS and RAF, but it also suggests that this activity is RAS-dependent. (3) NT-CNK rescues MAPK activation by RASV12G37. This finding is striking because it provides strong evidence that CNK function is intimately linked to the RAF activation mechanism. As for mammalian RAS, Drosophila RAS G37 or C40 effector mutants no longer interact with Drosophila RAF. Since only the G37 mutant is rescued by NT-CNK co-expression (a RAF-dependent event), it suggests that the mutant is either a weak loss-of-function with respect to RAF binding and/or that it retains another essential function that has been lost by the C40 mutant. The G37 mutant may thus prove useful to elucidate the role of CNK in RAF activation (Douziech, 2003).

CNK's inhibitory function has been mapped to a 30 amino acid region named the RIR. This region comprises at least two distinct, but co-required negative elements: the RIM and the IS elements that function together as an inhibitory unit. Although its mechanism of action is unknown, the RIR appears to block signal transmission from RAF to MEK through an association between the RIM and the RAF catalytic domain. Indeed, an isolated RAF catalytic domain or the Torso-RAF catalytic domain fusion protein (Tor4021-RAFc) have been found to associate with CNK. The association appears to be direct since it is detectable using a yeast two-hybrid interaction assay. The role of the IS element is unknown. It is not required for RAF binding, but it is essential for the inhibitory effect of the RIR. Interestingly, since only a catalytically competent RAF kinase domain associates with CNK, it is possible that the RIR works as a RAF pseudosubstrate to control MEK phosphorylation (Douziech, 2003).

What could be the purpose of CNK's bimodal effect? Several scenarios can be envisioned to explain the data and two of these are presented here. In quiescent cells, CNK could function together with 14-3-3 in preventing signal-independent MEK activation by RAF. This negative role might be important to ensure that no signal leaks through prior to genuine upstream activation, which otherwise might be sufficient to initiate a biological response. Upon RAS activation, CNK's N-terminal domains would then integrate RAS signals and thus convert CNK into a positive regulator of signal transmission. Since CNK's opposite action appears to augment the signal-to-noise ratio of the RAS/MAPK module, it might contribute to switch-like activation of the pathway. Alternatively, CNK's negative effect might have a similar role to RKIP, a RAF inhibitor. Namely, it might work as a rheostat to finely adjust the amount of MEK molecules activated by RAF to satisfy cell-specific requirements. For that matter, it will be interesting to determine whether the RIR is itself negatively regulated to increase signaling flow from RAF to MEK. This possibility is appealing given the large difference between the ability of WT CNK and RIR-inactivated CNK constructs to rescue the MAPK activation defect (Douziech, 2003).

CNK homologs are present in other metazoans. This evolutionary conservation strongly suggests that ERK/MAPK modules in other species are also regulated by a CNK activity at the level of RAF. Although the SAM and CRIC domains are relatively well conserved, the sequence corresponding to the RIR seems to be unique to Drosophila and Anopheles. Nonetheless, rat CNK2/maguin isoforms (CNK's closest homologs) have been shown to associate with c-RAF, which suggests that they contain a region functionally similar to the RIR. If that were the case, it would be important to verify whether mutations disrupting its presumed negative function have any oncogenic properties. In addition, given the significance the RAS/MAPK module plays in tumor formation in humans, the identification of a short inhibitory peptide against RAF catalytic function might open new avenues for anticancer drug development (Douziech, 2003).

Drosophila Src42 binding activity regulates RAF by a novel CNK-dependent derepression mechanism

Connector enhancer of KSR (CNK), an essential component of Drosophila receptor tyrosine kinase/mitogen-activated protein kinase pathways, negatively regulates RAF function. This bimodal property depends on the N-terminal region of CNK, which integrates RAS activity to stimulate RAF and a bipartite element, called the RAF-inhibitory region (RIR), which binds and inhibits RAF catalytic activity. The repressive effect of the RIR is counteracted by the ability of Src42 to associate, in an RTK-dependent manner, with a conserved region located immediately C-terminal to the RIR. Strikingly, several cnk loss-of-function alleles have mutations clustered in this area and provide evidence that these mutations impair Src42 binding. Surprisingly, the derepressing effect of Src42 does not appear to involve its catalytic function, but critically depends on the ability of its SH3 and SH2 domains to associate with CNK. Together, these findings suggest that the integration of RTK-induced RAS and Src42 signals by CNK as a two-component input is essential for RAF activation in Drosophila (Laberge, 2005).

