The KSR kinase domain has been reported to interact with MEK but not phosphorylate MEK. KSR has also been found to interact with RAF, but the regions involved have not been identified. It was important to examine whether forced association between KSR and RAF catalytic domains (KSRc and RAFc) would affect MEK activity. Advantage was taken of two Drosophila transgenic lines that express, during eye development, either RAFc or KSRc fused to the N-terminal portion of Torso4021, which comprises the extracellular and transmembrane regions of the Torso receptor tyrosine kinase (N-Tor4021). Tor4021 is a gain-of-function allele of torso. The aberrant Torso4021 protein has a Y327C change in the extracellular portion that is thought to promote ligand-independent receptor oligomerization (Roy, 2002).
N-Tor4021RAFc behaves as an activated form of RAF and transforms cone cells into additional R7 photoreceptor cells when expressed in the developing Drosophila eye (Dickson, 1992). In contrast, N-Tor4021KSRc is a potent dominant-negative form of KSR that blocks signaling through the MAPK pathway, presumably by sequestering MEK. It strongly antagonizes photoreceptor cell differentiation (Therrien, 1996). It was reasoned that crossing flies expressing N-Tor4021RAFc (BT98) to flies expressing N-Tor4021KSRc (KDN) should lead to the formation of hetero-oligomers, which might result in activation of endogenous MEK and MAPK. BT98 and KDN transgenic flies were crossed to a wild-type strain or to each other to assess the effect on MAPK compared with wild-type flies. Third instar eye discs of the different genotypes were stained with an anti-phospho-MAPK (anti-pMAPK) antibody; compared with wild-type pMAPK levels, BT98/+ flies had slightly elevated pMAPK levels posterior to the morphogenetic furrow at a position consistent with the expected transgene expression pattern. In agreement with elevated pMAPK, BT98/+ adult eyes have additional R7 photoreceptor cells that result in a rough eye phenotype. In contrast, the KDN/+ flies have reduced pMAPK levels, which is also consistent with the roughening of the external adult eye surface due to a block in photoreceptor cell differentiation. Strikingly, the BT98/KDN flies show a massive accumulation of pMAPK posterior to the morphogenetic furrow that correlates with a robust enhancement of activated RAF rough eye phenotype. These findings strongly suggest that N-Tor4021KSRc brings endogenous MEK to N-Tor4021RAFc, thus strongly enhancing endogenous MEK and MAPK activation (Roy, 2002).
To verify that the association between RAFc and KSRc is responsible for MEK phosphorylation, RAFc and KSRc were fused to the FK506-binding protein (FKBP) and FKBP-rapamycin-binding domain (FRB), respectively, to allow their heterodimerization in a rapamycin-dependent manner. A functional or inactivated myristoylation signal was introduced to examine the influence of membrane localization. RAFc and KSRc derivatives were tagged with the polyoma (pyo) and the Flag epitopes, respectively, to allow their detection. The FKBP-RAFc and FRB-KSRc fusion proteins were expressed separately or together along with myc-epitope-tagged MEKDA in the absence or the presence of rapamycin. Expression of KSRc or RAFc variants alone or together does not result in MEK phosphorylation in the absence of rapamycin. As expected, FRB-KSRc physically interacts with MEK as revealed by the associated mycMEKDA in anti-Flag immunoprecipitates, whereas the FKBP-RAFc did not interact with MEK. Rapamycin treatment does not affect the behavior of the KSRc and RAFc constructs when they are expressed alone, but promotes their heterodimerization when expressed together. This results in complex formation between RAF and MEK and leads to MEK phosphorylation. Interestingly, this effect is accompanied by a clear and strong mobility shift of FKBP-RAFc. Permutation of the myristoylation signal or no myristoylation signal on the two types of fusion proteins give identical results, which suggests that no additional membrane components are required in this event. Together, these results show that induced association between RAFc and KSRc results in MEK phosphorylation (Roy, 2002).
To address whether MEK phosphorylation depends on the catalytic function of RAFc or KSRc, the activity of two kinase-inactivated mutants, RAFcK498S and KSRcK705M was tested. As expected, kinase-inactivated RAFc does not support rapamycin-dependent MEK phosphorylation. Interestingly, this mutant does not display a mobility shift. In contrast, KSRcK705M, which interacts normally with MEK, does promote MEK phosphorylation, although not as efficiently as wild-type KSR. These results suggest that the ability of KSR to promote MEK phosphorylation does not absolutely require a catalytically intact function (Roy, 2002).
