BIOLOGICAL OVERVIEW
RAF is a critical effector of the small GTPa RAS in normal and malignant cells. Despite inten scrutiny, the mechanism regulating RAF (FlyBa name: Pole hole) activation remains partially understood. This study shows that the scaffold KSR (kina suppressor of RAS), a RAF homolog known to asmble RAF/MEK/ERK complexes, induces RAF activation in Drosophila by a mechanism mediated by its kina-like domain, but which is independent of its scaffolding property or putative kina 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; FlyBa name - Aveugle). Moreover, the data suggest that the interaction of KSR to CNK/HYP stimulates the RAS-dependent RAF-activating property of KSR. Together, the 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 kina) module, but may also function as a RAF activator. By analogy to catalytically impaired, but conformationally active B-RAF oncogenic mutants, the possibility is discusd that KSR reprents 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 kinas
e (MAPK) module, is a central event triggered by the small GTPa RAS to regulate a number of basic cellular process in metazoans, including cell proliferation, differentiation, and survival (Pearson, 2001). Unrestrained signaling through this pathway caud, for instance, by activating mutations in specific isoforms of either RAS or RAF, has been linked to veral types of cancer in humans and, for some of the, at an impressively high frequency (Malumbres, 2003; Wellbrock, 2004). Becau 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 rine/threonine kina (Douziech, 2006).
Three RAF members have been identified in mammals (A-RAF, B-RAF, and C-RAF/Raf-1) and homologs are prent in other metazoans, including Caenorhabditis elegans and Drosophila, where a single gene encoding a protein more cloly related to B-RAF has been identified (Dhillon, 2002; Chong, 2003). RAF proteins compri 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 prot
eins, which in turn mediates their cytoplasmic retention in unstimulated cells (Morrison, 1997). Upon receptor tyrosine kina (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 conrved rine/threonine residues (T599 and S602 in B-RAF) situated in the activation loop of the kina domain (Zhang, 2000; amino acid numbering of B-RAF is according to Wellbrock, 2004). Mutational analys as well as a recent crystallographic study of the B-RAF kina domain strongly suggest that phosphorylation of the 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 kina domain (Wan, 2004). The mechanism and kina(s) leading to the phosphorylation of the 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 kina suppressor of RAS (KSR) members, which are known to asmble RAF, MEK, and MAPK into functional complexes
(Morrison, 2003). Interestingly, the proteins are structurally related to RAF, although they have some key differences. For instance, they do not contain an RBD, but compri a conrved region called CA1 that was found in Drosophila to mediate an interaction between KSR and RAF (Roy, 2002). Further, they posss a kina-like domain that constitutively binds MEK, but which appears to be devoid of kina 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; Anlmo, 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 esntial 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 quence called the RAF-interacting motif (RIM) and modulates RAF activity according to the RTK signaling status (Douziech,
2003; Laberge, 2005). In the abnce of RTK signals, CNK-bound RAF is inhibited by a cond motif adjacent to the RIM, called the inhibitory quence (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 conrved region in CNK (CRIC) located in the N-terminal region of CNK (Douziech, 2003). The molecular role of the domains is currently unknown. In contrast, the binding of a Src family kina, Src42, to an RTK-dependent phospho-tyrosine residue (pY1163) located C-terminal to the IS motif appears to relea the inhibitory effect that the IS motif impos on RAF catalytic function (Douziech, 2006).
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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 kina domain appears to be the primary effector of this event, CNK participates in at least two ways: (1) It asmbles a KSR/RAF complex in vivo by int
eracting parately with the kina 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 conrved SAM domain-containing protein, Hyphen, who prence is esntial 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 propod 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, the 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 kina activity, as mutagenesis of an esntial 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 kina is recruited to accomplish this task or RAF itlf is executing it (Douziech, 2006).
橙子英文
Interestingly, CNK and HYP do not exhibit any positive activity unless KSR is prent, 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 expresd at sufficiently high levels as if it carries an intrinsic RAF-activating property that is unveiled when overexpresd 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 bad on the position in which KSR operates during this event and on the strong architectural and amino acid quence 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 t
he 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, decread intrinsic kina activity. As the mutations were capable of elevating endogenous ERK activity by their ability to stimulate endogenous wild-type RAF proteins, it has been propod that a catalytically impaired but conformationally derepresd RAF kina 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 kina 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 kina domain of RAF. This scenario might explain why the RAF-AL AA 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 bring
ing other RAF-activating proteins or questering inhibitory proteins from RAF. The identification of two mutations (KSR A696V–A703T and KSR R732H) 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 juxtapos a KSR/MEK complex to RAF and, owing to the IS of CNK, maintains this higher-order complex in an inactive state by lectively 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 releas 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 nds 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 asmbly 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 ud by KSR to drive RAF activation, it is likely that a similar functional interaction between the kina domains of KSR and RAF has been conrved 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 kina by GTP-bound Ras is a poorly understood step in receptor tyrosine kina 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 bad on its re
quirement for normal photoreceptor differentiation. The phenotypes of aveugle mutant cells in the eye and wing imaginal discs remble tho caud by reduction of EGFR pathway function. aveugle is required between ras and raf for EGFR signaling in the eye and for mitogen-activated protein kina 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 propod that Aveugle acts together with Cnk to promote Raf activation, perhaps by recruiting an activating kina (Roignant, 2006).
