ERK3信号通路的活化阻止细胞增殖

ERK3是ERK家族中结构较为独特的成员,尤其在分子生物学特征上与ERK家族其他成员明显不同,如基因结构中外显子之间的大内含子、蛋白质结构中活化环的丝氨酸单磷酸化位点以及激酶C端的延伸序列等.ERK3具有独特的丝氨酸单磷酸化位点,导致所有以苏氨酸/酪氨酸双磷酸化位点为磷酸化靶点的MEK分子均不能活化ERK3.ERK3的C端延伸序列能与细胞周期蛋白D3结合并调控ERK3的亚细胞定位,从而影响ERK3对细胞周期的调节.据目前文献推测,ERK3调控细胞周期的信号通路可能为:Ras→B-Raf→ERK3激酶→ERK3→G1期CDK复合物减少→S期抑制因子增多→细胞增殖阻滞于S期→细胞停止增殖,进入分化.此外,ERK3信号通路的活化与细胞分化、胚胎发育、胰岛素分泌以及肿瘤的发生密切相关.     

支架蛋白Gab2信号调控与乳腺癌

Gab2是支架蛋白Gabs家族中的重要成员.该家族蛋白通过介导膜受体与信号转运蛋白间的偶联及各信号分子间的整合参与信号传导.作为支架蛋白,Gab2可被酪氨酸激酶磷酸化激活,接受胞外多种因子刺激,招募富含SH2结构域的信号转运分子,活化下游SHP2/Ras/ERK和PI3K/AKT等一系列信号传导途径,在细胞增殖、分化、...     

柴胡提取物诱导人类白血病细胞HL-60的细胞凋亡及其机理研究

柴胡提取物诱导人类白血病细胞HL-60的细胞凋亡从而抑制其细胞生长.为了研究该过程的作用机理,我们研究了丝裂原活化蛋白激酶(MAPKs),包括胞外信号调节激酶(ERK1/2),c-jun氨基末端蛋白激酶(JNK)和p38丝裂原活化蛋白激酶(MAPK),在该过程中的磷酸化特征与动态变化.结果表明,柴胡提取物显著的增加了p38丝裂原活化蛋白激酶和胞外信号调节激酶(ERK1/2)的磷酸化作用,其增加值在测试范围内与测试剂量和作用时间成正相关,但在柴胡提取物诱导人类白血病细胞HL-60的细胞凋亡过程中,没有发现对氨基末端蛋白激酶(JNK)表现出磷酸化活性.柴胡提取物诱导白血病HL-60的细胞凋亡部分归结于对p38丝裂原活化蛋白激酶的上调节作用,这种上调节作用能够受到p38 MAPK特异性的抑制剂SB203580的部分逆转,而MEK的抑制剂U0126则对柴胡提取物诱导HL-60细胞凋亡过程中的胞外信号调节激酶(ERK1/2)的磷酸化具有显著的协同效应.这是首次报道柴胡提取物在诱导人白血病细胞HL-60细胞凋亡过程中参与p38丝裂原活化蛋白激酶的磷酸化,同时柴胡提取物作为胞外信号调节激酶(ERK1/2)抑制剂的协同作用物具有相应的药物学功能.     

血清通过调节mGluR1介导的信号通路调控细胞的生长与凋亡

血清因子能调节细胞的生长与凋亡,但是其分子机制尚不清楚.通过细胞培养基中血清存在与否,研究了代谢型谷氨酸受体1(mGluR1)介导的胞外信号调节激酶(ERK),蛋白激酶B(PKB/AKT)通路的活化及其对细胞生长与凋亡的影响.在过量表达mGluR1的HEK293细胞中,血清饥饿促进了mGluR1对ERK,AKT信号通路的活化;细胞凋亡剂STS应激损伤时,受体激动剂DHPG可降低细胞活性,促进细胞凋亡.在大鼠胶质瘤细胞中,与过表达mGluR1的HEK293细胞的结果相反,血清有助于mGluR1对ERK,AKT通路的活化作用;STS应激损伤时,内源性mGluR1的活化抑制了细胞凋亡.结果表明:血清中存在的细胞因子,通过细胞中表达水平不同的mGluR1受体,调节受体介导的信号通路,从而调控细胞生长与凋亡.本文可能揭示了一种血清调节细胞生长与凋亡的新机制.     

