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 ...     

Regulation of human immunodeficiency virus type 1 infectivity by the ERK mitogen-activated protein kinase signaling pathway

ERK1 and ERK2 mitogen-activated protein kinases (MAPK) play a critical role in regulation of cell proliferation and differentiation in response to mitogens and other extracellular stimuli. Mitogens and cytokines that activate MAPK in T cells have been shown to activate human immunodeficiency virus type 1 (HIV-1) replication. Little is known about the signal transduction pathways that activate HIV-1 replication in T cells upon activation by extracellular stimulation. Here, we report that activation of MAPK through the Ras/Raf/MEK signaling pathway enhances the infectivity of HIV-1 virions. Virus infectivity was enhanced by treatment of cells with MAPK stimulators, such as serum and phorbol myristate acetate, as well as by coexpression of constitutively activated Ras, Raf, or MEK (MAPK kinase) in the absence of extracellular stimulation. Treatment of cells with PD 098059, a specific inhibitor of MAPK activation, or with a MAPK antisense oligonucleotide reduced the infectivity of HIV-1 virions without significantly affecting virus production or the levels of virion-associated Gag and Env proteins. MAPK has been shown to regulate HIV-1 infectivity by phosphorylating Vif (X. Yang and D. Gabuzda, J. Biol. Chem. 273:29879-29887, 1998). However, MAPK activation enhanced virus infectivity in some cells lines that do not require Vif function. The HIV-1 Rev, Tat, p17(Gag), and Nef proteins were directly phosphorylated by MAPK in vitro, suggesting that other HIV-1 proteins are potential substrates for MAPK phosphorylation. These results suggest that activation of the ERK MAPK pathway plays a role in HIV-1 replication by enhancing the infectivity of HIV-1 virions through Vif-dependent as well as Vif-independent mechanisms. MAPK activation in producer cells may contribute to the activation of HIV-1 replication when T cells are activated by mitogens and other extracellular stimuli.     

细胞MEK1/2蛋白及其相关通路在单纯疱疹病毒Ⅱ型（HSV-2）复制中的作用

基于细胞Raf/MEK/ERK信号通路与病毒复制的关系,应用Western印迹检测 p-ERK1/2蛋白的表达、用终点滴定法测定病毒增殖量（TCID_５０）,以及观察感染细胞的细胞病变效应（CPE）等,揭示单纯疱疹病毒Ⅱ型(HSV-2)复制与 ERK通路的关系. 结果表明,HSV-2的复制可引起细胞ERK通路的活化;用U0126预先抑制ERK通路的活化,或用特异性siRNA敲减MEK1/2基因的表达可显著地抑制病毒复制.提示ERK信号通路以及MEK1/2蛋白对HSV-2的复制具有重要的作用.该研究对进一步阐明细胞ERK通路各激酶蛋白在病毒复制中的作用机制、寻找抗病毒作用靶标等奠定了良好的基础.     

MAP kinase: it's been longer than fifteen minutes

The review highlights evidence for different functions in the cell cycle of the two MAP kinase kinases, MEK1 and MEK2, and the two MAP kinases, ERK1 and ERK2. Functional differences may explain why instances of cell cycle arrest can be MEK1 or MEK2 dependent.     

MEK partner 1 (MP1): regulation of oligomerization in MAP kinase signaling

Specificity in signal transduction can be achieved through scaffolds, anchors, and adapters that assemble generic signal transduction components in specific combinations and locations. MEK Partner-1 (MP1) was identified as a potential "scaffold" protein for the mammalian extracellular signal-regulated kinase (ERK) pathway. To gain insight into the interactions of MP1 with the ERK pathway, we analyzed the ability of MP1 to bind to MEK1, ERK1, and to itself, and the regulation of these interactions. Gel filtration of cell lysates revealed two major MP1 peaks: a broad high molecular weight peak and a 28 kDa complex. An MP1 mutant that lost MEK1 binding no longer enhanced RasV12-stimulated ERK1 activity, and functioned as a dominant negative, consistent with the concept that MP1 function depends on facilitating these oligomerizations. Activation of the ERK pathway by serum or by RasV12 did not detectably affect MP1-MP1 dimerization or MP1-MEK1 interactions, but caused the dissociation of the MP1-ERK1 complex. Surprisingly, pharmacological inhibition of ERK activation did not restore the complex, suggesting that regulation of complex formation occurs independently of ERK phosphorylation. These results support the concept that MP1 functions as a regulator of MAP kinase signaling by binding to MEK1 and regulating its association with a larger signaling complex that may sequentially service multiple molecules of ERK.     

