InteractiveFly: GeneBrief

pole hole: Biological Overview | References


Gene name - pole hole

Synonyms - Raf

Cytological map position - 3A1-3A1

Function - signaling

Keywords - RAF/MEK/ERK pathway, eye, wing, terminal group, FGF signaling, oncogene

Symbol - phl

FlyBase ID: FBgn0003079

Genetic map position - X:2,227,994..2,237,904 [+]

Classification - serine/threonine-protein kinase

Cellular location - cytoplasmic



NCBI links: Precomputed BLAST | EntrezGene
BIOLOGICAL OVERVIEW

RAF is a critical effector of the small GTPase RAS in normal and malignant cells. Despite intense scrutiny, the mechanism regulating RAF (FlyBase name: Pole hole) activation remains partially understood. This study shows that the scaffold KSR (kinase suppressor of RAS), a RAF homolog known to assemble RAF/MEK/ERK complexes, induces RAF activation in Drosophila by a mechanism mediated by its kinase-like domain, but which is independent of its scaffolding property or putative kinase activity. Interestingly, it was found that KSR is recruited to RAF prior to signal activation by the RAF-binding protein CNK (connector enhancer of KSR) in association with a novel SAM (sterile α motif) domain-containing protein, named Hyphen (HYP; FlyBase name - Aveugle). Moreover, these data suggest that the interaction of KSR to CNK/HYP stimulates the RAS-dependent RAF-activating property of KSR. Together, these findings identify a novel protein complex that controls RAF activation and suggest that KSR does not only act as a scaffold for the MAPK (mitogen-activated protein kinase) module, but may also function as a RAF activator. By analogy to catalytically impaired, but conformationally active B-RAF oncogenic mutants, the possibility is discussed that KSR represents a natural allosteric inducer of RAF catalytic function (Douziech, 2006).

Signal transmission via the RAF/MEK/ERK pathway, also known as the mitogen-acitvated protein kinase (MAPK) module, is a central event triggered by the small GTPase RAS to regulate a number of basic cellular processes in metazoans, including cell proliferation, differentiation, and survival (Pearson, 2001). Unrestrained signaling through this pathway caused, for instance, by activating mutations in specific isoforms of either RAS or RAF, has been linked to several types of cancer in humans and, for some of these, at an impressively high frequency (Malumbres, 2003; Wellbrock, 2004). Because of potential benefits to human health, extensive efforts have been devoted to describe in molecular terms the signal transfer mechanism within the RAS/MAPK pathway. Despite significant progress, a number of events have proven particularly challenging. One notable example is the mechanism leading to the activation of the RAF serine/threonine kinase (Douziech, 2006).

Three RAF members have been identified in mammals (A-RAF, B-RAF, and C-RAF/Raf-1) and homologs are present in other metazoans, including Caenorhabditis elegans and Drosophila, where a single gene encoding a protein more closely related to B-RAF has been identified (Dhillon, 2002; Chong, 2003). RAF proteins comprise an N-terminal regulatory region, followed by a C-terminal catalytic domain. The N-terminal region includes a RAS-binding domain (RBD), a cysteine-rich domain (CRD), and an inhibitory 14–3–3-binding site encompassing Ser 259 (S259) in C-RAF. The binding of 14–3–3 to this latter site requires the phosphorylation of the S259-like residue in RAF proteins, which in turn mediates their cytoplasmic retention in unstimulated cells (Morrison, 1997). Upon receptor tyrosine kinase (RTK)-dependent activation, GTP-loaded RAS binds the RBD of RAF and facilitates the dephosphorylation of the S259-like residue, thereby releasing 14–3–3 and promoting the association of RAF to the membrane (Jaumot, 2001; Dhillon, 2002; Light, 2002). A number of phosphorylation events are then required to fully induce RAF catalytic activity (Chong, 2003). Although some are isozyme specific, two are probably common to all members and affect conserved serine/threonine residues (T599 and S602 in B-RAF) situated in the activation loop of the kinase domain (Zhang, 2000; amino acid numbering of B-RAF is according to Wellbrock, 2004). Mutational analyses as well as a recent crystallographic study of the B-RAF kinase domain strongly suggest that phosphorylation of these residues plays a critical role in the final stage of activation by destabilizing an inhibitory interaction that takes place between the P loop (subdomain I) and the DFG motif (subdomain VII)/activation loop of the kinase domain (Wan, 2004). The mechanism and kinase(s) leading to the phosphorylation of these residues are unknown (Douziech, 2006).

A number of scaffold proteins have also been suggested to regulate RAF activity (Kolch, 2000). However, their mode of action and functional interdependency is poorly understood. One example corresponds to the kinase suppressor of RAS (KSR) members, which are known to assemble RAF, MEK, and MAPK into functional complexes (Morrison, 2003). Interestingly, these proteins are structurally related to RAF, although they have some key differences. For instance, they do not contain an RBD, but comprise a conserved region called CA1 that was found in Drosophila to mediate an interaction between KSR and RAF (Roy, 2002). Further, they possess a kinase-like domain that constitutively binds MEK, but which appears to be devoid of kinase activity (Morrison, 2003). While the function of KSR as a scaffold of the MAPK module has been convincingly documented, genetic and biochemical characterization of the single Drosophila KSR isoform suggested that its activity is also required upstream of RAF (Therrien, 1995; Anselmo, 2002). This other role, however, has not been determined (Douziech, 2006).

Connector enhancer of KSR (CNK) is another scaffold protein acting as a putative regulator of RAF activity. As for KSR, its activity is essential for multiple RTK signaling events, where it appears to regulate the MAPK module at the level of RAF (Therrien, 1998). CNK homologs have been identified in other metazoans and evidence gathered in mammalian cell lines supports their participation in the regulation of B-RAF and C-RAF (Lanigan, 2003; Bumeister, 2004; Ziogas, 2005). A similar conclusion was also recently reached in C. elegans (Rocheleau, 2005). In flies, CNK associates directly with the catalytic domain of RAF through a short amino acid sequence called the RAF-interacting motif (RIM) and modulates RAF activity according to the RTK signaling status (Douziech, 2003; Laberge, 2005). In the absence of RTK signals, CNK-bound RAF is inhibited by a second motif adjacent to the RIM, called the inhibitory sequence (IS). In contrast, upon RTK activation, CNK integrates RAS and Src activity, which in turn leads to RAF activation. The ability of RAS to promote RAF activation was found to strictly depend on two domains: a sterile α motif (SAM) domain and the so-called conserved region in CNK (CRIC) located in the N-terminal region of CNK (Douziech, 2003). The molecular role of these domains is currently unknown. In contrast, the binding of a Src family kinase, Src42, to an RTK-dependent phospho-tyrosine residue (pY1163) located C-terminal to the IS motif appears to release the inhibitory effect that the IS motif imposes on RAF catalytic function (Douziech, 2006).

This study investigated the role of the SAM and CRIC domains of CNK during RAS-dependent RAF activation in Drosophila S2 cells. Strikingly, it was found that their activity is mediated by KSR and that KSR stimulates RAF catalytic function independently of its capacity to bridge RAF and MEK. This effect occurs at a step upstream of the activation loop phosphorylation, but downstream of the dephosphorylation of the S259-like residue, thus indicating that it regulates the final stage of RAF activation. While the catalytically devoid KSR kinase domain appears to be the primary effector of this event, CNK participates in at least two ways: (1) It assembles a KSR/RAF complex in vivo by interacting separately with the kinase domains of KSR and RAF through its SAM domain and RIM element, respectively, and (2) its CRIC region promotes CNK-bound KSR activity toward RAF in a RAS-dependent manner. Finally, It was found that the KSR/CNK interaction depends on a novel and evolutionarily conserved SAM domain-containing protein, Hyphen, whose presence is essential for RAS-induced signaling through the MAPK module at a step upstream of RAF. Together, this work unveils a network of interacting scaffolds that regulates the RAS-dependent catalytic function of RAF (Douziech, 2006).

Previously the ability of KSR to promote the formation of RAF/MEK complexes independently of RAS signals was demonstrated and it was proposed that this scaffolding effect is a key functional aspect of KSR (Roy, 2002). This study showed that KSR does not act alone to bring RAF and MEK together, but requires at least two other proteins, namely, CNK and HYP. Importantly, these data suggest that CNK/HYP-bound KSR activates RAF in a RAS-dependent manner and that this function occurs at a step regulating the activation loop of RAF. Given that Drosophila KSR does not appear to have intrinsic kinase activity, as mutagenesis of an essential residue for catalysis (i.e., K705M) still displays strong activity, it suggests that KSR does not phosphorylate the activation loop residues of RAF, and thus either another kinase is recruited to accomplish this task or RAF itself is executing it (Douziech, 2006).

Interestingly, CNK and HYP do not exhibit any positive activity unless KSR is present, while KSR overexpression can induce RAF-mediated MEK phosphorylation independently of RAS and CNK (Roy, 2002). It thus appears that KSR mediates the effect of RAS and CNK and that it can even bypass their requirement when expressed at sufficiently high levels as if it carries an intrinsic RAF-activating property that is unveiled when overexpressed along with wild-type RAF and MEK. Various models can be envisioned to explain the RAF-activating property of KSR. The one that is favored is based on the position in which KSR operates during this event and on the strong architectural and amino acid sequence homology between KSR and RAF members. A recent crystallographic study of the inactive B-RAF catalytic domain has uncovered an inhibitory interaction that takes place between the P loop and the DFG motif/activation loop (Wan, 2004). Structural analysis of this interaction strongly suggests that phosphorylation of the activation loop interferes with the interaction and thereby helps in switching and/or locking the DFG motif/activation loop into the active conformation. The importance of disrupting the inhibitory configuration is also strikingly suggested by the finding that up to 90% of a large number of B-RAF oncogenic mutations found in human melanomas affect a valine residue (V600) that stabilizes the inactive conformation (Davies, 2002; Wan, 2004). In fact, most of the other oncogenic B-RAF mutations recovered in melanoma cells could also be understood by their ability to disturb the inhibitory configuration. Surprisingly, some affected residues participating in catalysis, and hence, decreased intrinsic kinase activity. As these mutations were capable of elevating endogenous ERK activity by their ability to stimulate endogenous wild-type RAF proteins, it has been proposed that a catalytically impaired but conformationally derepressed RAF kinase domain transduces its effect to inactive RAF proteins, possibly via an allosteric process, and as a result promotes their catalytic activation. KSR may act through a similar mechanism. Its overexpression along with MEK and RAF may allow it to adopt a conformation that in turn disrupts the inhibited configuration of the RAF catalytic domain. This event would then position the activation loop of RAF in a suitable configuration for phosphorylation, which ultimately stabilizes the catalytically activated state. In physiological conditions, KSR may also operate via this process, but presumably in a regulated manner. For example, the conformation of the kinase domain of KSR might be controlled allosterically by the CNK/HYP complex in a RAS-dependent manner, which in turn induces an activating conformational change in the kinase domain of RAF. This scenario might explain why the RAF-ALAA mutant still responded to NT-CNK, as even if its activation state could not be stabilized by phosphorylation, its conformation might still be controllable allosterically, thereby resulting in detectable catalytic activity. Although not mutually exclusive, KSR may also work by bringing other RAF-activating proteins or sequestering inhibitory proteins from RAF. The identification of two mutations (KSRA696V–A703T and KSRR732H) that completely eliminate the RAF-activating property of KSR, but that do not affect its RAF/MEK scaffolding function, should prove valuable to ascertain this novel function biochemically and structurally (Douziech, 2006).

Collectively, this characterization of CNK’s functional elements/domains is providing novel insights as to how scaffold proteins can dynamically influence signaling within a given pathway. Indeed, it appears that prior to signal activation, the CNK/HYP pair juxtaposes a KSR/MEK complex to RAF and, owing to the IS of CNK, maintains this higher-order complex in an inactive state by selectively repressing RAF catalytic function (Douziech, 2003). Then, upon signal activation, CNK integrates two RTK-elicited signals that together leads to RAF activation. First, RTK-induced phosphorylation of the Y1163 residue of CNK allows the binding of Src42, which in turn releases the inhibitory effect of the IS motif (Laberge, 2005). Second, RTK-induced RAS activity not only acts through the RBD of RAF, but also via the SAM–CRIC region of CNK (Douziech, 2003), thereby enabling KSR to activate RAF. How the N-terminal domains of CNK integrate RAS activity is currently unknown. One possibility is that the SAM domain, in association with HYP, merely acts as a binding interface for KSR, while the CRIC region is the one that perceives RAS activity and communicates it to KSR. It is also conceivable that RAS sends signals to KSR independently of CNK, and as a result, allows KSR to respond to NT-CNK (Douziech, 2006).

In summary, this study has identified CNK as a molecular platform coordinating the assembly and activity of a RAF-activating complex and has unexpectedly found that KSR, which is recruited to CNK-bound RAF by the novel protein HYP, is a central component of the RAF activation process. Regardless of the exact mechanism used by KSR to drive RAF activation, it is likely that a similar functional interaction between the kinase domains of KSR and RAF has been conserved during evolution and, in fact, might be a basic feature governing RAF activation across metazoans (Douziech, 2006).

The novel SAM domain protein Aveugle is required for Raf activation in the Drosophila EGF receptor signaling pathway

Activation of the Raf kinase by GTP-bound Ras is a poorly understood step in receptor tyrosine kinase signaling pathways. One such pathway, the epidermal growth factor receptor (EGFR) pathway, is critical for cell differentiation, survival, and cell cycle regulation in many systems, including the Drosophila eye. A mutation in a novel gene, aveugle, has bee identified based on its requirement for normal photoreceptor differentiation. The phenotypes of aveugle mutant cells in the eye and wing imaginal discs resemble those caused by reduction of EGFR pathway function. aveugle is required between ras and raf for EGFR signaling in the eye and for mitogen-activated protein kinase phosphorylation in cell culture. aveugle encodes a small protein with a sterile motif (SAM) domain that can physically interact with the scaffold protein connector enhancer of Ksr (Cnk). It is proposed that Aveugle acts together with Cnk to promote Raf activation, perhaps by recruiting an activating kinase (Roignant, 2006).

