Inducible gene deletion reveals different roles for B-Raf and Raf-1 in B-cell antigen receptor signalling

Engagement of the B-cell antigen receptor (BCR) leads to activation of the Raf-MEK-ERK pathway and Raf kinases play an important role in the modulation of ERK activity. B lymphocytes express two Raf isoforms, Raf-1 and B-Raf. Using an inducible deletion system in DT40 cells, the contribution of Raf-1 and B-Raf to BCR signalling was dissected. Loss of Raf-1 has no effect on BCR-mediated ERK activation, whereas B-Raf-deficient DT40 cells display a reduced basal ERK activity as well as a shortened BCR-mediated ERK activation. The Raf-1/B-Raf double deficient DT40 cells show an almost complete block both in ERK activation and in the induction of the immediate early gene products c-Fos and Egr-1. In contrast, BCR-mediated activation of nuclear factor of activated T cells (NFAT) relies predominantly on B-Raf. Furthermore, complementation of Raf-1/B-Raf double deficient cells with various Raf mutants demonstrates a requirement for Ras-GTP binding in BCR-mediated activation of both Raf isoforms and also reveals the important role of the S259 residue for the regulation of Raf-1. Our study shows that BCR-mediated ERK activation involves a cooperation of both B-Raf and Raf-1, which are activated specifically in a temporally distinct manner.     

Activation of the Raf/MAP kinase cascade by the Ras-related protein TC21 is required for the TC21-mediated transformation of NIH 3T3 cells

TC21 is a member of the Ras superfamily of small GTP-binding proteins and, like Ras, has been implicated in the regulation of growth-stimulating pathways. Point mutations introduced into TC21 based on equivalent H-Ras oncogenic mutations are transforming in cultured cells, and oncogenic mutations in TC21 have been isolated from several human tumours. The mechanism of TC21 signalling in transformation is poorly understood. While activation of the serine/threonine kinases Raf-1 and B-Raf has been implicated in signalling pathways leading to transformation by H-Ras, it has been argued that TC21 does not activate Raf-1 or B-Raf. Since the Raf-signalling pathway is important in transformation by other Ras proteins, we assessed whether the Raf pathway is important to transformation by TC21. Raf-1 and B-Raf are constitutively active in TC21-transformed cells and the ERK/MAPK cascade is required for the maintenance of the transformed state. We demonstrate that oncogenic V23 TC21, like Ras, interacts with Raf-1 and B-Raf (but not with A-Raf), resulting in the translocation of the Raf proteins to the plasma membrane and in their activation. Furthermore, using point mutations in the effector loop of TC21, we show that the interaction of TC21 with Raf-1 is crucial for transformation.     

Extracellular signal-regulated kinase-dependent proliferation is mediated through the protein kinase A/B-Raf pathway in human uveal melanoma cells

Mutated B-Raf-mediated constitutive activation of ERK1/2 is involved in about 66% of cutaneous melanoma. By contrast, activating mutations in B-RAF are rare in ocular melanoma. This study aimed to determine the role of wild-type B-Raf ((WT)B-Raf) in uveal melanoma cell growth. We used cell lines derived from primary tumors of uveal melanoma to assess the role of (WT)B-Raf in cell proliferation and to characterize its upstream regulators and downstream effectors. Melanoma cell lines expressing (WT)B-Raf and (WT)Ras grew with similar proliferation rates, showed constitutive activation of ERK1/2, and had similar levels of B-Raf expression and B-Raf kinase activity as melanoma cell lines expressing the activating V600E mutation ((V600E)B-Raf). They were equally as sensitive to pharmacological inhibition of MEK1/2 for cell proliferation and transformation as (V600E)B-Raf cells. siRNA-mediated depletion of Raf-1 did not affect either ERK1/2 activation, whereas siRNA-mediated depletion of B-Raf reduced cell proliferation by up to 65% through the inhibition of ERK1/2 activation, irrespective of the mutational status of B-Raf. Pharmacological inhibition of cAMP-dependent protein kinase (PKA) and siRNA-mediated depletion of PKA greatly reduced B-Raf activity, ERK1/2 activation, and cell proliferation in (WT)B-Raf cells, whereas it did not affect (V600E)B-Raf cells, demonstrating a key role of PKA in mediating (WT)B-Raf/ERK signaling for uveal melanoma cell growth. Moreover, inactivation or depletion of PKA did not affect Rap-1 activity, and Rap-1 depletion did not affect either B-Raf activity or ERK1/2 activation. This ruled out a role for Rap1 in the PKA-mediated B-Raf/ERK activation in (WT)B-Raf cells. Finally, we demonstrated the importance of cyclin D1 in mediating PKA/(WT)B-Raf signaling for cell proliferation. Altogether, our results suggest that the PKA/B-Raf pathway is a potential target for therapeutic strategies against (WT)B-Raf-expressing uveal melanoma.     

