By screening for mutations that suppress the vulval defects caused by a constitutively active let-60 ras gene, six loss-of-function alleles of ksr-1, a novel C. elegans gene, were identified. ksr-1 positively mediates Ras signaling and functions downstream of or in parallel to let-60. In the absence of ksr-1 function, normal Ras signaling is impaired only slightly, suggesting ksr-1 may act to modulate the main signaling pathway, or in a branch that diverges from it. The predicted KSR-1 protein has a protein kinase domain and is most similar to a recently identified Drosophila protein involved in Ras signaling. It is proposed that the function of ksr-1 is evolutionarily conserved (Kornfeld, 1995).
Vulval induction in C. elegans is controlled by a highly conserved signaling pathway similar to the RTK-Ras-MAPK cascade in mammals. By screening for suppressors of the Multivulva phenotype caused by an activated let-60 ras allele, mutations in the ksr-1 gene were isolated that act as positive modifiers of vulval induction and are required for at least two other let-60 ras-mediated processes. Although ksr-1 mutations do not perturb vulval induction in an otherwise wild-type background, they have very strong effects on vulval induction in genetic backgrounds where Ras pathway activity is constitutively activated or compromised, suggesting that ksr-1 activity is required for maximal stimulation of vulval fates by the Ras pathway. Genetic epistasis analysis suggests that ksr-1 acts downstream of or in parallel to let-60 ras. ksr-1 encodes a novel putative protein kinase related to the Raf family of Ser/Thr kinases (Sundaram, 1995).
A regulatory B subunit of protein phosphatase 2A (PP2A) positively regulates an RTK-Ras-MAP kinase signaling cascade during Caenorhabditis elegans vulval induction. Although reduction of sur-6 PP2A-B function causes few vulval induction defects in an otherwise wild-type background, sur-6 PP2A-B mutations suppress the Multivulva phenotype of an activated ras mutation and enhance the Vulvaless phenotype of mutations in lin-45 raf, sur-8, or mpk-1. Double mutant analysis suggests that sur-6 PP2A-B acts downstream or in parallel to ras, but likely upstream of raf, and functions with ksr-1 in a common pathway to positively regulate Ras signaling (Sieburth, 1999).
KSR proteins positively regulate Ras signaling in C. elegans and Drosophila, as well as in Xenopus oocytes and certain mammalian cells. Murine KSR associates with several proteins in vivo, including Raf, MEK, and MAP kinase, and has been proposed to function as a scaffold protein involved in signal propagation through the Raf/MEK/MAP kinase cascade. Murine KSR is a phosphoprotein; although the role of phosphorylation in KSR regulation is unclear. Thus, KSR-1 is a potential target for regulation by PP2A during vulval induction. Alternatively, KSR-1 may act to regulate PP2A-B function. Another potential sur-6 PP2A-B-dependent PP2A target is LIN-45 Raf. The mechanism of Raf activation is still poorly understood, but there is evidence for both inhibitory and activating phosphates on Raf. Whereas in vitro studies suggest that PP2A can dephosphorylate Raf, it is probably not the major phosphatase to remove activating phosphates. However, a role for PP2A in removing inhibitory phosphates has not been ruled out. The placement of a B regulatory subunit of PP2A as a positive regulator of the Ras pathway, and the unexpected finding that it acts together with KSR-1, should lead to a better understanding of PP2A regulation and its physiological substrates (Sieburth, 1999).
Kinase suppressor of Ras (KSR) is an evolutionarily conserved component associated with Ras-dependent signaling pathways. Murine KSR (mKSR1) translocates from the cytoplasm to the plasma membrane in the presence of activated Ras. At the membrane, mKSR1 modulates Ras signaling by enhancing Raf-1 activity in a kinase-independent manner. The activation of Raf-1 is mediated by the mKSR1 cysteine-rich CA3 domain and involves a detergent labile cofactor that is not ceramide. These findings reveal another point of regulation for Ras-mediated signal transduction and further define a noncatalytic role for mKSR1 in the multistep process of Raf-1 activation (Michaud, 1997).
