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.     

Systems‐level interactions between insulin–EGF networks amplify mitogenic signaling

Crosstalk mechanisms have not been studied as thoroughly as individual signaling pathways. We exploit experimental and computational approaches to reveal how a concordant interplay between the insulin and epidermal growth factor (EGF) signaling networks can potentiate mitogenic signaling. In HEK293 cells, insulin is a poor activator of the Ras/ERK (extracellular signal‐regulated kinase) cascade, yet it enhances ERK activation by low EGF doses. We find that major crosstalk mechanisms that amplify ERK signaling are localized upstream of Ras and at the Ras/Raf level. Computational modeling unveils how critical network nodes, the adaptor proteins GAB1 and insulin receptor substrate (IRS), Src kinase, and phosphatase SHP2, convert insulin‐induced increase in the phosphatidylinositol‐3,4,5‐triphosphate (PIP₃) concentration into enhanced Ras/ERK activity. The model predicts and experiments confirm that insulin‐induced amplification of mitogenic signaling is abolished by disrupting PIP₃‐mediated positive feedback via GAB1 and IRS. We demonstrate that GAB1 behaves as a non‐linear amplifier of mitogenic responses and insulin endows EGF signaling with robustness to GAB1 suppression. Our results show the feasibility of using computational models to identify key target combinations and predict complex cellular responses to a mixture of external cues.     

Investigating Differential Dynamics of the MAPK Signaling Cascade Using a Multi-Parametric Global Sensitivity Analysis

Cell growth critically depends on signalling pathways whose regulation is the focus of intense research. Without utilizing a priori knowledge of the relative importance of pathway components, we have applied in silico computational methods to the EGF-induced MAPK cascade. Specifically, we systematically perturbed the entire parameter space, including initial conditions, using a Monte Carlo approach, and investigate which protein components or kinetic reaction steps contribute to the differentiation of ERK responses. The model, based on previous work by Brightman and Fell (2000), is composed of 28 reactions, 27 protein molecules, and 48 parameters from both mass action and Michaelis-Menten kinetics. Our multi-parametric systems analysis confirms that Raf inactivation is one of the key steps regulating ERK responses to be either transient or sustained. Furthermore, the results of amplitude-differential ERK phosphorylations within the transient case are mainly attributed to the balance between activation and inactivation of Ras while duration-differential ERK responses for the sustained case are, in addition to Ras, markedly affected by dephospho-/phosphorylation of both MEK and ERK. Our sub-module perturbations showed that MEK and ERK''s contribution to this differential ERK activation originates from fluctuations in intermediate pathway module components such as Ras and Raf, implicating a cooperative regulatory mode among the key components. The initial protein concentrations of corresponding reactions such as Ras, GAP, and Raf also influence the distinct signalling outputs of ERK activation. We then compare these results with those obtained from a single-parametric perturbation approach using an overall state sensitivity (OSS) analysis. The OSS findings indicate a more pronounced role of ERK''s inhibitory feedback effect on catalysing the dissociation of the SOS complex. Both approaches reveal the presence of multiple specific reactions involved in the distinct dynamics of ERK responses and the cell fate decisions they trigger. This work adds a mechanistic insight of the contribution of key pathway components, thus may support the identification of biomarkers for pharmaceutical drug discovery processes.     

Identification of signaling molecules mediating corticotropin-releasing hormone-R1alpha-mitogen-activated protein kinase (MAPK) interactions: the critical role of phosphatidylinositol 3-kinase in regulating ERK1/2 but not p38 MAPK activation