Regulation and role of Raf-1/B-Raf heterodimerization

The Ras-Raf-MEK-extracellular signal-regulated kinase (ERK) pathway participates in the control of many fundamental cellular processes including proliferation, survival, and differentiation. The pathway is deregulated in up to 30% of human cancers, often due to mutations in Ras and the B-Raf isoform. Raf-1 and B-Raf can form heterodimers, and this may be important for cellular transformation. Here, we have analyzed the biochemical and biological properties of Raf-1/B-Raf heterodimers. Isolated Raf-1/B-Raf heterodimers possessed a highly increased kinase activity compared to the respective homodimers or monomers. Heterodimers between wild-type Raf-1 and B-Raf mutants with low or no kinase activity still displayed elevated kinase activity, as did heterodimers between wild-type B-Raf and kinase-negative Raf-1. In contrast, heterodimers containing both kinase-negative Raf-1 and kinase-negative B-Raf were completely inactive, suggesting that the kinase activity of the heterodimer specifically originates from Raf and that either kinase-competent Raf isoform is sufficient to confer high catalytic activity to the heterodimer. In cell lines, Raf-1/B-Raf heterodimers were found at low levels. Heterodimerization was enhanced by 14-3-3 proteins and by mitogens independently of ERK. However, ERK-induced phosphorylation of B-Raf on T753 promoted the disassembly of Raf heterodimers, and the mutation of T753 prolonged growth factor-induced heterodimerization. The B-Raf T753A mutant enhanced differentiation of PC12 cells, which was previously shown to be dependent on sustained ERK signaling. Fine mapping of the interaction sites by peptide arrays suggested a complex mode of interaction involving multiple contact sites with a main Raf-1 binding site in B-Raf encompassing T753. In summary, our data suggest that Raf-1/B-Raf heterodimerization occurs as part of the physiological activation process and that the heterodimer has distinct biochemical properties that may be important for the regulation of some biological processes.     

Identification of Raf-1 S471 as a novel phosphorylation site critical for Raf-1 and B-Raf kinase activities and for MEK binding

The Ras-Raf-MAPK cascade is a key growth-signaling pathway and its uncontrolled activation results in cell transformation. Although the general features of the signal transmission along the cascade are reasonably defined, the mechanisms underlying Raf activation remain incompletely understood. Here, we show that Raf-1 dephosphorylation, primarily at epidermal growth factor (EGF)-induced sites, abolishes Raf-1 kinase activity. Using mass spectrometry, we identified five novel in vivo Raf-1 phosphorylation sites, one of which, S471, is located in subdomain VIB of Raf-1 kinase domain. Mutational analyses demonstrated that Raf-1 S471 is critical for Raf-1 kinase activity and for its interaction with mitogen-activated protein kinase kinase (MEK). Similarly, mutation of the corresponding B-Raf site, S578, resulted in an inactive kinase, suggesting that the same Raf-1 and B-Raf phosphorylation is needed for Raf kinase activation. Importantly, the naturally occurring, cancer-associated B-Raf activating mutation V599E suppressed the S578A mutation, suggesting that introducing a charged residue at this region eliminates the need for an activating phosphorylation. Our results demonstrate an essential role of specific EGF-induced Raf-1 phosphorylation sites in Raf-1 activation, identify Raf-1 S471 as a novel phosphorylation site critical for Raf-1 and B-Raf kinase activities, and point to the possibility that the V599E mutation activates B-Raf by mimicking a phosphorylation at the S578 site.     