Key steps in Raf activation include Raf translocation to the plasma membrane and release of its protein kinase domain from an intramolecular inhibitory domain through changes in the phosphorylation state of specific residues. These processes occur in the context of the essential scaffolding proteins Cnk and Ksr. Ave is required between Ras and Raf for EGFR signaling in differentiating photoreceptors and in S2 cells, and is present in the same complex as Cnk. Loss of ave in the eye disc disrupts normal photoreceptor differentiation; while R8 cells differentiate correctly, most of the other photoreceptors are missing. Although the mutation isolated is likely to be a null allele of ave, its phenotype is weaker than loss of function of core components of the EGFR pathway, including cnk. R8 is still able to recruit a few photoreceptors in the absence of ave, and only a small proportion of ave mutant cells die during the third larval instar. The reduced expression in ave mutant cells of PntP1, a direct target of the pathway, suggests that ave is required to increase the overall level of EGFR signaling. It is noted that MAPK phosphorylation is undetectable in the absence of ave in both eye disc cells and S2 cells, suggesting that examination of EGFR responses in vivo is more sensitive than detection of phospho-MAPK (Roignant, 2006).

If loss of ave simply reduces the level of EGFR signaling, it would imply that distinct thresholds of EGFR signaling recruit different subclasses of ommatidial cells, since ave has a stronger effect on recruitment of R1, R6, and cone cells than on R2–R5. The dependence of many different ommatidial cell fates on EGFR signaling has been taken to imply that the response of an undifferentiated cell to the EGFR signal changes over time. This change in cellular competence may be due to changes in transcription factor expression in signal-receiving cells. The intermediate phenotype of ave mutants suggests that specification of early differentiating photoreceptors such as R3 and R4 requires a lower level of EGFR signaling than specification of later differentiating cells such as R1, R6, and cone cells. Interestingly, phosphorylated MAPK levels are lower in the region of the eye disc in which R2–R5 differentiate than in more posterior regions. In addition, R7 differentiation has been shown to require both EGFR and Sevenless to signal through the Ras/MAPK module, suggesting that an elevated amount of signal is required for its specification. An alternative means of temporal control is the induction by EGFR activity of signaling molecules required to recruit later cell types; for instance, EGFR recruits cone cells in part by activating expression in photoreceptors of the Notch ligand Delta. ave might be required for the expression of specific EGFR target genes such as Delta that promote sequential induction of late-differentiating cell types (Roignant, 2006).

In addition to photoreceptor differentiation, EGFR signaling in the eye is required for cell survival and cell cycle arrest; these two functions have been proposed to require a lower level of EGFR activity than differentiation of R1–R7. The results support this conclusion, since it was found that some ave mutant cells that do not differentiate as photoreceptors are still able to arrest in G1. However, an increase was found in apoptosis in ave mutant clones, despite their ability to differentiate some photoreceptors in addition to R8. This result suggests that there may not be a sharp threshold between the differentiation and survival responses; the level of EGFR signaling achieved in the absence of ave can allow differentiation of some photoreceptors without preventing all apoptosis (Roignant, 2006).

The requirement for Ave in other EGFR-dependent processes appears to be variable. In the wing disc, ave is essential for notum growth and for expression of the EGFR target gene aos; aos is likely to be a high-threshold target, as it is expressed in cells containing high levels of phosphorylated MAPK. However, ave is not required for all signaling by EGFR or the RTK Torso during embryogenesis. Embryos lacking both the maternal and zygotic contribution of ave did not show any detectable change in midline aos-lacZ or terminal tailless expression. As in the wing disc, aos is thought to be activated by high levels of EGFR signaling,due to its overlap with phospho-MAPK staining. ave might be redundant with another molecule expressed at this stage of development, although no close sequence homolog is present in the Drosophila genome. Alternatively, the Ras/MAPK module may use a distinct mechanism for signal transduction during embryogenesis. In this regard, it will be interesting to test whether cnk is required for EGFR signaling in the embryo (Roignant, 2006).

Genetic and biochemical studies have shown that the scaffolding protein Cnk is required for RTK signaling downstream of Ras but upstream of Raf (Therrien, 1998; Douziech, 2003). Its N-terminal SAM and CRIC domains are essential for its function in promoting Raf activity (Douziech, 2003). SAM domains frequently act as homo- or hetero-dimerization motifs. The SAM domains of Ave and Cnk can directly interact in yeast, suggesting that the essential function of the SAM domain of Cnk may be to interact with Ave (Roignant, 2006).

How might the interaction of Ave with Cnk promote Raf activation? Since Cnk binds to Raf through a C-terminal Raf-interacting motif (RIM) (Therrien, 1998), this binding is unlikely to require Ave. In addition, the RIM is dispensable for the transduction of Ras signaling and, in fact, seems to have an inhibitory effect on Ras signaling (Douziech, 2003). No change in the strength of the interaction between Raf and Cnk has been observed when ave is removed by RNAi. A more likely possibility is that association of Ave with Cnk helps to bring an activator kinase into proximity with Raf. Raf activation in mammalian cells involves dephosphorylation of inhibitory sites followed by phosphorylation of activating sites (for review, see Dhillon, 2002; Chong, 2003). However, the identity of the activating kinases is still unclear; Ksr was a candidate, but the current view is that it acts as a scaffolding protein rather than an active kinase (Morrison, 2001). In C. elegans, epistasis tests suggest that Cnk promotes Raf activation after dephosphorylation but before the activating phosphorylation events (Rocheleau, 2005), consistent with a model in which Cnk in association with Ave attracts an activator kinase to Raf. Certain SAM domains have been shown to act as kinase-docking sites; for example, the SAM domain of ETS-1 provides a docking site for the ERK-2 MAPK, promoting phosphorylation of and transcriptional activation by ETS-1 (Seidel, 2002). Likewise, the ETS-2 SAM domain serves as a docking site for the Cdc2 family kinase Cdk10 (Kasten, 2001). A search for other binding partners of Ave may lead to the identification of the activating kinase for Raf (Roignant, 2006).

An alternative possibility is that association of Ave with Cnk could help to recruit Raf to the plasma membrane. In S2 cells, Cnk is required for membrane recruitment of Raf (Anselmo, 2002), but it may not be sufficient for this function, since overexpression in CHO cells of MAGUIN-1, the closest mammalian homolog of Drosophila Cnk, does not recruit Raf-1 to the plasma membrane (Yao, 2000). The SAM domain of human p73 has been shown to directly bind lipid membranes (Barrera, 2003), suggesting the possibility that Ave links Cnk or Raf directly to the plasma membrane. However, no clear change was seen in the subcellular localization of tagged Cnk when Ave is knocked down by RNAi (Roignant, 2006).

Another well-described property of SAM domains is their ability to polymerize, promoting the formation of homo- or hetero-oligomers. This mechanism underlies long-range transcriptional repression by the SAM domain proteins TEL and Polyhomeotic. In the context of Raf activation, it is possible that polymerization of Ave, together with Cnk and perhaps other SAM domain-containing proteins, leads to the formation of large scaffolding complexes in which the local concentration of Raf and/or its activators is increased. Interestingly, the yeast adaptor protein Ste50, which is required for the activation of a MAPKKK, Ste11 (Ramezani-Rad, 2003), induces polymerization of Ste11 through interactions between the SAM domains of the two molecules (Bhattacharjya, 2005). This may stabilize a complex in which the Ste20 kinase can phosphorylate Ste11 (Ramezani-Rad, 2003). A stabilizing function might explain why ave is not essential in all contexts in Drosophila, as high concentrations of the molecules it recruits could lead to Ave-independent signaling. The evolutionary conservation of Ave suggests that it is likely to regulate the Ras/Raf/MAPK module in other species (Roignant, 2006).

Drosophila Raf's N terminus contains a novel conserved region and can contribute to torso RTK signaling

Drosophila Raf (DRaf) contains an extended N terminus, in addition to three conserved regions (CR1-CR3); however, the function(s) of this N-terminal segment remains elusive. In this study, a novel region within Draf's N terminus that is conserved in BRaf proteins of vertebrates was identified and termed conserved region N-terminal (CRN). The N-terminal segment can play a positive role(s) in the Torso receptor tyrosine kinase pathway in vivo, and its contribution to signaling appears to be dependent on the activity of Torso receptor, suggesting this N-terminal segment can function in signal transmission. Circular dichroism analysis indicates that DRaf's N terminus (amino acids 1-117) including CRN (amino acids 19-77) is folded in vitro and has a high content of helical secondary structure as predicted by proteomics tools. In yeast two-hybrid assays, stronger interactions between DRaf's Ras binding domain (RBD) and the small GTPase Ras1, as well as Rap1, were observed when CRN and RBD sequences were linked. Together, these studies suggest that DRaf's extended N terminus may assist in its association with the upstream activators (Ras1 and Rap1) through a CRN-mediated mechanism(s) in vivo (Ding, 2010).

Amino acids 19-77) within Draf's N terminus, conserved for Raf genes of most invertebrates and BRaf genes of vertebrates, was identified and termed CRN. This conserved region has not been described by others, but potential roles for the extended N terminus have been proposed in two reports. One found that in HeLa cells, the N terminus of BRaf may mediate Raf dimerization to generate BRaf-BRaf or BRaf-CRaf complexes, and play an important regulatory role in calcium-induced BRaf activation. Another study reported that deletion of BRaf's N terminus did not affect BRaf-CRaf dimer formation. Instead, it was found that N-terminal residues appeared to facilitate interaction with HRas in vitro. In accordance with the previous study, stronger interactions between DRaf's RBD (Ras binding domain) and the small GTPase Ras1δCAAX were observed when N-terminal and RBD sequences were linked in a yeast two-hybrid analysis. This suggested that the N terminus might assist in Ras1 binding. Furthermore, the identity of specific residues in the N terminus that might participate in Ras1 binding were mapped to the CRN region (amino acids 19-77). Two known Raf motifs, RBD and CRD, are involved in Raf's interaction with Ras. This studies, and previous results using BRaf, suggest that the N-terminal residues of DRaf and BRaf proteins, particularly the CRN region, might be another element that plays a role(s) in Ras-Raf coupling (Ding, 2010).

The small GTPase Rap shares with Ras nearly identical Raf binding regions that comprise switch 1 and the lipid moiety. Rap functions as an antagonist of Ras in regulating CRaf activity, but can activate BRaf in a parallel way with Ras. Isoform-specific features of different Raf family members may explain their distinct responses to Rap. In flies, both Ras1 and Rap1 can interact with and activate DRaf. Thus, it was reasonable to test whether DRaf's N terminus including CRN might also assist in Rap1 binding. In agreement with this idea, stronger interaction between RBD and Rap1δCAAX was observed when DRaf's CRN and RBD sequences were linked in vitro, further suggesting that the N terminus may contribute to both Ras1 and Rap1 binding potentially through a CRN-mediated mechanism(s) in vivo (Ding, 2010).

No direct interaction between Ras1 or Rap1 and the isolated DRaf N-terminal segment (amino acids 1-117) was detected, or when the N terminus was linked with the Ras1/Rap1 binding-deficient RBDR174L. Thus, the contribution of DRaf's N-terminal residues to Ras1 and Rap1 binding requires the presence of RBD. It is possible that the CRN-containing N terminus may assist in Raf-Ras interaction by making RBD more accessible to Ras1 and/or in a sequential manner, subsequent to RBD-Ras1 interaction, by stabilizing the RBD-Ras1 complex. Deletion of CRN may result in conformational or structural changes that reduce Ras1 binding affinity. Structural analysis of these complexes may provide important clues and help to understand the molecular mechanism(s) by which CRN assists in Ras-Raf interaction. The computational analysis suggested conserved CRN has the propensity to form two α-helical structures (α1 and α2) and contains a putative phosphorylation motif T-S-K located in α2. In agreement, DRaf's N terminus (amino acids 1-117) was folded in vitro and had a high content of helical secondary structure. These findings may help to establish a basis for future determination of molecular structure (Ding, 2010).

Although no verified binding partner(s) for DRaf or BRaf's N terminus has been identified, it is still possible that CRN may interact with other regulatory factors in vivo, that may affect Ras or Rap binding and/or function in activation of DRaf and BRaf. If so, the conserved structural features of CRN most likely relate to these regulatory events in vivo. Site-directed mutagenesis of conserved sites/motifs could provide useful information regarding the molecular mechanism(s) of CRN's role in the activation of DRaf and BRaf (Ding, 2010).

This in vitro study of DRaf's N terminus was initiated on the basis of in vivo findings using both loss- and gain-of-function genetic assays that deletion of N-terminal residues consistently reduces DRaf's signal potential in the Torso pathway. When expressed at high levels, FL DRaf enhanced the gain-of-function effects of the torRL3 allele much more significantly than DRafδN114. In embryos from trk-/- mothers, addition of FL DRaf, but not DRAFδN114, partially restored the A8 denticle belt structure. These findings indicate that the N terminus can play a positive role(s) in Torso RTK signaling. Interestingly, the contribution of DRaf's N terminus in the Torso pathway appeared to be dependent on upstream receptor activity, suggesting its role in transmission of the signal. Together with yeast two-hybrid data it is proposed that the presence of N-terminal residues may facilitate the association of DRaf with the upstream regulators Ras1 and Rap1, thereby assisting in transmission of the RTK signal in vivo (Ding, 2010).

For instance, in the trk- background, a small amount of active GTP-Ras1 and GTP-Rap1 are likely present, mostly due to activation by residual upstream Trunk activity, the presence of Torso-like ligand, and/or the intrinsic activity of the Torso receptor. The trk1 mutation used in this analysis results in protein truncation at the last 16 amino acids. It is possible that overexpression of FL DRaf proteins in this background increases the likelihood of interaction between abundant DRaf proteins and membrane bound GTP-Ras1 or GTP-Rap1. This in turn, could elevate the RTK signal and partially restore development of the A8 denticle belt structure in some embryos. In contrast, deletion of the N terminus could destabilize Ras1-DRaf (or Rap1-DRaf) coupling or decrease the duration of interaction, resulting in reduced DRaf signal transmission. This may explain why expression of DRafδN114 failed to rescue the A8 denticle belt in embryos from trk-/- mothers (Ding, 2010).

