The Raf/MEK/ERK pathway: new concepts of activation.

The Raf/MEK/ERK signaling was the first MAP kinase cascade to be characterized. It is probably one of the most well known signal transduction pathways among biologists because of its implication in a wide variety of cellular functions as diverse -and occasionally contradictory- as cell proliferation, cell-cycle arrest, terminal differentiation and apoptosis. Discovery and understanding of this pathway have benefited from the combination of both genetic studies in worms and flies and biochemical studies in mammalian cells. However, ten years after, this field is still under debate and new molecular partners in the cascade continue to increase the complexity of its regulation. This review deals with the emergence of new concepts in the activation and regulation of the Raf/MEK/ERK module. In particular, the preponderant role of B-Raf is underlined, and the role of novel regulators such as KSR is discussed.     

Changed genome heterochromatinization upon prolonged activation of the Raf/ERK signaling pathway

The Raf/ERK (Extracellular Signal Regulated Kinase) signal transduction pathway controls numerous cellular processes, including growth, differentiation, cellular transformation and senescence. ERK activation is thought to involve complex spatial and temporal regulation, to achieve a high degree of specificity, though precisely how this is achieved remains to be confirmed. We report here that prolonged activation of a conditional form of c-Raf-1 (BXB-ER) leads to profound changes in the level and distribution of a heterochromatic histone mark. In mouse fibroblasts, the heterochromatic trimethylation of lysine 9 in histone H3 (H3K9Me3) is normally confined to pericentromeric regions. However, following ERK activation a genome-wide redistribution of H3K9Me3 correlates with loss of the histone modification from chromocentres and the appearance of numerous punctuate sites throughout the interphase nucleus. These epigenetic changes during interphase correlate with altered chromosome structure during mitosis, where robust H3K9Me3 signals appear within telomeric heterochromatin. This pattern of heterochromatinization is distinct from previously described oncogene induced senescence associated heterochromatin foci (SAHF), which are excluded from telomeres. The H3K9Me3 histone mark is known to bind the major heterochromatin protein HP1 and we show that the alterations in the distribution of this histone epistate correlate with redistribution of HP1β throughout the nucleus. Interestingly while ERK activation is fully reversible, the observed chromatin changes induced by epigenetic modifications are not reversible once established. We describe for the first time a link from prolonged ERK activation to stable changes in genome organization through redistribution of heterochromatic domains involving the telomeres. These epigenetic changes provide a possible mechanism through which prolonged activation of Raf/ERK can lead to growth arrest or the induction of differentiation, senescence and cancer.     

Growth arrest signaling of the Raf/MEK/ERK pathway in cancer

The Raf/MEK/extraceUular signal-regulated kinase （ERK） pathway has a pivotal role in facilitating cell proliferation, and its deregulated activation is a central signature of many epithelial cancers. However paradoxically, sustained activity of Raf/MEK/ERK can also result in growth arrest in many different cell types. This anti-proliferative Raf/MEK/ERK signaling also has physiological significance, as exemplified by its potential as a tumor suppressive mechanism. Therefore, significant questions include in which cell types and by what mechanisms this pathway can mediate such an opposing context of signaling. Particularly, our understating of the role of ERK1 and ERK2, the focal points of pathway signaling, in growth arrest signaling is still limited. This review discusses these aspects of Raf/MEK/ ERK-mediated growth arrest signaling.     

Mammalian Sprouty4 suppresses Ras-independent ERK activation by binding to Raf1

The signalling cascade including Raf, mitogen-activated protein kinase (MAPK) kinase and extracellular-signal-regulated kinase (ERK) is important in many facets of cellular regulation. Raf is activated through both Ras-dependent and Ras-independent mechanisms, but the regulatory mechanisms of Raf activation remain unclear. Two families of membrane-bound molecules, Sprouty and Sprouty-related EVH1-domain-containing protein (Spred) have been identified and characterized as negative regulators of growth-factor-induced ERK activation. But the molecular functions of mammalian Sproutys have not been clarified. Here we show that mammalian Sprouty4 suppresses vascular epithelial growth factor (VEGF)-induced, Ras-independent activation of Raf1 but does not affect epidermal growth factor (EGF)-induced, Ras-dependent activation of Raf1. Sprouty4 binds to Raf1 through its carboxy-terminal cysteine-rich domain, and this binding is necessary for the inhibitory activity of Sprouty4. In addition, Sprouty4 mutants of the amino-terminal region containing the conserved tyrosine residue, which is necessary for suppressing fibroblast growth factor signalling, still inhibit the VEGF-induced ERK pathway. Our results show that receptor tyrosine kinases use distinct pathways for Raf and ERK activation and that Sprouty4 differentially regulates these pathways.     