RKIP regulates MAP kinase signaling in cells with defective B-Raf activity

MAP kinase (MAPK) signaling results from activation of Raf kinases in response to external or internal stimuli. Here, we demonstrate that Raf kinase inhibitory protein (RKIP) regulates the activation of MAPK when B-Raf signaling is defective. We used multiple models including mouse embryonic fibroblasts (MEFs) and primary keratinocytes from RKIP- or Raf-deficient mice as well as allografts in mice to investigate the mechanism. Loss of B-Raf protein or activity significantly reduces MAPK activation in these cells. We show that RKIP depletion can rescue the compromised ERK activation and promote proliferation, and this rescue occurs through a Raf-1 dependent mechanism. These results provide formal evidence that RKIP is a bona fide regulator of Raf-1. We propose a new model in which RKIP plays a key role in regulating the ability of cells to signal through Raf-1 to ERK in B-Raf compromised cells.     

Serine and tyrosine phosphorylations cooperate in Raf-1, but not B-Raf activation

The Raf family of serine/threonine protein kinases couple growth factor receptor stimulation to mitogen activated protein kinase activation, but their own regulation is poorly understood. Using phospho-specific antisera, we show that activated Raf-1 is phosphorylated on S338 and Y341. Expression of Raf-1 with oncogenic Ras gives predominantly S338 phosphorylation, whereas activated Src gives predominantly Y341 phosphorylation. Phosphorylation at both sites is maximal only when both oncogenic Ras and activated Src are present. Raf-1 that cannot interact with Ras-GTP is not phosphorylated, showing that phosphorylation is Ras dependent, presumably occurring at the plasma membrane. Mutations which prevent phosphorylation at either site block Raf-1 activation and maximal activity is seen only when both are phosphorylated. Mutations at S339 or Y340 do not block Raf-1 activation. While B-Raf lacks a tyrosine phosphorylation site equivalent to Y341 of Raf-1, S445 of B-Raf is equivalent to S338 of Raf-1. Phosphorylation of S445 is constitutive and is not stimulated by oncogenic Ras. However, S445 phosphorylation still contributes to B-Raf activation by elevating basal and consequently Ras-stimulated activity. Thus, there are considerable differences between the activation of the Raf proteins; Ras-GTP mediates two phosphorylation events required for Raf-1 activation but does not regulate such events for B-Raf.     

Tumour cell responses to MEK1/2 inhibitors: acquired resistance and pathway remodelling

The Raf/MEK1/2 [mitogen-activated protein kinase/ERK (extracellular-signal-regulated kinase) kinase 1/2]/ERK1/2 signalling pathway is frequently activated in human tumours due to mutations in BRAF or KRAS. B-Raf and MEK1/2 inhibitors are currently undergoing clinical evaluation, but their ultimate success is likely to be limited by acquired drug resistance. We have used colorectal cancer cell lines harbouring mutations in B-Raf or K-Ras to model acquired resistance to the MEK1/2 inhibitor selumetinib (AZD6244). Selumetinib-resistant cells were refractory to other MEK1/2 inhibitors in cell proliferation assays and exhibited a marked increase in MEK1/2 and ERK1/2 activity and cyclin D1 abundance when assessed in the absence of inhibitor. This was driven by a common mechanism in which resistant cells exhibited an intrachromosomal amplification of their respective driving oncogene, B-Raf V600E or K-RasG13D. Despite the increased signal flux from Raf to MEK1/2, resistant cells maintained in drug actually exhibited the same level of ERK1/2 activity as parental cells, indicating that the pathway is remodelled by feedback controls to reinstate the normal level of ERK1/2 signalling that is required and sufficient to maintain proliferation in these cells. These results provide important new insights into how tumour cells adapt to new therapeutics and highlight the importance of homoeostatic control mechanisms in the Raf/MEK1/2/ERK1/2 signalling cascade.     