In most target cells, activation of the type 1 CRH receptor (CRH-R1) by CRH or urocortin (UCN I) leads to stimulation of the Gs-protein/adenylyl cyclase/protein kinase A cascade. Signal transduction of CRH-R1 also involves alternative pathways such as phosphorylation of ERK1/2 and p38 MAPK, two members of the MAPK family that mediate important pathophysiological responses. The intracellular pathways by which CRH-R1 activates these MAPK are only partially understood; here we characterized further signaling mechanisms and molecules involved in CRH-R1-mediated ERK1/2 and p38 MAPK activation. In human embryonic kidney 293 cells overexpressing recombinant CRH-R1alpha, UCN I induced ERK1/2 and p38 MAPK activation was dependent on signaling molecules involved in agonist-induced CRH-R1alpha trafficking and endocytosis. Furthermore, time course studies and use of selective inhibitors demonstrated that ERK1/2 activation occured within 5 min, was sustained for at least 60 min, and was dependent on both phosphatidylinositol 3-kinase (PI3-K)/Akt activation and epidermoid growth factor receptor transactivation involving matrix metelloproteinases. UCN I effect on p38 MAPK phosphorylation was more transient, returned to basal within 40 min and was dependent on epidermoid growth factor receptor transactivation, but not PI3-K/Akt activation. Overexpression of G(alpha-)transducin, showed that G(betagamma)-subunit activation is only partially required for ERK1/2 phosphorylation and does not play a role in p38 MAPK phosphorylation, whereas overexpression of a dominant-negative Ras (Ras N17) attenuated both ERK and p38 MAPK activation. In conclusion, a complex signaling network appears to mediate CRH-R1alpha-MAPK interactions; PI3-K might play a critical role in the regulation of CRH-R1alpha signaling selectivity and cellular responses.     

Distinct LPS-induced signals regulate LPS uptake and morphological changes in medfly hemocytes

Recently we demonstrated that lipopolysaccharide (LPS) promotes activation of the Ras/ERK cascade in medfly hemocytes and that phagocytosis of Escherichia coli by insect hemocytes is mediated by an integrin-dependent process via the activation of FAK/Src complex (J Biol Chem 273 (1998) 14813; FEBS Letters 496 (2001) 55). In the current study we wanted to further elucidate the effects of LPS on medfly hemocytes, in order to better understand the regulation of the evolutionary conserved signaling mechanisms between insects and mammals. We initially observed that different stimuli, including LPS, E. coli, RGD, fibronectin and heat shock activate hemocyte ERK. The response of hemocytes to these stimuli denoted that hemocyte ERK is evidently stimulated by at least an LPS receptor and via an integrin-mediated process. The medfly hemocytes respond to LPS by changing their morphology, inducing the activation of several signaling pathways, including Ras/MEK/ERK, PI-3K/ERK and Rho pathways and contributing to LPS uptake. Experiments based on inhibitors of specific signaling pathways, such as manumycin A, toxin A, U0126, PD98059 and wortmannin revealed that Ras, MEK and PI-3K are involved in the activation of ERK. Whether PI-3K is an intermediate of Ras/MEK/ERK pathway or activates ERK via other signaling pathway it remains to be elucidated. ERK is not activated via Rho pathway, denoting that Rho may not be an upstream effector molecule of ERK pathway. Regarding the role(s) that these kinases play in hemocytes, it can be suggested that PI-3K and Rho GTPases can modulate hemocyte shape changes, whereas ERK, Ras and MEK cannot. In addition, PI-3K as well as Ras and MEK through ERK activation participate in LPS endocytosis. Therefore, PI-3K shares a dual role; it is involved both in cell shape changes and in LPS endocytosis. Since ERK activation appears to be independent of the integrity of actin filaments, as cytochalasin D and latrunculin A did not block ERK activation, it can be concluded that LPS endocytosis is independent of actin cytoskeleton remodeling as is the case in mammalian systems.     

Phosphorylation of Serine 779 in Fibroblast Growth Factor Receptor 1 and 2 by Protein Kinase C? Regulates Ras/Mitogen-activated Protein Kinase Signaling and Neuronal Differentiation