Modulation of the Akt/Ras/Raf/MEK/ERK pathway by A3 adenosine receptor

Downstream A₃ receptor signalling plays an important role in the regulation of cell death and proliferation. Therefore, it is important to determine the molecular pathways involved through A₃ receptor stimulation. The phosphatidylinositide-3-OH kinase (PI3K)/Akt and the Raf/mitogen-activated protein kinase (MAPK/ERK) kinase (MEK)/mitogen-activated protein kinase (MAPK) pathways have central roles in the regulation of cell survival and proliferation. The crosstalk between these two pathways has also been investigated. The focus of this review centres on downstream mediators of A₃ adenosine receptor signalling.     

Modulation of the Ras/Raf/MEK/ERK pathway by Ca(2+), and calmodulin

Ras activation induces a variety of cellular responses that depend on the specific activated effector, the intensity and amplitude of its activation, and the cellular type. Transient activation followed by a sustained but low signal of the Ras/Raf/MEK/ERK pathway is a common feature of cell proliferation in many systems. On the contrary, sustained, high activation is linked with either senescence or apoptosis in fibroblasts and to differentiation in neurones and PC12 cells. The temporal regulation of the pathway is relevant and not only depends on the specific receptor activated but also on the presence of diverse modulators of the pathway. We review here evidence showing that calcium (Ca(2+)) and calmodulin (CaM) are able to regulate the Ras/Raf/MEK/ERK pathway. CaM-binding proteins (CaMBPs) as Ras-GRF and CaM-dependent protein kinase IV (CaMKIV) positively modulate ERK1/2 activation induced by either NGF or membrane depolarisation in neurones. In fibroblasts, CaM binding to EGF receptor and K-Ras(B) may be involved in the downregulation of the pathway after its activation, allowing a proliferative signalling.     

Contribution of the ERK5/MEK5 pathway to Ras/Raf signaling and growth control.

The activity of the catalytic domain of the orphan MAP kinase ERK5 is increased by Ras but not Raf-1 in cells, which suggests that ERK5 might mediate Raf-independent signaling by Ras. We found that Raf-1 does contribute to Ras activation of ERK5 but in a manner that does not correlate with Raf-1 catalytic activity. A clue to the mechanism of action of Raf-1 on ERK5 comes from the observation that endogenous Raf-1 binds to endogenous ERK5, suggesting the involvement of regulatory protein-protein interactions. This interaction is specific because Raf-1 binds only to ERK5 and not ERK2 or SAPK. Finally, we demonstrate the ERK5/MEK5 pathway is required for Raf-dependent cellular transformation and that a constitutively active form of MEK5, MEK5DD, synergizes with Raf to transform NIH 3T3 cells. These observations suggest that ERK5 plays a large role in Raf-1-mediated signal transduction.     

Roles of the Raf/MEK/ERK pathway in cell growth, malignant transformation and drug resistance

Growth factors and mitogens use the Ras/Raf/MEK/ERK signaling cascade to transmit signals from their receptors to regulate gene expression and prevent apoptosis. Some components of these pathways are mutated or aberrantly expressed in human cancer (e.g., Ras, B-Raf). Mutations also occur at genes encoding upstream receptors (e.g., EGFR and Flt-3) and chimeric chromosomal translocations (e.g., BCR-ABL) which transmit their signals through these cascades. Even in the absence of obvious genetic mutations, this pathway has been reported to be activated in over 50% of acute myelogenous leukemia and acute lymphocytic leukemia and is also frequently activated in other cancer types (e.g., breast and prostate cancers). Importantly, this increased expression is associated with a poor prognosis. The Ras/Raf/MEK/ERK and Ras/PI3K/PTEN/Akt pathways interact with each other to regulate growth and in some cases tumorigenesis. For example, in some cells, PTEN mutation may contribute to suppression of the Raf/MEK/ERK cascade due to the ability of activated Akt to phosphorylate and inactivate different Rafs. Although both of these pathways are commonly thought to have anti-apoptotic and drug resistance effects on cells, they display different cell lineage specific effects. For example, Raf/MEK/ERK is usually associated with proliferation and drug resistance of hematopoietic cells, while activation of the Raf/MEK/ERK cascade is suppressed in some prostate cancer cell lines which have mutations at PTEN and express high levels of activated Akt. Furthermore the Ras/Raf/MEK/ERK and Ras/PI3K/PTEN/Akt pathways also interact with the p53 pathway. Some of these interactions can result in controlling the activity and subcellular localization of Bim, Bak, Bax, Puma and Noxa. Raf/MEK/ERK may promote cell cycle arrest in prostate cells and this may be regulated by p53 as restoration of wild-type p53 in p53 deficient prostate cancer cells results in their enhanced sensitivity to chemotherapeutic drugs and increased expression of Raf/MEK/ERK pathway. Thus in advanced prostate cancer, it may be advantageous to induce Raf/MEK/ERK expression to promote cell cycle arrest, while in hematopoietic cancers it may be beneficial to inhibit Raf/MEK/ERK induced proliferation and drug resistance. Thus the Raf/MEK/ERK pathway has different effects on growth, prevention of apoptosis, cell cycle arrest and induction of drug resistance in cells of various lineages which may be due to the presence of functional p53 and PTEN and the expression of lineage specific factors.     

