Disruption of signaling by Yersinia effector YopJ, a ubiquitin-like protein protease

Homologs of the Yersinia virulence effector YopJ are found in both plant and animal bacterial pathogens, as well as plant symbionts. These YopJ family members were shown to act as cysteine proteases. The catalytic triad of the protease was required for inhibition of the mitogen-activated protein kinase (MAPK) and nuclear factor kappaB (NF-kappaB) signaling in animal cells and for induction of localized cell death in plants. The substrates for YopJ were shown to be highly conserved ubiquitin-like molecules, which are covalently added to numerous regulatory proteins. YopJ family members exert their pathogenic effect on cells by disrupting this posttranslational modification.     

植物MAPK级联途径及其功能研究进展

促分裂原活化蛋白激酶(Mitogen Actived Protein Kinase,MAPK)是一种蛋白激酶,蛋白激酶又称蛋白磷酸化酶,其可通过将ATP上的磷酸基团转移到底物蛋白质氨基酸残基上,来催化底物蛋白质磷酸化。MAPK级联途径广泛存在于真核生物中。植物中的MAPK级联途径与动物和酵母类似,都包括MAPKKK、MAPKK和MAPK 3种蛋白激酶。大量的研究表明,植物中的MAPK级联途径不仅能被多种生物与非生物胁迫所激活,同时也参与激素信号转导以及植物的生长发育进程。文章就植物中MAPK级联途径及其功能的研究情况进行了综述,并展望了相关研究的发展趋势。     

植物MAPK级联途径应答的非生物胁迫研究进展

促分裂原活化蛋白激酶（mitogen-activated protein kinase, MAPK）是一种高度保守的蛋白激酶，广泛存在于真核生物中。MAPK级联途径是一种重要的高度保守的细胞信号转导途径，主要包括MAPKKK、MAPKK和MAPK三种蛋白激酶，通过磷酸化顺序被激活。MAPK级联途径参与了植物生长发育、激素调节、生物胁迫以及非生物胁迫的应答响应。从植物MAPK级联途径成员的分类、结构特征及对非生物胁迫的响应等方面进行了综述，将为系统的理解植物MAPK级联途径及其在非生物逆境胁迫下的表达调控等提供参考。     

MAP kinase phosphatase as a locus of flexibility in a mitogen-activated protein kinase signaling network

Intracellular signaling networks receive and process information to control cellular machines. The mitogen-activated protein kinase (MAPK) 1,2/protein kinase C (PKC) system is one such network that regulates many cellular machines, including the cell cycle machinery and autocrine/paracrine factor synthesizing machinery. We used a combination of computational analysis and experiments in mouse NIH-3T3 fibroblasts to understand the design principles of this controller network. We find that the growth factor-stimulated signaling network containing MAPK 1, 2/PKC can operate with one (monostable) or two (bistable) stable states. At low concentrations of MAPK phosphatase, the system exhibits bistable behavior, such that brief stimulus results in sustained MAPK activation. The MAPK-induced increase in the amounts of MAPK phosphatase eliminates the prolonged response capability and moves the network to a monostable state, in which it behaves as a proportional response system responding acutely to stimulus. Thus, the MAPK 1, 2/PKC controller network is flexibly designed, and MAPK phosphatase may be critical for this flexible response.     

