The molecular scaffold KSR1 regulates the proliferative and oncogenic potential of cells

The specificity of signaling through mitogen-activated protein kinase pathways has been attributed to both the control of intensity and duration of signaling and the actions of protein scaffolds. Here we demonstrate that the molecular scaffold KSR1 regulates the intensity and duration of ERK activation to modulate a cell's proliferative and oncogenic potential. Deletion of KSR1 eliminates the prolonged phase of ERK activation induced by platelet-derived growth factor and blocks Ras(V12)-induced transformation. The introduction of KSR1 into KSR1(-/-) mouse embryo fibroblasts causes a concentration-dependent increase in signaling and transformation, to a maximum at 14 times the wild-type KSR1 expression levels, but inhibits these responses at higher expression levels. An increase in KSR1 expression to levels that are optimal for signaling leads to a threefold increase in proliferative capacity and is coincident with the level of KSR1 expression that maximally associates with all members of the Raf/MEK/ERK cascade. These data reveal that cells contain a reserve proliferative capacity that is accessible by the optimal expression of a noncatalytic signaling component and that altering the expression level of a molecular scaffold can modulate the actions of growth factors and oncogenes.     

Phosphorylation regulates KSR1 stability, ERK activation, and cell proliferation

Kinase suppressor of Ras (KSR) is a molecular scaffold that interacts with the components of the Raf/MEK/ERK kinase cascade and positively regulates ERK signaling. Phosphorylation of KSR1, particularly at Ser(392), is a critical regulator of KSR1 subcellular localization and ERK activation. We examined the role of phosphorylation of both Ser(392) and Thr(274) in regulating ERK activation and cell proliferation. We hypothesized that KSR1 phosphorylation is involved in generating signaling specificity through the Raf/MEK/ERK kinase cascade in response to stimulation by different growth factors. In fibroblasts, platelet-derived growth factor stimulation induces sustained ERK activation and promotes S-phase entry. Treatment with epidermal growth factor induces transient ERK activation but fails to drive cells into S phase. Mutation of Ser(392) and Thr(274) (KSR1.TVSA) promotes sustained ERK activation and cell cycle progression with either platelet-derived growth factor or epidermal growth factor treatment. KSR1(-/-) mouse embryo fibroblasts expressing KSR1.TVSA proliferate two times faster and grow to a higher density than cells expressing the same level of wild-type KSR1. In addition, KSR1.TVSA is more stable than wild-type KSR1. These data demonstrate that phosphorylation and stability of the molecular scaffold KSR1 are critical regulators of growth factor-specific responses that promote cell proliferation.     

Phosphorylation regulates the nucleocytoplasmic distribution of kinase suppressor of Ras.

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 caused 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 accumulated in the nucleus within 2 h of treatment with leptomycin B, suggesting that KSR cycles continuously through the nucleus. Nuclear import of KSR was blocked by mutations that inhibit the interaction of KSR with MEK. Coexpression of fluorescent forms of KSR and MEK in cells revealed that each protein promoted 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.     

The molecular scaffold kinase suppressor of Ras 1 (KSR1) regulates adipogenesis

Mitogen-activated protein kinase pathways are implicated in the regulation of cell differentiation, although their precise roles in many differentiation programs remain elusive. The Raf/MEK/extracellular signal-regulated kinase (ERK) kinase cascade has been proposed to both promote and inhibit adipogenesis. Here, we titrate expression of the molecular scaffold kinase suppressor of Ras 1 (KSR1) to regulate signaling through the Raf/MEK/ERK/p90 ribosomal S6 kinase (RSK) kinase cascade and show how it determines adipogenic potential. Deletion of KSR1 prevents adipogenesis in vitro, which can be rescued by introduction of low levels of KSR1. Appropriate levels of KSR1 coordinate ERK and RSK activation with C/EBPbeta synthesis leading to the phosphorylation and stabilization of C/EBPbeta at the precise moment it is required within the adipogenic program. Elevated levels of KSR1 expression, previously shown to enhance cell proliferation, promote high, sustained ERK activation that phosphorylates and inhibits peroxisome proliferator-activated receptor gamma, inhibiting adipogenesis. Titration of KSR1 expression reveals how a molecular scaffold can modulate the intensity and duration of signaling emanating from a single pathway to dictate cell fate.     