Key steps in Raf activation include Raf translocation to the plasma membrane and relea of its protein kina domain from an intramolecular inhibitory domain through changes in the phosphorylation state of specific residues. The process occur in the context of the esntial scaffolding proteins Cnk and Ksr. Ave is required between Ras and Raf for EGFR signaling in differentiating photoreceptors and in S2 cells, and is prent 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 abnce of ave, and onl住房出租模板
y 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 increa the overall level of EGFR signaling. It is noted that MAPK phosphorylation is undetectable in the abnce of ave in both eye disc cells and S2 cells, suggesting that examination of EGFR respons in vivo is more nsitive 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 subclass 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 respon 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 elev
冰柜什么牌子的好ated 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 quential induction of late-differentiating cell types (Roignant, 2006).拓跋氏
宋白In addition to photoreceptor differentiation, EGFR signaling in the eye is required for cell survival and cell cycle arrest; the two functions have been propod 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 increa 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 respons; the level of EGFR signaling achieved in the abnce of ave can allow differentiation of some photoreceptors without preventing all apoptosis (Roignant, 2006).
The requirement for Ave in other EGFR-dependent process appears to be variable. In the wing disc, ave is esntial for notum growth and for expression of the EGFR target gene aos; aos is likely
to be a high-threshold target, as it is expresd 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 expresd at this stage of development, although no clo quence homolog is prent in the Drosophila genome. Alternatively, the Ras/MAPK module may u 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 esntial 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 esntial 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, ems to have an inhibitory effect on Ras signaling (Douziech, 2003). No change in the strength of the interaction between Raf and Cnk has been obrved when ave is removed by RNAi. A more likely possibility is that association of Ave with Cnk helps to bring an activator kina into proximity with Raf. Raf activation in mammalian cells involves dephosphorylation of inhibitory sites followed by phosphorylation of activating sites (for review, e Dhillon, 2002; Chong, 2003). However, the identity of the activating kinas is still unclear; Ksr was a candidate, but the current view is that it acts as a scaffolding protein rather than an active kina (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 kina to Raf. Certain SAM domains have been shown to act as kina-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). Likewi, the ETS-2 SAM domain rves as a docking site for the Cdc2 family kina Cdk10 (Kasten, 2001). A arch for other binding partners of Ave may lead to the identification of the activating kina 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 (Anlmo, 2002), but it may not be sufficient for this function, since overexpression in CHO cells of MAGUIN-1, the clost 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 en 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 incread. 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). T
his may stabilize a complex in which the Ste20 kina can phosphorylate Ste11 (Ramezani-Rad, 2003). A stabilizing function might explain why ave is not esntial in all contexts in Drosophila, as high concentrations of the molecules it recruits could lead to Ave-independent signaling. The evolutionary conrvation 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 conrved region and can contribute to torso RTK signaling
中国梦心得体会Drosophila Raf (DRaf) contains an extended N terminus, in addition to three conrved regions (CR1-CR3); however, the function(s) of this N-terminal gment remains elusive. In this study, a novel region within Draf's N terminus that is conrved in BRaf proteins of vertebrates was identified and termed conrved region N-terminal (CRN). The N-terminal gment can play a positive role(s) in the Torso receptor tyrosine kina pathway in vivo, and its contribution to signaling appears to be dependent on the activity of Torso receptor, suggesting this N-terminal gment 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 condary structure as predicted by proteomics tools. In yeast two-hybrid assays, stronger interactions between
温州有什么好玩的地方旅游景点DRaf's Ras binding domain (RBD) and the small GTPa Ras1, as well as Rap1, were obrved when CRN and RBD quences were linked. Together, the 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, conrved for Raf genes of most invertebrates and BRaf genes of vertebrates, was identified and termed CRN. This conrved region has not been described by others, but potential roles for the extended N terminus have been propod 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 GTPa Ras1δCAAX were obrved when N-terminal and RBD quences 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 GTPa Rap shares with Ras nearly identical Raf binding regions that compri 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 respons 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