Conditionally oncogenic forms of the A-Raf and B-Raf protein kinases display different biological and biochemical properties in NIH 3T3 cells.

The protein kinase domains of mouse A-Raf and B-Raf were expressed as fusion proteins with the hormone binding domain of the human estrogen receptor in mammalian cells. In the absence of estradiol, 3T3 and rat1a cells expressing delta A-Raf:ER and delta B-Raf:ER were nontransformed, but upon the addition of estradiol the cells became oncogenically transformed. Morphological oncogenic transformation was more rapid and distinctive in cells expressing delta B-Raf:ER compared with cells expressing delta A-Raf:ER. Biochemical analysis of cells transformed by delta A-Raf:ER and delta B-Raf:ER revealed several interesting differences. The activation of delta B-Raf:ER consistently led to the rapid and robust activation of both MEK and p42/p44 MAP kinases. By contrast, the activation of delta A-Raf:ER led to a weak activation of MEK and the p42/p44 MAP kinases. The extent of activation of MEK in cells correlated with the ability of the different Raf kinases to phosphorylate and activate MEK1 in vitro. delta B-Raf:ER phosphorylated MEK1 approximately 10 times more efficiently than delta Raf-1:ER and at least 500 times more efficiently than delta A-Raf:ER under the conditions of the immune-complex kinase assays. These results were confirmed with epitope-tagged versions of the Raf kinase domains expressed in insect cells. The activation of all three delta Raf:ER proteins in 3T3 cells led to the hyperphosphorylation of the resident p74raf-1 and mSOS1 proteins, suggesting the possibility of "cross-talk" between the different Raf kinases and feedback regulation of intracellular signaling pathways. The activation of either delta B-Raf:ER or delta Raf-1:ER in quiescent 3T3 cells was insufficient to promote the entry of the cells into DNA synthesis. By contrast, the activation of delta A-Raf:ER in quiescent 3T3 cells was sufficient to promote the entry of the cells into S phase after prolonged exposure to beta-estradiol. The delta Raf:ER system has allowed us to reveal significant differences between the biological and biochemical properties of oncogenic forms of the Raf family of protein kinases. We anticipate that cells expressing these proteins and other estradiol-regulated protein kinases will be useful tools in future attempts to unravel the complex web of interactions involved in intracellular signal transduction pathways.     

B-Raf and Raf-1 are regulated by distinct autoregulatory mechanisms

B-Raf is a key regulator of the ERK pathway and is mutationally activated in two-thirds of human melanomas. In this work, we have investigated the activation mechanism of B-Raf and characterized the roles of Ras and of B-Raf phosphorylation in this regulation. Raf-1 is regulated by an N-terminal autoinhibitory domain whose actions are blocked by interaction with Ras and subsequent phosphorylation of Ser(338). We observed that B-Raf also contains an N-terminal autoinhibitory domain and that the interaction of this domain with the catalytic domain was inhibited by binding to active H-Ras. However, unlike Raf-1, the phosphorylation of B-Raf at Ser(445) was constitutive and was only moderately increased by expression of constitutively active H-Ras or constitutively active PAK1. Ser(445) phosphorylation is important to the B-Raf activation mechanism, however, because mutation of this site to alanine increased the affinity of the regulatory domain for the catalytic domain and increased autoinhibition. Similarly, expression of constitutively active PAK1 also decreased auto-inhibition. B-Raf autoinhibition was negatively regulated by acidic substitutions at phosphorylation sites within the activation loop of B-Raf and by the oncogenic substitution V599E. However, these substitutions did not affect the ability of the regulatory domain to co-immunoprecipitate with the catalytic domain. These data demonstrate that B-Raf activity is autoregulated, that constitutive phosphorylation of Ser(445) primes B-Raf for activation, and that a key feature of phosphorylation within the activation loop or of oncogenic mutations within this region is to block autoinhibition.     

