Raf kinase inhibitory protein regulates aurora B kinase and the spindle checkpoint

Raf kinase inhibitory protein (RKIP or PEBP) is an inhibitor of the Raf/MEK/MAP kinase signaling cascade and a suppressor of cancer metastasis. We now show that RKIP associates with centrosomes and kinetochores and regulates the spindle checkpoint in mammalian cells. RKIP depletion causes decreases in the mitotic index, the number of metaphase cells, and traversal times from nuclear envelope breakdown to anaphase, and an override of mitotic checkpoints induced by spindle poisons. Raf-1 depletion or MEK inhibition reverses the reduction in the mitotic index, whereas hyperactivation of Raf mimics the RKIP-depletion phenotype. Finally, RKIP depletion or Raf hyperactivation reduces kinetochore localization and kinase activity of Aurora B, a regulator of the spindle checkpoint. These results indicate that RKIP regulates Aurora B kinase and the spindle checkpoint via the Raf-1/MEK/ERK cascade and demonstrate that small changes in the MAP kinase (MAPK) pathway can profoundly impact the fidelity of the cell cycle.     

Raf激酶抑制蛋白的研究进展

Raf激酶抑制蛋白(RKIP)是磷脂酰乙醇胺结合蛋白家族的成员。RKIP通过与Raf-1结合,抑制了Ras/Raf-1/MEK/ERK信号转导通路,并在NF-κB及G蛋白偶联受体(GPCR)信号转导通路中也起重要调节作用。RKIP参与细胞凋亡、肿瘤转移、神经发育以及精子发生等病理生理过程,通过研究RKIP能为治疗相关疾病提供新思路新靶点。本文主要介绍RKIP的生物功能,着重于其在神经系统、肿瘤和生殖系统中的研究进展。     

Raf kinase inhibitor protein correlates with sensitivity of nasopharyngeal carcinoma to radiotherapy

Raf kinase inhibitory protein (RKIP) is a metastasis suppressor whose expression is reduced in nasopharyngeal carcinoma (NPC) tissues and is absent in NPC metastases. To investigate the effect of RKIP on radiosensitivity of NPC, high metastatic 5‐8F with low RKIP expression and non‐metastatic 6‐10B with high RKIP expression were stably transfected with plasmids that expressed sense and antisense RKIP cDNA. Overexpression of RKIP sensitized 5‐8F cells to radiation‐induced cell death, G₂‐M cell cycle arrest and apoptosis. In contrast, downexpression of RKIP in 6‐10B cells protected cells from radiation‐induced cell death, G₂‐M cell cycle arrest and apoptosis. In addition, RKIP expression altered the radiosensitivity of NPC cells through MEK and ERK phosphorylation changes of Raf‐1/MEK/ERK signaling pathway. We further investigated the RKIP expression in NPC patients and its association with patients' survival after radiotherapy. Downexpression of RKIP was significantly correlated with advanced clinical stage, lymph node metastasis and radioresistance. Furthermore, survival curves showed that patients with RKIP downexpression had a poor prognosis and induced relapse. Multivariate analysis confirmed that RKIP expression was an independent prognostic indicator. The data suggested that RKIP was a potential biomarker for the radiosensitivity and prognosis of NPC, and its dysregulation might play an important role in the radioresistance of NPC. J. Cell. Biochem. 110: 975–984, 2010. © 2010 Wiley‐Liss, Inc.     

The Raf/MEK/extracellular signal-regulated kinase 1/2 pathway can mediate growth inhibitory and differentiation signaling via androgen receptor downregulation in prostate cancer cells