RTK-induced activation of the small GTPase RAS was recognized early on as a critical event in RAF activation. RAS triggers plasma membrane anchoring of RAF through a direct contact between GTP-loaded RAS and RAF. However, this step is insufficient to induce RAF activation, but is a prerequisite for a complex series of regulatory events. For example, Ste20-like kinases and Src family kinases (SFKs) have been shown to collaborate with RAS in RTK-induced Raf-1 activation, owing to their ability to directly phosphorylate Raf-1 serine 338 (S338) and tyrosine 341 (Y341), respectively. However, these particular events are probably specific to Raf-1 as the equivalent S338 residue in B-RAF is constitutively phosphorylated, whereas the Y341-like residue is not conserved in B-RAF or in Drosophila and C. elegans RAF. Nonetheless, it remains possible that these kinases use different means to regulate RAF members. This would be consistent with genetic findings in Drosophila, which suggest that RAF is also regulated by an RTK-induced but RAS-independent pathway linked to SFKs (Laberge, 2005).

In addition to kinases and phosphatases regulating RAF activity, a number of apparently nonenzymatic proteins also modulate RAF function. One of these is Connector eNhancer of KSR (CNK), an evolutionarily conserved multidomain-containing protein originally identified in a KSR-dependent genetic screen in Drosophila. Genetic experiments in flies have indicated that CNK activity is required downstream of RAS, but upstream of RAF, thus suggesting that CNK regulates RAF activity. In agreement with this interpretation, CNK was found to interact directly with the catalytic domain of RAF and to modulate its function. The role of CNK, however, is probably not restricted to the MAPK pathway. Indeed, although mammalian CNK proteins have also been found to modulate the RAS/MAPK pathway, recent studies have indicated that they also control other events, including membrane/cytoskeletal rearrangement, Rho-mediated SRF transcriptional activity and RASSF1A-induced cell death. Given their ability to influence distinct signaling events, it is possible that CNK proteins act as signal integrators to orchestrate crosstalks between pathways (Laberge, 2005).

Intriguingly, although CNK activity is vital for RAS/MAPK signaling in Drosophila, it has opposite effects on RAF function. A structure/function analysis revealed that two domains (SAM and CRIC) located in the N-terminal region of CNK are integrating RAS signals, enabling RAF to phosphorylate MEK. However, the ability of CNK to associate with the RAF catalytic domain was mapped to a short bipartite element, named the RAF-inhibitory region (RIR), that strongly antagonizes MEK phosphorylation by RAF. Surprisingly, the RIR exerts its effect even in the presence of RAS signals, hence resulting in lower RAS-induced MAPK signaling output (Laberge, 2005).

The inhibitory effect of the RIR is relieved by an RTK-induced SFK signal. Specifically, a region located immediately C-terminal to the RIR including tyrosine 1163 (Y1163) is essential for CNK's positive function in vivo and for Sevenless (Sev) RTK-dependent MAPK activation. Upon SEV expression, one of the two SFKs found in Drosophila, Src42, associates and mediates (through the Y1163 region of CNK) RTK positive effects on the MAPK module. Remarkably, cnk loss-of-function mutations affecting the Y1163 region are fully compensated by inactivation of the RIR, thereby arguing that the Y1163 region is integrating the RTK-induced Src42 signal to counteract the RIR inhibitory function. Unexpectedly, genetic and molecular evidence has revealed that it is not Src42 catalytic function per se, but rather its binding capacity that is the key event in this process. Taken together, these results provide compelling evidence that CNK regulates RAF function by integrating both RAS and Src42 signals elicited by an RTK (Laberge, 2005).

This study shows that CNK integrates RAS and Src42 signals as a binary input, thereby allowing RAF to send signals to MEK. The RAS signal is received by the SAM and CRIC domains of CNK, which appears to enhance RAF catalytic function, whereas Src42 activity is integrated by the Y1163 region of CNK and seems to relieve the inhibitory effect that the RIR imposes on RAF's ability to phosphorylate MEK. Why would RAF activation depend on two distinct but corequired signals emitted by the same RTK? One possibility is that this requirement generates specificity downstream of an RTK. For example, only receptors that activate both RAS and Src42 would lead to activation of the MAPK module within discretely localized CNK complexes. Consequently, the combinatorial use of multifunctional signals might be a means to produce a specific output from generic signals (Laberge, 2005).