The FGF receptors (FGFRs) control a multitude of cellular processes both during development and in the adult through the initiation of signaling cascades that regulate proliferation, survival, and differentiation. Although FGFR tyrosine phosphorylation and the recruitment of Src homology 2 domain proteins have been widely described, we have previously shown that FGFR is also phosphorylated on Ser⁷⁷⁹ in response to ligand and binds the 14-3-3 family of phosphoserine/threonine-binding adaptor/scaffold proteins. However, whether this receptor phosphoserine mode of signaling is able to regulate specific signaling pathways and biological responses is unclear. Using PC12 pheochromocytoma cells and primary mouse bone marrow stromal cells as models for growth factor-regulated neuronal differentiation, we show that Ser⁷⁷⁹ in the cytoplasmic domains of FGFR1 and FGFR2 is required for the sustained activation of Ras and ERK but not for other FGFR phosphotyrosine pathways. The regulation of Ras and ERK signaling by Ser⁷⁷⁹ was critical not only for neuronal differentiation but also for cell survival under limiting growth factor concentrations. PKCϵ can phosphorylate Ser⁷⁷⁹ in vitro, whereas overexpression of PKCϵ results in constitutive Ser⁷⁷⁹ phosphorylation and enhanced PC12 cell differentiation. Furthermore, siRNA knockdown of PKCϵ reduces both growth factor-induced Ser⁷⁷⁹ phosphorylation and neuronal differentiation. Our findings show that in addition to FGFR tyrosine phosphorylation, the phosphorylation of a conserved serine residue, Ser⁷⁷⁹, can quantitatively control Ras/MAPK signaling to promote specific cellular responses.     

Chemoattractant stimulation of TORC2 is regulated by receptor/G protein–targeted inhibitory mechanisms that function upstream and independently of an essential GEF/Ras activation pathway in Dictyostelium

Global stimulation of Dictyostelium with different chemoattractants elicits multiple transient signaling responses, including synthesis of cAMP and cGMP, actin polymerization, activation of kinases ERK2, TORC2, and phosphatidylinositide 3-kinase, and Ras-GTP accumulation. Mechanisms that down-regulate these responses are poorly understood. Here we examine transient activation of TORC2 in response to chemically distinct chemoattractants, cAMP and folate, and suggest that TORC2 is regulated by adaptive, desensitizing responses to stimulatory ligands that are independent of downstream, feedback, or feedforward circuits. Cells with acquired insensitivity to either folate or cAMP remain fully responsive to TORC2 activation if stimulated with the other ligand. Thus TORC2 responses to cAMP or folate are not cross-inhibitory. Using a series of signaling mutants, we show that folate and cAMP activate TORC2 through an identical GEF/Ras pathway but separate receptors and G protein couplings. Because the common GEF/Ras pathway also remains fully responsive to one chemoattractant after desensitization to the other, GEF/Ras must act downstream and independent of adaptation to persistent ligand stimulation. When initial chemoattractant concentrations are immediately diluted, cells rapidly regain full responsiveness. We suggest that ligand adaptation functions in upstream inhibitory pathways that involve chemoattractant-specific receptor/G protein complexes and regulate multiple response pathways.     

Convergence of signaling pathways on the activation of ERK in B cells

The B cell receptor (BCR) initiates three major signaling pathways: the Ras pathway, which leads to extracellular signal-regulated kinase (ERK) activation; the phospholipase C-gamma pathway, which causes calcium mobilization; and the phosphoinositide 3-kinase (PI 3-kinase) pathway. These combine to induce different biological responses depending on the context of the BCR signal. Both the Ras and PI 3-kinase pathways are important for B cell development and activation. Several model systems show evidence of cross-regulation between these pathways. Here we demonstrate through the use of PI 3-kinase inhibitors and a dominant-negative PI 3-kinase construct that the BCR-induced phosphorylation and activation of ERK is dependent on PI 3-kinase. PI 3-kinase feeds into the Ras signaling cascade at multiple points, both upstream and downstream of Ras. We also show that ERK activation is dependent on phospholipase C-gamma, in keeping with its dependence on calcium mobilization. Last, the activation of PI 3-kinase itself is completely dependent on Ras. We conclude that the PI 3-kinase and Ras signaling cascades are intimately connected in B cells and that the activation of ERK is a signal integration point, since it requires simultaneous input from all three major signaling pathways.     