Raf proteins and cancer: B-Raf is identified as a mutational target

A recent report has shown that activating mutations in the BRAF gene are present in a large percentage of human malignant melanomas and in a proportion of colon cancers. The vast majority of these mutations represent a single nucleotide change of T-A at nucleotide 1796 resulting in a valine to glutamic acid change at residue 599 within the activation segment of B-Raf. This exciting new discovery is the first time that a direct association between any RAF gene and human cancer has been reported. Raf proteins are also indirectly associated with cancer as effectors of activated Ras proteins, oncogenic forms of which are present in approximately one-third of all human cancers. BRAF and RAS mutations are rarely both present in the same cancers but the cancer types with BRAF mutations are similar to those with RAS mutations. This has been taken as evidence that the inappropriate regulation of the downstream ERKs (the p42/p44 MAP kinases) is a major contributing factor in the development of these cancers. Recent studies in mice with targeted mutations of the raf genes have confirmed that B-Raf is a far stronger activator of ERKs than its better studied Raf-1 homologue, even in cell types in which the protein is barely expressed. The explanation for this lies in a number of key differences in the regulation of B-Raf and Raf-1 activity. Constitutive phosphorylation of serine 445 of B-Raf leads to this protein having a higher basal kinase activity than Raf-1. Phosphorylation of threonine 598 and serine 601 within the activation loop of B-Raf at the plasma membrane also regulates its activity. The V599E mutation is thought to mimic these phosphorylations, resulting in a protein with high activity, leading to constitutive ERK activation. B-Raf now provides a critical new target to which drugs for treating malignant melanoma can be developed and, with this in mind, it is now important to gain clear insight into the biochemical properties of this relatively little characterised protein.     

Characterization of Ser338 phosphorylation for Raf-1 activation

Raf kinases are essential for regulating cell proliferation, survival, and tumorigenesis. However, the mechanisms by which Raf is activated are still incompletely understood. Phosphorylation plays a critical role in Raf activation in response to mitogens. The present study characterizes phosphorylation of Ser338, a crucial event for Raf-1 activation. Here we report that mutation of Lys375 to Met diminishes phosphorylation of Ser338 on both wild type Raf-1 in cells treated with epidermal growth factor (EGF) or 12-O-tetradecanoylphorbol-13-acetate (TPA) and a constitutively active mutant in which Tyr340/Tyr341 are replaced by 2 aspartic acids, a conserved substitution present in natural B-Raf. The loss of Ser338 phosphorylation in these Raf mutants is not engendered by a mutation-induced conformational change, inasmuch as mutation of another site (Ser471 to Ala) in the activation segment also abolishes Ser338 phosphorylation, whereas both the kinase-dead mutants of Raf-1 are phosphorylated well by active Pak1. Furthermore, our data demonstrate that EGF-stimulated phosphorylation of Ser338 is inhibited by Sorafenib, a Raf kinase inhibitor, but not by the MEK inhibitor U0126. Interestingly, a kinase-dead mutation and Sorafenib also markedly reduce phosphorylation of Ser445 on B-Raf, a site equivalent to Raf-1 Ser338. Finally, our data reveal that Ser338 is phosphorylated on inactive Raf-1 by an active mutant of Raf-1 when they are dimerized in cells and that artificial dimerization of Raf-1 causes Ser338 phosphorylation, accompanied by activation of ERK1/2. Altogether, our data suggest that Ser338 on Raf-1 is autophosphorylated in response to mitogens.     

Essential role of B-Raf in oligodendrocyte maturation and myelination during postnatal central nervous system development

Mutations in the extracellular signal-regulated kinase (ERK) pathway, particularly in the mitogen-activated protein kinase/ERK kinase (MEK) activator B-Raf, are associated with human tumorigenesis and genetic disorders. Hence, B-Raf is a prime target for molecule-based therapies, and understanding its essential biological functions is crucial for their success. B-Raf is expressed preferentially in cells of neuronal origin. Here, we show that in mice, conditional ablation of B-Raf in neuronal precursors leads to severe dysmyelination, defective oligodendrocyte differentiation, and reduced ERK activation in brain. Both B-Raf ablation and chemical inhibition of MEK impair oligodendrocyte differentiation in vitro. In glial cell cultures, we find B-Raf in a complex with MEK, Raf-1, and kinase suppressor of Ras. In B-Raf-deficient cells, more Raf-1 is recruited to MEK, yet MEK/ERK phosphorylation is impaired. These data define B-Raf as the rate-limiting MEK/ERK activator in oligodendrocyte differentiation and myelination and have implications for the design and use of Raf inhibitors.     