Raf-1激酶与肿瘤治疗

自Raf激酶被证明为逆转录病毒致癌基因的产物以来,逐渐成为人们研究的热点。研究表明,Raf激酶既是Ras的效应物,又能作为ERK信号通路中的重要组分,成为活化的Ras和ERK之间的一个重要纽带。Ras-Raf-MEK-ERK信号通路参与了细胞增殖、分化和凋亡等生物学过程。作为这一信号通路上的节点蛋白,Raf激酶在肿瘤发生过程中起着关键作用。Raf家族成员Raf-1（cRaf）在调控细胞运动和凋亡过程中发挥关键作用,它既可以通过抑制促凋亡激酶ASK1和MST2活性来抑制细胞凋亡,也可以通过激活Rok-α的活性来促进细胞迁移。该文主要综述了Raf-1激酶的调控机制及其在肿瘤发生过程中的作用,同时也总结了以Raf-1为靶点的肿瘤治疗的最新进展。     

Raf激酶抑制剂及其耐药机制研究进展

Ras/Raf/MEK/ERK 通路是调节细胞生长与增殖的重要信号传导通路。在Ras/Raf/MEK/ERK 通路中某些成员的突变往往与恶性肿瘤的发生密切相关。B-Raf 激酶是该通路中Raf 家族最重要的亚型,其主要突变形式B-RafV600E 在黑色素瘤等多种肿瘤中高度表达。选择性B-RafV600E 抑制剂vemurafenib 和dabrafenib 的上市使得晚期黑色素瘤的治疗进入新纪元,但是耐药性和副作用依然限制了二者的使用。综述目前Raf 激酶抑制剂耐药性与副作用产生机制以及Raf 激酶抑制剂的最新研究进展。     

ASK1激活的分子机制与相关疾病

凋亡信号调节激酶1(Apoptosis signal-regulating kinase 1,ASK1)是细胞丝裂原活化蛋白激酶激酶激酶(mitogen-activated protein kinase kinase kinase,MAP3Ks)家族成员之一,在调节细胞凋亡过程中起到非常重要的作用.在正常细胞中,ASK1的活化受到严格的控制,如苏氨酸/丝氨酸磷酸化和去磷酸化、蛋白-蛋白相互作用等.多种应激和促炎因子能激活ASK1,因此在多种生理和病理过程中都有活化的ASK1的参与.     

JNK信号通路研究进展

c-Jun氨基末端激酶(JNK)家族是促分裂原活化蛋白激酶(MAPK)超家族成员之一,以JNK为中心的JNK信号通路可被细胞因子、生长因子、应激等多种因素激活,大量实验提示JNK信号通路在细胞分化、细胞凋亡、应激反应以及多种人类疾病的发生与发展中起着至关重要的作用。现对JNK信号通路的基本构成、调节方式及其与胞内其他信号通路间相互作用进行综述。     

JNK活化机制的研究进展

cJun氨基末端激酶(JNK)家族是促分裂原活化蛋白激酶(MAPK)超家族成员之一,MAPK信号通路是多级蛋白激酶的级联反应,包括三个关键的激酶:MAPK、MAPK的激酶(MAPKK)和MAPK激酶的激酶(MAPKKK).JNK信号通路中有许多支架蛋白,如:JIP、JAMP、POSH等,能够与JNK及JNK信号通路中相关成员结合成复合物,调节它们的活性和细胞内定位,JNK信号通路可被细胞因子、生长因子、应激等多种因素激活,大量实验提示JNK活化在细胞增殖、细胞凋亡、应激反应以及多种人类疾病的发生与发展中起着重要的作用.JNK信号通路与其他信号通路间也有着相互作用.现对JNK活化机制的研究进展进行综述.     