The physiological role of KSR in vertebrate signal transduction was examined using Xenopus laevis oocytes. Overexpression of KSR, in combination with overexpression of the intracellular dimeric protein 14-3-3, induces Xenopus oocyte meiotic maturation and cdc2 kinase activation; the effect of KSR and 14-3-3 on oocyte maturation was blocked by co-expression of dominant-negative Raf-1. It is noted that KSR contains multiple potential binding sites for 14-3-3, and the yeast two-hybrid system and co-immunoprecipitation experiments were used to show that KSR can bind to 14-3-3. Furthermore, KSR can form a complex with Raf kinase both in vitro and in cultured cells. Cell fractionation studies have revealed that KSR forms a complex with 14-3-3 in both the membrane and cytoplasmic fractions of cell lysates; however, KSR only forms a complex with Raf-1 in the membrane fraction. These findings suggest that KSR, 14-3-3 and Raf form an oligomeric signaling complex and that KSR positively regulates the Ras signaling pathway in vertebrate organisms (Xing, 1997).
A proline-directed serine/threonine ceramide-activated protein (CAP) kinase mediates transmembrane signaling through the sphingomyelin pathway. CAP kinase reportedly initiates proinflammatory TNF alpha action by phosphorylating and activating Raf-1. The present studies delineate kinase suppressor of Ras (KSR), identified genetically in C. elegans and Drosophila, as CAP kinase. Mouse KSR, like CAP kinase, renatures and autophosphorylates as a 100-kDa membrane-bound polypeptide. KSR overexpression constitutively activates Raf-1. TNF alpha or ceramide analogs markedly enhance KSR autophosphorylation and its ability to complex with, phosphorylate, and activate Raf-1. In vitro, low nanomolar concentrations of natural ceramide stimulate KSR to autophosphorylate, and transactivate Raf-1. Other lipid second messengers are ineffective. Thr269, the Raf-1 site phosphorylated by CAP kinase, is also recognized by KSR. Thus, by previously established criteria, KSR appears to be CAP kinase (Zhang, 1997).
Ksr (kinase suppressor of Ras) was identified as a regulator of the Ras-MAP kinase (mitogen-activated protein kinase) pathway by genetic screens in Drosophila and Caenorhabditis elegans. Ksr is a kinase with similarities to the three conserved regions of Raf kinases, especially within the kinase domain. To investigate whether these structural similarities correlate with common functional properties, an examination was made of the ability of mKsr-1, the murine homolog of Ksr, to interact with components of the vertebrate MAP kinase pathway. In the yeast two-hybrid interaction assay, mKsr-1 did not bind to either Ras, B-Raf or Raf-1, but interacted strongly with both MEK-1 and MEK-2, activators of MAP kinase. The Ksr-MEK interaction was confirmed by co-immunoprecipitation experiments. Ectopically expressed mKsr-1 co-precipitates with endogenous MEK-1 in COS-1 cells, and endogenous Ksr and MEK co-precipitates from PC12 cells. Phosphorylation of MEK by mKsr-1 was not detected, however. In contrast, the MEK subpopulation complexed with mKsr-1 in COS-1 cells or PC12 cells does not display kinase activity. This ability of Ksr to block MEK in an inactive form correlates with a biological response: mKsr-1 does not transform NIH3T3 cells, and, furthermore, mKsr-1 reduces Ras-induced transformation. Similarly, mKsr-1 inhibits the proliferation of embryonic neuroretina cells induced by Ras and B-Raf but not that induced by MEK. These results suggest a novel mechanism for Ksr in regulating the MAP kinase pathway, at least in part through an ability to interact with MEK (Denouel-Galy, 1997).
Genetic screens in Drosophila melanogaster and Caenorhabditis elegans have identified the kinase suppressor of Ras, Ksr, as a new component in the Ras intracellular signaling pathway. In these organisms, mutations in Ksr results in attenuation of Ras-mediated signaling. Homologs of Ksr have also been isolated from mice and humans; their precise role in Ras signaling is not yet well defined. Interactions between the murine form of Ksr (mKsr-1) and other components of the Ras pathway have now been studied. To gain insight into the biological function of Ksr, a yeast two-hybrid screen was used and an interaction between the carboxy-terminal region of mKsr-1 and mitogen-activated protein (MAP) kinase kinase 1 (MAPKK-1 or MEK-1) was found. An interaction was also detected between MAP kinase (also called extracellular signal-regulated kinase, or ERK), and the amino-terminal region of mKsr-1. These interactions are recapitulated in COS-7 cells. When COS-7 cells are transfected with either full-length mKsr-1 or just its carboxy-terminal region, an inhibition of serum-stimulated MAP kinase activation is observed. Microinjection of full-length mKsr-1 or its carboxy-terminal, but not its amino-terminal region, blocks serum-induced DNA synthesis in rat embryo fibroblasts. Co-injection of mKsr-1 with MEK-1 reverses the blockage. Together with the data from genetic analyses, these findings led to the proposal that mKsr-1 may control MAP kinase signaling by serving as a scaffold protein that links MEK and its substrate ERK (Yu, 1998).
