InteractiveFly: GeneBrief

connector enhancer of ksr: Biological Overview | References

BIOLOGICAL OVERVIEW

In Drosophila melanogaster, the receptor tyrosine kinase (RTK) Anaplastic lymphoma kinase (Alk) and its ligand Jelly belly (Jeb) are required to specify muscle founder cells in the visceral mesoderm. This study identified a critical role for the scaffolding protein Cnk (Connector enhancer of kinase suppressor of Ras) in this signaling pathway. Embryos that ectopically expressed the minimal Alk interaction region in the carboxyl terminus of Cnk or lacked maternal and zygotic cnk did not generate visceral founder cells or a functional gut musculature, phenotypes that resemble those of jeb and Alk mutants. Deletion of the entire Alk-interacting region in the cnk locus affected the Alk signaling pathway in the visceral mesoderm and not other RTK signaling pathways in other tissues. In addition, the Cnk-interacting protein Aveugle (Ave) was shown to be critical for Alk signaling in the developing visceral mesoderm. Alk signaling stimulates the MAPK/ERK pathway, but the scaffolding protein Ksr, which facilitates activation of this pathway, was not required to promote visceral founder cell specification. Thus, Cnk and Ave represent critical molecules downstream of Alk, and their loss genocopies the lack of visceral founder cell specification of Alk and jeb mutants, indicating their essential roles in Alk signaling (Wolfstetter, 2017).

Receptor tyrosine kinase (RTK) signaling plays an essential role in development by transducing external signals into the nucleus and other cellular compartments, thereby altering gene expression and promoting intracellular responses. The hallmarks of RTK signaling are conserved among eukaryotic organisms and involve ligand-dependent activation of a transmembrane receptor protein tyrosine kinase and the recruitment of canonical intracellular signaling modules and cascades, such as the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathway. Alk activation stimulates this pathway through the guanosine triphosphatase Ras and the serine-threonine kinases Raf [a MAPK kinase kinase (MAPKKK)], MEK (a MAPK kinase), and MAPK/ERK. Other factors that contribute to or modulate the activity of this pathway have been identified, such as the kinase suppressor of Ras (Ksr), which was identified by mutagenesis screens in Ras-sensitized genetic backgrounds in Drosophila melanogaster and Caenorhabditis elegans. Because of inconsistent findings regarding the catalytic activity of its kinase domain, the role of Ksr has remained controversial. Different models have proposed distinct roles for Ksr as an activator of Raf in parallel to or downstream of Ras or as a scaffolding protein for the assembly of Raf-MEK protein complexes. There is no evidence for a direct interaction between Ksr and Ras, but dimerization between Ksr and Raf can stimulate Raf activity in a manner that is independent of the kinase activity of Ksr, suggesting that Ksr may act as a scaffold in the context of RTK signaling (Wolfstetter, 2017).

A genetic modifier screen using ectopic expression of a dominant-negative, chimeric version of Ksr in the fly eye led to the identification of another critical factor for Ras-ERK signaling named Connector enhancer of kinase suppressor of Ras (Cnk). The cnk locus encodes a large protein of 1557 amino acids containing an N-terminal sterile α motif (SAM), followed by a conserved region in Cnk (CRIC), a PDZ domain, proline-rich motifs, and a pleckstrin homology (PH) domain. The protein structure suggests that Cnk acts as a multidomain protein scaffold. Like Ksr, Cnk functions downstream of various RTK signaling events including epidermal growth factor receptor (EGFR)-tribution of Cnk to embryonic Torso signaling, supporting the finding - and EGFR-dependent air sac development in the dorsal thorax. Ectopic expression of the Cnk N-terminal region enhances the effects of activated RasV12 independently of MAPK/ERK activation in the Drosophila eye. The C-terminal region contains a Raf inhibitory region (RIR) that binds to and represses Raf, which is released upon phosphorylation of Cnk by the Src family kinase Src42A. Thus, Cnk functions as a molecular scaffold to support Ksr-mediated Raf activation and to recruit and integrate additional signaling components such as Src42A (Wolfstetter, 2017).

During embryonic development in D. melanogaster, the visceral mesoderm (VM) gives rise to a lattice of midgut muscles that ensheaths the larval midgut. The VM consists of naïve myoblasts that become specified as either founder cells (FCs) or fusion competent myoblasts (FCMs). Subsequently, the FCs fuse one-to-one with FCMs and eventually form the binucleate visceral myotubes. Specification of VM cells requires the Drosophila ortholog of the receptor anaplastic lymphoma kinase (ALK), initially identified as part of a chimeric protein created by the 2;5 (p23:q35) translocation in human anaplastic large cell lymphoma cell lines. Drosophila Alk is expressed in the segmental clusters of the embryo that segregate from the dorsal trunk mesoderm to form the VM. Alk protein can be detected at the membrane of all VM cells, but only the distal arch within each cluster comes into direct contact with a secreted, small low-density lipoprotein domain ligand named Jelly belly (Jeb). Binding of Jeb to the extracellular part of Alk activates a downstream signaling cascade that results in ERK phosphorylation and triggers expression of an FC-specific subset of genes including Hand, optomotor-blind-related-gene-1 (org-1), and kin of irre (kirre; also referred to as dumbfounded or duf). Jeb-Alk signaling is crucial for visceral myoblasts to commit to the FC fate. In the absence of either ligand or receptor, neither ERK phosphorylation nor the expression of FC-specific marker genes in the VM occurs. Moreover, visceral cells fail to undergo myoblast fusion, and the VM subsequently disintegrates in jeb and Alk mutant embryos (Wolfstetter, 2017).