Previously, an auto-inhibitory role had been assigned to residues compromising the first half of the DRaf protein, in addition to their functions in promoting its activity. Deletion of the N-terminal amino acids 1-272 (including the N terminus and CR1) or 1-402 (including the N terminus, CR1, and CR2) of DRaf at least partially relieved these negative effects. In this study, although removal of the N-terminal 1-114 residues did not result in constitutive DRafδN114 activity in embryos lacking the maternal Torso receptor, it is still possible that the N terminus may contribute to auto-inhibitory effects. Together with CR1 and CR2, these N-terminal residues (1-114) may help maintain DRaf's inactive conformation. If so, the N terminus might play dual roles, both positively and negatively regulating DRaf. Therefore, its contribution to signaling may be neutralized by this auto-inhibition and consequently result in a subtle in vivo effect. If so, selective mutagenesis of the 'inhibitory' motifs/sites in the N-terminal region or removal of other cofactors involved in its negative regulation may amplify signaling differences between FL DRaf and DRafδN114. Ras binding has been thought crucial to recruit Raf to the membrane and promote its RTK signaling activity. However, the Drosophila Torso pathway appears tolerant of alterations in Ras1-DRaf coupling. Draf C110 has a R174L point mutation in the RBD domain and likely comprised for Ras1 binding. The RBDR174L is Ras binding deficient in the yeast two-hybrid assay. However, tll expression patterns and cuticles of the embryos derived from mothers with Draf C110/Draf C110 germ cells were indistinguishable from those of wild-type embryos, suggesting a mechanism(s) independent of RBD-Ras1 interaction might function in recruiting DRaf to the membrane. In agreement with this model, it has been found that membrane translocation of CRaf could be mediated by its interaction with phosphatidic acid (PA) and independent of Ras binding. This PA binding site is also conserved in ARaf, BRaf, and DRaf. Thus, DrafC110 could be recruited to the cell membranes by associating with PA. Moreover, it is known that Raf's CRD participates in Ras binding through its interaction with the lipid moiety of Ras. Once at the membrane, it is also possible that the interaction between DrafC110's CRD and Ras1 could further promote its membrane attachment and result in relatively normal Torso signal production. In this study, the presence of RBD, CRD, and the potential PA binding site may be sufficient to promote DRaf's activation in Torso signaling. This may explain why at approximately endogenous wild-type protein level maternally expressed DRafδN114 is able to rescue the embryonic terminal defects of Draf11-29 mutants. Together, considering the Torso pathway's tolerance of alterations in Ras1-DRaf coupling and the minor role DRaf's N terminus plays in Ras1 binding, it is reasonable that the phenotypic consequences of removing these N-terminal residues (DRafδN114) are not great in Torso signaling. The subtle phenotypic effects of DRaf's N terminus could also be due to compensation provided by potential autoregulatory feedback or alternative redundant processes in the in vivo system. In this study, the expression of DRaf proteins at a low level appeared to sensitize the assay system. It was found that deletion of the N terminus seemed to increase the threshold of DRaf protein levels required for normal signaling. Furthermore, by adding one copy of the ectopic torRL3 allele or removing wild-type maternal Trunk activity the sensitivity of the Torso pathway was apparently increased. These allowed the embryonic terminal system to display enhanced differences between FL DRaf and DRafδN114 proteins (Ding, 2010).

Why is this N terminus with its 'subtle' functional effects conserved during evolution, and what is its biological relevance? There are numerous RTK pathways functioning in Drosophila cellular and developmental processes. In spite of the identical Ras-Raf-MEK signal cassette they share, these RTK pathways can lead to different biological responses. Previous studies indicated that such specificity might be due to the difference in the intensity and/or duration of the signal. This suggested that the magnitude of Raf signal could function as a critical determinant of biological responses. Participation of multiple DRaf elements in Ras1 or Rap1 binding could be a good strategy to modulate its activity. Normally, tight association with Ras1 or Rap1 through RBD and CRD regions is required and sufficient to initiate the activation of DRaf, while minor adjustments/regulation of interaction by the CRN region could optimize signaling potential and reduce variability. Thus, the extended N terminus including CRN may play a role(s) as one element in a multidomain effort to promote DRaf's interaction with Ras1 and Rap1, participating and assisting in regulation to reliably attain maximal signal output (Ding, 2010).

CNK and HYP form a discrete dimer by their SAM domains to mediate RAF kinase signaling

Activation of RAF catalytic activity is facilitated by a regulatory complex comprising the proteins CNK (Connector enhancer of KSR), HYP (Hyphen), and KSR (Kinase Suppressor of Ras). The sterile alpha-motif (SAM) domain found in both CNK and HYP plays an essential role in complex formation. The x-ray crystal structure of the SAM domain of CNK in complex with the SAM domain of HYP was determined. The structure reveals a single-junction SAM domain dimer of 1:1 stoichiometry in which the binding mode is a variation of polymeric SAM domain interactions. Through in vitro and in vivo mutational analyses, it was shown that the specific mode of dimerization revealed by the crystal structure is essential for RAF signaling and facilitates the recruitment of KSR to form the CNK/HYP/KSR regulatory complex. Two docking-site models are presented to account for how SAM domain dimerization might influence the formation of a higher-order CNK/HYP/KSR complex (Rajakulendran, 2008)

Raf activation is regulated by tyrosine 510 phosphorylation in Drosophila

The proto-oncoprotein Raf is pivotal for mitogen-activated protein kinase (MAPK) signaling, and its aberrant activation has been implicated in multiple human cancers. However, the precise molecular mechanism of Raf activation, especially for B-Raf, remains unresolved. By genetic and biochemical studies, this study has demonstrated that phosphorylation of tyrosine 510 is essential for activation of Drosophila Raf (Draf), which is an ortholog of mammalian B-Raf. Y510 of Draf is phosphorylated by the c-src homolog Src64B. Acidic substitution of Y510 promotes and phenylalanine substitution impairs Draf activation without affecting its enzymatic activity, suggesting that Y510 plays a purely regulatory role. It was further shown that Y510 regulates Draf activation by affecting the autoinhibitory interaction between the N- and C-terminal fragments of the protein. Finally, it was shown that Src64B is required for Draf activation in several developmental processes. Together, these results suggest a novel mechanism of Raf activation via Src-mediated tyrosine phosphorylation. Since Y510 is a conserved residue in the kinase domain of all Raf proteins, this mechanism is likely evolutionarily conserved (Xia, 2008; full text of article).

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).

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).

KSR is a scaffold required for activation of the ERK/MAPK module

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 in Drosophila) by RAF. 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 characterization of KSR with respect to the ERK/MAPK pathway has been undertaken by a number of groups using mKSR1, a murine isoform. Like RAF, mKSR1 associates with HSP90 and p55/CDC37 as well as with 14-3-3 (see Drosophila Leonardo) proteins. Interestingly, mKSR1 has also been shown to interact constitutively with MEK and in a RAS-dependent manner with RAF and ERK/MAPK. These results led different groups to propose that mKSR1 might coordinate the assembly of the ERK/MAPK module. However, this hypothesis remains to be tested because the molecular relationship between mKSR1 and the three kinase components of the ERK/MAPK module is currently unknown. Furthermore, functional assays conducted by different groups have produced contradictory results: this results in a fragmented and somewhat controversial view of the role of KSR. A notable case focusses on the catalytic function of mKSR1. It has been reported that mKSR1 can phosphorylate and activate RAF in a TNFalpha- or EGF-dependent manner. Intriguingly, however, these results could not be reproduced by other laboratories. Instead, mKSR1 activity was reported to be independent of its putative catalytic function. Another discrepancy is the observation made by a number of groups that forced expression of mKSR1 strongly and specifically blocks signaling through the ERK/MAPK pathway, whereas others showed that mKSR1 strongly cooperated with activated RAS to induce meiotic maturation of Xenopus oocytes. This contradiction might be attributable to differences in mKSR1 expression levels. Given that mKSR1 interacts with several components of the ERK/MAPK module, it is possible that these components are sequestered from each other when mKSR1 levels are in excess. In any event, it remains unclear whether the information obtained using mKSR1 truly reflects the function of KSR as it had been genetically defined. It is possible that the experimental systems used could simply not support normal mKSR1 function. For example, it is intriguing that in the Xenopus oocyte maturation assay, mKSR1 activity mainly depended on its cysteine-rich motif, whereas several loss-of-function mutations affecting other parts of the KSR protein have been identified in Drosophila and C. elegans (Roy, 2002 and references therein).

A major problem hindering the elucidation of the role of KSR is the lack of an assay that faithfully recapitulates its function. A simple transfection protocol has now been used: a KSR-dependent functional assay has been reconstituted in a homologous system, that is, in Schneider (S2) cells using only Drosophila-derived components. KSR has shown to strongly promoted MEK phosphorylation by RAF in a RAS-dependent manner. Strikingly, KSR activity appears to depend mainly on its ability to associate independently with RAF and MEK, thereby allowing it to link the two kinases. Together, these findings suggest that KSR promotes signal propagation through the ERK/MAPK module by coordinating the assembly of a RAF/MEK complex (Roy, 2002).

KSR was originally recognized as an essential component of RAS-mediated signaling pathways in Drosophila and C. elegans. Although the characterization of mouse KSR1 linked it to the ERK module, its precise molecular function has not heretofore been elucidated. This study shows that Drosophila KSR is genuinely required for signal transmission through the MAPK pathway at a step upstream of MEK. In agreement with this, KSR directly participates in the phosphorylation of MEK by RAF and this event does not absolutely require the putative catalytic function of KSR. KSR associates independently with MEK and with RAF. These independent interactions lead to the formation of a stable RAF/MEK complex, thereby positioning the activator RAF in close proximity to its substrate MEK. Hence, KSR operationally behaves as a molecular scaffold that assembles the RAF/MEK functional pair. Given that MP1, a MEK binding partner that enhances enzymatic activation of the MAP kinase cascade, apparently coordinates the formation of MEK/ERK complexes, it will certainly be interesting to investigate whether KSR and MP1 function together to orchestrate the formation of a complete ERK/MAPK module (Roy, 2002).

Mouse KSR1 has been previously reported to associate constitutively with MEK and in a RAS-dependent manner with RAF. However, the physical consequences of these associations have not been explored further. This study shows that Drosophila KSR also interacts with MEK and RAF, but, in contrast to mKSR1, the Drosophila KSR/RAF association does not appear to depend on RAS activity. A reason for this difference might be that the association between mKSR1 and c-RAF does not resist the immunoprecipitation procedure. However, in the presence of activated RAS other contacts might have formed, which could stabilize the complex. RAS-induced oligomerization and/or other proteins might be involved in stabilizing the mKSR1/c-RAF complex. Regardless of the exact mechanism, these results clearly show that the associations between KSR and RAF and between KSR and MEK are mediated by independent parts of KSR and result in the association of RAF and MEK. Given that these data suggest that RAS is dispensable for these interactions, this implies that an inactive KSR/MEK/RAF ternary complex might exist in nonstimulated cells. Interestingly, STE5 has also been shown to assemble a three-kinase MAPK complex prior to signal transduction. This might be a general mechanism used by scaffolding proteins to allow highly efficient switch-like signal transmission. Consistent with this possibility, it was found that endogenous KSR, which apparently represents <1% of endogenous RAF and MEK protein levels, is predominantly associated with RAF and MEK in S2 cells (Roy, 2002).

The CA1 domain, a conserved region of KSR of ~40 amino acids at the N terminus, appears to be involved in connecting the KSR/MEK complex to RAF. It is still unclear whether or not the interaction is direct. Although the mutation affecting the CA1 domain reduces the KSR/RAF association, it does not abrogate it. This suggests that either the mutation does not fully disrupt the interaction with RAF or that another region(s) of KSR makes contact with RAF. This is consistent with the fact that this mutation allows to some extent the formation of a RAF/MEK association, albeit not as effectively as wild-type KSR and that it genetically behaves as a weak loss-of-function mutation (Therrien, 1995). Alternatively, it is possible that the mutation does not affect RAF binding per se, but aberrantly localizes the mutant KSR protein. Although MEK might stabilize the RAF/KSR interaction, it does not primarily mediate the interaction. Indeed, dsMEK RNA does not perturb the KSR/RAF association, and the two mutants, KSRD800A-D817A and KSRC922Y, which no longer interacted with MEK, still associated with RAF (Roy, 2002).

Systematic mutagenesis and deletion mutants of KSR should allow the identification of additional regions, if any, involved in the formation of the RAF/MEK complex. Given that mKSR1, like the RAF isozymes, interacts with the 14-3-3 proteins, HSP90, and p55/CDC37, it will be interesting to discover the contribution of these proteins in the KSR/RAF association and in the regulation of the KSR-dependent RAF/MEK complex (Roy, 2002).

Previous work in Drosophila has shown that ksr loss-of-function mutations suppresses activated RAS-mediated signaling, but does not alter activated RAF function (Therrien, 1995). These observations led to the proposal that KSR is required at a step between RAS and RAF or in a pathway that acts in parallel. However, the data were also consistent with the possibility that the activated RAF transgene was expressed to high levels, thereby bypassing the requirement for KSR function. The current results are consistent with the second interpretation. It was found that KSR promotes MEK phosphorylation when low amounts of the RAF construct are transfected, but that this effect declines upon increasing the amounts of transfected RAF. The genetic data also raise the possibility that KSR is involved in the RAS-dependent RAF activation mechanism. This latter process has been the subject of numerous studies, but remains largely enigmatic. A number of laboratories have tested the possibility that KSR directly phosphorylates RAF. Most of these attempts have failed to show catalytic function for KSR. The current data do not support the findings of a catalytic role for KSR, but may provide an explanation for the apparent contradictory evidence. Interestingly, there is a correlation between the ability of KSR to bind MEK and the appearance of a mobility shift in transfected RAF, which appears to be caused by phosphorylation. KSRD800A-D817A and KSRC922Y does not induce the mobility shift, whereas KSRK705M does. This suggests that this event does not depend on the putative catalytic function of KSR, but, rather on its ability to interact with MEK. Moreover, the mobility shift observed for FKBP-RAFc appears to depend on its autocatalytic function because FKBP-RAFcK498S does not display the mobility shift, even though it heterodimerizes with the FRB-KSRc/MEK complex upon rapamycin treatment. Together, these data suggest that RAF autophosphorylation is strongly stimulated when MEK is brought to RAF by KSR, and thus KSR might be involved indirectly in RAF activation, not by virtue of its catalytic function, but rather by the effect of recruiting MEK to RAF. Consistent with this, the only KSR-dependent phosphorylated residues in KSR that have been identified correspond to RAF autophosphorylation sites (Roy, 2002).