Genetic screens for modifiers of activated Ras phenotypes have identified a novel protein, kinase suppressor of Ras (KSR), which shares significant sequence homology with Raf family protein kinases. Studies using Drosophila melanogaster and Caenorhabditis elegans predict that KSR positively regulates Ras signaling; however, the function of mammalian KSR is not well understood. Two predicted kinase-dead mutants of KSR retain the ability to complement ksr-1 loss-of-function alleles in C. elegans, suggesting that KSR may have physiological, kinase-independent functions. Furthermore, murine KSR forms a multimolecular signaling complex in human embryonic kidney 293T cells composed of HSP90, HSP70, HSP68, p50(CDC37), MEK1, MEK2, 14-3-3, and several other, unidentified proteins. Treatment of cells with geldanamycin, an inhibitor of HSP90, decreases the half-life of KSR, suggesting that HSPs may serve to stabilize KSR. Both nematode and mammalian KSRs are capable of binding to MEKs, and three-point mutants of KSR, corresponding to C. elegans loss-of-function alleles, are specifically compromised in MEK binding. KSR does not alter MEK activity or activation. However, KSR-MEK binding shifts the apparent molecular mass of MEK from 44 to >700 kDa, and this results in the appearance of MEK in membrane-associated fractions. Together, these results suggest that KSR may act as a scaffolding protein for the Ras-mitogen-activated protein kinase pathway (Stewart, 1999).
Genetic and biochemical studies have identified kinase suppressor of Ras (KSR) to be a conserved component of Ras-dependent signaling pathways. To better understand the role of KSR in signal transduction, studies investigating the effect of phosphorylation and protein interactions on KSR function have been initiated. Five in vivo phosphorylation sites of KSR have been identified. In serum-starved cells, KSR contains two constitutive sites of phosphorylation (Ser297 and Ser392), which mediate the binding of KSR to the 14-3-3 family of proteins. In the presence of activated Ras, KSR contains three additional sites of phosphorylation (Thr260, Thr274, and Ser443), all of which match the consensus motif (Px[S/T]P) for phosphorylation by mitogen-activated protein kinase (MAPK). Treatment of cells with the MEK inhibitor PD98059 blocks phosphorylation of the Ras-inducible sites and activated MAPK associates with KSR in a Ras-dependent manner. Together, these findings indicate that KSR is an in vivo substrate of MAPK. Mutation of the identified phosphorylation sites does not alter the ability of KSR to facilitate Ras signaling in Xenopus oocytes, suggesting that phosphorylation at these sites may serve other functional roles, such as regulating catalytic activity. Interestingly, during the course of this study, it was found that the biological effect of KSR varies dramatically with the level of KSR protein expressed. In Xenopus oocytes, KSR functions as a positive regulator of Ras signaling when expressed at low levels, whereas at high levels of expression, KSR blocks Ras-dependent signal transduction. Likewise, overexpression of Drosophila KSR blocks R7 photoreceptor formation in the Drosophila eye. Therefore, the biological function of KSR as a positive effector of Ras-dependent signaling appears to be dependent on maintaining KSR protein expression at low or near-physiological levels (Cacace, 1999).