This study has identified the multidomain scaffolding protein Cnk as a potential Alk binding partner and essential component in the Alk signaling pathway. Cnk bound to the intracellular part of Alk by its C-terminal region. Loss of cnk function or expression of dominant-negative cnk constructs in Drosophila interfered with Alk signaling in multiple developmental contexts. Moreover, germline clone-derived embryos lacking maternal and zygotic Cnk failed to specify visceral FCs and did not develop a functional midgut. In agreement with its proposed function, epistasis experiments revealed that Cnk operates between Ras and Raf in the Alk signaling pathway. Further targeted deletion of a minimal Alk interaction region (AIR) in Cnk resulted in a specific decrease of Jeb-Alk-induced ERK phosphorylation within the visceral FC row. Deletion of the larger Alk interacting region blocked specification of visceral FCs in response to Alk activation. Although the SAM domain containing Cnk binding partner Aveugle (Ave) was essential for Alk signaling, it was found that Ksr is not essentially required to drive Alk signaling in the developing VM. Thus, Cnk and its binding partner Ave serve as critical components for Alk signaling in Drosophila (Wolfstetter, 2017).

This study uncovered an essential function for the protein scaffold Cnk in Alk signaling. The identification of multiple Cnk preys as AlkICD interactors in the Y2H analysis revealed a region in Cnk that likely mediates this interaction and allowed definition of a minimal AIR that was sufficient to bind Alk. The importance of Cnk in Alk signaling was supported by the loss of FCs in the VM of germline clone-derived cnk mutants [cnk (m-/z-)], which genocopied the embryonic Alk loss-of-function phenotype and the dominant-negative effect of ectopic CnkAIR expression on visceral FC specification. The tissue-specific decrease of ERK phosphorylation in the visceral FC row of cnkΔAIR mutants and the loss of visceral FCs upon deletion of the entire Alk interacting region identified by the Y2H approach further support a direct interaction between Cnk and Alk (Wolfstetter, 2017).

Various interactions between Cnk and membrane-associated factors (although not other RTKs) have been reported. In cultured mammalian cells, CNK1 promotes insulin signaling by binding to and localizing cytohesins at the plasma membrane, and binding of CNK1 to the transmembrane ligand EphrinB1 links fibronectin-mediated cell adhesion to EphrinB-associated JNK signaling. Moreover, mammalian CNK2 (also called MAGUIN), the mammalian CNK homolog most similar to Drosophila Cnk, binds to various members of the membrane-associated MAGUK family proteins and Densin-180. It will be interesting to determine whether the RTK binding capacities of Cnk are limited to Alk (Wolfstetter, 2017).

Cnk localizes close to the plasma membrane. Although the Alk-Cnk interaction appears to be important for ERK activation and visceral FC specification in the VM, Alk does not appear to be required for the subcellular localization of Cnk. Whether the Alk-Cnk interaction depends on Alk activity, potentially resulting in posttranslational modification of Cnk, will be interesting to pursue in further studies. Notably, Drosophila Cnk is tyrosine-phosphorylated upon coexpression with the activated form of the RTK Sevenless (SEVS11) in S2 cells, and activation of the platelet-derived growth factor receptor induces tyrosine phosphorylation of mammalian CNK1, leading to changes in CNK1 subcellular localization (Wolfstetter, 2017).

Cnk is generally abundant in the Drosophila embryo where its function is required in various RTK signaling pathways. However, the CnkAIR appears to be critical only for Alk signaling. Morphological analysis further reveals a minor contribution of Cnk to embryonic Torso signaling, supporting the finding that Torso signals are processed by three or more parallel branches. This finding also agrees with earlier analyses of embryonic Torso signaling in ave^m-/z- mutants, which form terminally derived structures. Notably, the differences in tracheal phenotypes caused by the cnk63F null allele and the strong, Raf-repressing cnk^sag alleles, which have C-terminal nonsense mutations and abrogate Alk signaling in the VM, indicate that distinct Cnk domains are involved in specific RTK-signaling pathways. This notion is also supported by the observation that the strong, Heartless-related phenotypes exhibited by cnk^m-/z- null mutants were barely apparent in cnkΔY2H ^m-/z- embryos, which, on the other hand, displayed a complete loss of Alk-driven FC specification in the VM (Wolfstetter, 2017).

Selectivity for a requirement of CNK by different RTKs has also been observed in mammalian cells because CNK2 appears to be required for nerve growth factor, but not EGF-induced ERK activation in PC12 cells. Therefore, Cnk contributes to multiple signaling events, but its importance to different RTKs varies, perhaps reflecting differential wiring of downstream signaling in different developmental processes, an aspect that will be interesting to explore in future studies. Cnk has been described as a protein scaffold that facilitates Ras-Raf-MAPK signaling at the plasma membrane, allowing signal integration to enhance Raf and MAPK activation. The epistatic analysis presented in this study shows that Cnk is required downstream of activated Alk and RasV12 but upstream of activated Raf in the VM, which agrees with previous studies in Drosophila. Thus, activated Ras seems to require Cnk to transmit signaling to Raf in the VM (Wolfstetter, 2017).

Alk activation at the membrane of a prospective visceral FC by the ligand Jeb, which is secreted from the neighboring somatic mesoderm, induces the Raf/MAPK/ERK signaling cascade, eventually leading to the transcriptional activation of downstream targets including kirre, org-1, and Hand. Cnk and Ave are core components of the Alk signaling pathway that are required downstream of the receptor and upstream of Raf to mediate visceral FC specification. Although not essential for Jeb-Alk signaling, Ksr appears to be required for full activation of ERK (Wolfstetter, 2017).

Ave directly binds to Cnk through an interface formed by their SAM domains. This interaction is thought to be necessary to recruit Ksr to a complex that in turn promotes Raf activation in the presence of activated Ras. Although this study identified Ave as a critical component for Cnk function downstream of Alk, the single Ksr in Drosophila was not required for Alk-mediated FC specification. Cnk was originally identified in a Ksr-dependent genetic screen in Drosophila, and its function has been proposed to mediate the association between Ksr and Raf, suggesting that Ksr should also play an important role in Alk signaling. However, the role of Ksr is unclear, with early reports suggesting an inhibitory function rather than an activating potential in RTK signaling). Ksr requires the presence of additional factors such as 14-3-3 proteins or activated Ras, and loss of Ksr-1 suppresses RasE13-induced but not wild-type signaling during C. elegans vulva formation, suggesting altered affinities of Ksr for different variants of Ras. Because this analyses revealed a function for ksr in driving robust ERK phosphorylation, it is plausible that, although nonessential, Ksr might enhance Alk signaling by integrating signals from activated Ras to Cnk-associated Raf (Wolfstetter, 2017).