B7家族成员反向信号调控的研究进展

共刺激分子免疫球蛋白家族-B7家族成员与CD28家族成员之间相互作用向T细胞传递共刺激信号,在T细胞充分活化和功能发挥中发挥了重要的功能.近几年研究表明,部分B7家族成员向T细胞传递免疫信号的同时,也向表达B7分子的抗原提呈细胞传递反向信号,增强或抑制了抗原提呈细胞的功能,并进一步在维持T细胞免疫和T细胞耐受中发挥重要的功能.     

Extracellular Signal-Regulated Kinase 7 (ERK7), a Novel ERK with a C-Terminal Domain That Regulates Its Activity, Its Cellular Localization, and Cell Growth

Mitogen-activated protein (MAP) kinases play distinct roles in a variety of cellular signaling pathways and are regulated through multiple mechanisms. In this study, a novel 61-kDa member of the MAP kinase family, termed extracellular signal-regulated kinase 7 (ERK7), has been cloned and characterized. Although it has the signature TEY activation motif of ERK1 and ERK2, ERK7 is not activated by extracellular stimuli that typically activate ERK1 and ERK2 or by common activators of c-Jun N-terminal kinase (JNK) and p38 kinase. Instead, ERK7 has appreciable constitutive activity in serum-starved cells that is dependent on the presence of its C-terminal domain. Interestingly, the C-terminal tail, not the kinase domain, of ERK7 regulates its nuclear localization and inhibition of growth. Taken together, these results elucidate a novel type of MAP kinase whereby interactions via its C-terminal tail, rather than extracellular signal-mediated activation cascades, regulate its activity, localization, and function.     

ERK7 is an autoactivated member of the MAPK family

Extracellular signal-regulated kinase 7 (ERK7) shares significant sequence homology with other members of the ERK family of signal transduction proteins, including the signature TEY activation motif. However, ERK7 has several distinguishing characteristics. Unlike other ERKs, ERK7 has been shown to have significant constitutive activity in serum-starved cells, which is not increased further by extracellular stimuli that typically activate other members of the mitogen-activated protein kinase (MAPK) family. On the other hand, ERK7's activation state and kinase activity appear to be regulated by its ability to utilize ATP and the presence of its extended C-terminal region. In this study, we investigated the mechanism of ERK7 activation. The results suggest that 1) MAPK kinase (MEK) inhibitors do not suppress ERK7 kinase activity; 2) intramolecular autophosphorylation is sufficient for activation of ERK7 in the absence of an upstream MEK; and 3) multiple regions of the C-terminal domain of ERK7 regulate its kinase activity. Taken together, these results indicate that autophosphorylation is sufficient for ERK7 activation and that the C-terminal domain regulates its kinase activity through multiple interactions.     

ERK7 expression and kinase activity is regulated by the ubiquitin-proteosome pathway

ERK7 is a unique member of the extracellular signal-regulated kinase (ERK) subfamily of MAP kinases. Although ERK7 shares a TEY motif in the activation loop of the kinase, it displays constitutive activation, nuclear localization, and growth inhibitory properties that are regulated by its C-terminal domain. Because ERK7 is expressed at low levels compared with ERK2 and its activity is dependent upon its expression level, we investigated the mechanism by which ERK7 expression is regulated. We now show that ERK7 expression is regulated by ubiquitination and rapid proteosomal turnover. Furthermore, both the kinase domain and the C-terminal tail are independently degraded at a rate comparable with that of the intact protein. Analysis of a series of chimeras between ERK2 and ERK7 reveal that the N-terminal 20 amino acids of the kinase domain are a primary determinant of ERK7 degradation. Fusion of the N-terminal 20 amino acids is both necessary and sufficient to cause proteolytic degradation of both ERK2 and green fluorescent protein. Finally, ERK7 is stabilized by an N-terminal mutant of Cullin-1 suggesting that ERK7 is ubiquitinated by the Skip1-Cullin-F box complex. These results indicate that ERK7 is a highly regulated enzyme whose cellular expression and kinase activation level is tightly controlled by the ubiquitin-proteosome pathway.     