The protein kinase KSR-1 is a recently identified participant in the Ras signaling pathway. The subcellular localization of KSR-1 is variable. In serum-deprived cultured cells, KSR-1 is primarily found in the cytoplasm; in serum-stimulated cells, a significant portion of KSR-1 is found at the plasma membrane. To identify the mechanism that mediates KSR-1 translocation, a yeast two-hybrid screen was performed. Three clones that interacted with KSR-1 were found to encode the full-length gamma10 subunit of heterotrimeric G-proteins. KSR-1 also interacts with gamma2 and gamma3 in a two-hybrid assay. Deletion analysis demonstrates that the isolated CA3 domain of KSR-1, which contains a cysteine-rich zinc finger-like domain, interacts with gamma subunits. Coimmunoprecipitation experiments have demonstrated that KSR-1 binds to beta1 gamma3 subunits when all three are transfected into cultured cells. Lysophosphatidic acid treatment of cells induces KSR-1 translocation to the plasma membrane from the cytoplasm; translocation is blocked by administration of pertussis toxin but not by dominant-negative Ras. Finally, transfection of wild-type KSR-1 inhibits beta1 gamma3-induced mitogen-activated protein kinase activation in cultured cells. These results demonstrate that KSR-1 translocation to the plasma membrane is mediated, at least in part, by an interaction with beta gamma and that this interaction may modulate mitogen-activated protein kinase signaling (Bell, 1999).
Kinase suppressor of Ras (KSR) is an evolutionarily conserved component of Ras-dependent signaling pathways. The identification of B-KSR1, a novel splice variant of murine KSR1 that is highly expressed in brain-derived tissues, is reported. B-KSR1 protein is detectable in mouse brain throughout embryogenesis, is most abundant in adult forebrain neurons, and is complexed with activated mitogen-activated protein kinase (MAPK) and MEK in brain tissues. Expression of B-KSR1 in PC12 cells results in accelerated nerve growth factor (NGF)-induced neuronal differentiation and detectable epidermal growth factor (EGF)-induced neurite outgrowth. Sustained MAPK activity is observed in cells stimulated with either NGF or EGF, and all effects on neurite outgrowth can be blocked by the MEK inhibitor PD98059. In B-KSR1-expressing cells, the MAPK-B-KSR1 interaction is inducible and correlates with MAPK activation, while the MEK-B-KSR1 interaction is constitutive. Further examination of the MEK-B-KSR1 interaction reveals that all genetically identified loss-of-function mutations in the catalytic domain severely diminish MEK binding. Moreover, B-KSR1 mutants defective in MEK binding are unable to augment neurite outgrowth. Together, these findings demonstrate the functional importance of MEK binding and indicate that B-KSR1 may function to transduce Ras-dependent signals that are required for neuronal differentiation or that are involved in the normal functioning of the mature central nervous system (Muller, 2000).
Kinase suppressor of Ras (KSR) is a conserved component of the Ras pathway that interacts directly with MEK and MAPK. KSR1 translocates from the cytoplasm to the cell surface in response to growth factor treatment and this process is regulated by Cdc25C-associated kinase 1 (C-TAK1). C-TAK1 constitutively associates with mammalian KSR1 and phosphorylates serine 392 to confer 14-3-3 binding and cytoplasmic sequestration of KSR1 in unstimulated cells. In response to signal activation, the phosphorylation state of S392 is reduced, allowing the KSR1 complex to colocalize with activated Ras and Raf-1 at the plasma membrane, thereby facilitating the phosphorylation reactions required for the activation of MEK and MAPK (Muller, 2000).
Genetic analysis of Ras signaling has unveiled the participation of non-enzymatic accessory proteins in signal transmission. These proteins, KSR, CNK, and Sur-8, can interact with multiple core components of the Ras/MAP kinase cascade and may contribute to the structural organization of this cascade. However, the precise biochemical nature of the contribution of these proteins to Ras signaling is currently unknown. This study shows that CNK and KSR are required for stimulus dependent Raf kinase activation. CNK is required for membrane recruitment of Raf, while KSR is likely required to couple Raf to upstream kinases. These results demonstrate that CNK and KSR are integral components of the cellular machinery mediating Raf activation (Anselmo, 2002).
KSR (kinase suppressor of Ras) has been proposed as a molecular scaffold regulating the Raf/MEK/ERK kinase cascade. KSR is phosphorylated on multiple phosphorylation sites by associated kinases. To identify potential mechanisms used by KSR to regulate ERK activation, green fluorescent protein was fused to intact and mutated KSR constructs lacking specific phosphorylation sites, and the subcellular distribution of each construct was observed in live cells. Mutation of a subset of KSR phosphorylation sites causes the redistribution of KSR to the nucleus. To determine whether intact KSR is normally imported to the nucleus, REF-52 fibroblasts expressing KSR were treated with 10 nm leptomycin B, which inhibits Crm1-dependent nuclear export. KSR accumulates in the nucleus within 2 h of treatment with leptomycin B, suggesting that KSR cycles continuously through the nucleus. Nuclear import of KSR is blocked by mutations that inhibit the interaction of KSR with MEK. Coexpression of fluorescent forms of KSR and MEK in cells reveals that each protein promotes the localization of the other in the cytoplasm. These data indicate that the subcellular distribution of KSR is dynamically regulated through phosphorylation and MEK interaction in a manner that may affect signaling through ERK (Brennan, 2002).