Oncogenic B-RafV600E inhibits apoptosis and promotes ERK-dependent inactivation of Bad and Bim

Recent studies have revealed that B-Raf mutations are very common in malignant melanoma and are required for tumor growth and maintenance. The majority of melanoma-associated B-Raf mutations involve a single point mutation, V600E, which results in greatly elevated B-Raf kinase activity and constitutive activation of MAPK/ERK downstream. Here we show that B-Raf(V600E) increases resistance to apoptosis induced by chemotherapeutic drugs and promotes ERK-dependent phosphorylation of the BH3-only proteins Bim and Bad that are involved in setting thresholds for apoptosis. ERK-dependent phosphorylation of Bim resulted in degradation of this BH3-only protein, whereas phosphorylation of Bad has previously been shown to result in its sequestration by 14-3-3 proteins. Consistent with this, inhibition of ERK activity in a panel of melanoma cell lines resulted in stabilization of Bim and dephosphorylation of Bad. Furthermore, apoptosis induced through overexpression of Bad or Bim was efficiently blocked by coexpression of mutant B-Raf(V600E). However, small interfering RNA-mediated silencing of Bim and Bad expression conferred only modest protection against cytotoxic drugs, whereas oncogenic B-Raf strongly protected against the same stimuli. These observations suggest that B-Raf-initiated inactivation of Bad and Bim only partly contributes to the anti-apoptotic activities of this oncogene and that other points within the cell death machinery are also targeted by deregulated ERK signaling.     

Raf-1 and B-Raf promote protein kinase C theta interaction with BAD

PKCtheta regulates the proliferation, survival and differentiation of T-cells. Here we show that PKCtheta interacts with Raf-1 and B-Raf kinases. Raf-1 enhanced the kinase activity of associated PKCtheta, while PKCtheta reduced the catalytic activity of associated Raf-1. In contrast, B-Raf binding did not affect PKCtheta kinase activity, and PKCtheta did not change B-Raf activity. Coexpression of mutationally activated Raf-1 in cells enhanced the phosphorylation of T538 in the PKCtheta activation loop. PKCtheta and Raf cooperated in terms of binding to BAD, a pro-apoptotic Bcl-2 family protein that is inactivated by phosphorylation. While neither Raf-1 nor B-Raf could phosphorylate BAD, they enhanced the ability of PKCtheta to interact with BAD and to phosphorylate BAD in vitro and in vivo, suggesting a new role for Raf proteins in T-cells by targeting PKCtheta to interact with and phosphorylate BAD.     

Ras-mutant Cancer Cells Display B-Raf Binding to Ras That Activates Extracellular Signal-regulated Kinase and Is Inhibited by Protein Kinase A Phosphorylation

The small G protein Ras regulates proliferation through activation of the mitogen-activated protein (MAP) kinase (ERK) cascade. The first step of Ras-dependent activation of ERK signaling is Ras binding to members of the Raf family of MAP kinase kinase kinases, C-Raf and B-Raf. Recently, it has been reported that in melanoma cells harboring oncogenic Ras mutations, B-Raf does not bind to Ras and does not contribute to basal ERK activation. For other types of Ras-mutant tumors, the relative contributions of C-Raf and B-Raf are not known. We examined non-melanoma cancer cell lines containing oncogenic Ras mutations and express both C-Raf and B-Raf isoforms, including the lung cancer cell line H1299 cells. Both B-Raf and C-Raf were constitutively bound to oncogenic Ras and contributed to Ras-dependent ERK activation. Ras binding to B-Raf and C-Raf were both subject to inhibition by the cAMP-dependent protein kinase PKA. cAMP inhibited the growth of H1299 cells and Ras-dependent ERK activation via PKA. PKA inhibited the binding of Ras to both C-Raf and B-Raf through phosphorylations of C-Raf at Ser-259 and B-Raf at Ser-365, respectively. These studies demonstrate that in non-melanocytic Ras-mutant cancer cells, Ras signaling to B-Raf is a significant contributor to ERK activation and that the B-Raf pathway, like that of C-Raf, is a target for inhibition by PKA. We suggest that cAMP and hormones coupled to cAMP may prove useful in dampening the effects of oncogenic Ras in non-melanocytic cancer cells through PKA-dependent actions on B-Raf as well as C-Raf.     