Although previous genetic and biochemical data on KSR have suggested its involvement in the MAPK module, they have never demonstrated it. The RNAi technique was used to deplete endogenous KSR levels in S2 cells as well as the levels of the other major components and examine the effect on MAPK activation. A stable S2 cell line expressing RASV12 under the control of a heavy metal-inducible promoter was used to activate the MAPK pathway. Addition of copper to cell culture media augmented total RAS1 levels, which resulted in a modest increase in phospho-MEK (pMEK) and a robust elevation of pMAPK (Roy, 2002).
To further unravel the molecular function of KSR, a KSR-dependent MEK phosphorylation assay was reconstituted using native KSR and RAF proteins. However, mouse KSR1 has been reported to block signaling through the ERK/MAPK pathway when overexpressed in various cell lines. This effect is likely owing to sequestration of specific components of the pathway that are in limiting amounts. RAF and MEK are prime candidates, as they have been reported to interact with mKSR1. To circumvent this problem, KSR was coexpressed together with RAF and MEK. RASV12 was also used to activate the MAPK pathway. To prevent possible MAPK-dependent negative-feedback effects, a kinase-inactivated version of MEK (MEKDA) was used throughout this work. Compared with myc-epitope-tagged MEKDA expressed alone, RASV12 coexpression slightly increases pMEK levels. Coexpression of KSR or pyo-epitope-tagged RAF in those two conditions (absence or presence of RASV12) does not significantly alter the pMEK profiles. Strikingly, coexpression of MEK with KSR and RAF without RASV12 induces MEK phosphorylation, which strongly increases upon addition of RASV12. These results suggest that native KSR promotes the ability of RAF to phosphorylate MEK. Although RAS activity strongly increases the effect of KSR, the fact that an effect of KSR is detected in the absence of RASV12 suggests among different possibilities that either endogenous RAS activity contributes to that effect or that the higher levels of the transiently expressed proteins somewhat bypassed the normal RAS-dependency of MEK phosphorylation by RAF (Roy, 2002).
Next, tests were performed to see whether the kinase function of RAF and the putative kinase function of KSR were required for MEK phosphorylation in this assay. Kinase-inactivated mutants for pyoRAF (K498M) and KSR (K705M) were generated, and their respective effect was tested. As expected, RAF kinase function is absolutely required for MEK phosphorylation. In contrast, KSRK705M still promotes MEK phosphorylation, although slightly less efficiently than wild-type KSR. The ability of KSR to stimulate MEK phosphorylation does not absolutely depend on its putative kinase function (Roy, 2002).
Advantage was taken of the simple cotransfection assay in S2 cells to examine the effect of five additional mutations in KSR. Evidence had been presented that mKSR1 can phosphorylate RAF. As a kinase-inactive mutant, two conserved aspartic residues were changed in subdomains VI and VII to alanine residues. Because the KSRK705M mutant is still active, a KSR kinase-defective mutant [similar to the reported mutants (D800A-D817A)] was generated to test its effect. The four other mutants affected independent regions of KSR. KSRL50S-R51G lies within the CA1 domain. This mutation has been recovered as a hypomorphic loss-of-function allele in a RAS-dependent genetic screen in Drosophila (Therrien, 1995). KSRC398S-C401S disrupts the integrity of the cysteine-rich motif. A similar mutation in mKSR1 has been shown to abrogate KSR's ability to promote RAS-dependent Xenopus oocyte maturation. KSR contains an FXFP motif that has been proposed to function as a MAPK docking site. The function of this motif in KSR is unknown. The first phenylalanine residue of the motif, KSRF518W, was changed to verify its functional relevance. Finally, a mutation in KSR isolated in a RAS-dependent genetic screen in C. elegans (Sundaram, 1995) has been reported to prevent the association between KSR and MEK (Stewart, 1999). A similar mutation, KSRC922Y, was generated to examine the effect of impairing the KSR/MEK interaction. Interestingly, in contrast to KSRK705M, which still supports MEK phosphorylation, KSRD800A-D817A and KSRC922Y completely abrogates KSR activity. In addition, KSRL50S-R51G shows a severe reduction of activity, whereas KSRC398S-C401S shows only a slight, but reproducible reduction in KSR activity, and KSRF518W is as active as wild-type KSR. Given that the two KSR mutations (L50S-R51G and C922Y) corresponding to loss-of-function mutations isolated in genetic screens also disrupts the ability of KSR to induce MEK phosphorylation by RAF, it strongly suggests that the assay recapitulates the normal function of KSR (Roy, 2002).