玉米大斑病菌MAPK超家族的全基因组鉴定及途径模型建立

【目的】从全基因组水平鉴定玉米大斑病菌（Setosphaeria turcica）的MAPK超家族基因,并对其进行系统进化、基因结构、多重序列比对及保守位点分析,构建玉米大斑病菌中的MAPK级联途径模型,为深入研究该植物病原菌中MAPK级联途径的功能奠定基础。【方法】利用玉米大斑病菌基因组数据库,通过基于隐马尔科夫模型的HMMER 3.0软件搜索基因组,鉴定并获得MAPK超家族成员序列、基因组定位信息;采用MEGA 5.0软件进行系统进化分析;通过GSDS工具进行基因结构分析;利用ClustalX、MEME工具分析MAPK蛋白激酶区的保守性及保守位点。【结果】在玉米大斑病菌基因组中发现了4个MAPK基因、3个MAPKK基因和3个MAPKKK基因。系统进化分析将MAPK分为Kss1/Fus3、Slt2、Hog1及Ime2 4类;MAPKK分为Pbs2、Ste7及Mkk1 3类;MAPKKK分为Ste11、Bck1及Ssk2 3类。基因组定位及基因结构分析表明,MAPK超家族散布在基因组中,且MAPK基因的结构最为复杂多样,MAPKK次之,MAPKKK结构最简单。多重序列比对与保守位点分析表明,玉米大斑病菌MAPK超家族的激酶结构域均高度保守,其中MAPK具有保守的“-TxY-”磷酸化位点,MAPKK含有保守的“-SD[V/I]WS-”磷酸化位点,MAPKKK含有“-G[S/T][V/P][F/M][W/Y]M[A/S]PEV-”特异性保守位点。【结论】全基因组分析表明,玉米大斑病菌中包括4个MAPK基因、3个MAPKK基因和3个MAPKKK基因。通过系统进化、基因结构及多重序列比对和保守位点分析,在玉米大斑病菌中构建了Fus3/Kss1-homolog、Slt2-homolog、Hog1-homolog和Ime2-homolog 4条MAPK级联途径,其中Ime2 homolog为一条新发现的MAPK级联途径。该信息为深入解析植物病原真菌MAPK超家族的功能奠定了基础。     

The protein kinase p90 rsk as an essential mediator of cytostatic factor activity

Persistent activation of p42 mitogen-activated protein kinase (p42 MAPK) during mitosis induces a "cytostatic factor" arrest, the arrest responsible for preventing the parthenogenetic activation of unfertilized eggs. The protein kinase p90 Rsk is a substrate of p42 MAPK; thus, the role of p90 Rsk in p42 MAPK-induced mitotic arrest was examined. Xenopus laevis egg extracts immunodepleted of Rsk lost their capacity to undergo mitotic arrest in response to activation of the Mos-MEK-1-p42 MAPK cascade of protein kinases. Replenishing Rsk-depleted extracts with catalytically competent Rsk protein restored the ability of the extracts to undergo mitotic arrest. Rsk appears to be essential for cytostatic factor arrest.     