PI3K is required for insulin-stimulated but not EGF-stimulated ERK1/2 activation

The Ras/Raf/extracellular signal-regulated kinase 1 and 2 (ERK1/2) signaling pathway is known to cross-talk with other signaling pathways, including phosphatidylinositol 3-kinase (PI3K)/Akt pathway. However, the role of PI3K in ERK-1/2 activation induced by tyrosine kinase receptors was not fully understood. Here, we report that two structurally distinct PI3K inhibitors, wortmannin and LY294002, inhibited insulin-induced activation of ERK1/2 but had no effect on EGF-induced activation of ERK1/2 in hepatocellular carcinoma BEL-7402 and SMMC-7721 cells, breast cancer MCF-7 cells, and prostate cancer LNCaP cells. Although protein kinase C could act as a mediator between PI3K and ERK1/2, protein kinase C inhibitor chelerythrine chloride did not inhibit insulin-induced ERK1/2 activation. Both insulin- and EGF-induced ERK1/2 activation are strictly dependent on Ras activation, however, wortmannin only inhibited insulin-induced, but not EGF-induced Ras activation. These results indicate that PI3K plays different roles in the activation of Ras/ERK1/2 signaling by insulin and EGF, and that insulin-stimulated, but not EGF-stimulated, ERK1/2 and Akt signalings diverge at PI3K.     

The metastasis suppressor Nm23 as a modulator of Ras/ERK signaling

NM23-H1 (also known as NME1) was the first identified metastasis suppressor, which displays a nucleoside diphosphate kinase (NDPK) and histidine protein kinase activity. NDPKs are linked to many processes, such as cell migration, proliferation, differentiation, but the exact mechanism whereby NM23-H1 inhibits the metastatic potential of cancer cells remains elusive. However, some recent data suggest that NM23-H1 may exert its anti-metastatic effect by blocking Ras/ERK signaling. In mammalian cell lines NDPK-mediated attenuation of Ras/ERK signaling occurs through phosphorylation (thus inactivation) of KSR (kinase suppressor of Ras) scaffolds. In this review I summarize our knowledge about KSR’s function and its regulation in mammals and in C. elegans. Genetic studies in the nematode contributed substantially to our understanding of the function and regulation of the Ras pathway (i.e. KSR’s discovery is also linked to the nematode). Components of the RTK/Ras/ERK pathway seem to be highly conserved between mammals and worms. NDK-1, the worm homolog of NM23-H1 affects Ras/MAPK signaling at the level of KSRs, and a functional interaction between NDK-1/NDPK and KSRs was first demonstrated in the worm in vivo. However, NDK-1 is a factor, which is necessary for proper MAPK activation, thus it activates rather than suppresses Ras/MAPK signaling in the worm. The contradiction between results in mammalian cell lines and in the worm regarding NDPKs’ effect exerted on the outcome of Ras signaling might be resolved, if we better understand the function, structure and regulation of KSR scaffolds.     

Lose and gain: impacts of ERK5 and JNK cascades on each other

Kinase cascades in ERK5 (Extracellular signal-regulated kinases) and JNK (c-Jun N-terminal kinases) signaling pathways mediate the sensing and processing of stimuli. Cross-talks between signaling cascades is a likely phenomenon that can cause apparently different biological responses from a single pathway, on its activation. Feedback loops have the potential to greatly alter the properties of a pathway and its response to stimuli. Based on enzyme kinetic reactions, mathematical models have been developed to predict and analyze the impacts of cross-talks and feedback loops in ERK5 and JNK cascades. It has been observed that, there is no significant impact on neither ERK5 activation nor JNKs’ activation due to cross-talks between them. But it is due to cross-talks and feedback loops in ERK5 and JNK cascade, ERK5 gets activated in a transient manner in the absence of input signals. Planning to obtain the parameter values from the experimentalist and the result should be validated by experimental verification.     

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.     

Prolactin-stimulated activation of ERK1/2 mitogen-activated protein kinases is controlled by PI3-kinase/Rac/PAK signaling pathway in breast cancer cells