The importance of ERK activation in RTK-mediated signaling in the Drosophila embryo is difficult to address directly because the rolled locus (encoding the only MAPK/ERK1-2 ortholog in Drosophila) is located close to the centromere and therefore not accessible for standard germline clone analysis. The removal of the 42-amino acid AIR from Cnk by genomic editing of the cnk locus suggests that ERK phosphorylation, even at decreased amounts, is sufficient to promote Alk-induced specification of visceral FCs in vivo. Whereas the AIR is sufficient to mediate the Alk-Cnk interaction in vitro, the ability of the cnkΔAIR mutant to support Alk-driven FC specification in the developing embryo highlights additional requirements. The Y2H analysis supports a direct interaction between Alk and Cnk mediated by a more extensive binding interface within the Alk-Y2H binding region of Cnk. Accordingly, deletion of this region in the cnk locus (cnkΔY2H) blocks VM specification in vivo. However, the possibility cannot be excluded that the Y2H region of Cnk may form interactions with additional Alk binding proteins, which could mediate the Alk-Cnk interaction in an indirect manner (Wolfstetter, 2017).

In summary, this study has identified an interaction between Alk and Cnk mediated by an Alk binding region in Cnk. This region was specifically required for the activation of ERK and formation of FCs downstream of the activated Alk receptor in the Drosophila VM. Together with Ave, Cnk represents an important signaling module that is required for Alk-mediated signaling during embryogenesis. Cnk and Ave represent molecules identified downstream of Alk, whose loss genocopies the lack of visceral FC specification of Alk and jeb mutants. Further work should allow a better understanding of the importance of Cnk in Alk signaling and whether this is conserved in mammalian systems (Wolfstetter, 2017).

Apical accumulation of the Sevenless receptor tyrosine kinase during Drosophila eye development is promoted by the small GTPase Rap1

The Ras/MAPK-signaling pathway plays pivotal roles during development of metazoans by controlling cell proliferation and cell differentiation elicited, in several instances, by receptor tyrosine kinases (RTKs). While the internal mechanism of RTK-driven Ras/MAPK signaling is well understood, far less is known regarding its interplay with other co-required signaling events involved in developmental decisions. In a genetic screen designed to identify new regulators of RTK/Ras/MAPK signaling during Drosophila eye development, the small GTPase Rap1, PDZ-GEF, and Canoe as components contributing to Ras/MAPK-mediated R7 cell differentiation. Rap1 signaling has recently been found to participate in assembling cadherin-based adherens junctions in various fly epithelial tissues. This study shows that Rap1 activity is required for the integrity of the apical domains of developing photoreceptor cells and that reduced Rap1 signaling hampers the apical accumulation of the Sevenless RTK in presumptive R7 cells. It thus appears that, in addition to its role in cell-cell adhesion, Rap1 signaling controls the partitioning of the epithelial cell membrane, which in turn influences signaling events that rely on apico-basal cell polarity (Baril, 2014).

This report describes a genetic screen in Drosophila for dominant modifiers of a CNK-dependent rough eye phenotype. Two of those modifiers, Rap1 and PDZ-GEF, were characterized to further shed light on the mechanism by which Rap1-mediated events influence photoreceptor cell development (Baril, 2014).

Given the role Connector eNhancer of KSR (CNK) plays in RTK-elicited Ras/MAPK signaling, mutations in loci encoding general components of this pathway in flies were recovered in a CNK C-terminal-dependent (CCT) screen. They correspond to Star [S; trafficking factor for the EGFR ligand Spitz, Egfr, daughter of sevenless (dos; Gab2 homolog), Son of sevenless (Sos; RasGEF), Ras85D, rl/mapk, ksr, and pointed (pnt; ETS domain transcription factor mediating MAPK activity). Mutations in two genes previously identified in RTK-dependent screens, but of unclear function, were also isolated. These are kismet [kis; chromatin remodeler] and multiple ankyrin repeats single KH domain [mask; putative RNA-binding protein (Baril, 2014).

Mutant alleles not identified in classical RTK/MAPK-dependent genetic screens, but for which a functional link to RTK signaling in flies or in other organisms had been established were also recovered. These include Btk family kinase at 29A [Btk29A; the single representative of Tec family kinases]; Delta (Dl); and three Rap1 pathway loci, PDZ-GEF, Rap1, and canoe (cno). Finally, mutations were isolated in four additional loci not previously reported to influence RTK/MAPK signaling: Pre-mRNA-processing factor 19 (Prp19), BREFeldin A sensitivity 1 (Bre1), NEM sensitive factor 2 (Nsf2), and second mitotic wave missing (swm) (Baril, 2014).

The Prp19 locus encodes a core spliceosome component. A specific role for these proteins has been unvailed in the Ras/MAPK pathway. Indeed, several splicing factors, including Prp19 and Prp8, were identified in a genome-wide RNAi screen in S2 cells for modulators of Ras-induced MAPK activation. Incidentally, a single mutant allele of Prp8 was also recovered in the CCT screen. Characterization of their implication in the pathway revealed that they specifically regulate MAPK protein levels by controlling the alternative splicing of selected introns of the mapk pre-mRNAs. It is thus likely that Prp19 alleles were recovered in the CCT screen because of their impact on endogenous MAPK levels during eye development (Baril, 2014).

The association that the last three genes (Bre1, Nsf2, and swm) might have with respect to CCT activity is less clear. Bre1 encodes a RING finger-containing E3 ligase mediating histone H2B monoubiquitination. This modification contributes to specific histone epigenetic changes such as histone H3K4 and H3K79 methylation that correlate with transcriptional activation. A role for Bre1 in Notch- and Wingless-dependent gene expression has been reported, but whether it acts similarly downstream of RTK signaling is not known. Given the concerted, yet distinct role Notch and EGFR signaling play in morphogenetic furrow progression and thereby in eye development, it could well be that, as for Dl, Bre1 was recovered primarily for its function in Notch signaling (Baril, 2014).