RNA viruses and the mitogenic Raf/MEK/ERK signal transduction cascade

The Raf/MEK/ERK signal transduction cascade belongs to the mitogen-activated protein kinase (MAPK) cascades. Raf/MEK/ERK signaling leads to stimulus-specific changes in gene expression, alterations in cell metabolism or induction of programmed cell death (apoptosis), and thus controls cell differentiation and proliferation. It is induced by extracellular agents, including pathogens such as RNA viruses. Many DNA viruses are known to induce cellular signaling via this pathway. As these pathogens partly use the DNA synthesis machinery for their replication, they aim to drive cells into a proliferative state. In contrast, the consequences of RNA virus-induced Raf/MEK/ERK signaling were less clear for a long time, but since the turn of the century the number of publications on this topic has rapidly increased. Research on this virus/host-interaction will broaden our understanding of its relevance in viral replication. This important control center of cellular responses is differently employed to support the replication of several important human pathogenic RNA viruses including influenza, Ebola, hepatitis C and SARS corona viruses.     

The Raf/MEK/ERK pathway can govern drug resistance,apoptosis and sensitivity to targeted therapy

The effects of the Raf/MEK/ERK and PI3K/PTEN/Akt/mTOR signaling pathways on proliferation, drug resistance, prevention of apoptosis and sensitivity to signal transduction inhibitors were examined in FL/ΔAkt-1:ER*(Myr⁺) + ΔRaf-1:AR cells which are conditionally-transformed to grow in response to Raf and Akt activation. Drug resistant cells were isolated from FL/ΔAkt-1:ER*(Myr⁺) + ΔRaf-1:AR cells in the presence of doxorubicin. Activation of Raf-1, in the drug resistant FL/ΔAkt-1:ER*(Myr⁺) + ΔRaf-1:AR cells, increased the IC₅₀ for doxorubicin 80-fold, whereas activation of Akt-1, by itself, had no effect on the doxorubicin IC₅₀. However, Akt-1 activation enhanced cell proliferation and clonogenicity in the presence of chemotherapeutic drugs. Thus the Raf/MEK/ERK pathway had profound effects on the sensitivity to chemotherapeutic drugs, and Akt-1 activation was required for the long-term growth of these cells as well as resistance to chemotherapeutic drugs. The effects of doxorubicin on the induction of apoptosis in the drug resistant cells were enhanced by addition of either mTOR and MEK inhibitors. These results indicate that targeting the Raf/MEK/ERK and PI3K/Akt/mTOR pathways may be an effective approach for therapeutic intervention in drug resistant cancers that have mutations activating these cascades.     

The Raf/MEK/ERK pathway can govern drug resistance,apoptosis and sensitivity to targeted therapy

The effects of the Raf/MEK/ERK and PI3K/PTEN/Akt/mTOR signaling pathways on proliferation, drug resistance, prevention of apoptosis and sensitivity to signal transduction inhibitors were examined in FL/ΔAkt-1:ER*(Myr⁺) + ΔRaf-1:AR cells which are conditionally-transformed to grow in response to Raf and Akt activation. Drug resistant cells were isolated from FL/ΔAkt-1:ER*(Myr⁺) + ΔRaf-1:AR cells in the presence of doxorubicin. Activation of Raf-1, in the drug resistant FL/ΔAkt-1:ER*(Myr⁺) + ΔRaf-1:AR cells, increased the IC₅₀ for doxorubicin 80-fold, whereas activation of Akt-1, by itself, had no effect on the doxorubicin IC₅₀. However, Akt-1 activation enhanced cell proliferation and clonogenicity in the presence of chemotherapeutic drugs. Thus the Raf/MEK/ERK pathway had profound effects on the sensitivity to chemotherapeutic drugs, and Akt-1 activation was required for the long-term growth of these cells as well as resistance to chemotherapeutic drugs. The effects of doxorubicin on the induction of apoptosis in the drug resistant cells were enhanced by addition of either mTOR and MEK inhibitors. These results indicate that targeting the Raf/MEK/ERK and PI3K/Akt/mTOR pathways may be an effective approach for therapeutic intervention in drug resistant cancers that have mutations activating these cascades.     