It was then investigated whether the effect of the mutations could be caused by defects in KSR's ability to associate with MEK and/or RAF. To verify this, KSR or the mutant variants were immunoprecipitated from cell lysates used to analyze pMEK levels and the amount of coimmunoprecipitated mycMEKDA and pyoRAF was examined. As reported for mKSR1, Drosophila KSR also associates with MEK and RAF. Two mutants, KSRD800A-D817A and KSRC922Y, do not interact with MEK. Interestingly, they also fail to stimulate MEK phosphorylation. In contrast, KSRK705M, which is almost as active as wild-type KSR, is not significantly affected by its association with MEK. These results suggest that the ability of KSR to interact with MEK is critical to stimulate MEK phosphorylation. Intriguingly, KSRL50S-R51G associates normally with MEK, yet is severely diminished in its capacity to promote MEK phosphorylation. Although no drastic effect regarding the binding properties of this mutant was observed, there is a very reproducible demonstration of an approximately twofold decrease in KSR's capacity to associate with RAF. This observation suggests that the CA1 domain mediates an association with RAF. KSRD800A-D817A and KSRC922Y also show a similar RAF-association defect. Their reduced interaction with RAF might be caused by their inability to bind MEK, which normally might stabilize the KSR/RAF interaction. Alternatively, these mutations might induce structural perturbations that reduce the KSR/RAF association independent of the inability of MEK to bind KSR. Taken together, these data indicate that KSR associates independently with MEK and RAF, and that these interactions appear to be critical for KSR activity (Roy, 2002).
Previous work has reported that the association between mKSR1 and RAF is RAS-dependent (Therrien, 1996; Xing, 1997). Interestingly, Drosophila KSR can associate with RAF without coexpressing an activated form of RAS. It was therefore of interest to determine whether endogenous RAS1 activity might contribute to this interaction. To verify this, pyoRAF and KSR were coexpressed in the presence of dsRNA for GFP, RAS1, MEK, or MAPK; then the levels of immunoprecipitated RAF and the levels of associated KSR were measured. As for the negative controls, addition of dsRAS1 RNA did not perturb the KSR/RAF association, which suggests that the Drosophila KSR/RAF interaction can occur in the absence of RAS-mediated signals. The inability of dsMEK RNA to alter the KSR/RAF association is another indication that KSR interacts with RAF independently of MEK (Roy, 2002).
Given that KSR appears to associate independently with RAF and MEK, tests were performed to see whether KSR might physically link RAF and MEK together. Fixed amounts of RAF and MEK were coexpressed alone or in the presence of increasing amounts of KSR, and the levels of associated MEK were evaluated by probing the anti-pyo immunoprecipitates with anti-myc. Coexpression of RAF and MEK alone does not result in the formation of a stable RAF/MEK association. In sharp contrast, expression of KSR allows the formation of a RAF/MEK complex. The levels of MEK associated with RAF increase upon augmenting the expression levels of KSR as predicted if KSR physically connects RAF and MEK. If this model is correct, KSRC922Y should not induce the formation of a RAF/MEK complex because it no longer binds to MEK. Interestingly, when coexpressed with RAF and MEK, wild-type KSR shows greater protein stability compared with KSRC922Y. Therefore more DNA for the mutant construct was transfected to compare the effect of equal amounts of proteins. As predicted, KSRC922Y can not promote the formation of a RAF/MEK complex (Roy, 2002).
The fact that KSR induces the formation of a RAF/MEK complex in the absence of cotransfected RASV12 is intriguing. Whether endogenous RAS1 activity is involved in the RAF/MEK complex formation was therefore verified by eliminating RAS's contribution using dsRAS1 RNA. Surprisingly, dsRAS1 RNA does not affect the complex. Similarly, coexpression of RASV12 does not significantly enhance its formation. Together, these findings strongly suggest that KSR connects RAF and MEK and that the assembly does not require RAS activity (Roy, 2002).
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, the 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 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).
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