Nsf2 encodes an AAA ATPase involved in vesicular trafficking and synaptic vesicle release. Previous genetic studies have also associated this gene with Notch and Wingless signaling, and thus this could be the basis for the isolation of Nsf2 alleles. Alternatively, Nsf2 activity could be required in trafficking events directly involved in RTK signaling (Baril, 2014).

The swm gene [aka Su(Rux)2B encodes a novel protein that comprises a CCCH zinc finger and a RNA recognition motif. SWM localizes to the nucleus and was found to play multiple roles during Drosophila development, although its precise molecular function is not known. During eye development, Swm regulates the proliferation of undifferentiated cells by controlling their G1/S transition. In particular, third instar eye discs deprived of SWM activity are reduced in size and, as epitomized by the gene name, they lack the second mitotic wave, which corresponds to a row of cells located at a few cells distance posterior to the morphogenetic furrow that undergo a unique and synchronous round of cell division. This event increases the pool of uncommitted cells used for completing ommatidial assembly. Both Notch and EGFR signaling are essential for cell cycle progression of the uncommitted cells in the second mitotic wave, but act at distinct steps. Whether the swm alleles were recovered because of their impact on the second mitotic wave or for another role of SWM in differentiating cells remains to be investigated (Baril, 2014).

The ability of the CCT screen to identify mutations in three loci linked to Rap1 signaling strongly suggests a functional relationship between CNK and Rap1 activity. Yet, no evidence was found for physical association between CNK and Rap1 or PDZ-GEF, and thus the molecular underpinning of this relationship is currently not known. One possibility for their genetic interactions could be through their separate roles in RTK-mediated events. Rap1 signaling promotes adherens junction formation in differentiating photoreceptor cells, which contributes to their clustering. This phenomenon, in turn, is thought to enable the cells to respond to extracellular cues promoting differentiation. By lowering Rap1 activity, cohesive contacts between differentiating cells would be suboptimal and thereby would impede ommatidial assembly to some degree. In this scenario, the impact of CCT expression on photoreceptor cell differentiation would be exacerbated by heterozygous mutations in Rap1-signaling components as these would reduce the sensitivity of developing cells to differentiation cues (Baril, 2014).

Interestingly, it has been noted that loss of Rap1 activity does not prevent EGFR-induced MAPK activation per se, and thus Rap1 does not appear to work like Ras as a direct RAF activator. However, the data for this conclusion were based on small Rap1 mutant clones that were close or within the morphogenetic furrow. This work was extended by producing larger clones depleted in Rap1 or PDZ-GEF activity. These clones covered the zone where R7 cell commitment normally occurs. Markedly, it was found that reduced Rap1 signaling in this area considerably decreased MAPK activity as well as global pTyr levels and thereby mimicked the loss of RTK activity. Consistent with this, a strong impairment in R7 cell fate specification was observed (Baril, 2014).

In agreement with thes findings, Mavromatakis (2012) recently showed by genetic means that R7 cell fate specification had an absolute requirement in Rap1 activity. According to their model, R7 cell precursors sense higher Notch signaling owing to their position in the developing ommatidium, which at this stage is antagonistic to Ras/MAPK-mediated neuronal differentiation. To counteract Notch signaling, Mavromatakis (2012) proposed that presumptive R7 cells turn on two RTKs (EGFR and SEV) to produce higher MAPK activity. Intriguingly, their work suggested that Rap1 was required downstream of SEV, although they could not distinguish whether Rap1 acted through the canonical MAPK pathway or parallel to it. This was investigated and it was found Rap1 does not seem to work directly through the MAPK pathway since ectopic expression of Rap1V12 during eye development or in cultured S2 cells did not promote MAPK phosphorylation. Moreover, depletion of Rap1 or PDZ-GEF by RNAi in S2 cells had no consequence on MAPK activation induced by SEV or EGFR. Although the precise mechanism by which Rap1 influences signaling downstream of SEV remains to be delineated, the combined data suggest that Rap1 works at two distinct levels in SEV-mediated signaling, that is, upstream of SEV by modulating the apical localization of SEV and downstream of SEV by a mechanism that has yet to be characterized (Baril, 2014).

Adherens junctions form a belt-like microdomain that encircles epithelial cells apically and that play a major role in cell-cell adhesion, motility, and polarity. One of the core structural components of adherens junctions is Ecad, which is a transmembrane glycoprotein that forms Ca2+-dependent homophilic interactions between adjacent cells. The intracellular portion of Ecad is complexed to the catenins that, in turn, mediate linkage to the actomyosin cytoskeleton. Studies conducted over the past 10 years in both vertebrate and invertebrate organisms demonstrate the critical role that Rap1 signaling plays in modulating the connections of adherens junctions to the actomyosin network, which then influence cell-cell adhesion, cell shape, and cell migration (Baril, 2014).

Although the current data are consistent with this view, they also hint at a new role for Rap1 signaling that is to control apical domain formation in developing photoreceptor cells. Given that adherens junctions may act as physical barriers between apical and basolateral membrane compartments, the influence of Rap1 on adherens junction dynamics could represent the mechanism by which Rap1 exerts its effect on the apical domain compartment. A more exciting alternative would be that Rap1 activity directly controls the formation of the apical domain. Work conducted in fly embryos by Choi (2013) recently provided evidence supporting this model. Indeed, not only did that study find that Rap1 activity is essential for establishing the apico-basal polarity of cellularizing embryos, but their data also suggest that it has a direct impact on the apical localization of Bazooka, a member of the Par complex, which then orchestrates apical domain assembly. Whether Rap1 signaling has a direct influence on cell polarity during eye development is still unclear. Nonetheless, further characterization of the impact that Rap1 signaling has on apical domain formation/maintenance should reveal novel aspects by which cell compartmentalization is brought about and regulated as well as how it connects to downstream signaling events in epithelial cells (Baril, 2014).