GnIH通过p38MAPK信号通路对猪卵巢颗粒细胞自噬与凋亡的影响

【目的】细胞自噬和凋亡存在着相互制约,p38MAPK信号通路作为细胞凋亡的主要调控通路之一,也对细胞自噬存在促进和抑制的双重作用。已有研究表明,促性腺激素抑制激素(gonadotropin-inhibitory hormone , GnIH)对细胞自噬与凋亡均有影响,但作用机制尚不明确。故探究GnIH通过p38MAPK信号通路对猪卵巢颗粒细胞（pGCs）自噬与凋亡的影响及其机理,为解决母猪的产子率以及同期发情等问题提供参考。【方法】min、10 min、30 min、60 min、90 min)分组,用Western blot检测猪卵巢颗粒细胞p38与p-p38的蛋白表达量变化;2、验证GnIH对p38MAPK信号通路的影响：按（空白对照、GnIH、p38激活剂（U-46619）、U-46619+GnIH）分组,用Western blot检测p38与p-p38的蛋白表达量变化;3、探究不同浓度GnIH对自噬和凋亡的影响：按（空白对照、10 ^-6mol·L ^-1 GnIH、10 ^-8mol·L ^-1 GnIH、10 ^-10mol·L ^-1 GnIH、10 ^-12mol·L ^-1 GnIH）分组,用Western blot检测自噬与凋亡标志性蛋白的表达量变化;4、验证不同浓度GnIH通过p38信号通路对自噬和凋亡的影响：将细胞分成6组（空白对照、U-46619、U-46619+10 ^-6 mol·L ^-1 GnIH、U-46619+10 ^-8mol·L ^-1 GnIH、U-46619+10 ^-10mol·L ^-1 GnIH、U-46619+10 ^-12mol·L ^-1 GnIH）,用Western blot检测自噬与凋亡标志性蛋白的表达量变化。【结果】1. GnIH孵育10 min后,显著降低p38与p-p38的蛋白表达量（P<0.05）,提示,GnIH对p38MAPK信号通路的最佳作用时间为10 min;2. U-46619显著促进pGCs的p38磷酸化水平（P<0.05）,GnIH显著抑制pGCs的p38磷酸化水平（P<0.05）,提示,U-46619使p38MAPK信号通路活化,GnIH对p38MAPK信号通路的活化有抑制作用;3. 当 GnIH的浓度为10 ^-6 mol·L ^-1时,pGCs的自噬和凋亡水平显著升高（P<0.05）,随着GnIH浓度的降低,pGCs的自噬水平逐渐升高（P<0.05）,pGCs的凋亡水平逐渐降低（P<0.05）,提示,高浓度GnIH可以促进自噬和凋亡,随着GnIH浓度的降低,自噬水平逐渐升高,而凋亡水平逐渐下降;4. 加入U-46619后,GnIH使pGCs的自噬显著上调（P<0.05）,并且使pGCs的凋亡显著下调（P<0.05）,提示,不同浓度GnIH通过p38MAPK信号通路影响pGCs的自噬和凋亡。【结论】GnIH可能通过抑制p38MAPK信号通路的活化,上调pGCs的自噬,减少pGCs的凋亡。     

藜CaMAPKK2的表达分析及盐胁迫信号通路互作组分的筛选

【目的】通过对抗逆植物藜的丝裂原活化蛋白激酶（MAPK）级联途径中MAPKK的胁迫表达模式分析及信号转导途径互作组分的筛选,探索植物藜响应外界胁迫信号诱发逆境耐受的机制。【方法】以藜叶片总RNA为模板,利用定量PCR方法对NaCl、H2O2和ABA胁迫下藜MAPKK表达规律进行了分析。利用RT-PCR结合RACE技术获得了藜MAPKK的全长cDNA序列。利用酵母双杂交技术对MAPKK盐胁迫信号通路互作组分进行了分析。【结果】获得一个藜MAPKK的全长cDNA序列,命名为CaMAPKK2,其开放阅读框为1 089 bp,编码一个由362个氨基酸组成的丝裂原活化蛋白激酶。定量PCR显示CaMAPKK2受盐胁迫诱导明显上调表达,同时受外源H2O2和ABA调控。H2O2合成抑制剂DPI与ABA合成抑制剂Na2WO4显著抑制了300 mmol•L-1 NaCl处理下CaMAPKK2的表达。以全长CaMAPKK2为诱饵蛋白,利用酵母双杂交技术筛选到5个可能与CaMAPKK2相互作用的蛋白。测序结果显示,其中1个序列可通读,该cDNA序列长794 bp,与欧洲赤杨（Alnus glutinosa）和拟南芥的噻唑合成酶（thiazole biosynthetic enzyme）基因AgTHI1和AtTHI1核酸序列相似度达79%和78%,其它4个序列没有连续的读码框。【结论】CaMAPKK2受NaCl和H2O2诱导上调表达,暗示盐胁迫可能通过诱导H2O2和ABA的积累从而导致CaMAPKK2表达增加。要进一步筛选CaMAPKK2互作组分需获得更多阳性克隆并开展相关功能验证试验。     

Essential cytoplasmic translocation of a cytokine receptor-assembled signaling complex

