The Rho1p Exchange Factor Rgf1p Signals Upstream from the Pmk1 Mitogen-activated Protein Kinase Pathway in Fission Yeast

The Schizosaccharomyces pombe exchange factor Rgf1p specifically regulates Rho1p during polarized growth. Rgf1p activates the β-glucan synthase (GS) complex containing the catalytic subunit Bgs4p and is involved in the activation of growth at the second end, a transition that requires actin reorganization. In this work, we investigated Rgf1p signaling and observed that Rgf1p acted upstream from the Pck2p-Pmk1p MAPK signaling pathway. We noted that Rgf1p and calcineurin play antagonistic roles in Cl⁻ homeostasis; rgf1Δ cells showed the vic phenotype (viable in the presence of immunosuppressant and chlorine ion) and were unable to grow in the presence of high salt concentrations, both phenotypes being characteristic of knockouts of the MAPK components. In addition, mutations that perturb signaling through the MAPK pathway resulted in defective cell integrity (hypersensitivity to caspofungin and β-glucanase). Rgf1p acts by positively regulating a subset of stimuli toward the Pmk1p-cell integrity pathway. After osmotic shock and cell wall damage HA-tagged Pmk1p was phosphorylated in wild-type cells but not in rgf1Δ cells. Finally, we provide evidence to show that Rgf1p regulates Pmk1p activation in a process that involves the activation of Rho1p and Pck2p, and we demonstrate that Rgf1p is unique in this signaling process, because Pmk1p activation was largely independent of the other two Rho1p-specific GEFs, Rgf2p and Rgf3p.     

Regulation of Cell Motility by Mitogen-activated Protein Kinase

Cell interaction with adhesive proteins or growth factors in the extracellular matrix initiates Ras/ mitogen-activated protein (MAP) kinase signaling. Evidence is provided that MAP kinase (ERK1 and ERK2) influences the cells' motility machinery by phosphorylating and, thereby, enhancing myosin light chain kinase (MLCK) activity leading to phosphorylation of myosin light chains (MLC). Inhibition of MAP kinase activity causes decreased MLCK function, MLC phosphorylation, and cell migration on extracellular matrix proteins. In contrast, expression of mutationally active MAP kinase kinase causes activation of MAP kinase leading to phosphorylation of MLCK and MLC and enhanced cell migration. In vitro results support these findings since ERK-phosphorylated MLCK has an increased capacity to phosphorylate MLC and shows increased sensitivity to calmodulin. Thus, we define a signaling pathway directly downstream of MAP kinase, influencing cell migration on the extracellular matrix.     

Regulation of G2/M Progression by the STE Mitogen-activated Protein Kinase Pathway in Budding Yeast Filamentous Growth

Inoculation of diploid budding yeast onto nitrogen-poor agar media stimulates a MAPK pathway to promote filamentous growth. Characteristics of filamentous cells include a specific pattern of gene expression, elongated cell shape, polar budding pattern, persistent attachment to the mother cell, and a distinct cell cycle characterized by cell size control at G2/M. Although a requirement for MAPK signaling in filamentous gene expression is well established, the role of this pathway in the regulation of morphogenesis and the cell cycle remains obscure. We find that ectopic activation of the MAPK signal pathway induces a cell cycle shift to G2/M coordinately with other changes characteristic of filamentous growth. These effects are abrogated by overexpression of the yeast mitotic cyclins Clb1 and Clb2. In turn, yeast deficient for Clb2 or carrying cdc28-1N, an allele of CDK defective for mitotic functions, display enhanced filamentous differentiation and supersensitivity to the MAPK signal. Importantly, activation of Swe1-mediated inhibitory phosphorylation of Thr-18 and/or Tyr-19 of Cdc28 is not required for the MAPK pathway to affect the G2/M delay. Mutants expressing a nonphosphorylatable mutant Cdc28 or deficient for Swe1 exhibit low-nitrogen-dependent filamentous growth and are further induced by an ectopic MAPK signal. We infer that the MAPK pathway promotes filamentous growth by a novel mechanism that inhibits mitotic cyclin/CDK complexes and thereby modulates cell shape, budding pattern, and cell-cell connections.     