The Drosophila Arf GEF Steppke controls MAPK activation in EGFR signaling

Guanine nucleotide exchange factors (GEFs) of the cytohesin protein family are regulators of GDP/GTP exchange for members of the ADP ribosylation factor (Arf) of small GTPases. They have been identified as modulators of various receptor tyrosine kinase signaling pathways including the insulin, the vascular epidermal growth factor (VEGF) and the epidermal growth factor (EGF) pathways. These pathways control many cellular functions, including cell proliferation and differentiation, and their misregulation is often associated with cancerogenesis. In vivo studies on cytohesins using genetic loss of function alleles are lacking, however, since knockout mouse models are not available yet. Recently studies have identified mutants for the single cytohesin Steppke (Step) in Drosophila, and an essential role of Step in the insulin signaling cascade has been demonstrated. The present study provides in vivo evidence for a role of Step in EGFR signaling during wing and eye development. By analyzing step mutants, transgenic RNA interference (RNAi) and overexpression lines for tissue specific as well as clonal analysis, it was found that Step acts downstream of the EGFR and is required for the activation of mitogen-activated protein kinase (MAPK) and the induction of EGFR target genes. It was further demonstrated that step transcription is induced by EGFR signaling whereas it is negatively regulated by insulin signaling. Furthermore, genetic studies and biochemical analysis show that Step interacts with the Connector Enhancer of KSR (CNK). It is proposed that Step may be part of a larger signaling scaffold coordinating receptor tyrosine kinase-dependent MAPK activation (Hahn, 2013).

The proper development of multicellular organisms requires the coordination of proliferation and differentiation, which is a particular challenge during the formation of the tissues and organs of the body. Numerous studies have shown that receptor tyrosine kinases such as the vascular growth factor receptor (VEGFR) epidermal growth factor receptor (EGFR) and insulin/insulin-like growth factor receptors (InR/IGF-Rs) play prominent roles in signaling cell proliferation and differentiation. Misregulation of both pathways is often causative for tumor development and progression through their effects on uncontrolled cell growth, inhibition of apoptosis, angiogenesis, and tumor-associated inflammation. Determining how growth and differentiation are coordinated by these pathways is thus essential to understanding normal development, as well as disease states such as cancer (Hahn, 2013).

Steppke (Step) has been identified as a new and essential component of the insulin signaling pathway in Drosophila. The insulin signaling cascade is conserved from flies to humans and was shown to regulate cell and organismal growth in response to extrinsic signals such as growth factors and nutrient availability. In Drosophila, activation of a unique insulin-like receptor (InR) stimulates a conserved downstream cascade that includes the Phosphatidylinositol-3-kinase (PI3K) and Protein Kinase B (PKB or AKT). AKT is involved in enhancement of glucose absorption and glycogen synthesis, and regulates the activity of the Forkhead box O (FoxO) transcription factor, a negative regulator of cell growth. Step acts downstream of the insulin receptor and upstream of PI3K in the insulin/IGF-like signaling (IIS) cascade. Step is a member of the cytohesin family of guanine nucleotide exchange factors (GEFs) which regulate small GTPases of the ADP-ribosylation factor (ARF) family. Small ARF GTPases are involved in the regulation of many cellular processes including vesicle transport, cell adhesion and migration. Studies in mice have confirmed an evolutionary conserved role of cytohesin family members in IIS (Hahn, 2013 and references therein).

Whereas previous studies focused on the role of Step in IIS-dependent larval growth control, this study examined its function in the Drosophila wing, which develops from an epithelial sheet during larval and pupal stages. The wing is an ectodermal structure formed by a dorsal and ventral epithelium, interspersed with cuticular ectodermal tubes, the so called wing veins. Stereotypical arrangement of wing veins is determined in the imaginal wing disc in late larval and pupal stages by several signaling pathways including the EGFR cascade. EGFR activation by EGF-like ligands Spitz or Vein results in the activation of the small GTPase RAS by its loading with guanosine triphosphate (GTP), which as a result triggers the activation of a number of downstream effector proteins including the Ser/Thr-kinase RAF [mitogen-activated protein kinase (MAPK) kinase kinase]. Once activated, RAF phosphorylates and activates MEK (MAPK kinase), which in turn phosphorylates and activates MAPK/ERK. Phosphorylated MAPK exerts its role in the cytoplasm as well as in the nucleus, where it controls expression of EGFR target genes like pointed (pnt), argos (aos), rhomboid (rho) and ventral nervous system defective (vnd). The scaffolding protein Connector enhancer of KSR (CNK) has been described to facilitate RAS/RAF/MAPK signaling by providing a protein scaffold at the plasma membrane that integrates Src and RAS activities to enhance RAF and MAPK activation. EGFR/MAPK signaling is crucial quite early during wing vein differentiation, where phosphorylation of MAPK determines the positioning of proveins and later during development for maintenance of longitudinal veins. In addition to patterning, both EGFR/RAS/MAPK signaling and IIS control general cell proliferation and cell growth during wing development. Thus, EGFR/RAS/MAPK signaling controls both cell fate (vein versus intervein) and general cell proliferation along with IIS at similar times within the wing tissue (Hahn, 2013).

Recent studies in human lung and breast adenosarcoma cancer cell lines indicated a function of cytohesins in ErbB (EGFR) signaling, where they facilitate signaling by stabilizing an asymmetric ErbB receptor dimer. This study provides the first in vivo model that the cytohesin Step, in addition to its previously characterized function as component of IIS, regulates EGFR signaling dependent wing growth and vein differentiation. Genetic, immunohistochemical and biochemical experiments indicate that Step acts downstream of the EGF receptor in the EGFR signaling cascade and is necessary and sufficient for MAPK activation and the induction of EGFR target genes. Whereas step transcription is negatively regulated by IIS, it is induced by EGFR signaling. Evidences are further provided that Step might directly interact with the Connector Enhancer of KSR (CNK) protein that is part of a protein scaffold known to coordinate RAS-dependent RAF and MAPK signaling from tyrosine kinase receptors (Hahn, 2013).