There is strong evidence that deregulation of prolactin (PRL) signaling contributes to pathogenesis and chemoresistance of breast cancer. Therefore, understanding cross-talk between distinct signal transduction pathways triggered by activation of the prolactin receptor (PRL-R), is essential for elucidating the pathogenesis of metastatic breast cancer.In this study, we applied a sequential inhibitory analysis of various signaling intermediates to examine the hierarchy of protein interactions within the PRL signaling network and to evaluate the relative contributions of multiple signaling branches downstream of PRL-R to the activation of the extracellular signal-regulated kinases ERK1 and ERK2 in T47D and MCF-7 human breast cancer cells.Quantitative measurements of the phosphorylation/activation patterns of proteins showed that PRL simultaneously activated Src family kinases (SFKs) and the JAK/STAT, phosphoinositide-3 (PI3)-kinase/Akt and MAPK signaling pathways. The specific blockade or siRNA-mediated suppression of SFK/FAK, JAK2/STAT5, PI3-kinase/PDK1/Akt, Rac/PAK or Ras regulatory circuits revealed that (1) the PI3-kinase/Akt pathway is required for activation of the MAPK/ERK signaling cascade upon PRL stimulation; (2) PI3-kinase-mediated activation of the c-Raf-MEK1/2-ERK1/2 cascade occurs independent of signaling dowstream of STATs, Akt and PKC, but requires JAK2, SFKs and FAK activities; (3) activated PRL-R mainly utilizes the PI3-kinase-dependent Rac/PAK pathway rather than the canonical Shc/Grb2/SOS/Ras route to initiate and sustain ERK1/2 signaling. By interconnecting diverse signaling pathways PLR may enhance proliferation, survival, migration and invasiveness of breast cancer cells.     

PI3K‐dependent cross‐talk interactions converge with Ras as quantifiable inputs integrated by Erk

Although it is appreciated that canonical signal‐transduction pathways represent dominant modes of regulation embedded in larger interaction networks, relatively little has been done to quantify pathway cross‐talk in such networks. Through quantitative measurements that systematically canvas an array of stimulation and molecular perturbation conditions, together with computational modeling and analysis, we have elucidated cross‐talk mechanisms in the platelet‐derived growth factor (PDGF) receptor signaling network, in which phosphoinositide 3‐kinase (PI3K) and Ras/extracellular signal‐regulated kinase (Erk) pathways are prominently activated. We show that, while PI3K signaling is insulated from cross‐talk, PI3K enhances Erk activation at points both upstream and downstream of Ras. The magnitudes of these effects depend strongly on the stimulation conditions, subject to saturation effects in the respective pathways and negative feedback loops. Motivated by those dynamics, a kinetic model of the network was formulated and used to precisely quantify the relative contributions of PI3K‐dependent and ‐independent modes of Ras/Erk activation.     

Vav mediates Ras stimulation by direct activation of the GDP/GTP exchange factor Ras GRP1

Here we describe a new signaling cross-talk between the Vav/Rac1 and Ras pathways that is established through the stimulation of RasGRP1, an exchange factor for Ras subfamily GTPases. This interaction is crucial for Ras activation in lymphoid cells, since this GTPase cannot become activated in the absence of Vav proteins. The activation of RasGRP1 requires both the generation of diacylglycerol via phospho lipase C-gamma and the induction of actin polymerization, two responses induced by Vav and Rac1 that facilitate the translocation of RasGRP1 to juxtamembrane areas of the cell. Consistent with this, the cross-talk can be activated by tyrosine-phosphorylated wild-type Vav, oncogenic Vav and constitutively active Rac1. Conversely, Ras activation can be blocked in lymphocytes and ectopic systems using inhibitors affecting either phospholipase C-gamma or F-actin polymerization. These results indicate that a relay mechanism exists in lymphoid and other cells helping in the generation of robust signaling responses by the Rac/Rho and Ras pathways upon receptor engagement.     

Increased Protein Expression of the PTEN Tumor Suppressor in the Presence of Constitutively Active Notch-1