Stress Activated Protein Kinase Pathway Modulates Homologous Recombination in Fission Yeast

Rad52 is a key player in homologous recombination (HR), a DNA repair pathway that is dedicated to double strand breaks repair and recovery of perturbed replication forks. Here we show that fission yeast Rad52 homologue is phosphorylated when S phase cells are exposed to ROS inducers such as ultraviolet A radiation or hydrogen peroxide, but not to ultraviolet C or camptothecin. Phosphorylation does not depend on kinases Chk1, Rad3, Tel1 or Cdc2, but depends on a functional stress activated protein kinase (SAPK) pathway and can be partially prevented by anti-oxidant treatment. Indeed, cells lacking Sty1, the major fission yeast MAP kinase of the SAPK pathway, do not display Rad52 phosphorylation and have UVA induced Rad52 foci that persist longer if compared to wild type cells. In addition, spontaneous intrachromosomal HR is diminished in cells lacking Sty1 and, more precisely, gene conversion is affected. Moreover, HR induced by site-specific arrest of replication forks is twice less efficient in cells that do not express Sty1. Importantly, impairing HR by deletion of the gene encoding the recombinase Rhp51 leads to Sty1 dependent Rad52 phosphorylation. Thus, SAPK pathway impinges on early step of HR through phosphorylation of Rad52 in cells challenged by oxidative stress or lacking Rhp51 and is required to promote spontaneous gene conversion and recovery from blocked replication forks.     

A Link between Aurora Kinase and Clp1/Cdc14 Regulation Uncovered by the Identification of a Fission Yeast Borealin-Like Protein

The chromosomal passenger complex (CPC) regulates various events in cell division. This complex is composed of a catalytic subunit, Aurora B kinase, and three nonenzymatic subunits, INCENP, Survivin, and Borealin. Together, these four subunits interdependently regulate CPC function, and they are highly conserved among eukaryotes. However, a Borealin homologue has never been characterized in the fission yeast, Schizosaccharomyces pombe. Here, we isolate a previously uncharacterized S. pombe protein through association with the Cdc14 phosphatase homologue, Clp1/Flp1, and identify it as a Borealin-like member of the CPC. Nbl1 (novel Borealin-like 1) physically associates with known CPC components, affects the kinase activity and stability of the S. pombe Aurora B homologue, Ark1, colocalizes with known CPC subunits during mitosis, and shows sequence similarity to human Borealin. Further analysis of the Clp1–Nbl1 interaction indicates that Clp1 requires CPC activity for proper accumulation at the contractile ring (CR). Consistent with this, we describe negative genetic interactions between mutant alleles of CPC and CR components. Thus, this study characterizes a fission yeast Borealin homologue and reveals a previously unrecognized connection between the CPC and the process of cytokinesis in S. pombe.     

Glucose Activates TORC2-Gad8 Protein via Positive Regulation of the cAMP/cAMP-dependent Protein Kinase A (PKA) Pathway and Negative Regulation of the Pmk1 Protein-Mitogen-activated Protein Kinase Pathway

The target of rapamycin (TOR) kinase belongs to the highly conserved eukaryotic family of phosphatidylinositol 3-kinase-related kinases. TOR proteins are found at the core of two evolutionary conserved complexes, known as TORC1 and TORC2. In fission yeast, TORC2 is dispensable for proliferation under optimal growth conditions but is required for starvation and stress responses. TORC2 has been implicated in a wide variety of functions; however, the signals that regulate TORC2 activity have so far remained obscure. TORC2 has one known direct substrate, the AGC kinase Gad8, which is related to AKT in human cells. Gad8 is phosphorylated by TORC2 at Ser-546 (equivalent to AKT Ser-473), leading to its activation. Here, we show that glucose is necessary and sufficient to induce Gad8 Ser-546 phosphorylation in vivo and Gad8 kinase activity in vitro. The glucose signal that activates TORC2-Gad8 is mediated via the cAMP/PKA pathway, a major glucose-sensing pathway. By contrast, Pmk1, similar to human extracellular signal-regulated kinases and a major stress-induced mitogen activated protein kinase (MAPK) in fission yeast, inhibits TORC2-dependent Gad8 phosphorylation and activation. Inhibition of TORC2-Gad8 also occurs in response to ionic or osmotic stress, in a manner dependent on the cAMP/PKA and Pmk1-MAPK signaling pathways. Our findings highlight the significance of glucose availability in regulation of TORC2-Gad8 and indicate a novel link between the cAMP/PKA, Pmk1/MAPK, and TORC2-Gad8 signaling.     