Different domains of the mitogen-activated protein kinases ERK3 and ERK2 direct subcellular localization and upstream specificity in vivo.

Extracellular signal-regulated kinase 3 (ERK3) is a member of the mitogen-activated protein (MAP) kinase family. ERK3 is most similar in its kinase catalytic domain to ERK2, yet it displays many unique properties. Among these, unlike ERK2, which translocates to the nucleus following activation, ERK3 is constitutively localized to the nucleus, despite the lack of a defined nuclear localization sequence. We created two chimeras between ERK2 and the catalytic domain of ERK3 (ERK3DeltaC), and some mutants of these chimeras, to examine the basis for the different behaviors of these two MAP kinase family members. We find the following: 1) the N-terminal folding domain of ERK3 functions in phosphoryl transfer reactions with the C-terminal folding domain of ERK2; 2) the C-terminal halves of ERK2 and ERK3DeltaC are primarily responsible for their subcellular localization in resting cells; and 3) the N-terminal folding domain of ERK2 is required for its activation in cells, its interaction with MEK1, and its accumulation in the nucleus.     

Functional characterization of human RSK4, a new 90-kDa ribosomal S6 kinase, reveals constitutive activation in most cell types

The 90-kDa ribosomal S6 kinases (RSK1-3) are important mediators of growth factor stimulation of cellular proliferation, survival, and differentiation and are activated via coordinated phosphorylation by ERK and 3-phosphoinositide-dependent protein kinase-1 (PDK1). Here we performed the functional characterization of a predicted new human RSK homologue, RSK4. We showed that RSK4 is a predominantly cytosolic protein with very low expression and several characteristics of the RSK family kinases, including the presence of two functional kinase domains and a C-terminal docking site for ERK. Surprisingly, however, in all cell types analyzed, endogenous RSK4 was maximally (constitutively) activated under serum-starved conditions where other RSKs are inactive due to their requirement for growth factor stimulation. Constitutive activation appeared to result from constitutive phosphorylation of Ser232, Ser372, and Ser389, and the low basal ERK activity in serum-starved cells appeared to be sufficient for induction of approximately 50% of the constitutive RSK4 activity. Finally experiments in mouse embryonic stem cells with targeted deletion of the PDK1 gene suggested that PDK1 was not required for phosphorylation of Ser232, a key regulatory site in the activation loop of the N-terminal kinase domain, that in other RSKs is phosphorylated by PDK1. The unusual regulation and growth factor-independent kinase activity indicate that RSK4 is functionally distinct from other RSKs and may help explain recent findings suggesting that RSK4 can participate in non-growth factor signaling as for instance p53-induced growth arrest.     

90-kDa ribosomal S6 kinase is phosphorylated and activated by 3-phosphoinositide-dependent protein kinase-1.

90-kDa ribosomal S6 kinase-2 (RSK2) belongs to a family of growth factor-activated serine/threonine kinases composed of two kinase domains connected by a regulatory linker region. The N-terminal kinase of RSK2 is involved in substrate phosphorylation. Its activation requires phosphorylation of the linker region at Ser(369), catalyzed by extracellular signal-regulated kinase (ERK), and at Ser(386), catalyzed by the C-terminal kinase, after its activation by ERK. In addition, the N-terminal kinase must be phosphorylated at Ser(227) in the activation loop by an as yet unidentified kinase. Here, we show that the isolated N-terminal kinase of RSK2 (amino acids 1-360) is phosphorylated at Ser(227) by PDK1, a constitutively active kinase, leading to 100-fold stimulation of kinase activity. In COS7 cells, ectopic PDK1 induced the phosphorylation of full-length RSK2 at Ser(227) and Ser(386), without involvement of ERK, leading to partial activation of RSK2. Similarly, two other members of the RSK family, RSK1 and RSK3, were partially activated by PDK1 in COS7 cells. Finally, our data indicate that full activation of RSK2 by growth factor requires the cooperation of ERK and PDK1 through phosphorylation of Ser(227), Ser(369), and Ser(386). Our study extend recent findings which implicate PDK1 in the activation of protein kinases B and C and p70(S6K), suggesting that PDK1 controls several major growth factor-activated signal transduction pathways.     