Upregulated ERK1/2 activity is correlated with androgen receptor (AR) downregulation in certain prostate cancer (PCa) that exhibits androgen deprivation-induced neuroendocrine differentiation, but its functional relevance requires elucidation. We found that sustained ERK1/2 activation using active Raf or MEK1/2 mutants is sufficient to induce AR downregulation at mRNA and protein levels in LNCaP. Downregulation of AR protein, but not mRNA, was blocked by proteasome inhibitors, MG132 and bortezomib, indicating that the pathway regulation is mediated at multiple points. Ectopic expression of a constitutively active AR inhibited Raf/MEK/ERK-mediated regulation of the differentiation markers, neuron-specific enolase and neutral endopeptidase, and the cyclin-dependent kinase inhibitors, p16^INK4A and p21^CIP1, but not Rb phosphorylation and E2F1 expression, indicating that AR has a specific role in the pathway-mediated differentiation and growth inhibitory signaling. However, despite the sufficient role of Raf/MEK/ERK, its inhibition using U0126 or ERK1/2 knockdown could not block androgen deprivation-induced AR downregulation in an LNCaP neuroendocrine differentiation model, suggesting that additional signaling pathways are involved in the regulation. We additionally report that sustained Raf/MEK/ERK activity can downregulate full length as well as hormone binding domain-deficient AR isoforms in androgen-refractory C4-2 and CWR22Rv1, but not in LAPC4 and MDA-PCa-2b. Our study demonstrates a novel role of the Raf/MEK/ERK pathway in regulating AR expression in certain PCa types and provides an insight into PCa responses to its aberrant activation.     

Raf激酶抑制蛋白在信号传导通路及凋亡中的关键作用

细胞维持自身的完整性存在众多调控,避免它从一个生物学状态到另一个状态随意的转换。Raf激酶抑制蛋白（Rafkinase inhibitor protein,RKIP）,属于磷脂酰乙醇胺结合蛋白（phosphatidylethanolamine binding protein,PEBP）家族的成员,是新的一类信号级联传导的调节器来维持细胞自身平衡。RKIP可以抑制MAP激酶途径（Raf-1／MEK／ERK）,G蛋白偶联途径（G—protein coupled receptor,GPCR）,以及NF-κb途径。正因为RKIP在这些信号传导中的作用,使其在细胞凋亡过程中发挥了关键性的作用。更加深入的了解RKIP如何在肿瘤细胞调控表达,了解它如何对细胞凋亡、信号传导产生影响可以为人类治疗癌症起到极大的推动作用。     

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.     

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.     

Molecular mechanism of cofilin dephosphorylation by ouabain

We previously reported that phosphorylated cofilin-triosephosphate isomerase (TPI) complex interacts with Na,K-ATPase and enhances the pump activity through the phosphorylation of cofilin via Rho-mediated signaling pathway. In this study, we tested the hypothesis that the dephosphorylation of cofilin may be induced through Na,K-ATPase inhibition by ouabain. The phosphorylation level of cofilin by ouabain which decreases in a time- and dose-dependent manner in various human cell lines, remains unchanged by pretreatment with Src inhibitor, PP2; epidermal growth factor receptor (EGFR) inhibitor, AG1478; Raf-1 kinase (Raf) inhibitor, GW5074; and ERK kinase (MEK) inhibitor, PD98059, and by transfection of Ras dominant negative mutant (RasN17). This suggests that ouabain dephosphorylates cofilin through the Src/EGFR/Ras/Raf/MEK pathway. Ouabain activates Ras/Raf/MEK pathway, but down-regulates Rho kinase (ROCK)/LIM kinase (LIMK)/cofilin pathway, implying that there may be a cross-talk by ouabain between the Ras/Raf/MEK and the ROCK/LIMK/cofilin pathways. Immunofluorescence and flow cytometry suggest that ouabain-induced active form of cofilin may be involved in cytoskeletal reorganization and cell volume regulation. Thus, these findings demonstrate a new molecular mechanism for the dephosphorylation of cofilin through the inhibition of Na,K-ATPase by ouabain.     

Induction of Raf kinase inhibitor protein contributes to macrophage differentiation

Differential gene expression analysis of human blood monocytes has identified the Raf kinase inhibitor protein (RKIP) as a continuously upregulated gene in macrophage and dendritic cell maturation. Using realtime RT-PCR and Western blot analysis we were able to confirm the initial DNA-microarray findings of RKIP induction on mRNA and protein levels. RKIP upregulation in primary cells and overexpression in THP-1 cells did not alter ERK activity but strongly reduced the amount of the NFkappaB subunit p65 in the nucleus. mRNA levels and cell surface expression of maturation markers including the integrin CD11c and the scavenger receptor CD36 were significantly increased in RKIP transfected THP-1 cells. Our data show for the first time that RKIP is upregulated during macrophage and dendritic cell differentiation on mRNA and protein levels and we conclude that RKIP contributes to the monocytic differentiation process via inhibition of the NFkappaB signaling cascade independent from the canonical Ras/Raf/MEK/ERK pathway.     