Cytokine signaling is thought to require assembly of multicomponent signaling complexes at cytoplasmic segments of membrane-embedded receptors, in which receptor-proximal protein kinases are activated. Indeed, CD40, a tumor necrosis factor receptor (TNFR) family member, forms a complex containing adaptor molecules TRAF2 and TRAF3, ubiquitin-conjugating enzyme Ubc13, cellular inhibitor of apoptosis proteins 1 and 2 (c-IAP1/2), IkappaB kinase regulatory subunit IKKgamma (also called NEMO), and mitogen-activated protein kinase (MAPK) kinase kinase MEKK1 upon ligation. TRAF2, Ubc13, and IKKgamma were required for complex assembly and activation of MEKK1 and MAPK cascades. However, these kinases were not activated unless the multicomponent signaling complex translocated from CD40 to the cytosol upon c-IAP1/2-induced degradation of TRAF3. This two-stage signaling mechanism may apply to other innate immune receptors, accounting for spatial and temporal separation of MAPK and IKK signaling.     

Natural killer cell signaling pathways

Natural killer (NK) cells are lymphocytes of the innate immune system that are involved in the early defenses against foreign cells, as well as autologous cells undergoing various forms of stress, such as microbial infection or tumor transformation. NK cell activation is controlled by a dynamic balance between complementary and antagonistic pathways that are initiated upon interaction with potential target cells. NK cells express an array of activating cell surface receptors that can trigger cytolytic programs, as well as cytokine or chemokine secretion. Some of these activating cell surface receptors initiate protein tyrosine kinase (PTK)-dependent pathways through noncovalent associations with transmembrane signaling adaptors that harbor intracytoplasmic ITAMs (immunoreceptor tyrosine-based activation motifs). Additional cell surface receptors that are not directly coupled to ITAMs also participate in NK cell activation. These include NKG2D, which is noncovalently associated to the DAP10 transmembrane signaling adaptor, as well as integrins and cytokine receptors. NK cells also express cell surface inhibitory receptors that antagonize activating pathways through protein tyrosine phosphatases (PTPs). These inhibitory cell surface receptors are characterized by intracytoplasmic ITIMs (immunoreceptor tyrosine-based inhibition motifs). The tyrosine-phosphorylation status of several signaling components that are substrates for both PTKs and PTPs is thus key to the propagation of the NK cell effector pathways. Understanding the integration of these multiple signals is central to the understanding and manipulation of NK cell effector signaling pathways.     

A network of control mediated by regulator of calcium/calmodulin-dependent signaling

Calmodulin (CaM) is a major effector for the intracellular actions of Ca2+ in nearly all cell types. We identified a CaM-binding protein, designated regulator of calmodulin signaling (RCS). G protein-coupled receptor (GPCR)-dependent activation of protein kinase A (PKA) led to phosphorylation of RCS at Ser55 and increased its binding to CaM. Phospho-RCS acted as a competitive inhibitor of CaM-dependent enzymes, including protein phosphatase 2B (PP2B, also called calcineurin). Increasing RCS phosphorylation blocked GPCR- and PP2B-mediated suppression of L-type Ca2+ currents in striatal neurons. Conversely, genetic deletion of RCS significantly increased this modulation. Through a molecular mechanism that amplifies GPCR- and PKA-mediated signaling and attenuates GPCR- and PP2B-mediated signaling, RCS synergistically increases the phosphorylation of key proteins whose phosphorylation is regulated by PKA and PP2B.     