The MAP kinase ERK5 binds to and phosphorylates p90 RSK

We showed previously that p90 RSK was activated in cells expressing an activated mutant of MEK5, the activator of the MAP kinase ERK5. Based on the following evidence, we suggest that ERK5 can directly activate RSK in cells. ERK5 binds to RSK in vitro and co-immunoprecipitates from cell extracts; activation of ERK5 weakens its binding to RSK, suggesting that RSK is released upon activation. Phosphorylation of RSK by ERK5 in vitro causes its activation, indicating that RSK is a substrate of ERK5. In cells activation of ERK5 but not p38 or the c-Jun N-terminal kinase is associated with RSK activation. The large C-terminal domain of ERK5 is not required for binding or activation of RSK by ERK5; however, the common docking or CD domain of ERK5 and the docking or D domain of RSK are important for their association.     

Human JIK, a novel member of the STE20 kinase family that inhibits JNK and is negatively regulated by epidermal growth factor

Mammalian members related to Saccharomyces cerevisiae serine/threonine kinase STE20 can be divided into two subfamilies based on their structure and function. The PAK subfamily is characterized by an N-terminal p21-binding domain (also known as CRIB domain), a C-terminal kinase domain, and is regulated by the small GTP-binding proteins Rac1 and Cdc42Hs. The second group is represented by the GCK-like members, which contain an N-terminal catalytic domain and lack the p21-binding domain. Some of them have been demonstrated to induce c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) cascade, while others have been shown to be activated by a subset of stress conditions or apoptotic agents, although little is known about their specific function. Here, we have identified a novel human STE20-related serine/threonine kinase, belonging to the GCK-like subfamily. This kinase does not induce the JNK/SAPK pathway, but, instead, inhibits the basal activity of JNK/SAPK, and diminishes its activation in response to human epidermal growth factor (EGF). Therefore, we designated this molecule JIK for JNK/SAPK-inhibitory kinase. The inhibition of JNK/SAPK signaling pathway by JIK was found to occur between the EGF receptor and the small GTP-binding proteins Rac1 and Cdc42Hs. In contrast, JIK does not activate nor does it inhibit ERK2, ERK6, p38, or ERK5. Furthermore, JIK kinase activity is not modulated by any exogenous stimuli, but, interestingly, it is dramatically decreased upon EGF receptor activation. Thus, JIK might represent the first member of the STE20 kinase family whose activity can be negatively regulated by tyrosine kinase receptors, and whose downstream targets inhibit, rather than enhance, JNK/SAPK activation.     

The MAP kinase kinase kinase MLK2 co-localizes with activated JNK along microtubules and associates with kinesin superfamily motor KIF3.

The MLK (mixed lineage) ser/thr kinases are most closely related to the MAP kinase kinase kinase family. In addition to a kinase domain, MLK1, MLK2 and MLK3 each contain an SH3 domain, a leucine zipper domain and a potential Rac/Cdc42 GTPase-binding (CRIB) motif. The C-terminal regions of the proteins are essentially unrelated. Using yeast two-hybrid analysis and in vitro dot-blots, we show that MLK2 and MLK3 interact with the activated (GTP-bound) forms of Rac and Cdc42, with a slight preference for Rac. Transfection of MLK2 into COS cells leads to strong and constitutive activation of the JNK (c-Jun N-terminal kinase) MAP kinase cascade, but also to activation of ERK (extracellular signal-regulated kinase) and p38. When expressed in fibroblasts, MLK2 co-localizes with active, dually phosphorylated JNK1/2 to punctate structures along microtubules. In an attempt to identify proteins that affect the activity and localization of MLK2, we have screened a yeast two-hybrid cDNA library. MLK2 and MLK3 interact with members of the KIF3 family of kinesin superfamily motor proteins and with KAP3A, the putative targeting component of KIF3 motor complexes, suggesting a potential link between stress activation and motor protein function.