Intriguingly, despite the fact that the second Drosophila SFK, Src64, is naturally expressed in S2 cells, it does not act like Src42 in response to Sev, Egfr and InsR activation. Although the reason for this difference is not immediately clear, it was found that, unlike Tec29, overexpression of an Src64YF variant is nonetheless capable of associating with CNK and inducing its tyrosine phosphorylation. It is thus possible that Src64 fulfills a similar role to Src42, but downstream of other RTKs or in response to other types of stimuli and that difference in either their subcellular localization, requirement for specific cofactors or additional regulatory events account for their distinct behavior (Laberge, 2005).

The mechanism by which the binding of Src42 to CNK deactivates the RIR is currently unknown and a number of scenarios can be envisioned. For example, it might induce a conformational change that suppresses the inhibitory effect that the RIR imposes on RAF catalytic activity. Alternatively, it is possible that Src42 binding displaces an inhibitory protein interacting with CNK or facilitates the relocalization of a CNK/RAF complex to a subcellular compartment that is required for RAS-dependent RAF activation. However, it is not believed that this mechanism involves displacing CNK away from RAF since neither Sev expression nor Src42 depletion alters the CNK/RAF interaction (Laberge, 2005).

Although several questions are left unanswered regarding the Src42/CNK association, collectively, the data suggest a subtle binding mode reminiscent of the mammalian Src/FAK interaction. Indeed, it appears that CNK is phosphorylated on the Y1163 residue not by Src42 itself, but either by the receptor or by another kinase and that this step generates a high-affinity binding site for the SH2 domain of Src42 thereby triggering its recruitment. This event is presumably not sufficient for a stable association and/or derepression of the RIR, but also requires the binding of the SH3 domain to an unidentified sequence element within CNK. The engagement of the SH3 and SH2 domains of Src42 on CNK would not only relieve the RIR's inhibitory effect, but would also derepress Src42 autoinhibited configuration and possibly orient favorably Src42 to phosphorylate one or a few specific tyrosine residues on CNK. This scenario is certainly plausible considering that CNK has a total of 39 tyrosine residues. This would explain why depletion of endogenous Src42 led to a reduction, but not a complete elimination, of SEV-induced CNK tyrosine phosphorylation or why the Y1163F mutation impairs CNK phosphorylation mediated by Src42Y511F, since a disruption of the Src42/CNK association would prevent Src42 from phosphorylating the other sites. Although these Src42-dependent phosphorylated residues do not appear to play a role in activating the MAPK module, their concerted regulation suggests that CNK is coordinating signaling between the MAPK module and at least one other pathway (Laberge, 2005).

Leonardo and 14-3-3eta, 14-3-3 isoforms

The two Drosophila 14-3-3 isoforms, Leonardo and 14-3-3eta, have been shown to positively regulate Ras-mediated signaling in the development of the compound eye. 14-3-3eta was identified as a suppressor of activated Ras1 in eye development. Suppressor of Ras1 3-9 (SR3-9) alleles act as dominant suppressors of sev-Ras1, that is, of Ras1 expressed ectopically in the eye disc using a sevenless promoter, a treatment that produces a rough eye phenotype. The eyes of flies carrying SR3-9 are less rough than those of flies expressing sev-Ras1 (Ras1 expressed ectopically using a sevenless promoter, producing a rough eye phenotype). It is known that SR3-9 functions either downstream of or in parallel to Raf kinase, since SR3-9 dominantly suppresses the rough eye defect caused by an activated raf expression construct (Chang, 1997).

Leonardo exhibits similar involvement in the Raf/Ras pathway. Clones of mutant leonardo show a loss of photoreceptors. Ommatidia lack outer as well as inner photoreceptors. This phenotype is reminiscent of clones homozygous for Drosophila ras or raf hypomorphic alleles. When Leonardo antisense RNA is expressed in eye imaginal discs in postmitotic photoreceptors, a weakly penetrant but reproducible loss-of-photoreceptor phenotype results. Since the artificial activation of Raf rescues the nonviability caused by leonardo mutation and permits photoreceptor development, it has been concluded that leonardo acts downstream of Ras and upstream of Raf in the signaling pathway that controls cell proliferation in the Drosophila eye imaginal disc (Kockel, 1997a).