水稻纹枯病菌MAPK级联信号途径分析及信号通路模型预测

MAPK级联信号途径在植物病原真菌的生长、发育、繁殖及致病性等方面起着重要作用。根据已公布的5个水稻纹枯病菌(Rhizoctonia solani)全基因组数据利用生物信息学方法对该病原菌的MAPK(mitogenactivated protein kinase)级联信号途径基因进行了鉴定,并预测水稻纹枯病菌MAPK级联途径图。蛋白结构域和motif分析结果表明,水稻纹枯病菌的MAPK信号途径基因均含有丝氨酸/苏氨酸蛋白激酶结构域,并且基因序列具有较高保守性。多重序列比对结果表明,水稻纹枯病菌基因组中存在11个MAPKKK基因,分属于Ste11、Bck1和Ssk2类;10个MAPKK基因,分别为Ste7、Pbs2及Mkk1类;12个MAPK基因,被分类为Kss1/Fus3、Hog1、Slt2和Ime2。在5个水稻纹枯病菌基因组中,水稻纹枯病菌(taxid:456999)和AG-3 Rhs 1 AP(taxid:1086054)基因组中存在Kss1/Fus3-MAPK、Hog1-MAPK、Slt2-MAPK和Ime2-MAPK信号通路,AG-1 IB(taxid:1108050)基因组中存在Kss1/Fus3-MAPK、Hog1-MAPK和Slt2-MAPK通路,AG-8 WAC10335(taxid:1287689)基因组中仅存在Hog1-MAPK通路,而AG-1 IA(taxid:983506)基因组中只有2个MAPKKK基因未找到完整的信号通路。依据上述研究结果预测了水稻纹枯病菌的Kss1/Fus3、Hog1、Slt2和Ime2级联途径模型,此结果为深入研究水稻纹枯病菌的MAPK家族功能奠定了重要的理论基础。     

MAP激酶在植物信号传递网络中的功能

促分裂素原活化蛋白激酶(mitogen-activated protein kinases,MAP激酶,MAPK)链是真核生物信号传递网络中的重要途径之一.MAPK链由3类蛋白激酶MAP3K-MAP2K-MAPK组成,通过依次磷酸化将上游信号传递至下游应答分子.本文主要阐述MAPK链在植物的逆境反应、抗病反应和激素调控等信号传递网络中的功能.     

Signaling life and death in the thymus: timing is everything

T lymphocytes are generated in the thymus, where developing thymocytes must accept one of two fates: They either differentiate or they die. These fates are chiefly determined by signals that originate from the T cell receptor (TCR), a single receptor complex with a remarkable capacity to decide between distinct cell fates. This review explores TCR signaling in thymocytes and focuses on the kinetic aspects of ligand binding, coreceptor involvement, protein phosphorylation, and mitogen-activated protein kinase (MAPK) activation. Understanding the logic of TCR signaling may eventually explain how thymocytes and T cells distinguish self from nonself, a phenomenon that has fascinated immunologists for 50 years.     

Plant mitogen-activated protein kinases and their roles in mediation of signal transduction in abiotic stresses

Mitogen-activated protein kinase (MAPK) cascade plays a central role in transfer information from diverse receptors/sensors to a wide range of cellular responses in plants. MAP kinases are organized into a complex network for efficient transmission of specific stimuli, including the abiotic stress signaling. In recent years, the mutants of loss-offunction and gain-of-function, and other additional tools are used to investigate the plant MAPK cascades. This review has summarized the recent progress on the MAPK cascade involved in mediation of the transduction of several pronounced abiotic stress signalings, such as salt, drought, low and high temperature, wound, hormone, and deficient nutrients. Currently, although part of the components of the MAPK cascade responding to the abiotic stresses have been identified, the integral molecular mechanisms of the abiotic stresses signaling transduction mediated viaMAPK cascade are largely unknown and need to be elucidated further in the future.     

Localized stabilization of microtubules by integrin- and FAK-facilitated Rho signaling

Microtubule (MT) stabilization is regulated by the small guanosine triphosphate (GTP)-binding protein Rho and its effector, mammalian homolog of Diaphanous (mDia), in migrating cells, but factors responsible for localized stabilization at the leading edge are unknown. We report that integrin-mediated activation of focal adhesion kinase (FAK) at the leading edge is required for MT stabilization by the Rho-mDia signaling pathway in mouse fibroblasts. MT stabilization also involved FAK-regulated localization of a lipid raft marker, ganglioside GM1, to the leading edge. The integrin-FAK signaling pathway may facilitate Rho-mDia signaling through GM1, or through a specialized membrane domain containing GM1, to stabilize MTs in the leading edge of migrating cells.