While scaffold proteins are thought to be key components of signaling pathways, their exact function is unknown. By preassembling multiple components of signaling cascades, scaffolds are predicted to influence the efficiency and/or specificity of signaling events. A potential scaffold of the Ras/mitogen-activated protein kinase (MAPK) pathway, kinase suppressor of Ras (KSR), has been examined by generating KSR-deficient mice. KSR-deficient mice are grossly normal even though ERK kinase activation is attenuated to a degree sufficient to block T-cell activation and inhibit tumor development. Consistent with its role as a scaffold, high-molecular-weight complexes containing KSR, MEK, and ERK are lost in the absence of KSR. This demonstrates that KSR is a bona fide scaffold that, while not required, enhances signaling via the Ras/MAPK signaling pathway (Nguyen, 2002).
All KSR proteins contain a conserved cysteine-rich C1 domain, and studies have implicated this domain in the regulation of KSR1 subcellular localization and function. To further elucidate the biological role of the KSR1 C1 domain, its three-dimensional solution structure was determined using nuclear magnetic resonance (NMR). While the overall topology of the KSR1 C1 domain is similar to the C1 domains of Raf-1 and PKCgamma, the predicted ligand-binding region and the surface charge distribution are unique. Moreover, by generating chimeric proteins in which these domains have been swapped, it has been found that the C1 domains of Raf-1, PKCgamma, and KSR1 are not functionally interchangeable. The KSR1 C1 domain does not bind with high affinity or respond biologically to phorbol esters or ceramide, and it does not interact directly with Ras, indicating that the putative ligand(s) for the KSR1 C1 domain are distinct from those that interact with PKCgamma and Raf-1. In addition, analysis of the chimeric proteins supports the model that Raf-1 is a ceramide-activated kinase and that its C1 domain is involved in the ceramide-mediated response. Finally, the findings demonstrate an absolute requirement of the KSR1 C1 domain in mediating the membrane localization of KSR1, a crucial feature of its scaffolding activity. Together, these results underscore the functional specificity of these important regulatory domains and demonstrate that the structural features of the C1 domains can provide valuable insight into their ligand-binding properties (Zhou, 2002).
Kinase suppressor of Ras (KSR) is a loss-of-function allele that suppresses the rough eye phenotype of activated Ras in Drosophila and the multivulval phenotype of activated Ras in Caenorhabditis elegans. Genetic and biochemical studies suggest that KSR is a positive regulator of Ras signaling that functions between Ras and Raf or in a pathway parallel to Raf. The effect of mammalian KSR expression was examined on the activation of extracellular ligand-regulated (ERK) mitogen-activated protein (MAP) kinase in fibroblasts. Ectopic expression of KSR inhibits the activation of ERK MAP kinase by insulin, phorbol ester, or activated alleles of Ras, Raf, and mitogen and extracellular-regulated kinase. Expression of deletion mutants of KSR demonstrates that the KSR kinase domain is necessary and sufficient for the inhibitory effect of KSR on ERK MAP kinase activity. KSR inhibits cell transformation by activated RasVal-12 but has no effect on the ability of RasVal-12 to induce membrane ruffling. These data indicate that KSR is a potent modulator of a signaling pathway essential to normal and oncogenic cell growth and development (Joneson, 1998).