The biology of a prostate cancer metastasis suppressor protein: Raf kinase inhibitor protein

Raf kinase inhibitor protein (RKIP) was originally identified as a protein that bound membrane phospholipids and was named phosphatidylethanolamine binding protein-2 (PEBP-2). RKIP was than identified as a protein that bound Raf and blocked its ability to phosphorylate MEK, thus earning its new name of RKIP. Subsequent to identification of its role in the Raf:MEK pathway, RKIP has been demonstrated to regulate several other signaling pathways including G-protein signaling and NF-kappaB signaling. Its involvement in several signaling pathways has engendered RKIP to contribute to several physiological processes including membrane biosynthesis, spermatogenesis, neural development, and apoptosis. RKIP is expressed in many tissues including brain, lung, and liver and thus, dysregulation of RKIP expression or function has potential to contribute to pathophysiology in these tissues. Loss of RKIP expression in prostate cancer cells confers a metastatic phenotype on them. Additionally, restoration of RKIP expression in a metastatic prostate cancer cell line does not effect primary tumor growth, but it does inhibit prostate cancer metastasis. These parameters identify RKIP as a metastasis suppressor gene. In this review, the biology and pathophysiology of RKIP is described.     

MEK1-ERKs级联激活方式是调控单纯疱疹病毒Ⅱ型复制的重要机制

本研究通过阐明MEK1和MEK2亚型在单纯疱疹病毒Ⅱ型(herpes simplex virus type 2,HSV2)复制中介导的Raf/MEK/ERK(简称ERK)通路活化中的作用,以期进一步阐明该通路调控病毒复制的机制.研究中应用了MEK抑制剂U0126、针对MEK1和MEK2的特异性小干扰RNA(small ...     

The RKIP (Raf-1 Kinase Inhibitor Protein) conserved pocket binds to the phosphorylated N-region of Raf-1 and inhibits the Raf-1-mediated activated phosphorylation of MEK

The Raf-MEK-ERK pathway regulates many fundamental biological processes, and its activity is finely tuned at multiple levels. The Raf kinase inhibitory protein (RKIP) is a widely expressed negative modulator of the Raf-MEK-ERK signaling pathway. We have previously shown that RKIP inhibits the phosphorylation of MEK by Raf-1 through interfering with the formation of a kinase-substrate complex by direct binding to both Raf-1 and MEK. Here, we show that the evolutionarily conserved ligand-binding pocket of RKIP is required for its inhibitory activity towards the Raf-1 kinase mediated activation of MEK. Single amino acid substitutions of two of the conserved residues form the base and the wall of the pocket confers a loss-of-function phenotype on RKIP. Loss-of-function RKIP mutants still appear to bind to Raf-1. However the stability of the complexes formed between mutants and the N-region Raf-1 phosphopeptide were drastically reduced. Our results therefore suggest that the RKIP conserved pocket may constitute a novel phosphoamino-acid binding motif and is absolutely required for RKIP function.     

Structural basis for RKIP binding with its substrate Raf1 kinase

Raf1 kinase inhibitor protein (RKIP) negatively regulates the Raf1/MEK/ERK pathway which is vital for cell growth and differentiation. It is also a biomarker in clinical cancer diagnosis. RKIP binds to the N-terminus of Raf1 kinase but little is known about the structural basis of RKIP binding with Raf1. Here, we demonstrate that the N-terminus of human Raf1 kinase (hRaf1_1-147aa) binds with human RKIP (hRKIP) at its ligand-binding pocket, loop “127–149”, and the C-terminal helix by NMR experiments. D70, D72, E83, Y120, and Y181 were further verified as the key residues participating in the interaction of hRKIP and hRaf1_1-147aa. G143-R146 fragment was also critical for hRKIP binding with hRaf1_1-147aa, for its deletion decreased the binding affinity around 300 times, from 154 to 0.46 mM^?1. Our results provide important structural clues for designing the lead compound that disrupts RKIP–Raf1 interaction.     