Surprisingly, the simple coexpression of KSR with MEK, RAF, and activated RAS, is sufficient to reconstitute a KSR-dependent assay. The reliability of the assay is supported by the observation that wild-type KSR behaves as a positive component of the pathway, as genetics predicted, and KSR mutations corresponding to previously identified ksr loss-of-function alleles are inert. The critical aspect of the assay is the simultaneous expression of RAF and MEK with KSR. When KSR is overexpressed in a cell line, RAF and MEK also need to be coexpressed to high enough levels to prevent their uncoupling on separate KSR molecules. A similar situation has been observed for JIP-1, which was first thought to be an inhibitor of the JNK pathway. The results thus provide an explanation as to why many laboratories found that KSR inhibits signaling through the ERK pathway when overexpressed in various cell lines. When expressed at very low levels, mKSR1 has been shown to accelerate RAS-dependent Xenopus oocyte meiotic maturation, a process that depends on activation of the ERK module. Intriguingly, mKSR1 activity in this system appears to be mediated mainly by the cysteine-rich motif (CRM). Although this region is probably required for normal KSR activity, it does not account for its entire function. Therefore, this assay probably recapitulates only partially the normal function of KSR. Biochemical analysis of the CRM has revealed that it is involved in targeting mKSR1 to the plasma membrane in a RAS-dependent manner. Nonetheless, it is unclear why the CRM alone is capable of stimulating RAS-dependent Xenopus oocyte maturation. KSRC398S-C401S is reproducibly less active than wild-type KSR in the S2 cell assay, thus confirming the functional relevance of the CRM. However, the effect is weak. This could be caused by the fact that the main participants were overexpressed, thereby making the system less dependent on signals normally concentrating the various components to the plasma membrane (Roy, 2002).

Intriguingly, besides the lysine to arginine change in subdomain II of the mammalian homologs, KSR proteins have a highly conserved kinase domain (Therrien, 1995). It is formally possible that this domain is enzymatically active, but the proper conditions and/or substrates to detect its activity have not been found. The observation that the two KSRK705M mutants used in this study are slightly less active than their wild-type counterparts, even though they associate with MEK to the same extent as wild-type KSR, suggests that KSR might possess a catalytic function that is required in concert with its scaffolding property for full activity. Alternatively, it is possible that their lesser activity is due to structural changes in KSR that do not perturb the KSR-MEK interaction but affect the way KSR presents MEK to RAF. Stringent sequence conservation might thus be required to maintain a particular kinase domain conformation to allow highly specific and robust interaction with MEK for the sole purpose of presenting a particular portion of MEK to RAF. Given their structural relatedness and the good homology between their kinase domains, RAF and KSR probably evolved from a common ancestral kinase by gene duplication. One of the descendents of this hypothetical duplication event might have given rise to the three RAF kinase family members, which retained catalytic function, whereas the other descendent might have eventually led to the two KSR genes found in mammals that evolved as scaffolds specialized in bridging RAF and MEK proteins together. Although it is currently unclear whether the functional shift observed for the KSR kinase domain will also be observed in other uncharacterized kinases for a similar purpose, this certainly highlights the importance of showing the catalytic activity of a kinase or any other enzyme before assuming it performs an enzymatic step in a given process (Roy, 2002).

It is becoming increasingly clear that components for several signaling pathways are specifically organized by scaffolding proteins. However, very little is known about the way they operate. The genetic and molecular data gathered so far on Drosophila KSR are consistent with its involvement in signaling efficiency, that is, when KSR is nonfunctional, low amounts of signal reach MAPK. The chain of events is, however, not severed because activated RAF can rescue ksr loss-of-function phenotypes in Drosophila. The results show a role for KSR as a molecular scaffold coordinating the RAF/MEK interaction. This is, however, one part of the signal propagation mechanism through the ERK/MAPK module. Once MEK is activated, it must relay the signal to MAPK. It will be interesting to determine whether KSR also participates in this process or whether another molecule, such as MP1, executes that step independently (Roy, 2002).

A dimerization-dependent mechanism drives RAF catalytic activation

The ERK (extracellular signal-regulated kinase) pathway is an evolutionarily conserved signal transduction module that controls cellular growth, differentiation and survival. Activation of receptor tyrosine kinases (RTKs) by the binding of growth factors initiates GTP loading of RAS, which triggers the initial steps in the activation of the ERK pathway by modulating RAF family kinase function. Once activated, RAF participates in a sequential cascade of phosphorylation events that activate MEK, and in turn ERK. Unbridled signalling through the ERK pathway caused by activating mutations in RTKs, RAS or RAF has been linked to several human cancers. Of note, one member of the RAF family, BRAF, is the most frequently mutated oncogene in the kinase superfamily. Not surprisingly, there has been a colossal effort to understand the underlying regulation of this family of kinases. In particular, the process by which the RAF kinase domain becomes activated towards its substrate MEK remains of topical interest. Using Drosophila Schneider S2 cells, this study demonstrates that RAF catalytic function is regulated in response to a specific mode of dimerization of its kinase domain, which is termed the side-to-side dimer. Moreover, the RAF-related pseudo-kinase KSR (kinase suppressor of Ras) also participates in forming side-to-side heterodimers with RAF and can thereby trigger RAF activation. This mechanism provides an elegant explanation for the longstanding conundrum about RAF catalytic activation, and also provides an explanation for the capacity of KSR, despite lacking catalytic function, to directly mediate RAF activation. RAF side-to-side dimer formation is essential for aberrant signalling by oncogenic BRAF mutants, and an oncogenic mutation was identified that acts specifically by promoting side-to-side dimerization. Together, these data identify the side-to-side dimer interface of RAF as a potential therapeutic target for intervention in BRAF-dependent tumorigenesis (Rajakulendran, 2009).

Together, this study indicates that dimerization of the RAF kinase domain with KSR or with other RAF molecules is central to its activation mechanism. It is posited that other regulatory proteins that impinge on RAF activation, such as RAS, CNK and HYP, may also act by modulating dimerization. A case in point is the role of 14-3-3 proteins, which are known to activate RAF through promoting dimerization. Interestingly, the 14-3-3 consensus binding site in RAF is also conserved in KSR, suggesting that 14-3-3 could also act to promote KSR-RAF heterodimers. Consistent with this possibility, it was found that depletion of endogenous 14-3-3 proteins perturbed KSR-dependent RAF activation, as did mutation of the consensus 14-3-3 binding site in KSR or RAF (Rajakulendran, 2009).

The mapping of oncogenic mutations to the activation segment of BRAF proved unequivocally that the activation segment of RAF is a key modulator of its catalytic function. If dimerization is also a critical modulator of RAF catalytic function, it was wondered whether certain oncogenic RAF mutations might promote kinase activity by promoting dimerization. Oncogenic mutations were sought that mapped to the side-to-side dimer interface and one such mutation, Glu558Lys, was identified that promoted kinase domain dimerization in solution. To investigate how the Glu558Lys mutation functions to hyperactivate RAF in vivo, the FRB-FKBP-rapamycin system was used to assess RAF activation in S2 cells. When the Glu558Lys mutation was tested in a kinase-dead background, it strongly hyperactivated wild-type RAF in trans in a rapamycin-dependent manner. It was reasoned that the added hydrogen-bonding potential of Glu558Lys with Ser 561 on the opposite protomer might promote dimerization. Consistent with this possibility, the hyperactivity of Glu558Lys in trans was selectively attenuated towards the RAF(Ser561Lys) counterpart. These results indicate that the mechanism by which the oncogenic RAF(Glu558Lys) mutation acts is by promoting side-to-side dimers (Rajakulendran, 2009).

Given the in trans mechanism of action of the Glu558Lys mutation, it was questioned whether the more prevalent class of oncogenic RAF mutations that impinge on modulation of the activation segment might also act to promote kinase domain dimerization. In contrast to the Glu558Lys mutant, the kinase-inactive version of a RAF activation segment gain-of-function mutant [RAF(Thr571Glu/Thr574Asp) did not show an enhanced ability to activate a wild-type counterpart in trans in the FRB-FKBP-rapamycin system. This suggests that the prevalent class of oncogenic mutations affecting the activation segment act exclusively in cis to promote intrinsic RAF activity. Consistent with the notion that dimerization is a critical modulator of RAF catalytic function, the hyperactivity of the RAF activation segment mutant in cis is ablated by a dimer interface mutation. These results raise the possibility that small molecule strategies directed at preventing the formation of side-to-side dimers by RAF could act as a therapeutic for RAF-dependent human tumours, one that would complement conventional strategies at present directed at inhibiting RAF enzymatic activity by blocking the catalytic cleft (Rajakulendran, 2009).

Identification of autosomal regions involved in Drosophila Raf function

Raf is an essential downstream effector of activated Ras in transducing proliferation or differentiation signals. Following binding to Ras, Raf is translocated to the plasma membrane, where it is activated by a yet unidentified 'Raf activator.' In an attempt to identify the Raf activator or additional molecules involved in the Raf signaling pathway, a genetic screen was conducted to identify genomic regions that are required for the biological function of Drosophila Raf (Draf). A collection of chromosomal deficiencies representing ~70% of the autosomal euchromatic genomic regions was examined for the abilities of these regions to enhance the lethality associated with a hypomorphic viable allele of Draf, DrafSu2. Of the 148 autosomal deficiencies tested, 23 behaved as dominant enhancers of DrafSu2, causing lethality in DrafSu2 hemizygous males. Four of these deficiencies identified genes known to be involved in the Drosophila Ras/Raf (Ras1/Draf) pathway: Ras1, rolled (rl, encoding a MAPK), 14-3-3epsilon, and bowel (bowl). Two additional deficiencies removed the Drosophila Tec and Src homologs, Tec29A and Src64B. Src64B interacts genetically with Draf and an activated form of Src64B, when overexpressed in early embryos, causes ectopic expression of the Torso (Tor) receptor tyrosine kinase-target gene tailless. In addition, a mutation in Tec29A partially suppresses a gain-of-function mutation in tor. These results suggest that Tec29A and Src64B are involved in Tor signaling, raising the possibility that they function to activate Draf. Finally, a genetic interaction was discovered between DrafSu2 and Df(3L)vin5 that reveals a novel role for Draf in limb development. Loss of Draf activity causes limb defects, including pattern duplications, consistent with a role for Draf in regulation of engrailed (en) expression in imaginal discs (Li, 2000).

Src64B and Tec29A are removed by two deficiencies that each dominantly enhance the lethality of DrafSu2. They were selected as candidate genes for these two deficiencies because a survey of FlyBase for genes in the regions removed by the deficiencies did not yield other genes more likely to be involved in Draf function. The Src64BDelta17 allele in homozygotes enhances DrafSu2, confirming that Src64B genetically interacts with DrafSu2. Overexpression of an activated form of Src64B in early embryos can cause activation of the Tor target gene tll and cuticular defects similar to those caused by gain-of-function mutations in tor. These results are consistent with a role for Src64B in Tor signaling and/or Draf activation. It was not possible to demonstrate that Tec29A could enhance Draf using an available mutant allele of Tec29A. However, indirect evidence has been obtained suggesting a requirement of Tec29A in Tor signaling. (1) Tec29A206 homozygous mutant embryos exhibit defects in the terminal structures that are specified by the Tor pathway. Specifically, they show defective mouth parts and shortened Filzkörper, phenotypes consistent with disruption of Tor signaling. (2) Reducing the activity of Tec29A suppresses a gain-of-function tor allele. Most strikingly, embryos zygotically homozygous for Tec29A206 that are derived from torY9 mothers exhibit mouth parts and Filzkörper indistinguishable from those of Tec29A206 embryos. (3) Mutation of Tec29A restores most of the ventral denticle bands that would have been deleted due to torY9, suggesting that Tec29A is genetically epistatic to tor. However, many of the embryos still exhibit minor disruptions in the ventral denticle bands, a defect reminiscent of weak tor gain-of-function mutations. This suggests that homozygosity for Tec29A206 cannot completely suppress torY9. (4) Possibly, while Tec29A may be required for Tor signaling, Tec29A206 may not be a null allele and therefore cannot completely suppress torY9. This would be consistent with the inability of this allele to enhance DrafSu2. Alternatively, the maternally contributed Tec29A may be able to partially mediate signaling by the mutant TorY9 protein. (5) Tec29A may not be an absolute requirement for Tor signaling, but rather it may function in a separate pathway that in conjunction with Tor is required for the differentiation of terminal structures (Li, 2000).

The likelihood that Src64B and Tec29A are involved in Draf activation is based upon data from in vitro studies of mammalian c-Src function. Src kinases can phosphorylate and activate Raf-1 in vitro, and the tyrosine residues phosphorylated by Src are important for Raf-1 activation. Tec kinases are very similar to Src kinases in the kinase domain, but lack the C-terminal regulatory tyrosine and the N-terminal myristylation site that are specific for Src family members. Tec kinases interact with and are activated by Src through phosphorylation. It has been shown in Drosophila that Tec29A is regulated by Src64B and that both are required for the growth of ring canals of the egg chamber. Although it has not been documented that Tec can phosphorylate Raf in vivo, given the similarities in the kinase domain, it is not unreasonable to propose that Tec could do so. Finally, consistent with these results, the two genomic regions containing Src64B and Tec29A have also been identified as required for the function of Corkscrew (Csw) in a similar screen for modifiers of a partial loss-of-function csw allele (Li, 2000).