Oligonol, an oligomerized lychee fruit-derived polyphenol, activates the Ras/Raf-1/MEK1/2 cascade independent of the IL-6 signaling pathway in rat primary adipocytes

Oligonol is a lychee fruit-derived low-molecular form of polyphenol. In this study, the effect of Oligonol on the mitogen activated-protein kinase (MAPK) signaling pathway in primary adipocytes was investigated to examine the mechanism underlying the enhanced levels of phosphorylated extracellular-signaling regulatory kinase1/2 (ERK1/2) that accompany an in vitro increase in lipolysis. Oligonol significantly elevated the levels of activated Ras and the phosphorylation of Raf-1 and MAPK/ERK kinase1/2 (MEK1/2) with no increase in pan-Raf-1 and -MEK1/2 proteins. The increase in phosphorylation of Raf-1 and MEK1/2 with Oligonol was inhibited completely by pretreatment with GW5074, a selective Raf-1 inhibitor, or PD98059, a selective MEK1/2 inhibitor. IL-6 also activated the MAPK signaling pathway in adipocytes through the association with its receptor. IL-6-induced phosphorylation of Raf-1 and MEK1/2 was significantly inhibited by pretreatment with the IL-6 receptor antibody. Under such a condition, however, the levels of phosphorylated Raf-1 and MEK1/2 with Oligonol still remained significantly higher, and there was a significant decrease in secretion of IL-6 from adipocytes, compared with untreated control cells. These results suggest that Oligonol activates the Ras/Raf-1/MEK1/2 signaling pathway, independent of the IL-6 signaling pathway, leading to activation of ERK1/2 proteins in primary adipocytes.     

Regulation of ERK Kinase by MEK1 Kinase Inhibition in the Brain

Metabotropic (slow) and ionotropic (fast) neurotransmission are integrated by intracellular signal transduction mechanisms involving protein phosphorylation/dephosphorylation to achieve experience-dependent alterations in brain circuitry. ERK is an important effector of both slow and fast forms of neurotransmission and has been implicated in normal brain function and CNS diseases. Here we characterize phosphorylation of the ERK-activating protein kinase MEK1 by Cdk5, ERK, and Cdk1 in vitro in intact mouse brain tissue and in the context of an animal behavioral paradigm of stress. Cdk5 only phosphorylates Thr-292, whereas ERK and Cdk1 phosphorylate both Thr-292 and Thr-286 MEK1. These sites interact in a kinase-specific manner and inhibit the ability of MEK1 to activate ERK. Thr-292 and Thr-286 MEK1 are phosphorylated in most mouse brain regions to stoichiometries of ∼5% or less. Phosphorylation of Thr-292 MEK1 is regulated by cAMP-dependent signaling in mouse striatum in a manner consistent with negative feedback inhibition in response to ERK activation. Protein phosphatase 1 and 2A contribute to the maintenance of the basal phosphorylation state of both Thr-292 and Thr-286 MEK1 and that of ERK. Activation of the NMDA class of ionotropic glutamate receptors reduces inhibitory MEK1 phosphorylation, whereas forced swim, a paradigm of acute stress, attenuates Thr-292 MEK1 phosphorylation. Together, the data indicate that these inhibitory MEK1 sites phosphorylated by Cdk5 and ERK1 serve as mechanistic points of convergence for the regulation of ERK signaling by both slow and fast neurotransmission.     

p21-Activated Kinase 1 (PAK1) Can Promote ERK Activation in a Kinase-independent Manner

PAK1 plays an important role in proliferation and tumorigenesis, at least partially by promoting ERK phosphorylation of C-RAF (Ser-338) or MEK1 (Ser-298). We observed how that overexpression of a kinase-dead mutant form of PAK1 increased phosphorylation of MEK1/2 (Ser-217/Ser-221) and ERK (Thr-202/Tyr-204), although phosphorylation of B-RAF (Ser-445) and C-RAF (Ser-338) remained unchanged. Furthermore, increased activation of the PAK1 activator Rac1 induced the formation of a triple complex of Rac1, PAK1, and MEK1 independent of the kinase activity of PAK1. These data suggest that PAK1 can stimulate MEK activity in a kinase-independent manner, probably by serving as a scaffold to facilitate interaction of C-RAF.     