Canoe, a scaffolding protein

The molecular structure of Canoe suggests its direct association with Ras. It has significant homology with a mammalian Ras-binding protein AF-6 (Kuriyama, 1996), which as been cloned as a fusion partner of All-1, a protein involved in acute myeloid leukemias in humans and a homolog of Drosophila Trithorax. Cno and AF-6 share two putative Ras-binding domains (RA1 and RAs), located at the N-terminus, as well as a kinesin-like and a myosin-V-like domain, and a Discs large homologous region, (DHR, also known as the GLGF or PDA motif) (Matsuo, 1997 and Kuriyama, 1996). The direct interaction of Raf, phosphatidylinositol-3-OH kinase, Ral GDS and Rin1 with activated Ras has been demonstrated. There is no obvious homology among Ras-interacting interfaces of these proteins with either AF-6 or Canoe, indicating that activated Ras can recognize a variety of target interfaces. The recombinant N-terminal domain of AF-6 and Canoe specifically interact with GTP Ha-Ras. The known Ras target c-Raf-1 inhibits the interaction of AF-6 with GTP gamma Ha-Ras. The recombinant N-terminal domain of AF-6 and Canoe specifically interact with GTP Ha-Ras. The known Ras target c-Raf-1 inhibits the interaction of AF-6 with GTP gamma Ha-Ras (Kuriyama, 1996 and references)

Sieglitz, F., Matzat, T., Yuva-Adyemir, Y., Neuert, H., Altenhein, B. and Klambt, C. (2013). Antagonistic feedback loops involving rau and sprouty in the Drosophila eye control neuronal and glial differentiation. Sci Signal 6: ra96. PubMed ID: 24194583

Antagonistic feedback loops involving rau and sprouty in the Drosophila eye control neuronal and glial differentiation

During development, differentiation is often initiated by the activation of different receptor tyrosine kinases (RTKs), which results in the tightly regulated activation of cytoplasmic signaling cascades. In the differentiation of neurons and glia in the developing Drosophila eye, this study found that the proper intensity of RTK signaling downstream of fibroblast growth factor receptor (FGFR) or epidermal growth factor receptor require two mutually antagonistic feedback loops. A positive feedback loop was identified mediated by the Ras association (RA) domain-containing protein Rau (CG8965) that sustains Ras activity and counteracts the negative feedback loop mediated by Sprouty. Rau has two RA domains that together show a binding preference for GTP (guanosine 5'-triphosphate)-loaded (active) Ras. Rau homodimerizes and is found in large-molecular weight complexes. Deletion of rau in flies decreases the differentiation of retinal wrapping glia and induces a rough eye phenotype, similar to that seen in alterations of Ras signaling. Further, the expression of sprouty is repressed and that of rau is increased by the COUP transcription factor Seven-up in the presence of weak, but not constitutive, activation of FGFR. Together, these findings reveal another regulatory mechanism that controls the intensity of RTK signaling in the developing neural network in the Drosophila eye (Sieglitz, 2013).

During development, often single bursts of RTK activity suffice to direct important cellular decisions. In other cases, multiple rounds of RTK activation are required to trigger a certain reaction profile, and in yet other cases, such as in the developing eye imaginal disc, a sustained low level of EGFR activity is needed. This study identified the Drosophila RA domain containing protein Rau, which constitutes the first cell-autonomous positive feedback regulator acting on both EGFR- and FGFR-induced signaling. In the developing fly compound eye, it was found that sustained RTK activity is modulated through a positive feedback loop initiated by Rau, which is counterbalanced by the negative regulator Sprouty. The balance of these two regulatory mechanisms ensures the correct activity of EGFR- and FGFR-dependent signaling pathways in the developing eye (Sieglitz, 2013).