The proper expression of en in the posterior compartment of imaginal discs is essential for maintaining compartmental boundaries and patterning of Drosophila limbs. Despite much insight into the events required for Hh signaling, little is known about the mechanism(s) by which en expression is controlled in the posterior compartment. Two instances have been identified where a further reduction in Draf function, due to the presence of a deficiency, results in defects in posterior pattern elements in the limbs. DrafSu2/Y; Df(3L)vin5/+ male survivors exhibit notching only in the posterior region of the wing, and partial pattern duplications in the posterior compartment. Since no specific role for Draf has been described in the limbs, the requirements for Draf in the imaginal discs were examined. Since clonal analysis with null alleles is uninformative, because Draf mutant clones do not develop, Draf was conditionally provided to the developing animals in a Draf null background (Li, 2000).

As a result of withholding Draf during the second and early third larval instars, animals with anterior pattern element duplications in the posterior compartment were frequently observed. By examining the imaginal discs of these animals, it could be determined that when there are insufficient levels of Draf, en expression is no longer restricted to the normal posterior compartment, which suggests that Draf may act to repress/restrict En expression. Along with ectopic expression of En in the anterior compartment and increased levels of En in the posterior compartment, a new mirror image anterior compartment devoid of en expression was induced. This observation is consistent with the observation that when En is ectopically expressed, ectopic anterior pattern elements are induced. Also, ectopic expression of En in the anterior compartment induces expression of high levels of Hh and Dpp, which are responsible for overgrowth and the duplication of anterior pattern elements. Indeed, when Hh was examined in the partially rescued Draf null males, it was found to be widely ectopically expressed. The posteriorly restricted wing notching observed in DrafSu2/Y; Df(3L)vin5/+ male survivors is also consistent with a requirement for Draf in negatively regulating en, since elevated levels of En expression in the posterior compartment partially inactivate both en and inv, which are necessary for the development and terminal differentiation of posterior fates. Taken together, these observations suggest that the Df(3L)vin5 deficiency contains a gene that participates with Draf in patterning of the limbs (Li, 2000) (Douziech, 2006).

Dual function of Ras in Raf activation

The small guanine nucleotide binding protein p21Ras plays an important role in the activation of the Raf kinase. However, the precise mechanism by which Raf is activated remains unclear. It has been proposed that the sole function of p21Ras in Raf activation is to recruit Raf to the plasma membrane. Two basic observations constitute the basis of this model: (1) purified Raf-1 cannot be activated by Ras-GTP in vitro, and (2) in cultured cells, Raf-1 can be activated by being targeted to the membrane without co-transfection of activated Ras. This activation of membrane-targeted Raf cannot be inhibited by a dominant negative form of Ras (Li, 1998 and references).

All Raf proteins share three conserved regions: CR1, CR2, and CR3. CR1 and CR2 are located in the N-terminal regulatory region, which acts to suppress the catalytic activity of the C-terminal CR3 kinase domain. Removal of the N terminus (including CR1 and CR2) results in constitutive activation of Raf-1 in mammalian cells. The activation of Raf requires its direct interaction with Ras in its activated GTP-bound form. Two regions within CR1 have been identified that bind Ras: the Ras-binding domain (RBD) and a cysteine-rich domain (CRD). The RBD associates with the Ras effector domain with high affinity, and Arg89, located in the RBD of Raf-1, is a key residue that mediates this interaction. A mutation in this residue, R89L, abolishes the association between Ras and Raf-1, as well as Ras-dependent activation of Raf-1. The CRD also associates with Ras through residues different from those that contact the RBD. Interestingly, it was demonstrated that the CRD:Ras interaction only occurs once the association of RBD with Ras has occurred. The Drosophila Draf protein is structurally and functionally homologous to mammalian Raf-1. Human RAF-1 is 46% identical in amino acid sequence to Draf, and is able to substitute in Drosophila for Draf for viability and signal transduction. A Drosophila mutation, DrafC110, associated with a reduced level of Draf activity, has been isolated that cannot support the survival of the animal. This mutation is an amino acid change in Arg217, equivalent to Arg89 of Raf-1. The DrafC110 R217L mutation prevents the Draf:Ras1 interaction, suggesting that like Arg89 in Raf-1, Arg217 is essential for Ras1:Draf interaction. Dominant intragenic suppressors of DrafC110 have been identified that restore viability to DrafC110 flies. Two of these suppressors, Su2 and Su3, identify mutations P308L and F290I, respectively, in the CRD of Draf. When the analogous DrafC110 mutation along with either of its intragenic suppressors are introduced into mammalian Raf-1, the suppressors do not function to restore the lost Ras:Raf association. Rather, they increase the basal level activity of Raf-1 (Li, 1998 and references).

To further understand the mechanism of Raf activation by Ras, the activity of a number of Raf mutations were examined in the complete absence of Ras activity. These experiments are feasible in Drosophila because it is possible to generate eggs completely devoid of Ras1 or Draf activity using the mosaic 'FLP-DFS' technique. This is achieved following generation of germline mosaics that allow production of eggs derived from germline cells homozygous for either Ras1 or Draf protein null mutations. The embryos derived from females that lack maternal Draf or Ras1 activities are referred to as Draf or Ras1 embryos, respectively. Draf plays a central role in Tor signal transduction because embryos lacking maternal Draf gene activity have phenotypes identical to those of tor null embryos, resulting in the complete absence of posterior tll expression. Weaker Draf alleles exhibit reduced levels of tll expression. Therefore, visualization of tll expression allows one to determine the level of activity of Tor, Draf, or its downstream signal transducer. Previously, it had been proposed that N-terminal truncation or membrane targeting of Raf results in Ras-independent 'constitutive activation' of Raf. The signaling activity of an N-terminally truncated Draf (DrafdeltaN) mutation expressed in embryos completely devoid of Ras1 activity has been reexamined. Contrary to the current model, it is demonstrated that Ras1 is necessary for the activation of DrafdeltaN. Further, it is shown that the activity of membrane-targeted DrafdeltaN is still sensitive to the presence of Ras1. Finally, the role of Ras1 in Draf activation was examined using point mutations in Draf that disrupt its association with Ras1. DrafC110 in combination with one of its intragenic suppressors, Su3, is still regulated by Ras1 and Tor, although Su3 does not restore the Draf C110:Ras1 molecular interaction. The unregulated enzymatic activity of Raf-1, due to the Su3 mutation, is not sufficient for the mutant Draf to transduce Tor signals, suggesting that Ras1, which is not physically interacting with the mutant Draf protein, is required for the activation of the mutant Draf. Taken together, these results suggest that Ras has an essential function in Raf activation beyond translocating Raf to the plasma membrane (Li, 1998).

Currently, the exact mechanism by which Raf is regulated is still unknown. In addition to Ras, other Raf-interacting proteins have been isolated as potential Raf activators. These include 14-3-3 and KSR. Studies with the Drosophila 14-3-3 genes indicate that they cannot encode the 'Raf activator' since these proteins are necessary, but not sufficient for Draf signaling. The situation with KSR is less clear. KSR associates with 14-3-3 and also with Raf-1 at the membrane in a Ras-dependent manner. It has been suggested that KSR is a ceramide-activated protein (CAP) kinase that phosphorylates and activates Raf-1 in vitro. However, it has been found that KSR plays a structural role in modulating Raf/MEK/MAPK signal propagation and it does not appear to phosphorylate Raf-1. A 'two-step' mechanism by which Ras activates Raf is proposed. It is suggested that first activated Ras binds to Raf at the membrane and brings Raf into close proximity to its activator. The conformational change that results from the binding of Ras to the N-terminal region of Raf relieves the inhibitory effects of the N terminus, exposing the CR3 kinase domain to the 'Raf activator'. Since it has been shown that Ras-GTP does not directly activate Raf, the existence of a 'Raf activator' has to be postulated. As a second step, it is proposed that, independent of the binding of Ras to Raf, activated Ras activates the unknown 'Raf activator'. A test of this model obviously awaits the identification of the 'Raf activator' (Li, 1998 and references).

Somatic control over the germline stem cell lineage during Drosophila spermatogenesis

Stem cells divide both to produce new stem cells and to generate daughter cells that can differentiate. The underlying mechanisms are not well understood, but conceptually are of two kinds. Intrinsic mechanisms may control the unequal partitioning of determinants leading to asymmetric cell divisions that yield one stem cell and one differentiated daughter cell. Alternatively, extrinsic mechanisms, involving stromal cell signals, could cause daughter cells that remain in their proper niche to stay stem cells, whereas daughter cells that leave this niche differentiate. Drosophila spermatogenesis has been used as a model stem cell system to show that there are excess stem cells and gonialblasts in testes that are deficient for Raf activity. In addition, the germline stem cell population remains active for a longer fraction of lifespan than in wild type. Finally, raf is required in somatic cells that surround germ cells. It is concluded that a cell-extrinsic mechanism regulates germline stem cell behaviour (Tran, 2000).

The testis proliferation center in Drosophila melanogaster includes the germline and somatic stem cells that maintain spermatogenesis. As a germline stem cell divides, one daughter becomes a gonialblast, while the other remains a stem cell. To amplify the germline population, each gonialblast executes four divisions as 2° spermatogonia, which exit the mitotic cycle and enter a meiotic and differentiation program as a clone of 16 spermatocytes. The gonialblast and its progeny are encysted by somatic cells derived from cyst progenitor stem cells. Mutants affecting 2° spermatogonia have been characterized: signal transduction pathways were tested for possible involvement at earlier decision points in this germline stem cell lineage. Null mutations in raf, encoding a serine/threonine kinase involved in receptor tyrosine kinase pathways, are lethal in larvae, but such flies carrying a heat shock (HS)-Raf transgene can survive to fertile adults by using daily heatshocks. By withdrawing heat shock when adults eclose, animals become progressively raf deficient as the protein decays. Testes from such hypomorphic raf-deficient males 5 days after eclosion were indistinguishable from controls. By day 7, however, 43 of 44 raf mutant testes show great expansion of the proliferation center. The increase in cell number is due to excess, early stage germ cells, and continues such that on day 15, raf -deficient testes are filled with these cells at the expense of post-mitotic cells (Tran, 2000).

To identify the earliest defect in germ-cell progression, testing was carried out on the fusome, a membrane and cytoskeletal organelle specific to the germline that is spheroid throughout stem cells and gonialblasts, but that branches extensively throughout interconnected 2° spermatogonia. In raf-deficient testes, fusome structure is not normal. Unbranched fusomes are found in many cells, even those located some distance from the hub, where, in wild-type testes, only branched fusomes interconnecting 2° spermatogonia are found. Although branched fusomes do appear in raf-deficient testes, unlike wild-type testes, these fusomes usually appear only after the appearance of an intervening region containing many excess germ cells that have only spheroid fusomes. These excess germ cells do not result from an increased frequency of germline stem cell divisions, because the M-phase index for the tier of cells adjacent to the hub in raf-deficient testes is almost identical to that in the wild type (Tran, 2000).

Cytoplasmic Bag-of-marbles protein (Bam-C), which first accumulates in 2° spermatogonial cells was tested and was found to be required for their progression into spermatocytes. In the wild type, the total population of germline stem cells and gonialblasts comprises the few non-staining cells between the hub and the first rows of 2° spermatogonia. In raf-deficient testes, the first Bam-C-expressing germ cells are located much further from the hub. Also, most of the intervening non-staining cells contain unbranched fusomes, consistent with these cells representing excess gonialblasts and/or germline stem cells (Tran, 2000).

To verify this, M5-4 and S1-33, markers expressed in hub cells, germline stem cells and gonialblasts, but not in 2° spermatogonia, were examined. In raf-deficient testes, while hub cells appear normal, the number of germ cells expressing these markers is greatly increased. Furthermore, marker expression persists for a significantly longer fraction of adult lifespan. For instance, by day 19, all control testes have M5-4 marker expression in fewer than three germ cells, rather than the average 5 to 9 germline stem cells plus associated gonialblasts contained in testes from young adults. In contrast, only 17% of raf-deficient testes on day 19 show loss of germ cell expression, whereas 48% maintain expression at least equivalent to that of day 1, and a further 35% still show increased expression. These data suggest that germline stem cells and gonialblasts remain active for a significantly longer period than in the wild type. This was further tested by directly counting the number of cycling germline stem cells, which were judged to be cells that are located adjacent to the hub and contain a spherical fusome and nuclear anillin, a late interphase marker. Whereas wild-type testes averaged 3.1 late interphase germline stem cells per testis on day 1, on day 19 aged cohorts average only 0.8. This suggests an age-induced quiescence of the germline stem cell population. In contrast to this, in raf-deficient testes, late interphase germline stem cell numbers are maintained over a 19 day period suggesting that germline stem cells as a population remain active for a longer period than in wild-type (Tran, 2000).

To determine whether raf function is required in the germ line, which exhibits the phenotypes described above, or in the surrounding somatic cells, raf null mutant clones were generated. Persistent germline clones indicate the existence of a raf null germline stem cell. In all cases, progression through spermatogenesis is normal as judged by groups of 16 mutant germ cells that are morphologically indistinguishable from surrounding wild-type spermatocytes. Thus, raf function is dispensable in germ cells. In contrast, no cyst cell clones were recovered, suggesting that raf is necessary for viability or proliferation of cyst progenitor cells. Since the previous analysis was under hypomorphic conditions for raf, the mosaic analysis was repeated, introducing a HS-Raf transgene to provide a basal level of raf function so that raf-deficient cyst cells might survive. Persistent raf-deficient cyst cell clones indicate the existence of a raf-deficient cyst progenitor cell. Such testes have excess early stage, raf+ germ cells. Thus, raf is required in the cyst cell lineage. In addition, in raf-deficient testes two cyst cell markers normally expressed in later-stage cyst cells, LacZ600 and Eyes absent, are now expressed prematurely in somatic cells adjacent to the hub, probably the cyst progenitor cells. This shows that raf function is required in somatic cells surrounding germline stem cells (Tran, 2000).