Baicalein protects against cardiac hypertrophy through blocking MEK‐ERK1/2 signaling

Baicalein, a flavonoid present in the root of Scutellaria baicalensis, is well known for its antibacterial, antiviral, anti‐inflammatory, antithrombotic, and antioxidant effects. Here we show that baicalein also attenuates cardiac hypertrophy. Aortic banding (AB) was performed to induce cardiac hypertrophy secondary to pressure overload in mice. Mouse chow containing 0.05% baicalein (dose: 100 mg/kg/day baicalein) was begun 1 week prior to surgery and continued for 8 weeks after surgery. Our data demonstrated that baicalein prevented cardiac hypertrophy and fibrosis induced by AB, as assessed by echocardiographic and hemodynamic parameters and by pathological and molecular analysis. The inhibitory action of baicalein on cardiac hypertrophy was mediated by effects on mitogen‐activated protein kinase kinase (MEK)‐extracellular signal‐regulated kinases (ERK1/2) signaling and GATA‐4 activation. In vitro studies performed in rat cardiac H9c2 cells confirmed that baicalein attenuated cardiomyocyte hypertrophy induced by angiotensin II, which was associated with inhibiting MEK‐ERK1/2 signaling. In conclusion, our results suggest that baicalein has protective potential for targeting cardiac hypertrophy and fibrosis through suppression of MEK‐ERK1/2 signaling. Baicalein warrants further research as a potential antihypertrophic agent that might be clinically useful to treat cardiac hypertrophy and heart failure. J. Cell. Biochem. 114: 1058–1065, 2013. © 2012 Wiley Periodicals, Inc.     

Glycogen synthase kinase-3beta is tyrosine-phosphorylated by MEK1 in human skin fibroblasts

Glycogen synthase kinase-3beta (GSK-3beta) can be associated with several proteins in cell. We analyzed the immunoprecipitates by an anti-GSK-3beta antibody from cell lysate of human fibroblasts and found that this protein was co-precipitated with mitogen-activated protein kinase kinase (MEK1/2). U0126, a MEK1/2 inhibitor, inhibited tyrosine phosphorylation of GSK-3beta, suggesting that MEK1/2 was involved in the phosphorylation of Tyr(216) in GSK-3beta. In vitro kinase assay was carried out using a recombinant human active MEK1 and we found that GSK-3beta was phosphorylated on Tyr(216) by this kinase in a dose- and time-dependent manner. Further, the pretreatment of fibroblasts with U0126 inhibited serum-induced nuclear translocation of GSK-3beta. These results suggested that MEK1/2 induces tyrosine phosphorylation of GSK-3beta and this cellular event might induce nuclear translocation of GSK-3beta. This is the first report to suggest that MEK1/2 phosphorylates not only ERK1/2 but also GSK-3beta.     

MEK1 plays contrary stage-specific roles in skeletal myogenic differentiation

The role of extracellular signal-regulated kinase (ERK) signaling in skeletal myogenesis has been reported to be both stimulatory and inhibitory. We propose that this discrepancy may arise from the stage-specific, different roles of mitogen-activated protein kinase kinase 1 (MEK1). We found that the phosphorylated MEK1 level of differentiating C2C12 cells was low on day 1 (early-stage) and reached a maximum on days 2–3 (mid-stage). Cells treated at early stage with the MEK-specific inhibitors, PD184352 and U0126, reduced both the MHC protein level and MCK promoter activity, demonstrating that high MEK1 activity at the mid-stage is required for myogenic differentiation. In contrast, treatment with the ERK-specific inhibitors, FR180204 and ERK inhibitor I, had no effect. However, because the sustained overexpression of constitutively active MEK1 inhibits myogenic differentiation, we further analyzed the stage-specific role of MEK1 using the Tet-Off expression system. The results demonstrated that myogenic differentiation was inhibited if active MEK1 expression was induced earlier than day 1 in differentiation condition, but stimulated if induced after that, demonstrating that activated MEK1 plays differential roles depending on activation time. In addition, the induction of active MEK1 at 12 h enhanced the Id2 protein level, while the induction at 36 h resulted in reduction. Thus, MEK1 plays stage-specific and contrary roles in myogenesis, and MEK1 activated at the mid-stage promotes muscle differentiation independent of ERK.     