This study demonstrates an in vivo function of the Arf GEF Step as an essential component of the EGFR signaling pathway which acts downstream of the EGFR. Step is necessary and sufficient for activation of MAPK and the induction of EGFR target genes in the Drosophila wing. Based on biochemical, immunohistochemical and the genetic data a mechanistic model is proposed in which Step and dCNK interaction is important for EGFR signaling. dCNK is the single member of the CNK protein family in Drosophila. CNK proteins are scaffolding proteins that have been linked with RAS, Rho, Rac, Ral and Arf GTPases and are proposed to act as general regulators of GTPase-mediated events downstream of receptor tyrosine kinases, including EGFR and InR/insulin-like growth factor receptors. Together with the kinase suppressor of RAS (KSR), CNK was shown to assemble a signaling complex including RAF and MEK which promotes RAS-dependent RAF activation and the subsequent phosphorylation of MAPK. It is suggested that Step is a functional part of this scaffolding complex via its direct interaction with CNK. This is also consistent with recent data in HeLa and 393T cells showing that human CNK1 directly interacts with cytohesin-2 to coordinate PI3K/AKT signaling downstream of InR/IGF-R. It was proposed that CNK1 recruits cytohesin-2 to the plasma membrane, where activity of plasma membrane bound GTPases leads to a PIP2 rich microenvironment, which enhances IRS1 recruitment and hence facilitates PI3K/AKT signaling. Similarly, Drosophila cytohesin Step was shown to be required for PI3K activation. Together, several lines of evidence support a role of cytohesins and CNK in similar signaling contexts (RAS/RAF/MAPK and PI3K/AKT signaling), where a direct interaction of both proteins as part of a signaling platform might promote downstream signaling events like MAPK phosphorylation and PI3K activation. This does not exclude other functions of cytohesins, e.g. the stabilization of asymmetric ErbB (EGFR) dimers, as shown recently in human lung and breast adenosarcoma cancer cell lines. The data indicate, however, that a major function of the Drosophila cytohesin Step in EGFR signaling resides downstream of the EGFR and upstream of MAPK (Hahn, 2013).

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

RAF kinase functions in the mitogen-activated protein kinase (MAPK) pathway to transmit growth signals to the downstream kinases MEK and ERK. Activation of RAF catalytic activity is facilitated by a regulatory complex comprising the proteins Cbk (Connector enhancer of KSR), Hyp (Hyphen/Aveugle), 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. This study has determined the x-ray crystal structure of the SAM domain of Cnk in complex with the SAM domain of Hyp. 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, this study shows 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).

A KSR/CNK complex mediated by HYP, a novel SAM domain-containing protein, regulates RAS-dependent RAF activation in Drosophila

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. 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. 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. 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. A number of phosphorylation events are then required to fully induce RAF catalytic activity. 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. 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. 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. 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 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. Further, they possess a kinase-like domain that constitutively binds MEK, but which appears to be devoid of kinase activity. 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. 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. 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. 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. 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-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 bringing other RAF-activating proteins or sequestering 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 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).

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 Src64^YF 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 Src42^Y511F, 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 RAS^V12, but not by activated RAF. (2) NT-CNK (deleted of the C-terminal repressive function) cooperates with RAS^V12 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 RAS^V12G37. 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).

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

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

To determine whether cnk has a role during Ras signaling, the ability of cnk to modify the rough eye phenotype caused by overexpression of Ras1^V12 (constitutively active Ras), sev^S11 (activated Sevenless RTK, and Raf^Tor4021 (activated RAF) was tested. cnk dominantly suppresses both Ras1^V12 and sev^S11 rough eye phenotypes but is unable to modify the Raf^Tor4021 rough eye phenotype. Analysis of apical sections of adult fly retinae support this conclusion, and multiple cnk alleles have the same effect. The ability of cnk to enhance KDN (dominant negative Kinase suppressor of Ras) and to suppress both Ras1^V12 and sev^S11 phenotypes suggests that the EK2-3 locus encodes a positive component of RAS signaling. In addition, the finding that cnk does not modify the Raf^Tor4021 phenotype suggests that cnk is required upstream or in parallel to Raf. The finding that cnk loss-of-function alleles modify sensitized genetic backgrounds like those produced by KDN or sev-Ras1^V12 suggests that this gene is normally required for photoreceptor differentiation. This conclusion is supported by examination of the mild rough eyes of a hypomorphic allele. Analysis of apical eye sections of hypomorphic allele homozygotes reveals the absence of R7 photoreceptors in more than 80% of the ommatidia, with roughly 15% of the ommatidia also missing an outer photoreceptor. This phenotype is similar to that observed for hypomorphic alleles of several components of the Ras pathway. Because R7 and outer photoreceptor cell differentiation depend on Sevenless and Epidermal growth factor, respectively, this observation suggests that cnk functions downstream of both receptors (Therrien, 1998).

In Drosophila, components of the RAS pathway are generally required for cell proliferation and/or survival. To test whether cnk is similarly required, large clones of cnk homozygous mutant tissue were generated in the eye. cnk appears to be required for cell proliferation and/or survival, since mutation of this gene prevents normal eye development in a similar manner as is observed when loss-of-function mutations in Ras1 or ksr are tested. Evidence is found indicating that cnk is required for RAS-dependent signaling pathways outside the eye. For example, like other components of this pathway, cnk loss-of-function alleles are synthetic lethal with a hypomorphic allele of Raf, Raf^HM7. Induction of cnk mutant clones in the wing prevents wing vein formation. Wing vein formation is controlled by Egfr, indicating a role for cnk in other RAS-dependent pathways (Therrien, 1998).