Cardiac hypertrophy: Targeting Raf/MEK/ERK1/2-signaling

Over the past two decades, basic research has revealed a complex network of regulatory mechanisms that control the ERK1/2-signaling cascade. ERK1/2 mediate cardiac hypertrophy, a major risk factor for the development of arrhythmias, heart failure and sudden death, but also beneficial effects, e.g. protection of the heart from cell death and ischemic injury. Selective targeting of these ambiguous ERK functions could provide a powerful tool in the treatment of cardiac disease. This short review will discuss new mechanistic insights into ERK1/2-dependent development of cardiac hypertrophy and the prospect to translate this knowledge into future therapeutic strategies.     

Chlamydial Antiapoptotic Activity Involves Activation of the Raf/MEK/ERK Survival Pathway

Chlamydiae are obligate intracellular bacteria that cause variety of human diseases. Chlamydia-infected host cells are profoundly resistant to apoptosis induced by many different apoptotic stimuli. The inhibition of apoptosis is thought to be an important immune escape mechanism allowing chlamydiae to productively complete their obligate intracellular growth cycle. Infection with chlamydiae can activate the Raf/MEK/ERK pathway. Because the survival pathway can modulate apoptosis, we used MEK-specific inhibitor U0126 and Raf-specific inhibitor GW5074 to examine the role of Raf/MEK/ERK pathway in chlamydial antiapoptotic activity. Apoptosis was induced by staurosporine (STS) and detected by morphology, DNA fragmentation, caspase-3 activation, and poly (ADP-ribose) polymerase cleavage. Inhibition of the pathway sensitized Chlamydia-infected cells to STS-mediated cell apoptosis. The data indicate that chlamydial antiapoptotic activity involves activation of the Raf/MEK/ERK survival pathway.     

主动脉弓缩窄诱导大鼠心肌肥大Raf/MEK/ERK通路的变化

目的:通过观察心肌肥大大鼠加速纤维肉瘤/丝裂素活化蛋白激酶激酶/胞外信号调节蛋白激酶(Raf/MEK/ERK)通路关键因子的基因和蛋白表达及蛋白磷酸化修饰水平上的变化,了解Raf/MEK/ERK通路在心肌肥大调控中的作用。方法: 20只SD大鼠随机分为假手术组和模型组,通过主动脉弓缩窄(TAC)法建立心肌肥大模型,12周后颌下静脉取血分离血清,检测氨基末端脑钠肽前体(NT-proBNP)含量,之后进行超声心动图测定和麻醉下的血流动力学测定,收集心肌标本,观察心肌组织的病理学改变,检测心肌组织Raf/MEK/ERK通路的关键因子基因、蛋白表达水平及蛋白磷酸化水平的变化。结果:与假手术组比较,TAC模型组大鼠超声心动图的左室舒张末期室间隔厚度(IVSd)、左室收缩末期室间隔厚度(IVSs)、左室后壁舒张末期厚度(LVPWd)、左室后壁收缩末期厚度(LVPWs)显著增厚(P＜0.05,P＜0.01),左室收缩末期内径(LVIDs)显著减小(P＜0.01),左心室质量(LV Mass)、左心系数LW(LV Mass/Weight)比值显著增加(P＜0.05,P＜0.01);大鼠心率(HR)、左心室最大收缩速率(+dp/dtmax)、左心室最大舒张速率(-dp/dtmax) 均显著降低(P＜0.01),血清中NT-pro BNP含量显著增加(P＜ 0.01);心肌细胞排列杂乱,心肌细胞肥大、胞质明显增多,炎症细胞浸润,出现大量胶原纤维沉积,大面积心肌细胞呈现蓝色;大鼠心肌组织中c-Raf在Ser259和Ser338上的磷酸化蛋白phospho-c-Raf (Ser259)和phospho-c-Raf (Ser338) 表达水平显著升高(P＜0.01),其下游MEK1/2、ERK1/2的磷酸化蛋白phospho-MEK1/2(Ser217/Ser221)和phospho-ERK1/2 (Thr202/Tyr204)表达水平也显著增高(P＜0.01)。结论: Raf/MEK/ERK通路在心肌肥大中的调控作用,可能通过激活关键因子c-Raf、MEK1、MEK2、ERK1和ERK2特异性位点的磷酸化实现的。     