Role of Polo Kinase and Mid1p in Determining the Site of Cell Division in Fission Yeast

The fission yeast Schizosaccharomyces pombe divides symmetrically using a medial F-actin– based contractile ring to produce equal-sized daughter cells. Mutants defective in two previously described genes, mid1 and pom1, frequently divide asymmetrically. Here we present the identification of three new temperature-sensitive mutants defective in localization of the division plane. All three mutants have mutations in the polo kinase gene, plo1, and show defects very similar to those of mid1 mutants in both the placement and organization of the medial ring. In both cases, ring formation is frequently initiated near the cell poles, indicating that Mid1p and Plo1p function in recruiting medial ring components to the cell center. It has been reported previously that during mitosis Mid1p becomes hyperphosphorylated and relocates from the nucleus to a medial ring. Here we show that Mid1p first forms a diffuse cortical band during spindle formation and then coalesces into a ring before anaphase. Plo1p is required for Mid1p to exit the nucleus and form a ring, and Pom1p is required for proper placement of the Mid1p ring. Upon overexpression of Plo1p, Mid1p exits the nucleus prematurely and displays a reduced mobility on gels similar to that of the hyperphosphorylated form observed previously in mitotic cells. Genetic and two-hybrid analyses suggest that Plo1p and Mid1p act in a common pathway distinct from that involving Pom1p. Plo1p localizes to the spindle pole bodies and spindles of mitotic cells and also to the medial ring at the time of its formation. Taken together, the data indicate that Plo1p plays a role in the positioning of division sites by regulating Mid1p. Given its previously known functions in mitosis and the timing of cytokinesis, Plo1p is thus implicated as a key molecule in the spatial and temporal coordination of cytokinesis with mitosis.     

MAPK级联途径参与ABA信号转导调节的植物生长发育过程

植物激素ABA参与调控植物生长发育和生理代谢以及多种胁迫应答过程,促分裂原活化蛋白激酶（MAPK）级联途径应答于多种生物和非生物胁迫,广泛参与调控植物的生长发育。MAPK级联途径与ABA信号转导协同作用参与调控植物种子萌发、气孔运动和生长发育,本文主要归纳了植物中受ABA调控激活的MAPK级联途径成员,阐述了它们参与ABA信号转导调控植物生理反应和生长发育的过程,并对MAPK级联途径与ABA信号转导的研究方向作出了展望,指出对MAPK下游底物的筛选是完善MAPK级联途径的重要组成部分。     

Dynamic Ubiquitination of the Mitogen-activated Protein Kinase Kinase (MAPKK) Ste7 Determines Mitogen-activated Protein Kinase (MAPK) Specificity

Ubiquitination is a post-translational modification that tags proteins for proteasomal degradation. In addition, there is a growing appreciation that ubiquitination can influence protein activity and localization. Ste7 is a prototype MAPKK in yeast that participates in both the pheromone signaling and nutrient deprivation/invasive growth pathways. We have shown previously that Ste7 is ubiquitinated upon pheromone stimulation. Here, we show that the Skp1/Cullin/F-box ubiquitin ligase SCF^Cdc4 and the ubiquitin protease Ubp3 regulate Ste7 ubiquitination and signal specificity. Using purified components, we demonstrate that SCF^Cdc4 ubiquitinates Ste7 directly. Using gene deletion mutants, we show that SCF^Cdc4 and Ubp3 have opposing effects on Ste7 ubiquitination. Although SCF^Cdc4 is necessary for proper activation of the pheromone MAPK Fus3, Ubp3 is needed to limit activation of the invasive growth MAPK Kss1. Finally, we show that Fus3 phosphorylates Ubp3 directly and that phosphorylation of Ubp3 is necessary to limit Kss1 activation. These results reveal a feedback loop wherein one MAPK limits the ubiquitination of an upstream MAPKK and thereby prevents spurious activation of a second competing MAPK.     