Germline stem cell divisions lead to a distinction between a self-renewing daughter cell and a sister cell committed to differentiation as a gonialblast. These data suggest that a somatic signal influences this decision by limiting the self-renewing potential. It cannot yet be said how this potential is encoded. It is hypothesized is that in raf-deficient testes, where cyst progenitor cell identity is disturbed, the signal is lost, and excess stem cell potential is produced. Upon division, both daughters of the germline stem cell inherit some stem cell character. At steady state, this increases the number of cells that become gonialblasts, and somehow prolongs the active state of the stem cell population. Thus, a somatic cell defect leads to a tumor in the germline stem cell lineage, which suggests that some tumors of progenitor cell populations could be initiated by genetic lesions in support cells, rather than in the tumorous cells themselves. In the testis, the Raf-dependent signal may be delivered by cyst progenitor cells, or their cyst-cell daughters. Mosaic analysis shows that depletion of Raf from just one of the two cyst progenitor cells surrounding a germline stem cell causes a defect. This may indicate a dose effect, where one heterozygous somatic cell is not sufficient to allow normal signaling. It is likely that the signal transducer Raf is engaged, owing to activation of the Epidermal growth factor receptor pathway in somatic cells. It is noted that in raf-deficient testes, differentiation of 2° spermatogonia is blocked because they do not transit to the spermatocyte stage. It is believed that this is a secondary effect owing to the defective cyst lineage, since a cyst cell signal governs this later transition from 2° spermatogonia to spermatocytes (Tran, 2000).

Somatic signals have been postulated to affect germline stem cell behaviour in the Drosophila ovary. However, as has been found here, the characterized signals are necessary to maintain germline stem cells, rather than restrict their self-renewing potential. Additionally, raf-deficient ovaries exhibit no increases in early stage germ cells. Thus, despite the superficial similarities of early germ cell development in ovary and testis, oogenesis and spermatogenesis are emerging as complementary systems from which different principles of stem cell regulation will emerge (Tran, 2000).

Raf acts to elaborate dorsoventral pattern in the ectoderm of developing embryos

In the early Drosophila embryo the activity of the EGF-receptor (Egfr) is required to instruct cells to adopt a ventral neuroectodermal fate. Using a gain-of-function mutation it has been shown that D-raf acts to transmit this and other late-acting embryonic Egfr signals. A novel role for D-raf was also identified in lateral cell development using partial loss-of-function D-raf mutations. Thus, evidence is provided that zygotic D-raf acts to specify cell fates in two distinct pathways that generate dorsoventral pattern within the ectoderm. These functional requirements for D-raf activity occur subsequent to its maternal role in organizing the anterioposterior axis. The consequences of eliminating key D-raf regulatory domains and specific serine residues in the transmission of Egfr and lateral epidermal signals were also addressed in this study (Radke, 2001).

In the Drosophila embryo, Egfr activity is required to instruct a field of cells that lie on either side of the ventral furrow to adopt a ventral ectodermal fate. It is from this neuroectodermal cell population that the ventral nervous system and epidermis arise. At later times, Egfr functions in germband retraction and cuticle formation. Embryos that develop without Egfr activity fail to form ventral cuticular structures and show the 'faint little ball' phenotype. A constitutively active form of the D-raf protein, D-raftor4021, was used to bypass the requirements for Egfr function in embryos that lacked Egfr gene activity. For the generation of hyperactive D-raftor4021-proteins, the extracellular and transmembrane domains of the torso RTK gene were fused to the D-raf kinase domain. Chimera D-raftor4021proteins were shown to act independently of sevenless RTK gene function in developing photoreceptor cells: the chimeric proteins exhibited gain-of-function effects in the Tor signaling pathway (Radke, 2001).

Would this activated D-raf protein act independently of Egfr to rescue the embryonic lethality associated with homozygous mutations in the Egfr gene? In the case of noninjected control, 25% of the embryos derived from heterozygous Egfr parents (Egfr-/+) failed to hatch, showed the faint little ball phenotype, and were homozygous for the Egfr mutation. D-rafWT mRNA was used as a control for the injection procedure, and it was found that after injection 27% of the embryos from heterozygous Egfr parents failed to hatch. These embryos showed the Egfr mutant phenotype at 24 hr. When D-raftor4021 mRNA was injected into the central region of embryos collected from heterozygous Egfr parents, all aspects of defective Egfr signaling were rescued for some of the mutant Egfr embryos. Of the 258 embryos that received injection, 217 (84%) hatched out of their egg cases as larvae, while 41 (16%) remained within their eggshells. Thus, an increase in embryonic hatching and suppression of Egfr-induced lethality was observed after injection of D-raftor4021 mRNA. Partial rescue of the Egfr phenotype was found in unhatched embryos that had received D-raftor4021 mRNAs with ventral cuticular structures observed. It was concluded that constitutively active D-raftor4021 molecules can bypass the requirement for Egfr activity in the embryo and direct cells of the embryonic ectoderm to adopt a ventral fate. These results show that D-raf participates downstream of Egfr in developing embryos (Radke, 2001).

Once it had been found that an activated form of the D-raf protein could suppress the effects of a loss-of-function Egfr allele, it was reasoned that embryos lacking maternal and zygotic D-raf activity would exhibit an Egfr-like phenotype. These embryos would also be expected to show defects associated with the loss of maternal D-raf function in Tor signaling. To determine whether the identities of cells in the ventral ectoderm were dependent on D-raf activity, marker gene expression patterns and cuticles produced by D-raf embryos were compared to those of wild-type and Egfr embryos. To generate these D-raf embryos, mosaic D-raf females were produced whose eggs lacked maternal D-raf proteins. Once fertilized, these eggs gave rise to two classes of embryos: the first class was composed of the paternally rescued D-raf torso embryos (D-raf-/+) that had inherited a wild-type D-raf gene from their fathers: they were defective in Tor RTK signaling and were missing head and tail structures at 24 hr. These D-raf torso embryos lacked maternal but not zygotic D-raf activity. The second phenotypic class was composed of the D-raf null embryos (D-raf-/Y) whose exoskeletons consisted of what appeared to be a small patch of dorsal cuticle. These embryos lacked maternal and zygotic D-raf activity throughout development. It was anticipated that this D-raf null embryonic class would exhibit the phenotypic characteristics consistent with defective Egfr signaling, a consequence of defective D-raf protein activity (Radke, 2001).

Initially, to determine whether the establishment of ventral cell identity by the maternal dorsal gene system occurred normally in D-raf embryos, the accumulation of rhomboid (rho) mRNAs between 4 and 6 hr (stages 9-12) of development was assayed. As visualized by in situ hybridization, a column of cells ~2-3 wide on either side of the ventral midline showed the accumulation of rho mRNAs. This temporal and spatial pattern of rho expression was observed in all embryos in the D-raf collections, with each embryo a member of either the D-raf torso (lacking maternal but not zygotic D-raf activity) or null class. An equivalent rho expression pattern was observed in wild-type and Egfr embryos. Thus, the initial step in the establishment of ventral cell identity, by dorsal and other maternal genes that act to define the dorsoventral embryonic axis, is not perturbed when these events take place in the absence of maternal or zygotic D-raf activity (Radke, 2001).

To determine whether EGR-receptor signaling occurs normally in D-raf embryos, expression of the orthodenticle (otd) gene was monitored. In wild-type control embryos, at 6 hr (stage 11) otd mRNAs accumulate in cells adjacent to the ventral midline and in the head. In embryos lacking Egfr activity, otd expression occurred only in those cells within the embryonic head. In D-raf embryo collections, two patterns of embryo staining were observed with approximately one-half of the embryos showing otd expression in cells along the ventral midline and in the head. For the remaining D-raf embryos, the accumulation of otd mRNAs was observed only in the head, similar to Egfr embryos (Radke, 2001).

To distinguish between torso and null embryos in D-raf collections, a ftz-ß-gal marker gene located on the paternal X chromosome was used. Males with the ftz-ß-gal gene were allowed to fertilize eggs from mosaic females that lacked D-raf activity. In this double-labeling experiment, embryos that showed a ftz pattern of ß-gal expression were assigned to the D-raf torso class. These embryos also displayed a wild-type pattern of otd expression. In those D-raf null embryos lacking ß-gal expression, otd mRNAs were detected only in cells of the head, similar to Egfr embryos (Radke, 2001).

Between 4 and 7 hr (stages 9-11) of development, wild-type and Egfr embryos accumulated decapentaplegic (dpp) mRNAs in cells that formed two lateral stripes, when embryos were viewed ventrally. A similar pattern of dpp mRNA accumulation is seen in D-raf mutant embryos at this developmental stage. However, the ventral distance between dpp stripes becomes smaller in Egfr embryos as they develop. The distance between lateral dpp stripes was recorded and compared in wild-type, Egfr, and D-raf embryos at 10 hr (stage 13) of development. For wild-type embryos the average stripe distance was 0.111 units. In the collection of Egfr embryos, ~75% showed an average dpp lateral stripe distance of 0.118 units, similar to wild type. This phenotypic class contained embryos that were heterozygous mutant (Egfr-/+) or wild type with respect to the Egfr gene. In the remaining 25% of the embryos the average dpp stripe distance was reduced to 0.075 units as anticipated for homozygous mutant Egfr embryos (Radke, 2001).

Two phenotypic classes of D-raf embryos were also distinguished on the basis of a statistically relevant difference in dpp stripe distance. In approximately one-half of the embryos the average dpp lateral stripe distance was 0.120 units, with the remaining embryos showing an average separation of 0.064 units. It was speculated that this second phenotypic class contained the D-raf null embryos. To test this idea, the marker ftz-ß-gal X chromosome was again employed in a double-labeling experiment to distinguish between D-raf torso and null embryos. As anticipated, it was the male D-raf null embryonic class that showed the decrease in distance between lateral dpp stripes, indicative of a loss in ventral cell fates (Radke, 2001).

On the basis of this analysis of rho, otd, and dpp gene expression patterns in D-raf null embryos, it has been concluded that ventral ectoderm cells are specified incorrectly in the absence of D-raf activity. This loss results in the production of a mature D-raf null exoskeleton that is severely reduced in size and devoid of ventral structures, consistent with the Egfr embryonic phenotype. However, the distance between lateral dpp stripes in Egfr (0.075 units) and D-raf null (0.064 units) embryos was compared: it was smaller in D-raf null embryos. In addition, after cursory inspection, the size of the exoskeleton patch produced by D-raf null embryos appeared smaller than that from Egfr embryos. These differences could be biologically significant and the analysis was expanded to address this potentially interesting finding (Radke, 2001).

To better understand the role that D-raf plays in the ectoderm and to access its regulation in various developmental pathways partial loss-of-function alleles of D-raf generated in vitro were used. D-raf shares homology with family members in CR1 that contain (1) D-ras binding motifs; (2) CR2, a region rich in serine and threonine residues, and (3)the CR3 kinase domain. CR1 is thought to exhibit positive control in the regulation of the D-raf protein via its interaction with D-Ras, while CR2 appears to be involved in the negative regulation of the molecule. Whether conserved subdomains (CR1 and CR2) or putative phosphorylation sites (serine 388 or 743) are essential for the activity of D-raf in the embryo or involved in its positive or negative regulation was tested. These modifications of D-raf often result in decreased D-raf activity. Thus, by expressing partial loss-of-function D-raf alleles in D-raf null embryos the role D-raf plays in developing embryos could be deciphered (Radke, 2001).

Using a structure-function strategy, several modified forms of the D-raf protein were generated. The D-rafWT and D-rafK497M genes were constructed as positive and negative controls, respectively, with the D-rafWT allele a full-length copy of a D-raf cDNA. D-rafK497M lysine 497, which was shown to be critical for D-raf protein kinase activity and likely involved in ATP binding, was replaced with a methionine. The N-terminal and CR1 deletion mutation, D-rafDelta315, was likely to show a partial loss-of-function in D-raf null embryos. For the D-rafDelta445 mutation both positive (CR1) and negative (CR2) control elements were lost, and it was predicted that this form of D-raf would act in a manner similar to wild type or, on the basis of its structural similarity to oncogenic forms of Raf-1, and show a gain-of-function effect in the embryo. Of the five phosphorylation sites identified for the human Raf-1 kinase, two are conserved in the D-raf protein. Serine to alanine substitutions at these sites were generated and it has been shown that S388 (CR2) plays a negative role while S743 (CR3) is involved in the positive control of D-raf in the Tor pathway. It was predicted that the D-rafS388A and D-rafS743A proteins would show similar phenotypic consequences for developing cells in the embryo (Radke, 2001).

Using P-element-mediated transformation, Drosophila lines were generated that contained an insertion of the D-rafWT, D-rafK497M, D-rafDelta315, D-rafDelta445, D-rafS388A, or D-rafS743A gene on either the second or third chromosome. Each of these modified D-raf genes were paternally introduced into D-raf embryos lacking maternal D-raf protein. The level and stability of D-raf proteins produced by expression of each paternally inherited D-rafmodified gene was tested. In this assay 100 embryos were collected for each sample and processed for Western analysis. Since the expression of each D-rafmodified gene was under the control of the hsp70 promoter, samples were processed from non-heat-shocked or heat-shocked embryos at 5 and 10 hr of development. These D-rafmodified proteins are variably stable and in D-raf null embryos show differences in the rescue of dorsoventral cuticular defects caused by the loss of D-raf maternal and zygotic function. The degree of phenotypic rescue observed in D-raf null embryos was as follows: D-rafWT > D-rafS388A > D-rafDelta445 > D-rafS743A > D-rafDelta315 > D-rafK497M (Radke, 2001).