Activation of Raf/MEK/ERK/cPLA2 signaling pathway is essential for chlamydial acquisition of host glycerophospholipids

Chlamydiae, a diverse group of obligate intracellular pathogens replicating within cytoplasmic vacuoles of eukaryotic cells, are able to acquire lipids from host cells. Here we report that activation of the host Raf-MEK-ERK-cPLA2 signaling cascade is required for the chlamydial uptake of host glycerophospholipids. Both the MAP kinase pathway (Ras/Raf/MEK/ERK) and Ca(2+)-dependent cytosolic phospholipase A2 (cPLA2) were activated in chlamydia-infected cells. The inhibition of cPLA2 activity resulted in the blockade of the chlamydial uptake of host glycerophospholipids and impairment in chlamydial growth. Blocking either c-Raf-1 or MEK1/2 activity prevented the chlamydial activation of ERK1/2, leading to the suppression of both chlamydial activation of the host cPLA2 and uptake of glycerophospholipids from the host cells. The chlamydia-induced phosphorylation of cPLA2 was also blocked by a dominant negative ERK2. Furthermore, activation of both ERK1/2 and cPLA2 was dependent on chlamydial growth and restricted within chlamydia-infected cells, suggesting an active manipulation of the host ERK-cPLA2 signaling pathway by chlamydiae.     

Metformin induces differentiation in acute promyelocytic leukemia by activating the MEK/ERK signaling pathway

Recent studies have shown that metformin, a widely used antidiabetic agent, may reduce the risk of cancer development. In this study, we investigated the antitumoral effect of metformin on both acute myeloid leukemia (AML) and acute promyelocytic leukemia (APL) cells. Metformin induced apoptosis with partial differentiation in an APL cell line, NB4, but only displayed a proapoptotic effect on several non-M3 AML cell lines. Further analysis revealed that a strong synergistic effect existed between metformin and all-trans retinoic acid (ATRA) during APL cell maturation and that metformin induced the hyperphosphorylation of extracellular signal-regulated kinase (ERK) in APL cells. U0126, a specific MEK/ERK activation inhibitor, abrogated metformin-induced differentiation. Finally, we found that metformin induced the degradation of the oncoproteins PML-RARα and c-Myc and activated caspase-3. In conclusion, these results suggest that metformin treatment may contribute to the enhancement of ATRA-induced differentiation in APL, which may deepen the understanding of APL maturation and thus provide insight for new therapy strategies.     

Ultrasound stimulates NF‐κB activation and iNOS expression via the Ras/Raf/MEK/ERK signaling pathway in cultured preosteoblasts

It has been shown that ultrasound (US) stimulation accelerates fracture healing in the animal models and non‐operatively clinical uses. Nitric oxide (NO) is a crucial early mediator in mechanically induced bone formation. Here we found that US‐mediated inducible nitric oxide synthase (iNOS) expression was attenuated by Ras inhibitor (manumycin A), Raf‐1 inhibitor (GW5074), MEK inhibitor (PD98059), NF‐κB inhibitor (PDTC), and IκB protease inhibitor (TPCK). US‐induced Ras activation was inhibited by manumycin A. Raf‐1 phosphorylation at Ser³³⁸ by US was inhibited by manumycin A and GW5074. US‐induced MEK and ERK activation was inhibited by manumycin A, GW5074, and PD98059. Stimulation of preosteoblasts with US activated IκB kinase α/β (IKK α/β), IκBαphosphorylation, p65 phosphorylation at Ser²⁷⁶, p65, and p50 translocation from the cytosol to the nucleus, and κB‐luciferase activity. US‐mediated an increase of IKK α/β, IκBα, and p65 phosphorylation, κB‐luciferase activity and p65 and p50 binding to the NF‐κB element was inhibited by manumycin A, GW5074, and PD98059. Our results suggest that US increased iNOS expression in preosteoblasts via the Ras/Raf‐1/MEK/ERK/IKKαβ and NF‐κB signaling pathways. J. Cell. Physiol. 220: 196–203, 2009. © 2009 Wiley‐Liss, Inc.