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

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

EVOLUTIONARY HOMOLOGS

Membrane localization of acetylated CNK1 mediates a positive feedback on RAF/ERK signaling

Spatiotemporal control is a common mechanism that modulates activity and function of signal transducers in the signaling network. This study identified acetylation of CNK1 (connector enhancer of kinase suppressor of Ras-1) as a late step in the activation of CNK1 signaling, accompanied with prolonged stimulation of extracellular signal-regulated kinase (ERK). The acetyltransferase CREB (cyclic adenosine 3',5'-monophosphate response element-binding protein)-binding protein and the deacetylase SIRT2 (sirtuin type 2) were identified as novel binding partners of CNK1, modulating the acetylation state of CNK1. Acetylation of CNK1 at position Lys(414) located in the pleckstrin homology domain drives membrane localization of CNK1 in growth factor-stimulated cells. Inhibition of ERK signaling abolishes CNK1 acetylation. Cosmic database search identified CNK1 mutants at position Arg(426) near the acetylation site in several human tumor types. These mutants show constitutive acetylation and membrane localization. CNK1 mutants substituting Arg(426), the acetylation mimetic mutant CNK1-K414Q, and membrane-anchored CNK1 mutants all interact with the protein kinase CRAF and stimulate ERK-dependent cell proliferation and cell migration. In RAS-transformed cells, CNK1 is acetylated and membrane-bound and drives cell proliferation. Thus, growth factor-stimulated ERK signaling induces CNK1 acetylation, and acetylated CNK1 promotes ERK signaling, demonstrating a novel function of CNK1 as positive feedback regulator of the RAF/MEK (mitogen-activated protein kinase kinase)/ERK pathway. In addition, acetylation of CNK1 is an important step in oncogenic signaling, promoting cell proliferation and migration (Fischer, 2017a).

AKT-dependent phosphorylation of the SAM domain induces oligomerization and activation of the scaffold protein CNK1

Scaffold proteins are hubs for the coordination of intracellular signaling networks. The scaffold protein CNK1 promotes several signal transduction pathway. This study demonstrate that sterile motif alpha (SAM) domain-dependent oligomerization of CNK1 stimulates CNK1-mediated signaling in growth factor-stimulated cells. We identified Ser22 located within the SAM domain as AKT-dependent phosphorylation site triggering CNK1 oligomerization. Oligomeric CNK1 increased the affinity for active AKT indicating a positive AKT feedback mechanism. A CNK1 mutant lacking the SAM domain and the phosphorylation-defective mutant CNK1S22A antagonizes oligomerization and prevents CNK1-driven cell proliferation and matrix metalloproteinase 14 promoter activation. The phosphomimetic mutant CNK1S22D constitutively oligomerizes and stimulates CNK1 downstream signaling. Searching the COSMIC database revealed Ser22 as putative target for oncogenic activation of CNK1. Like the phosphomimetic mutant CNK1S22D, the oncogenic mutant CNK1S22F forms clusters in serum-starved cells comparable to clusters of CNK1 in growth factor-stimulated cells. CNK1 clusters induced by activating Ser22 mutants correlate with enhanced cell invasion and binding to and activation of ADP ribosylation factor 1 associated with tumor formation. Mutational analysis indicate that EGF-triggered phosphorylation of Thr8 within the SAM domain prevents AKT binding and antagonizes CNK1-mediated AKT signaling. These findings reveal SAM domain-dependent oligomerization by AKT as switch for CNK1 activation (Fischer, 2017b).

Optogenetic clustering of CNK1 reveals mechanistic insights in RAF and AKT signalling controlling cell fate decisions

Scaffold proteins such as the multidomain protein CNK1 orchestrate the signalling network by integrating and controlling the underlying pathways. Using an optogenetic approach to stimulate CNK1 uncoupled from upstream effectors, this study identified selective clusters of CNK1 that either stimulate RAF-MEK-ERK or AKT signalling depending on the light intensity applied. OptoCNK1 implemented in MCF7 cells induces differentiation at low light intensity stimulating ERK activity whereas stimulation of AKT signalling by higher light intensity promotes cell proliferation. CNK1 clustering in response to increasing EGF concentrations revealed that CNK1 binds to RAF correlating with ERK activation at low EGF dose. At higher EGF dose active AKT binds to CNK1 and phosphorylates and inhibits RAF. Knockdown of CNK1 protects CNK1 from this AKT/RAF crosstalk. In C2 skeletal muscle cells CNK1 expression is induced with the onset of differentiation. Hence, AKT-bound CNK1 counteracts ERK stimulation in differentiated but not in proliferating cells. Ectopically expressed CNK1 facilitates C2 cell differentiation and knockdown of CNK1 impaired the transcriptional network underlying C2 cell differentiation. Thus, CNK1 expression, CNK1 clustering and the thereto related differential signalling processes decide on proliferation and differentiation in a cell type- and cell stage-dependent manner by orchestrating AKT and RAF signalling (Fischer, 2016).

EphrinB1 interacts with CNK1 and promotes cell migration through c-Jun N-terminal kinase (JNK) activation

The Eph receptors and their membrane-bound ligands, ephrins, play important roles in various biological processes such as cell adhesion and movement. The transmembrane ephrinBs transduce reverse signaling in a tyrosine phosphorylation-dependent or -independent, as well as PDZ-dependent manner. This study shows that ephrinB1 interacts with Connector Enhancer of KSR1 (CNK1) in an EphB receptor-independent manner. In cultured cells, cotransfection of ephrinB1 with CNK1 increases JNK phosphorylation. EphrinB1/CNK1-mediated JNK activation is reduced by overexpression of dominant-negative RhoA. Overexpression of CNK1 alone is sufficient for activation of RhoA; however, both ephrinB1 and CNK1 are required for JNK phosphorylation. Co-immunoprecipitation data showed that ephrinB1 and CNK1 act as scaffold proteins that connect RhoA and JNK signaling components, such as p115RhoGEF and MKK4. Furthermore, adhesion to fibronectin or active Src overexpression increases ephrinB1/CNK1 binding, whereas blocking Src activity by a pharmacological inhibitor decreases not only ephrinB1/CNK1 binding, but also JNK activation. EphrinB1 overexpression increases cell motility, however, CNK1 depletion by siRNA abrogates ephrinB1-mediated cell migration and JNK activation. Moreover, Rho kinase inhibitor or JNK inhibitor treatment suppresses ephrinB1-mediated cell migration. Taken together, these findings suggest that CNK1 is required for ephrinB1-induced JNK activation and cell migration (Cho, 2014).