The accumulation of D-raf protein was assayed in D-raf embryos that had inherited the D-rafWT gene. For these embryos the accumulation of D-raf proteins after heat induction was approximately twofold greater than that found in wild-type embryos at 5 hr. At 10 hr, the level of the D-rafWT protein was unchanged. The effect of D-rafWT proteins on otd and dpp gene expression patterns was determined in D-raf embryos. As anticipated, induction of the D-rafWT gene results in 100% of the D-raf null class showing wild-type ventral otd stripe expression and a normal pattern of dpp expression. Embryonic cuticles were examined at 24 hr to assess the ability of the D-rafWT gene to promote signaling in the late-stage Egfr pathway responsible for epidermal differentiation and the final cuticular pattern. Of these D-raf null embryos that had inherited the D-rafWT gene, 99% developed cuticles indistinguishable from their D-raf torso sisters. Thus, all ectodermal signaling pathways dependent on D-raf activity could be fully restored in null embryos by expression of the D-rafWT gene (Radke, 2001).

In the phenotypic analysis, 84% of D-rafS388A expressing D-raf null embryos showed rescue of Egfr-induced otd expression in ventral cells and the distance between dpp stripes appeared normal. By the completion of embryonic development, 97% of the D-raf null embryos showed the torso phenotype, while the remaining 3% showed a composite 'imperfect torso' phenotype. In addition to showing head and tail defects associated with the torso phenotype, embryos of the 'imperfect torso' class were twisted and had denticle bands of reduced width, indicative of partial loss of signaling in ventral cells that depend on the Egfr pathway for development. Since all of the D-raf null embryos showed some phenotypic rescue by D-rafS388A, it was concluded that serine 388 is not essential for the function of D-raf in the ectoderm. Instead, it was thought likely that S388 plays a negative role in the regulation of D-raf similar to its function in Tor signaling (Radke, 2001).

For D-raf null embryos that inherited the D-rafDelta445 gene, 52% showed rescue of the Egfr-induced otd expression pattern. This was approximately one-half the percentage rescued by the D-rafWTgene, although the quantity of truncated ~38-kD D-raf protein in these embryos was equivalent to that observed for D-raf embryos expressing the D-rafWT gene at 5 hr. For the human Raf-1 protein, removal of CR1 and CR2 resulted in unregulated kinase activity. Whether the D-rafDelta445 protein acted ectopically to create a wide ventral otd stripe was tested, but all of the otd stripes were of wild-type width. When dpp mRNA patterns were analyzed in D-rafDelta445 expressing null embryos the distance between lateral stripes in the third thoracic segment at 10 hr was similar to those that had inherited the D-rafWT gene (Radke, 2001).

In the analysis of 24-hr cuticular patterns 52% of the D-rafDelta445 embryos were rescued and showed the torso phenotype. For the remaining embryos, partial rescue was observed with signaling by the D-rafDelta445 protein defective in the determination of the ventral ectoderm. Of these embryos, 18% showed the 'imperfect torso' phenotype and 30% showed the 'null with denticles' phenotype. These 'null with denticles' embryos were twisted, had faint cuticles with narrow denticle bands, and were phenotypically similar to Egfr embryos homozygous for intermediate defective alleles of Egfr. Overall, it was found that signal transmission by D-rafDelta445 was less reliable when compared with D-rafWT, although the D-rafDelta445 protein had the potential to rescue all aspects of the embryonic D-raf null phenotype (Radke, 2001).

Analysis of D-raf embryos expressing the D-rafS743A gene was somewhat complicated by the insertion of D-rafS743A on the TM2 balancer chromosome. Thus, only one-half of the D-raf null embryos fertilized by D-rafS743A transgenic males inherited the D-rafS743A gene. The amount of D-rafS743A protein that accumulated in D-raf embryos with the D-rafS743A gene was determined; the D-rafS743A protein was ~1.5-fold greater than that observed for those embryos that had inherited the D-rafWT gene. Although greater levels of this modified D-raf protein accumulated in D-raf null embryos expressing the D-rafS743A gene, otd stripe expression was not observed. Also, the distance between lateral dpp stripes in these D-rafS743A embryos was diminished when compared with wild type, but not to the degree observed for embryos expressing the D-rafDelta315 or D-rafK497M genes. Thus, the specification of ventral cell fates at the midline requires the positive regulation of the D-raf protein at serine 743 (Radke, 2001).

Accordingly, 99% of the D-raf null embryos expressing the D-rafS743A gene showed the 'imperfect torso' phenotype. To better assess the pattern deletions generated by the loss of epidermal cell fates in these D-rafS743A embryos, epidermal sensory organs that develop in ventral and lateral domains of the embryo were scored. The separation between Keilin's organs and ventral black dots on the ventral surface was measured. Also, to determine whether patterning in lateral cells was normal for these embryos the distance between ventral and dorsal black dots was recorded. When compared with wild type, D-rafS743A embryonic cuticles showed a decreased distance between Keilin's organs and ventral black dots. A decrease in the distance between ventral and dorsal black dot material was also observed. This latter finding proved very informative for it led to the hypothesis that a novel pathway, dependent upon the D-raf protein, was operating for signal transmission in cells undergoing lateral epidermal development. It appears that cell fate specification in the ventralmost ectoderm via the EGR receptor and proper development of a subpopulation of lateral cells requires an optimal level of D-raf activity that is not achieved by the D-rafS743A protein (Radke, 2001).

Rescue of epidermal patterning defects was further diminished in D-raf null embryos that expressed the D-rafDelta315 gene. Using Western analysis it was found that the D-rafDelta315 protein migrated as an ~60-kD band detected at a level equivalent to that of the 90-kD D-rafWT protein at 5 hr. Approximately 80% of this D-rafDelta315 protein was present at 10 hr. When D-raf null embryos that inherited the D-rafDelta315 gene were assayed for otd and dpp stripe expression, ventral otd expression was not observed and the distance between lateral dpp stripes was much reduced when compared with embryos expressing the D-rafWT gene. Thus, a substantial decrease in the output of the Egfr-induced signal was detected. By the completion of development, 83 (81%) of the expected 102 D-raf null embryos with D-rafDelta315 protein showed cuticles with the 'null with denticles' phenotype (Radke, 2001).

Epidermal sensory organs were scored in D-raf null embryos expressing the D-rafDelta315 gene and their relative positions noted. Significantly, an absence of Keilin's organs was recorded and a corresponding expansion in the size of ventral black dot material was observed. The distance between these enlarged ventral dots was substantially reduced when compared with wild-type embryos. A reduction in the distance between ventral and dorsal black dot sensory organs was also observed. This finding again implicates D-raf in a pathway required for the development of lateral cells. Thus, by reducing the ability of the D-raf protein to act in signaling its role in the Egfr pathway has been verified and its function in a novel pathway involved in lateral cell development has also been uncovered (Radke, 2001).

As anticipated, D-raf-dependent pathways were not rescued when D-raf null embryos expressed the kinase defective D-rafK497M gene (Radke, 2001). Thus, D-raf acts downstream of the Egfr for the specification of ventral ectodermal cell fates. D-raf also plays a second role in a novel pathway that is required for lateral cell development. In particular the D-rafS743A and D-rafDelta315 alleles generated in vitro proved useful in defining the function of D-raf in cells of the lateral epidermis. It is hypothesized that this novel pathway acts to specify cells of the lateral ectoderm subsequent to instructions received by nuclei from the dorsal maternal gene product. Thus, dorsoventral patterning in the embryo is likely dependent on the activity of three zygotic signaling pathways with Dpp that acts in dorsal cells, Egfr that directs cells in the ventral ectoderm, and a novel RTK pathway that specifies lateral cell fates (Radke, 2001).

The lateral epidermis consists of two narrow stripes of tissue on the left and right sides of the embryo extending from the anterior head to the posterior tail region. For the meta- and meso-thoracic regions this lateral tissue gives rise to epidermal cuticular structures that form between dorsal and ventral black dot sensilla. Along the circumference of each abdominal segment these two regions of lateral cuticle can be subdivided into dorsolateral and ventrolateral domains. Normally in late-stage embryos the dorsolateral region is characterized by numerous discontinuous rows of long slender hairs that have a pattern similar to that found for region b of the dorsal epidermis. These dorsolateral hairs are most similar in size and morphology to a subset of dorsal hairs, the 4° hairs. The ventrolateral domain is characterized by a segmental organization of naked cuticle alternating with two to three sparse rows of denticles similar to those found in the ventral belts although not as strongly pigmented (Radke, 2001).

Several findings have indicated that a novel pathway acts in the determination of lateral ectodermal cell fates and are consistent with a role for D-raf in this pathway. Embryos that developed in the absence of dpp and dorsal activity are lateralized. Mutations in the Drosophila dCREB-A gene are also important for defining lateral embryonic regions. In the absence of dCREB-A gene function, embryos show development of only lateral epidermal structures. Two consequences of lateral cell induction have also been identified: activation of the MAP kinase protein and expression of the msh gene encoding a homeodomain protein product. Using D-raf proteins with partial function it has been found that D-raf also participates in the development of the lateral epidermis most likely to specify cellular fates in the lateral ectoderm (Radke, 2001 and references therein).

Is there a receptor tyrosine kinase responsible for triggering the activation of the D-raf protein and MAP kinase in cells of the lateral ectoderm? In mammalian systems, mitogenic signaling by insulin in fetal rat, brown adipocyte, and primary cultures involves the activation of Ras and Raf-1 proteins. Insulin also triggers an increase in Raf-1 activity in several cell lines that expressed large numbers of insulin receptors (Radke, 2001 and references therein).

The Raf-MEK-MAP kinase cascade acts in a variety of cells to transmit RTK-generated signals during Drosophila development. The protein kinase activity of D-raf is required to elicit distinct ventral cell fates specified by the EGR receptor in early embryos. Using partial loss-of-function mutations in D-raf, cell fates normally specified by high levels of Egfr activity were lost while those that required lower receptor activity appeared normal or were expanded (Radke, 2001).

How is a graded pattern of cell types within a developmental field generated by a receptor tyrosine kinase? It has been hypothesized that the main function of the Raf-MEK-MAPK phosphorylation cascade is to amplify RTK-initiated signals. In this case, the quantity of activated Raf, MEK, and MAPK molecules is directly proportional to the number of receptor molecules activated, in the absence of feedback mechanisms. This information is then translated into position-dependent gene expression patterns that lead to morphological changes and cellular development. In this model, the quantity of activated RTK receptors defines the determined state of the cell. However, a number of studies in Drosophila reveal the existence of parallel signaling pathways emanating from a receptor during embryonic development. To extend the amplification hypothesis, the Raf-MEK-MAP kinase cascade may also act to integrate signals received from these parallel pathways and ultimately define precise transcriptional outcomes using a multistep mechanism. In mammalian cells, Raf-1 is regulated by a variety of inputs including the enzymatic function of PKC, Src, and Jnk kinases that upregulate activity. Autophosphorylation also plays a role in regulating Raf-1, as well as binding to Ras, 14-3-3, KSR, hsp90, and p50 proteins. In addition, PKA, Atk (PKB), and phosphatases have been implicated in the downregulation of Raf-1 function (Radke, 2001).

This study has addressed the consequences of eliminating key D-raf regulatory domains or specific serine residues that might act to integrate distinct signaling pathways in the Egfr pathway for ventral cell determination. In general, signal transmission was less reliable for D-raf proteins that lacked the negative regulatory site S388 (D-rafS388A) or the regulatory sequences CR1 and CR2 associated with the N-terminal one-half of the molecule (D-rafDelta445). However, both proteins showed the potential to transmit the highest level of ventral signal. This phenomenon was perhaps indicative of an important role played by the D-raf protein in the assembly of multiprotein complexes with components derived from parallel pathways. The full-length wild-type D-raf molecule, which contains several conserved motifs, may serve to bring parallel-signaling components together. Thus, the structural integrity of the D-raf protein may be important for the efficiency of complex assembly or its stability. In this model only complete and stable-signaling complexes achieve the highest level of signal output. It is speculated that in the case of D-rafS388A and more often for D-rafDelta445 proteins, complete signaling complexes were not built, leading to the phosphorylation of fewer D-MEK molecules, decreased signal output, and fewer cell fate choices specified within the Egfr developmental field (Radke, 2001).

In contrast, the Egfr signal was severely compromised when transmitted by either D-rafS743A or D-rafDelta315 proteins. The range of cell types specified by these mutant D-raf molecules was dramatically reduced from the wild type. In both cases, the establishment of cell fates that require the highest level of Egfr activity was consistently lost. Serine 743 may be important for the formation of D-raf dimers or oligomers as has been suggested for Raf-1. This type of complex may be essential for the generation of the highest level of ventral signal. In embryos that developed with D-rafDelta315 proteins, cell fates were generated that required substantially lower levels of Egfr activity. It is speculated that the wild-type D-raf protein undergoes release from negative regulation imparted by the CR2 domain via its N-terminal and CR1 sequences. In the case of the D-rafDelta315 protein, maintenance of the negative regulatory function of CR2 severely limited the ability of D-raf molecules to activate D-MEK. These results point to a multistep process in the generation of active D-raf molecules with multiple upstream factors acting in parallel. The highest level of D-raf signal was generated when all inputs were received. In the absence of one or several interactions the signaling potential of the D-raf protein was reduced, but not abolished (Radke, 2001).

Role of the EGFR/Ras/Raf pathway in specification of photoreceptor cells in the Drosophila retina

The Drosophila Egfr receptor is required for differentiation of many cell types during eye development. Mosaic analyses with definitive null mutations were used to analyze the effects of complete absence of Egfr, Ras or Raf proteins during eye development. The Egfr, ras and raf genes are each found to be essential for recruitment of R1-R7 cells. In addition Egfr is autonomously required for MAP kinase activation. Egfr is not essential for R8 cell specification, either alone or redundantly with any other receptor that acts through Ras or Raf, or by activating MAP kinase. As with Egfr, loss of ras or raf perturbs the spacing and arrangement of R8 precursor cells. R8 cell spacing is not affected by loss of argos in posteriorly juxtaposed cells, which rules out a model in which Egfr acts through argos expression to position R8 specification in register between adjacent columns of ommatidia. The R8 spacing role of the Egfr is partially affected by simultaneous deletion of spitz and vein, two ligand genes, but the data suggest that Egfr activation independent of spitz and vein is also involved. The results prove that R8 photoreceptors are specified and positioned by distinct mechanisms from photoreceptors R1-R7 (Yang, 2001).