Topological analysis of MAPK cascade for kinetic ErbB signaling

Ligand-induced homo- and hetero-dimer formation of ErbB receptors results in different biological outcomes irrespective of recruitment and activation of similar effector proteins. Earlier experimental research indicated that cells expressing both EGFR (epidermal growth factor receptor) and the ErbB4 receptor (E1/4 cells) induced E1/4 cell-specific B-Raf activation and higher extracellular signal-regulated kinase (ERK) activation, followed by cellular transformation, than cells solely expressing EGFR (E1 cells) in Chinese hamster ovary (CHO) cells. Since our experimental data revealed the presence of positive feedback by ERK on upstream pathways, it was estimated that the cross-talk/feedback pathway structure of the Raf-MEK-ERK cascade might affect ERK activation dynamics in our cell system. To uncover the regulatory mechanism concerning the ERK dynamics, we used topological models and performed parameter estimation for all candidate structures that possessed ERK-mediated positive feedback regulation of Raf. The structure that reliably reproduced a series of experimental data regarding signal amplitude and duration of the signaling molecules was selected as a solution. We found that the pathway structure is characterized by ERK-mediated positive feedback regulation of B-Raf and B-Raf-mediated negative regulation of Raf-1. Steady-state analysis of the estimated structure indicated that the amplitude of Ras activity might critically affect ERK activity through ERK-B-Raf positive feedback coordination with sustained B-Raf activation in E1/4 cells. However, Rap1 that positively regulates B-Raf activity might be less effective concerning ERK and B-Raf activity. Furthermore, we investigated how such Ras activity in E1/4 cells can be regulated by EGFR/ErbB4 heterodimer-mediated signaling. From a sensitivity analysis of the detailed upstream model for Ras activation, we concluded that Ras activation dynamics is dominated by heterodimer-mediated signaling coordination with a large initial speed of dimerization when the concentration of the ErbB4 receptor is considerably high. Such characteristics of the signaling cause the preferential binding of the Grb2-SOS complex to heterodimer-mediated signaling molecules.     

K-Ras and B-Raf oncogenes inhibit colon epithelial polarity establishment through up-regulation of c-myc

KRAS, BRAF, and PI3KCA are the most frequently mutated oncogenes in human colon cancer. To explore their effects on morphogenesis, we used the colon cancer-derived cell line Caco-2. When seeded in extracellular matrix, individual cells proliferate and generate hollow, polarized cysts. The expression of oncogenic phosphatidylinositol 3-kinase (PI3KCA H1047R) in Caco-2 has no effect, but K-Ras V12 or B-Raf V600E disrupts polarity and tight junctions and promotes hyperproliferation, resulting in large, filled structures. Inhibition of mitogen-activated protein/extracellular signal-regulated kinase (ERK) kinase blocks the disruption of morphology, as well as the increased levels of c-myc protein induced by K-Ras V12 and B-Raf V600E. Apical polarity is already established after the first cell division (two-cell stage) in Caco-2 three-dimensional cultures. This is disrupted by expression of K-Ras V12 or B-Raf V600E but can be rescued by ribonucleic acid interference-mediated depletion of c-myc. We conclude that ERK-mediated up-regulation of c-myc by K-Ras or B-Raf oncogenes disrupts the establishment of apical/basolateral polarity in colon epithelial cells independently of its effect on proliferation.