Within the RTK signaling pathway, different positive and negative feedback mechanisms have been identified. A prominent negative feedback mechanism is triggered by the secreted protein Argos. Argos expression is induced by RTK activation, and secreted Argos protein can sequester the activating ligand Spitz. In addition, intracellular proteins have been identified to exert a negative feedback function. Sprouty is the most prominent inhibitor of RTK activity and was shown in this study to act downstream of FGFR signaling as well as downstream of EGFR signaling. However, the precise point at which Sprouty intercepts RTK signaling is variable. In the developing fly eye, Sprouty acts upstream of Ras, whereas in the developing wing, Sprouty functions at the level of Raf. An additional negative feedback loop is mediated by the cell surface protein Kekkon, which is specific to EGFR signaling (Sieglitz, 2013).

Positive feedback loops are less frequent and may act through the transcriptional activation of genes that encode activating ligands. This is found in the ventral ectoderm of Drosophila embryos or in follicle epithelium, where the activity of the EGFR pathway is amplified by induction of the expression of its ligand Vein. In addition, the expression of rhomboid may be triggered, which subsequently facilitates the release of activating ligands. Together, these mechanisms ensure a paracrine-mediated amplification of the RTK signal and are thus likely not as effective in regulating RTK activity in single cells (Sieglitz, 2013).

Rau is a previously unidentified positive regulator of RTK signaling that acts within the cell. This study found that Rau function sustains both EGFR and FGFR signaling activity. Rau is a small 51-kD protein that harbors two RA domains, which are found in several RasGTP effectors such as guanine nucleotide–releasing factors. Pull-down experiments demonstrated that Rau preferentially binds GTP-loaded (activated) Ras. The Rau protein is characterized by two RA domains. Although both RA domains are able to bind Ras individually, single RA domains do not show any selectivity toward the GTP-bound form of Ras. Thus, the clustering of two RA domains promotes the selection of RasGTP. In agreement with this notion, it was found that Rau can form dimers or, possibly, multimers. In lysates from embryos, Rau is found in high–molecular weight protein complex, suggesting that it could interact with other components of the RTK signalosome. This way, RA domains are further clustered, and thus, GTP-loaded Ras may be sequestered. In addition, this may also contribute to the clustering of Raf, which is more active in a dimerized state. Moreover, it was recently shown that Ras signaling depends on the formation of nanoclusters at the membrane. This local aggregation may further promote interaction of Ras with Son of sevenless, which can trigger additional activation of the RTK signaling cascade. In addition, Rau harbors a class II PDZ-binding motif, suggesting that Rau can integrate further signals to modulate RTK signaling (Sieglitz, 2013).

The activity of the EGFR and the FGFR signaling cascades is conveyed in part through the transcription factor Pointed. Heterozygous loss of pointed significantly increases the rough eye phenotype evoked by loss of Rau function. Moreover, upon overexpression of the constitutively active PointedP1, rau expression was also increased. In line with this notion, CG8965/rau was also identified in a screen for receptor tyrosine signaling targets. Thus, the data suggest that Rau activation occurs after initial RTK stimulation through direct transcriptional activation through Pointed, which is similar to the activation of the secreted EGFR antagonist Argos (Sieglitz, 2013).

This study has dissected the role of Rau in differentiating glial cells of the fly retina. These glial cells are borne out of the optic stalk and need to migrate onto the eye imaginal disc where some of these cells differentiate into wrapping glial cells upon contacting axonal membranes. The development of these glial cells is under the control of FGFR signaling. Initially, low activity of FGFR signaling in these glial cells is permissive for expression of seven-up, which encodes an orphan nuclear receptor of the COUP-TF (COUP transcription factor) family that suppresses sprouty, but not rau, expression. Activation of Rau requires greater activity of FGFR, which is achieved only through interaction with axons. High activation of FGFR signaling also inhibits seven-up expression and thus relieves the negative regulation of sprouty. This negative regulation of COUP-TFII transcription factors by RTKs is also seen during photoreceptor development in the fly eye and appears to be conserved during evolution (Sieglitz, 2013).

In conclusion, the Rau/Sprouty signaling module provides effective means to sustain a short RTK activation pulse, for example, during cellular differentiation. It is proposed that Rau dimers or multimers assemble a scaffold that favors the recruitment of RasGTP, which then could more efficiently activate the MAPK cascade. Thus, ultimately, Rau may promote the formation of Raf dimers, which might confer robustness and increased signaling intensity. Future studies will reveal the precise conformations and complexes that enable Rau to modulate RTK signaling in fly development (Sieglitz, 2013).

Table of contents

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

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