It is thought that EGFR activity is required for recruiting R1- R7 photoreceptor cells to ommatidia, probably through Ras, Raf and MAPK but the role of this pathway in R8 specification has been less clear. Loss-of-function studies with putative Egfr null clones or temperature sensitivity have suggested that Egfr is dispensable for R8 specification (although involved in R8 spacing); studies with dominant negative approaches have suggested that Egfr is essential for R8 specification. There is also a particular class of Egfr mutants, the Elp alleles, that prevent R8 specification, and there is evidence that R8 specification might depend on Egfr-independent Raf activation. A study of null mutations in the Egfr/Ras/Raf pathway has been undertaken to resolve some of these issues. Two prior studies of Egfr mutant clones used the genetically amorphic point mutations flb1K35 and topCO. For topCO the molecular defect is unknown; flb1K35 corresponds to Gln267 in Ochre, which truncates the Egfr early in the extracellular domain. Although it is a reasonable assumption that these are both null alleles, it is worth noting that another mutation encoding Gln430 in Amber (top38) retains significant function, so the possibility of residual function in topCO or flb1K35 caused by readthrough, translational reinitiation or other mechanisms cannot be completely excluded. However, these possibilities can be excluded for the allele top18A, which deletes all Egfr-coding sequences from the genome. The phenotype of top18A clones is similar to flb1K35 and topCO. ato is expressed in top18A clones. It is concluded that cells completely lacking Egfr-coding capacity can still differentiate R8 photoreceptor cells, although their patterning is abnormal and they later die. Cells that completely lack Egfr are not recruited as any other photoreceptor type (Yang, 2001).

By the late third instar, cells in mutant clones have lacked Egfr gene function for approximately 120 hours. It is possible that cells might have a homeostatic mechanism (such as upregulation of another receptor) that compensates for sustained absence of Egfr function, and that some processes that would be Egfr-dependent in normal eye cells have been rescued in the clones. There is experimental evidence for such homeostasis from studies of the Egfrts2 allele. When Egfr function is interrupted, MAP kinase activation is lost from eye discs within 30 minutes, but levels of activated MAP kinase rebound within a few hours, even in the continued absence of Egfr function. MAP kinase activation was examined within clones of Egfr mutant cells. MAP kinase activation is undetectable. Thus, specification of R8 cells in Egfr mutant clones is not associated with MAP kinase reactivation via an alternative pathway. This finding indicates that the restored dpERK staining seen in the Egfrts2 allele must depend nonautonomously on loss of Egfr function in other cells. For example, loss of Egfr function from the whole animal may lead to changes in humoral signals that nonautonomously affect MAPK by some mechanism (Yang, 2001).

Genetic studies suggest that specification of most ommatidial cells depends on activation of Ras and Raf by Egfr (or by Egfr and Sevenless in the case of R7). R8 cell specification in the absence of Egfr might indicate activation of Ras and Raf by another receptor. Clones of cells null for Ras or Raf have been examined to test this. The null phenotype of Ras closely resembles that of Egfr. Ato expression initiated normally but patterning is affected and more cells than normal retain atonal expression posterior to the furrow. R8 cells are specified and express the R8 protein Senseless. No other Elav-expressing photoreceptor cells are recruited (Yang, 2001).

The phenotype of clones mutant for raf is similar. R8 cell specification begins relatively normally, as indicated by onset of Ato and Senseless expression. R8 cell precursors are improperly spaced, however. More posteriorly, raf mutant R8 cells express the neural protein Elav only transiently. These results also confirm directly that Ras and Raf are required for R1-R7 recruitment, and show that after these clones are induced in the first larval instar, Ras and Raf play no essential roles in the proliferation, survival or maintenance of eye disc identity of most eye disc cells (Yang, 2001).

Since null clones for Egfr, ras, and raf each permit R8 specification, although they affect R8 spacing, it is concluded that R8 specification can occur independently of Egfr, and is also independent of any other receptor that acts through Ras and Raf. Although the requirement for MAP kinase has not been tested directly (since the MAP kinase gene rolled maps proximal to all extant flip recombination target [FRT] sites), it was found that MAP kinase activation is undetectable in Egfr-null clones (Yang, 2001).

For both Egfr and ras, there is a nonautonomous delay of morphogenetic furrow movement and loss of ato, especially in large clones with substantial areas of mutant cells posterior to the furrow. This suggests Egfr and ras are required for expression of factors that push the morphogenetic furrow across the eye disc. Two such factors are Hh and Dpp. Hh is reported to be expressed by photoreceptor cells; therefore, fewer cells are expected to express Hh in ras or Egfr clones. There were some differences between clones mutant for raf and clones mutant for ras or Egfr. Less Elav is detected in raf mutant cells. In Egfr or ras mutant clones, Elav protein is detected in the mutant R8 cells, although at lower levels than in nearby wild type cells. In Egfr mutant clones, normal levels of Elav protein are restored by expression of baculovirus p35, indicating that low Elav levels reflect commitment of Egfr mutant cells to apoptosis. It is possible that Elav is lost more rapidly in raf mutant cells because of more rapid apoptosis than Egfr or ras mutant clones. Delayed furrow progression was not seen in raf mutant clones, but this may be because they were too small (Yang, 2001).

The differences between raf clones and Egfr or ras clones could indicate ras-independent signaling to raf, as has been proposed to occur during the determination of the embryonic termini. Such signaling to permit Elav expression in more R8 precursor cells (or preserve R8 precursor cells from apoptosis for longer) would have to be independent of Egfr as well, whereas all raf activity in the embryonic termini is dependent on torso, the relevant receptor. An alternative explanation is that these apparent differences relate to the much smaller size of raf clones compared with Egfr and ras clones. For the autosomal Egfr and ras mutations, the Minute technique was used to compensate for the growth disadvantage of the homozygous cells. This is not readily possible for the X-linked raf mutation. As a consequence, the raf clones examined were much smaller than the Egfr and ras clones, and grew at a reduced rate relative to neighboring wild-type cells. In the similar situation of Minute heterozygous clones growing slowly in wild-type backgrounds, nonautonomous interactions have been demonstrated, prolonging the cell doubling time of the slow-growing M/+ cells, and accelerating the doubling time of neighboring wild-type cells. If changes in cellular properties are also induced by the differential growth of neighboring homozygous raf mutant and wild-type cells, it is possible that faster loss of Elav might not indicate additional roles for raf in differentiation or survival, but an indirect effect of competition by the nearby wild-type cells on the raf minus cells. At present, experimental evidence to distinguish these models is not available (Yang, 2001).

The common requirements for Egfr, ras and raf in R8 spacing are not shown by null mutations in spi, which codes for an Egfr ligand required for recruitment of R1-R7. It is possible that spi is required redundantly with vn, another ligand with no essential role in ommatidium development. It was found that R8 precursor specification occurs in clones doubly mutant for both spi and vn. R8 spacing occurs almost normally, although there are rare cases of multiple R8 cells like those that occur more frequently in Egfr mutant clones. This raises the possibility that spi and vn do have redundant roles in R8 precursor spacing, but if this is so, there must be another ligand, or ligand-independent process, that is also involved. It has been found that the Drosophila genome sequence predicts another Spi-like protein. Cells doubly mutant for two putative ligand processing molecules encoded by rhomboid and roughoid resemble cells mutant for the Egfr. This suggests that rhomboid and roughoid redundantly process spi and spi-like, which act redundantly on Egfr in R8 spacing. The spi, spi-like double- and spi, spi-like, vn triple-mutant combinations that would directly test the relative contributions of all three ligands have yet to be examined (Yang, 2001).

The inhibitory ligand Argos is also required nonautonomously for R8 spacing. It had been suggested that Argos could diffuse from proneural intermediate groups, where it is expressed in response to Egfr activation, creating an 'exclusion zone' for further Egfr activation that will position future intermediate groups precisely out of phase. It was found, however, that Argos function can be performed by protein secreted several ommatidia away, which questions whether Argos conveys precise spatial information. Crucially, proneural intermediate groups are positioned normally even if immediately posterior regions are null mutant for argos, refuting the 'exclusion zone' model for argos action. Larger argos clones do affect R8 spacing distant from the clone boundary, suggesting that argos may be globally necessary in an unpatterned way to keep Egfr activity in check. An alternative is that argos is required indirectly through its effect on photoreceptor differentiation. Accordingly, ectopic photoreceptor cells in argos mutant territories might alter the expression of furrow progression signals such as Dpp and Hh (Yang, 2001).

The main result of this study is that R8 precursor specification occurs in cells null for Egfr, ras or raf. This is consistent with the proposed Egfr/Ras/Raf pathway of recruitment for photoreceptors R1-R7. These results appear definitively to exclude essential roles for Egfr, ras, raf, spi or vn, in R8 specification (although they support roles in R8 spacing), and show that argos is dispensable for the proposed signaling by each pair of proneural intermediate groups; each pair positions R8 specification in the next most anterior column. It is thought that R8 specification instead relies on autoregulatory transcription of the proneural ato gene promoted by two other DNA-binding proteins, daughterless and senseless that can occur without Egfr signaling. Defects in arrangement of R8 cell precursors show that the Egfr/Ras/Raf pathway nevertheless plays a role in the patterning of R8 cells. The increased number of R8 cells in mutants indicates that Egfr normally activates Ras and Raf to suppress R8 specification in certain locations. The Egfr pathway might modulate Notch. However, the Egfr requirement for R8 spacing was found to be more autonomous than the Egfr requirement for E(spl) expression, raising the possibility of another target. One candidate is the homeobox gene rough (Yang, 2001).


REFERENCES

Search PubMed for articles about Drosophila Raf

Anselmo A. N., Bumeister R., Thomas J. M. and White M.A. (2002). Critical contribution of linker proteins to Raf kinase activation. J. Biol. Chem. 277: 5940-5943. PubMed ID: 11741918

Barrera F. N., Poveda J. A., Gonzalez-Ros J. M. and Neira, J. L. (2003). Binding of the C-terminal sterile motif (SAM) domain of human p73 to lipid membranes. J. Biol. Chem. 278: 46878-46885. PubMed ID: 12954612

Bhattacharjya, S., Xu, P., Chakrapani, M., Johnston, L. and Ni, F. (2005). Polymerization of the SAM domain of MAPKKK Ste11 from the budding yeast: Implications for efficient signaling through the MAPK cascades. Protein Sci. 14: 828-835. PubMed ID: 15689513

Bumeister R., Rosse C., Anselmo A., Camonis J. and White M. A. (2004). CNK2 couples NGF signal propagation to multiple regulatory cascades driving cell differentiation. Curr. Biol. 14: 439-445. PubMed ID: 15028221

Chong, H., Vikis, H. G. and Guan, K. L. (2003). Mechanisms of regulating the Raf kinase family. Cell. Signal. 15: 463-469. PubMed ID: 12639709

Davies, H., et al. (2002). Mutations of the BRAF gene in human cancer. Nature 417: 949-954. PubMed ID: 12068308

Dhillon, A. S. and Kolch, W. (2002). Untying the regulation of the Raf-1 kinase. Arch. Biochem. Biophys. 404: 3-9. PubMed ID: 12127063

Ding, J., Tchaicheeyan, O. and Ambrosio, L. (2010). Drosophila Raf's N terminus contains a novel conserved region and can contribute to torso RTK signaling. Genetics 184(3): 717-29. PubMed ID: 20008569

Douziech, M., et al., (2003). Bimodal regulation of RAF by CNK in Drosophila. EMBO J. 22: 5068-5078. PubMed ID: 14517245

Douziech, M., Sahmi, M., Laberge, G. and Therrien, M. (2006). A KSR/CNK complex mediated by HYP, a novel SAM domain-containing protein, regulates RAS-dependent RAF activation in Drosophila. Genes Dev. 20(7): 807-19. PubMed ID: PubMed ID; Online text

Jaumot, M. and Hancock, J. F. (2001). Protein phosphatases 1 and 2A promote Raf-1 activation by regulating 14-3-3 interactions. Oncogene 20: 3949-3958. PubMed ID: 11494123

Kasten, M. and Giordano, A. (2001). Cdk10, a Cdc2-related kinase, associates with the Ets2 transcription factor and modulates its transactivation activity. Oncogene 20: 1832-1838. PubMed ID: 11313931

Kolch, W. (2000). Meaningful relationships: The regulation of the Ras/Raf/MEK/ERK pathway by protein interactions. Biochem. J. 351: 289-305. PubMed ID: 11023813

Laberge, G., Douziech, M. and Therrien, M. (2005). Src42 binding activity regulates Drosophila RAF by a novel CNK-dependent derepression mechanism. EMBO J. 24(3): 487-98. 15660123

Lanigan, T. M., et al. (2003). Human homologue of Drosophila CNK interacts with Ras effector proteins Raf and Rlf. FASEB J. 17: 2048-2060. PubMed ID: 14597674

Li, W., Melnick, M. and Perrimon, N. (1998). Dual function of Ras in Raf activation. Development 125: 4999-5008. PubMed ID: PubMed ID; Online text

Li, W., Noll, E. and Perrimon. N. (2000). Identification of autosomal regions involved in Drosophila Raf function. Genetics 156: 763-774. PubMed ID: PubMed ID; Online text

Light, Y., Paterson, H. and Marais, R. (2002). 14-3-3 antagonizes Ras-mediated Raf-1 recruitment to the plasma membrane to maintain signaling fidelity. Mol. Cell. Biol. 22: 4984-4996. PubMed ID: 12077328

Malumbres M. and Barbacid M. (2003). RAS oncogenes: The first 30 years. Nat. Rev. Cancer 3: 459-465. PubMed ID: 12778136

Morrison D. K. and Cutler R. E. (1997). The complexity of Raf-1 regulation. Curr. Opin. Cell Biol. 9: 174-179. PubMed ID: 9069260

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Biological Overview

date revised: 15 July 2011

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