KSR1 is a functional protein kinase capable of serine autophosphorylation and direct phosphorylation of MEK1

The extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) pathway is a highly conserved signaling pathway that regulates diverse cellular processes including differentiation, proliferation, and survival. Kinase suppressor of Ras-1 (KSR1) binds each of the three ERK cascade components to facilitate pathway activation. Even though KSR1 contains a C-terminal kinase domain, evidence supporting the catalytic function of KSR1 remains controversial. In this study, we produced recombinant wild-type or kinase-inactive (D683A/D700A) KSR1 proteins in Escherichia coli to test the hypothesis that KSR1 is a functional protein kinase. Recombinant wild-type KSR1, but not recombinant kinase-inactive KSR1, underwent autophosphorylation on serine residue(s), phosphorylated myelin basic protein (MBP) as a generic substrate, and phosphorylated recombinant kinase-inactive MAPK/ERK kinase-1 (MEK1). Furthermore, FLAG immunoprecipitates from KSR1^−/− colon epithelial cells stably expressing FLAG-tagged wild-type KSR1 (+KSR1), but not vector (+vector) or FLAG-tagged kinase-inactive KSR1 (+D683A/D700A), were able to phosphorylate kinase-inactive MEK1. Since TNF activates the ERK pathway in colon epithelial cells, we tested the biological effects of KSR1 in the survival response downstream of TNF. We found that +vector and +D683A/D700A cells underwent apoptosis when treated with TNF, whereas +KSR1 cells were resistant. However, +KSR1 cells were sensitized to TNF-induced cell loss in the absence of MEK kinase activity. These data provide clear evidence that KSR1 is a functional protein kinase, MEK1 is an in vitro substrate of KSR1, and the catalytic activities of both proteins are required for eliciting cell survival responses downstream of TNF.     

Nm23-H1 metastasis suppressor expression level influences the binding properties, stability, and function of the kinase suppressor of Ras1 (KSR1) Erk scaffold in breast carcinoma cells

Metastatic disease is a significant contributor to cancer patient mortality. We previously reported that the Kinase Suppressor of Ras1 (KSR1) scaffold protein for the Erk mitogen-activated protein kinase pathway coimmunoprecipitated the metastasis suppressor protein Nm23-H1. We now hypothesize that altered expression levels of Nm23-H1 influence the binding properties, stability, and function of the KSR1 scaffold. Increased coimmunoprecipitation of Hsp90 with KSR1 was observed in either stable or transient transfectants of nm23-H1 in MDA-MB-435 human breast carcinoma cells. Similar trends were also observed in the cytoplasmic and nuclear fractions of cells. Cells expressing high levels of Nm23-H1 exhibited increased KSR1 degradation in the presence of either cycloheximide or an Hsp90-directed drug currently in clinical trial, 17-allylamino-17-demethoxygeldanamycin (17-AAG). In agreement with KSR1 degradation data, high-Nm23-H1-expression cells were preferentially inhibited in anchorage-independent colonization assays by 17-AAG. KSR1 scaffold binding patterns are dynamic in both the cytoplasmic and nuclear compartments, modulated by metastasis suppressor expression. Metastasis suppressor expression levels can impact traditional signaling pathways, such as the Erk pathway, resulting in altered tumor cell sensitivity to cancer therapeutics.     