In Drosophila and C. elegans, kinase suppressor of Ras (KSR) functions as a positive modulator of Ras-dependent signaling either upstream of or parallel to Raf. Attempts to characterize the biochemical and biological properties of mammalian KSR, however, have had limited success. Although some studies have demonstrated a requirement of KSR kinase activity for its action, others indicate the kinase function of KSR is dispensable and suggest that KSR acts primarily as a scaffold protein. Interpretations of KSR function are further hampered by the lack of a standardized assay for its kinase activity in vitro. To address this issue, a two-stage in vitro kinase assay was established in which KSR never comes in contact with any recombinant kinases other than c-Raf-1. Using this assay, KSR has been shown to be inactive when immunoprecipitated from quiescent COS-7 cells that overexpress Flag-tagged KSR, but KSR activity is rapidly and markedly induced upon epidermal growth factor treatment. Moreover, KSR-reconstituted mitogen-activated protein kinase activation is detected in KSR immunoprecipitates depleted of all contaminating kinases (c-Raf-1, MEK1, ERK2) by multiple high salt washes. Only full-length kinase-active KSR is capable of signaling c-Raf-1-dependent activity, since kinase inactive and C- and N-terminal deletion mutants are without effect. Furthermore, endogenous KSR isolated from A431 cells, which contain high levels of activated EGF receptor, displays constitutively enhanced kinase activity. Hence, KSR kinase activity is not an artifact of overexpression but a property intrinsic to this protein. The recognition of EGF as a potent activator of KSR kinase activity and the availability of a well defined in vitro kinase assay should facilitate the definition of the function of KSR as a Ras-effector molecule (Xing, 2000).
A two-stage in vitro assay for KSR kinase activity has been established in which KSR never comes in contact with any recombinant kinase other than c-Raf-1, and EGF has been defined as a potent activator of KSR kinase activity. However, these studies do not address the mechanism of c-Raf-1 stimulation by activated KSR. Here it is shown that phosphorylation of c-Raf-1 on Thr269 by KSR is necessary for optimal activation in response to EGF stimulation. In vitro, KSR specifically phosphorylates c-Raf-1 on threonine residues during the first stage of the two-stage kinase assay. Using purified wild type and mutant c-Raf-1 proteins, it has been demonstrated that Thr269 is the major c-Raf-1 site phosphorylated by KSR in vitro, and that phosphorylation of this site is essential for c-Raf-1 activation by KSR. KSR acts via transphosphorylation, not by increasing c-Raf-1 autophosphorylation, because kinase inactive c-Raf-1K375M serves as an equally effective KSR substrate. In vivo, low physiologic doses of EGF (0.001-0.1 ng/ml) stimulate KSR activation, and induce Thr269 phosphorylation and activation of c-Raf-1. Low dose EGF does not induce serine or tyrosine phosphorylation of c-Raf-1. High dose EGF (10-100 ng/ml) induces no additional Thr269 phosphorylation but rather increases c-Raf-1 phosphorylation on serine residues and tyrosines340/341. A Raf-1 mutant with valine substituted for Thr269 is unresponsive to low dose EGF, but this mutant is serine and Tyr340/341 phosphorylated and partially activated at high EGF doses. These studies show that Thr269 is the major c-Raf-1 site phosphorylated by KSR. Further, phosphorylation of this site is essential for c-Raf-1 activation by KSR in vitro and for optimal c-Raf-1 activation in response to physiologic EGF stimulation in vivo (Xing, 2001).
Kinase Suppressor of Ras (KSR) is a molecular scaffold that interacts with the core kinase components of the ERK cascade, Raf, MEK, and ERK and provides spatial and temporal regulation of Ras-dependent ERK cascade signaling. In this report, the heterotetrameric protein kinase, casein kinase 2 (CK2), has been identified as a new KSR1-binding partner. Moreover, the KSR1/CK2 interaction is required for KSR1 to maximally facilitate ERK cascade signaling and contributes to the regulation of Raf kinase activity. Binding of the CK2 holoenzyme is constitutive and requires the basic surface region of the KSR1 atypical C1 domain. Loss of CK2 binding does not alter the membrane translocation of KSR1 or its interaction with ERK cascade components; however, disruption of the KSR1/CK2 interaction or inhibition of CK2 activity significantly reduces the growth-factor-induced phosphorylation of C-Raf and B-Raf on the activating serine site in the negative-charge regulatory region (N-region). This decrease in Raf N-region phosphorylation further correlates with impaired Raf, MEK, and ERK activation. These findings identify CK2 as a novel component of the KSR1 scaffolding complex that facilitates ERK cascade signaling by functioning as a Raf family N-Region kinase (Ritt, 2006).
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 identified that interacts constitutively with the coiled-coil domain of the cytohesins; CNK1 facilitates the membrane recruitment of cytohesin-2 following insulin stimulation. Moreover, through protein depletion and rescue experiments, it was 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).
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