Mammalian Notch-1 is part of an evolutionarily conserved family of transmembrane receptorsbest known for involvement in cell fate decisions. Mutations that result in Notch-1 activation result inT-lineage oncogenesis. In other cell lineages, however, studies have indicated that cooperation withcellular signaling pathways, such as Ras, is necessary for Notch-mediated oncogenesis and in somesettings, Notch-1 has been reported to function as a tumor suppressor. In order to test the hypothesisthat the Notch-1 pathway exhibits cross-talk with Ras/Raf/MEK/ERK, the constitutively activecytoplasmic portion of Notch-1 was introduced into 293 HEK fibroblasts via retroviral transduction.ERK-1,-2 activation was markedly increased in cells expressing constitutively active Notch-1. Thesecells exhibited a more rounded morphology as compared to 293 cells transduced with an empty vectoror parental 293 cells. These observations correlated with decreased total and phosphorylated focaladhesion kinase protein (FAK). Subsequent examination of phosphatase and tensin homolog deletedon chromosome 10 (PTEN) revealed that total and phosphorylated PTEN protein was elevated in cellsexpressing constitutively active Notch-1. Loss of Akt phosphorylation was also observed in cellsbearing activated Notch-1. Two potential binding sites for the Notch effector CBF-1 were identified inthe human PTEN promoter sequence. A PTEN promoter luciferase reporter exhibited increasedactivity in the presence of Notch-1 signaling. These data indicate that Notch-1 can participate incross-talk with other signaling pathways such as Ras/Raf/MEK/ERK through the regulation of thePTEN tumor suppressor.     

Impact of oncogenic driver mutations on feedback between the PI3K and MEK pathways in cancer cells

Inhibition of the PI3K (phosphoinositide 3-kinase)/Akt/mTORC1 (mammalian target of rapamycin complex 1) and Ras/MEK [MAPK (mitogen-activated protein kinase)/ERK (extracellular-signal-regulated kinase) kinase]/ERK pathways for cancer therapy has been pursued for over a decade with limited success. Emerging data have indicated that only discrete subsets of cancer patients have favourable responses to these inhibitors. This is due to genetic mutations that confer drug insensitivity and compensatory mechanisms. Therefore understanding of the feedback mechanisms that occur with respect to specific genetic mutations may aid identification of novel biomarkers that predict patient response. In the present paper, we show that feedback between the PI3K/Akt/mTORC1 and Ras/MEK/ERK pathways is cell-line-specific and highly dependent on the activating mutation of K-Ras or overexpression c-Met. We found that cell lines exhibited differential signalling and apoptotic responses to PD184352, a specific MEK inhibitor, and PI103, a second-generation class I PI3K inhibitor. We reveal that feedback from the PI3K/Akt/mTORC1 to the Ras/MEK/ERK pathway is present in cancer cells harbouring either K-Ras activating mutations or amplification of c-Met but not the wild-type counterparts. Moreover, we demonstrate that inhibition of protein phosphatase activity by OA (okadaic acid) restored PI103-mediated feedback in wild-type cells. Together, our results demonstrate a novel mechanism for feedback between the PI3K/Akt/mTORC1 and the Ras/MEK/ERK pathways that only occurs in K-Ras mutant and c-Met amplified cells but not the isogenic wild-type cells through a mechanism that may involve inhibition of a specific endogenous phosphatase(s) activity. We conclude that monitoring K-Ras and c-Met status are important biomarkers for determining the efficacy of PI103 and other PI3K/Akt inhibitors in cancer therapy.     

The Scaffold Protein Shoc2/SUR-8 Accelerates the Interaction of Ras and Raf

Shoc2/SUR-8 positively regulates Ras/ERK MAP kinase signaling by serving as a scaffold for Ras and Raf. Here, we examined the role of Shoc2 in the spatio-temporal regulation of Ras by using a fluorescence resonance energy transfer (FRET)-based biosensor, together with computational modeling. In epidermal growth factor-stimulated HeLa cells, RNA-mediated Shoc2 knockdown reduced the phosphorylation of MEK and ERK with half-maximal inhibition, but not the activation of Ras. For the live monitoring of Ras binding to Raf, we utilized a FRET biosensor wherein Ras and the Ras-binding domain of Raf were connected tandemly and sandwiched with acceptor and donor fluorescent proteins for the FRET measurement. With this biosensor, we found that Shoc2 was required for the rapid interaction of Ras with Raf upon epidermal growth factor stimulation. To decipher the molecular mechanisms underlying the kinetics, we developed two computational models that might account for the action of Shoc2 in the Ras-ERK signaling. One of these models, the Shoc2 accelerator model, provided a reasonable explanation of the experimental observations. In this Shoc2 accelerator model, Shoc2 accelerated both the association and dissociation of Ras-Raf interaction. We propose that Shoc2 regulates the spatio-temporal patterns of the Ras-ERK signaling pathway primarily by accelerating the Ras-Raf interaction.