RGS19 inhibits Ras signaling through Nm23H1/2-mediated phosphorylation of the kinase suppressor of Ras

Besides serving as signal terminators for G protein pathways, several regulators of G protein signaling (RGS) can also modulate cell proliferation. RGS19 has previously been shown to enhance Akt signaling despite impaired Ras signaling. The present study examines the mechanism by which RGS19 inhibits Ras signaling. In HEK293 cells stably expressing RGS19, serum-induced Ras activation and phosphorylations of Raf/MEK/ERK were significantly inhibited, while cells expressing RGS2, 4, 7, 8, 10, or 20 did not exhibit this inhibitory phenotype. Conversely, siRNA-mediated knockdown of RGS19 enabled partial recovery of serum-induced ERK phosphorylation. Interestingly, two isoforms of the tumor metastasis suppressor Nm23 (H1 and H2) were upregulated in 293/RGS19 cells. As a nucleoside diphosphate kinase, Nm23H1 can phosphorylate the kinase suppressor of Ras (KSR). Elevated levels of phosphorylated KSR were indeed detected in the nuclear fractions of 293/RGS19 cells. Co-immunoprecipitation assays revealed that Nm23H1/2 can form complexes with RGS19, Ras, or KSR. siRNA-mediated knockdown of Nm23H1/2 allowed 293/RGS19 cells to partially recover their ERK responses to serum treatment, while overexpression of Nm23H1/2 in HEK293 cells suppressed the serum-induced ERK response. This study demonstrates that expression of RGS19 can suppress Ras-mediated signaling via upregulation of Nm23.     

Kinase suppressor of Ras (KSR) is a scaffold which facilitates mitogen-activated protein kinase activation in vivo

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. Here we analyze a potential scaffold of the Ras/mitogen-activated protein kinase (MAPK) pathway, kinase suppressor of Ras (KSR), by generating KSR-deficient mice. KSR-deficient mice were grossly normal even though ERK kinase activation was 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 were lost in the absence of KSR. This demonstrates that KSR is a bona fide scaffold that is not required for but enhances signaling via the Ras/MAPK signaling pathway.     

Kinase Suppressor of Ras 2 (KSR2) Regulates Tumor Cell Transformation via AMPK

Kinase suppressor of Ras 1 (KSR1) and KSR2 are scaffolds that promote extracellular signal-regulated kinase (ERK) signaling but have dramatically different physiological functions. KSR2(-/-) mice show marked deficits in energy expenditure that cause obesity. In contrast, KSR1 disruption has inconsequential effects on development but dramatically suppresses tumor formation by activated Ras. We examined the role of KSR2 in the generation and maintenance of the transformed phenotype in KSR1(-/-) mouse embryo fibroblasts (MEFs) expressing activated Ras(V12) and in tumor cell lines MIN6 and NG108-15. KSR2 rescued ERK activation and accelerated proliferation in KSR1(-/-) MEFs. KSR2 expression alone induced anchorage-independent growth and synergized with the transforming effects of Ras(V12). Similarly, RNA interference (RNAi) of KSR2 in MIN6 and NG108-15 cells inhibited proliferation and colony formation, with concomitant defects in AMP-activated protein kinase (AMPK) signaling, nutrient metabolism, and metabolic capacity. While constitutive activation of AMPK was sufficient to complement the loss of KSR2 in metabolic signaling and anchorage-independent growth, KSR2 RNAi, MEK inhibition, and expression of a KSR2 mutant unable to interact with ERK demonstrated that mitogen-activated protein (MAP) kinase signaling is dispensable for the transformed phenotype of these cells. These data show that KSR2 is essential to tumor cell energy homeostasis and critical to the integration of mitogenic and metabolic signaling pathways.     

Kinase Suppressor of Ras 1 Is Not Required for the Generation of Regulatory and Memory T Cells

The mammalian target of rapamycin (mTOR) kinase is a critical regulator of the differentiation of helper and regulatory CD4+ T cells, as well as memory CD8+ T cells. In this study, we investigated the role of the ERK signaling pathway in regulating mTOR activation in T cells. We showed that activation of ERK following TCR engagement is required for sustained mTOR complex 1 (mTORC1) activation. Absence of kinase suppressor of Ras 1 (KSR1), a scaffold protein of the ERK signaling pathway, or inhibition of ERK resulted in decreased mTORC1 activity following T cell activation. However, KSR1-deficient mice displayed normal regulatory CD4+ T cell development, as well as normal memory CD8+ T cell responses to LCMV and Listeria monocytogenes infection. These data indicate that despite its role in mTORC1 activation, KSR1 is not required in vivo for mTOR-dependent T cell differentiation.     