Multiple Roles of HIV-1 Capsid during the Virus Replication Cycle

Human immunodeficiency virus-1 capsid(HIV-1 CA) is involved in different stages of the viral replication cycle. During virion assembly, CA drives the formation of the hexameric lattice in immature viral particles, while in mature virions CA monomers assemble in cone-shaped cores surrounding the viral RNA genome and associated proteins. In addition to its functions in late stages of the viral replication cycle, CA plays key roles in a number of processes during early phases of HIV-1 infection including trafficking, uncoating, recognition by host cellular proteins and nuclear import of the viral preintegration complex. As a result of efficient cooperation of CA with other viral and cellular proteins, integration of the viral genetic material into the host genome, which is an essential step for productive viral infection, successfully occurs. In this review, we will summarize available data on CA functions in HIV-1 replication, describing in detail its roles in late and early phases of the viral replication cycle.     

Activation and nuclear translocation of PKCdelta,Pyk2 and ERK1/2 by gonadotropin releasing hormone in HEK293 cells

The mechanism of agonist-induced activation of Pyk2 and its relationship with ERK1/2 phosphorylation was analyzed in HEK293 cells stably expressing the gonadotropin releasing hormone (GnRH) receptor. GnRH stimulation caused rapid and sustained phosphorylation of ERK1/2 and Pyk2 that was accompanied by their nuclear translocation. Pyk2 was also localized on cell membranes and at focal adhesions. Dominant negative Pyk2 (PKM) had no effect on GnRH-induced ERK1/2 phosphorylation and c-fos expression. These actions of GnRH on ERK1/2 and Pyk2 were mimicked by activation of protein kinase C (PKC) and were abolished by its inhibition. GnRH caused translocation of PKC and δ, but not of , ι and λ, to the cell membrane, as well as phosphorylation of Raf at Ser338, a major site in the activation of MEK/ERK1/2. Stimulation of HEK293 cells by EGF caused marked ERK1/2 phosphorylation that was attenuated by the selective EGFR receptor (EGF-R) kinase inhibitor, AG1478. However, GnRH-induced ERK1/2 activation was independent of EGF-R activation. These results indicate that activation of PKC is responsible for GnRH-induced phosphorylation of both ERK1/2 and Pyk2, and that Pyk2 activation does not contribute to GnRH signaling. Moreover, GnRH-induced phosphorylation of ERK1/2 and expression of c-fos in HEK293 cells is independent of Src and EGF-R transactivation, and is mediated through the PKC/Raf/MEK cascade.     

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.     

PARP-1-induced cell death through inhibition of the MEK/ERK pathway in MNNG-treated HeLa cells

Poly(ADP-ribose) polymerase-1 (PARP-1) hyper-activation promotes cell death but the signaling events downstream of PARP-1 activation are not fully identified. To gain further information on the implication of PARP-1 activation and PAR synthesis on signaling pathways influencing cell death, we exposed HeLa cells to the DNA alkylating agent N-methyl-N′-methyl-nitro-N-nitrosoguanidine (MNNG). We found that massive PAR synthesis leads to down-regulation of ERK1/2 phosphorylation, Bax translocation to the mitochondria, release of cytochrome c and AIF and subsequently cell death. Inhibition of massive PAR synthesis following MNNG exposure with the PARP inhibitor PJ34 prevented those events leading to cell survival, whereas inhibition of ERK1/2 phosphorylation by inhibiting MEK counteracted the cytoprotective effect of PJ34. Together, our results provide evidence that PARP-1-induced cell death by MNNG exposure in HeLa cells is mediated in part through inhibition of the MEK/ERK signaling pathway and that inhibition of massive PAR synthesis by PJ34, which promotes sustained activation of ERK1/2, leads to cytoprotection.