The CNK1 scaffold binds cytohesins and promotes insulin pathway signaling

Protein scaffolds play an important role in signal transduction, regulating the localization of signaling components and mediating key protein interactions. This study reports that the major binding partners of the Connector Enhancer of KSR 1 (CNK1) scaffold are members of the cytohesin family of Arf guanine nucleotide exchange factors, and that the CNK1/cytohesin interaction is critical for activation of the PI3K/AKT cascade downstream from insulin and insulin-like growth factor 1 (IGF-1) receptors. A domain located in the C-terminal region of CNK1 was identifed that interacts constitutively with the coiled-coil domain of the cytohesins; CNK1 was found to facilitate the membrane recruitment of cytohesin-2 following insulin stimulation. Moreover, through protein depletion and rescue experiments, this study found that the CNK1/cytohesin interaction promotes signaling from plasma membrane-bound Arf GTPases to the phosphatidylinositol 4-phosphate 5-kinases (PIP5Ks) to generate a PIP(2)-rich microenvironment that is critical for the membrane recruitment of insulin receptor substrate 1 (IRS1) and signal transmission to the PI3K/AKT cascade. These findings identify CNK1 as a new positive regulator of insulin signaling (Lim, 2010).

CNK1 is a scaffold protein that regulates Src-mediated Raf-1 activation

Raf-1 is a regulator of cellular proliferation, differentiation and apoptosis. Activation of the Raf-1 kinase activity is tightly regulated and involves targeting to the membrane by Ras and phosphorylation by various kinases including the tyrosine kinase Src. The connector enhancer of Ksr1, CNK1 (See Drosophila), mediates Src-dependent tyrosine phosphorylation and activation of Raf-1. CNK1 binds preactivated Raf-1 and activated Src and forms a trimeric complex. CNK1 regulates the activation of Raf-1 by Src in a concentration-dependent manner typical for a scaffold protein. Down-regulation of endogenously expressed CNK1 by small inhibitory RNA interferes with Src-dependent activation of ERK. Thus, CNK1 allows a cross-talk between Src and Raf-1 and is essential for full activation of Raf-1 (Ziogas, 2005).

REFERENCES

Search PubMed for articles about Drosophila Cnk

Baril, C., Lefrancois, M., Sahmi, M., Knaevelsrud, H. and Therrien, M. (2014). Apical accumulation of the Sevenless receptor tyrosine kinase during Drosophila eye development is promoted by the small GTPase Rap1. Genetics 197(4): 1237-1250. PubMed ID: 24899161

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 citation: 15028221

Cho, H. J., Hwang, Y. S., Mood, K., Ji, Y. J., Lim, J., Morrison, D. K. and Daar, I. O. (2014). EphrinB1 interacts with CNK1 and promotes cell migration through c-Jun N-terminal kinase (JNK) activation. J Biol Chem 289(26): 18556-18568. PubMed ID: 24825906

Choi, W., Harris, N. J., Sumigray, K. D. and Peifer, M. (2013). Rap1 and Canoe/afadin are essential for establishment of apical-basal polarity in the Drosophila embryo. Mol Biol Cell 24(7): 945-963. PubMed ID: 23363604

Douziech, M., et al. (2003). Bimodal regulation of RAF by CNK in Drosophila. EMBO J. 22: 5068-5078. 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. 16600912

Fischer, A., Warscheid, B., Weber, W. and Radziwill, G. (2016). Optogenetic clustering of CNK1 reveals mechanistic insights in RAF and AKT signalling controlling cell fate decisions. Sci Rep 6: 38155. PubMed ID: 27901111

Fischer, A., Muhlhauser, W. W. D., Warscheid, B. and Radziwill, G. (2017a). Membrane localization of acetylated CNK1 mediates a positive feedback on RAF/ERK signaling. Sci Adv 3(8): e1700475. PubMed ID: 28819643

Fischer, A., Weber, W., Warscheid, B. and Radziwill, G. (2017b). AKT-dependent phosphorylation of the SAM domain induces oligomerization and activation of the scaffold protein CNK1. Biochim Biophys Acta 1864(1): 89-100. PubMed ID: 27769899

Hahn, I., Fuss, B., Peters, A., Werner, T., Sieberg, A., Gosejacob, D. and Hoch, M. (2013). The Drosophila Arf GEF Steppke controls MAPK activation in EGFR signaling. J Cell Sci 126: 2470-2479. PubMed ID: 23549788

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 citation: 14597674

Lim, J., Zhou, M., Veenstra, T. D. and Morrison, D. K. (2010). The CNK1 scaffold binds cytohesins and promotes insulin pathway signaling. Genes Dev. 24(14): 1496-506. PubMed Citation: 20634316

Mavromatakis, Y. E. and Tomlinson, A. (2012). The role of the small GTPase Rap in Drosophila R7 photoreceptor specification. Proc Natl Acad Sci U S A 109(10): 3844-3849. PubMed ID: 22355117

Rajakulendran, T., Sahmi, M., Kurinov, I., Tyers, M., Therrien, M. and Sicheri, F. (2008). CNK and HYP form a discrete dimer by their SAM domains to mediate RAF kinase signaling. Proc Natl Acad Sci U S A 105(8): 2836-2841. PubMed ID: 18287031

Rocheleau, C. E., Ronnlund, A., Tuck, S. and Sundaram, M.V. (2005). Caenorhabditis elegans CNK-1 promotes Raf activation but is not essential for Ras/Raf signaling. Proc. Natl. Acad. Sci. 102: 11757-11762. PubMed citation: 16085714

Therrien, M., Wong, A. M. and Rubin, G. M. (1998). CNK, a RAF-binding multidomain protein required for RAS signaling. Cell 95(3): 343-53. PubMed Citation: 9814705

Wolfstetter, G., Pfeifer, K., van Dijk, J. R., Hugosson, F., Lu, X. and Palmer, R. H. (2017). The scaffolding protein Cnk binds to the receptor tyrosine kinase Alk to promote visceral founder cell specification in Drosophila. Sci Signal 10(502). PubMed ID: 29066538

Ziogas, A., Moelling, K. and Radziwill G. (2005). CNK1 is a scaffold protein that regulates Src-mediated Raf-1 activation. J. Biol. Chem. 280: 24205-24211. PubMed citation: 15845549