MAP kinase meets mitosis: A role for Raf Kinase Inhibitory Protein in spindle checkpoint regulation

Raf Kinase Inhibitory Protein (RKIP) is an evolutionarily conserved protein that functions as a modulator of signaling by the MAP kinase cascade. Implicated as a metastasis suppressor, Raf Kinase Inhibitory Protein depletion correlates with poor prognosis for breast, prostate and melanoma tumors but the mechanism is unknown. Recent evidence indicates that Raf Kinase Inhibitory Protein regulates the mitotic spindle assembly checkpoint by controlling Aurora B Kinase activity, and the mechanism involves Raf/MEK/ERK signaling. In contrast to elevated MAP kinase signaling during the G1, S or G2 phases of the cell cycle that activates checkpoints and induces arrest or senescence, loss of RKIP during M phase leads to bypass of the spindle assembly checkpoint and the generation of chromosomal abnormalities. These results reveal a role for Raf Kinase Inhibitory Protein and the MAP kinase cascade in ensuring the fidelity of chromosome segregation prior to cell division. Furthermore, these data highlight the need for precise titration of the MAP kinase signal to ensure the integrity of the spindle assembly process and provide a mechanism for generating genomic instability in tumors. Finally, these results raise the possibility that RKIP status in tumors could influence the efficacy of treatments such as poisons that stimulate the Aurora B-dependent spindle assembly checkpoint.     

Suppression of coronavirus replication by inhibition of the MEK signaling pathway

We previously demonstrated that infection of cultured cells with murine coronavirus mouse hepatitis virus (MHV) resulted in activation of the mitogen-activated protein kinase (Raf/MEK/ERK) signal transduction pathway (Y. Cai et al., Virology 355:152-163, 2006). Here we show that inhibition of the Raf/MEK/ERK signaling pathway by the MEK inhibitor UO126 significantly impaired MHV progeny production (a reduction of 95 to 99% in virus titer), which correlated with the phosphorylation status of ERK1/2. Moreover, knockdown of MEK1/2 and ERK1/2 by small interfering RNAs suppressed MHV replication. The inhibitory effect of UO126 on MHV production appeared to be a general phenomenon since the effect was consistently observed in all six different MHV strains and in three different cell types tested; it was likely exerted at the postentry steps of the virus life cycle because the virus titers were similarly inhibited from infected cells treated at 1 h prior to, during, or after infection. Furthermore, the treatment did not affect the virus entry, as revealed by the virus internalization assay. Metabolic labeling and reporter gene assays demonstrated that translation of cellular and viral mRNAs appeared unaffected by UO126 treatment. However, synthesis of viral genomic and subgenomic RNAs was severely suppressed by UO126 treatment, as demonstrated by a reduced incorporation of [3H]uridine and a decrease in chloramphenicol acetyltransferase (CAT) activity in a defective-interfering RNA-CAT reporter assay. These findings indicate that the Raf/MEK/ERK signaling pathway is involved in MHV RNA synthesis.     

E3 ligases and deubiquitinating enzymes regulating the MAPK signaling pathway in cancers

The mitogen-activated protein kinase (MAPK) signaling pathway is the primary regulatory module of various cellular processes such as cell proliferation, differentiation, and stress responses. This pathway converts external stimuli to cellular responses via three major kinases: mitogen-activated protein kinase (MAPK), mitogen-activated protein kinase kinase (MAPKK), and mitogen-activated protein kinase kinase kinase (MAPKKK). Ubiquitination is a post-translational modification of proteins with ubiquitin, which results in the formation of mono- or poly-ubiquitin chains of substrate proteins. Conversely, removal of the ubiquitin by deubiquitinating enzymes (DUBs) is known as deubiquitination. This review summarizes mechanisms of the MAPK signaling pathways (ERK1/2, ERK5, p38, and JNK1/2/3 signaling pathway) in cancers, and of E3 ligases and DUBs that target the MAPK signaling components such as Raf, MEK1/2, ERK1/2, MEKK2/3, MEKK1-4, TAK1, DLK1, MLK1-4, ASK1/2, and MKK3-7.