CK2 Is a component of the KSR1 scaffold complex that contributes to Raf kinase activation

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, we identify the heterotetrameric protein kinase, casein kinase 2 (CK2), as a new KSR1-binding partner. Moreover, we find that 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.     

Epidermal growth factor treatment enhances the kinase activity of kinase suppressor of Ras

In Drosophila melanogaster and Caenorhabditis 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 demonstrated a requirement of KSR kinase activity for its action, others indicated the kinase function of KSR is dispensable and suggested 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, we established a two-stage in vitro kinase assay in which KSR never comes in contact with any recombinant kinases other than c-Raf-1. Using this assay, we show that KSR immunoprecipitated from quiescent COS-7 cells overexpressing Flag-tagged KSR was inactive, but its activity was rapidly and markedly induced upon epidermal growth factor treatment. Moreover, KSR-reconstituted mitogen-activated protein kinase activation was 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 was capable of signaling c-Raf-1-dependent activity as kinase inactive and C- and N-terminal deletion mutants were 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.     

C. elegans ksr-1 and ksr-2 have both unique and redundant functions and are required for MPK-1 ERK phosphorylation

Kinase Suppressor of Ras (KSR) is a conserved protein that positively regulates Ras signaling and may function as a scaffold for Raf, MEK, and ERK. However, the precise role of KSR is not well understood, and some observations have suggested that KSR might act in a parallel pathway. In C. elegans, ksr-1 is only required for a specific Ras-mediated process (sex myoblast migration) and is a nonessential positive regulator of other Ras-mediated developmental events. We report the existence of a second C. elegans ksr gene, ksr-2, which is required for Ras-mediated signaling during germline meiotic progression and functions redundantly with ksr-1 during development of the excretory system, hermaphrodite vulva, and male spicules. Thus, while the ksr-1 and ksr-2 genes are individually required only for specific Ras-dependent processes, together these two genes appear necessary for most aspects of Ras-mediated signaling in C. elegans. The finding that ksr-2; ksr-1 double mutants have strong ras-like phenotypes and severely reduced or absent levels of diphosphorylated MPK-1 ERK strongly supports models where KSR acts to promote the activation or maintenance of the Raf/MEK/ERK kinase cascade.     

Targeting cysteine rich C1 domain of Scaffold protein Kinase Suppressor of Ras (KSR) with anthocyanidins and flavonoids – a binding affinity characterization study

Kinase Suppressor of Ras (KSR) is a molecular scaffold that interacts with the core kinase components of the ERK cascade, Raf, MEK, ERK to provide spatial and temporal regulation of Ras-dependent ERK cascade signaling. Interruption of this mechanism can have a high influence in inhibiting the downstream signaling of the mutated tyrosine kinase receptor kinase upon ligand binding. Still none of the studies targeted to prevent the binding of Raf, MEK binding on kinase suppressor of RAS. In that perspective the cysteine rich C1 domain of scaffold proteins kinase suppressor of Ras-1 was targeted rather than its ATP binding site with small ligand molecules like flavones and anthocyanidins and analyzed through insilico docking studies. The binding energy evaluation shows the importance of hydroxyl groups at various positions on the flavone and anthocyanidin nucleus. Over all binding interaction shows these ligands occupied the potential sites of cysteine rich C1 domain of scaffold protein KSR.     

RAF inhibitor-induced KSR1/B-RAF binding and its effects on ERK cascade signaling

RAF kinase inhibitors can induce ERK cascade signaling by promoting dimerization of RAF family members in the presence of oncogenic or normally activated RAS. This interaction is mediated by a dimer interface region in the RAF kinase domain that is conserved in members of the ERK cascade scaffold family, kinase suppressor of RAS (KSR). In this study, we find that most RAF inhibitors also induce the binding of KSR1 to wild-type and oncogenic B-RAF proteins, including V600E B-RAF, but promote little complex formation between KSR1 and C-RAF. The inhibitor-induced KSR1/B-RAF interaction requires direct binding of the drug to B-RAF and is dependent on conserved dimer interface residues in each protein, but, unexpectedly, is not dependent on binding of B-RAF to activated RAS. Inhibitor-induced KSR/B-RAF complex formation can occur in the cytosol and is observed in normal mouse fibroblasts, as well as a variety of human cancer cell lines. Strikingly, we find that KSR1 competes with C-RAF for inhibitor-induced binding to B-RAF and, as a result, alters the effect of the inhibitors on ERK cascade signaling.     

Kinase suppressor of Ras1 compartmentalizes hippocampal signal transduction and subserves synaptic plasticity and memory formation

The ERK/MAP kinase cascade is important for long-term memory formation and synaptic plasticity, with a myriad of upstream signals converging upon ERK activation. Despite this convergence of signaling, neurons routinely activate appropriate biological responses to different stimuli. Scaffolding proteins represent a mechanism to achieve compartmentalization of signaling and the appropriate targeting of ERK-dependent processes. We report that kinase suppressor of Ras (KSR1) functions biochemically in the hippocampus to scaffold the components of the ERK cascade, specifically regulating the cascade when a membrane fraction of ERK is activated via a PKC-dependent pathway but not via a cAMP/PKA-dependent pathway. Specificity of KSR1-dependent signaling also extends to specific downstream targets of ERK. Behaviorally and physiologically, we found that the absence of KSR1 leads to deficits in associative learning and theta burst stimulation-induced LTP. Our report provides novel insight into the endogenous scaffolding role of KSR1 in controlling kinase activation within the nervous system.     

Kinase suppressor of ras 1 (KSR1) regulates PGC1α and estrogen-related receptor α to promote oncogenic Ras-dependent anchorage-independent growth

Kinase suppressor of ras 1 (KSR1) is a molecular scaffold of the Raf/MEK/extracellular signal-regulated kinase (ERK) cascade that enhances oncogenic Ras signaling. Here we show KSR1-dependent, but ERK-independent, regulation of metabolic capacity is mediated through the expression of peroxisome proliferator-activated receptor gamma coactivator 1α (PGC1α) and estrogen-related receptor α (ERRα). This KSR1-regulated pathway is essential for the transformation of cells by oncogenic Ras. In mouse embryo fibroblasts (MEFs) expressing H-Ras(V12), ectopic PGC1α was sufficient to rescue ERRα expression, metabolic capacity, and anchorage-independent growth in the absence of KSR1. The ability of PGC1α to promote anchorage-independent growth required interaction with ERRα, and treatment with an inhibitor of ERRα impeded anchorage-independent growth. In contrast to PGC1α, the expression of constitutively active ERRα (CA-ERRα) was sufficient to enhance metabolic capacity but not anchorage-independent growth in the absence of KSR1. These data reveal KSR1-dependent control of PGC1α- and ERRα-dependent pathways that are necessary and sufficient for signaling by oncogenic H-Ras(V12) to regulate metabolism and anchorage-independent growth, providing novel targets for therapeutic intervention.     

Amplification of apoptosis through sequential caspase cleavage of the MET tyrosine kinase receptor

Activation of the MET tyrosine kinase receptor by hepatocyte growth factor/scatter factor is classically associated with cell survival. Nonetheless, stress stimuli can lead to a caspase-dependent cleavage of MET within its juxtamembrane region, which generate a proapoptotic 40 kDa fragment (p40 MET). We report here that p40 MET is in fact generated through an additional caspase cleavage of MET within its extreme C-terminal region, which removes only few amino acids. We evidenced a hierarchical organization of these cleavages, with the C-terminal cleavage favoring the juxtamembrane one. As a functional consequence, the removal of the last amino acids of p40 MET increases its apoptotic capacity. Finally, cells expressing a MET receptor mutated at the C-terminal caspase site are unable to generate p40 MET and are resistant to apoptosis, indicating that generation of p40 MET amplifies apoptosis. These results revealed a two-step caspase cleavage of MET resulting in the reshaping of this survival receptor to a proapoptotic factor.