A Role for Mitogen-activated Protein Kinase in the Spindle Assembly Checkpoint in XTC Cells

The spindle assembly checkpoint prevents cells whose spindles are defective or chromosomes are misaligned from initiating anaphase and leaving mitosis. Studies of Xenopus egg extracts have implicated the Erk2 mitogen-activated protein kinase (MAP kinase) in this checkpoint. Other studies have suggested that MAP kinases might be important for normal mitotic progression. Here we have investigated whether MAP kinase function is required for mitotic progression or the spindle assembly checkpoint in vivo in Xenopus tadpole cells (XTC). We determined that Erk1 and/or Erk2 are present in the mitotic spindle during prometaphase and metaphase, consistent with the idea that MAP kinase might regulate or monitor the status of the spindle. Next, we microinjected purified recombinant XCL100, a Xenopus MAP kinase phosphatase, into XTC cells in various stages of mitosis to interfere with MAP kinase activation. We found that mitotic progression was unaffected by the phosphatase. However, XCL100 rendered the cells unable to remain arrested in mitosis after treatment with nocodazole. Cells injected with phosphatase at prometaphase or metaphase exited mitosis in the presence of nocodazole—the chromosomes decondensed and the nuclear envelope re-formed—whereas cells injected with buffer or a catalytically inactive XCL100 mutant protein remained arrested in mitosis. Coinjection of constitutively active MAP kinase kinase-1, which opposes XCL100's effects on MAP kinase, antagonized the effects of XCL100. Since the only known targets of MAP kinase kinase-1 are Erk1 and Erk2, these findings argue that MAP kinase function is required for the spindle assembly checkpoint in XTC cells.     

Phosphorylation-Independent Regulation of Atf1-Promoted Meiotic Recombination by Stress-Activated,p38 Kinase Spc1 of Fission Yeast

Background

Stress-activated protein kinases regulate multiple cellular responses to a wide variety of intracellular and extracellular conditions. The conserved, multifunctional, ATF/CREB protein Atf1 (Mts1, Gad7) of fission yeast binds to CRE-like (M26) DNA sites. Atf1 is phosphorylated by the conserved, p38-family kinase Spc1 (Sty1, Phh1) and is required for many Spc1-dependent stress responses, efficient sexual differentiation, and activation of Rec12 (Spo11)-dependent meiotic recombination hotspots like ade6-M26.

Methodology/Principal Findings

We sought to define mechanisms by which Spc1 regulates Atf1 function at the ade6-M26 hotspot. The Spc1 kinase was essential for hotspot activity, but dispensable for basal recombination. Unexpectedly, a protein lacking all eleven MAPK phospho-acceptor sites and detectable phosphorylation (Atf1-11M) was fully proficient for hotspot recombination. Furthermore, tethering of Atf1 to ade6 in the chromosome by a heterologous DNA binding domain bypassed the requirement for Spc1 in promoting recombination.

Conclusions/Significance

The Spc1 protein kinase regulates the pathway of Atf1-promoted recombination at or before the point where Atf1 binds to chromosomes, and this pathway regulation is independent of the phosphorylation status of Atf1. Since basal recombination is Spc1-independent, the principal function of the Spc1 kinase in meiotic recombination is to correctly position Atf1-promoted recombination at hotspots along chromosomes. We also propose new hypotheses on regulatory mechanisms for shared (e.g., DNA binding) and distinct (e.g., osmoregulatory vs. recombinogenic) activities of multifunctional, stress-activated protein Atf1.     

Linkers of Cell Polarity and Cell Cycle Regulation in the Fission Yeast Protein Interaction Network

The study of gene and protein interaction networks has improved our understanding of the multiple, systemic levels of regulation found in eukaryotic and prokaryotic organisms. Here we carry out a large-scale analysis of the protein-protein interaction (PPI) network of fission yeast (Schizosaccharomyces pombe) and establish a method to identify ‘linker’ proteins that bridge diverse cellular processes - integrating Gene Ontology and PPI data with network theory measures. We test the method on a highly characterized subset of the genome consisting of proteins controlling the cell cycle, cell polarity and cytokinesis and identify proteins likely to play a key role in controlling the temporal changes in the localization of the polarity machinery. Experimental inspection of one such factor, the polarity-regulating RNB protein Sts5, confirms the prediction that it has a cell cycle dependent regulation. Detailed bibliographic inspection of other predicted ‘linkers’ also confirms the predictive power of the method. As the method is robust to network perturbations and can successfully predict linker proteins, it provides a powerful tool to study the interplay between different cellular processes.     

丝裂原活化蛋白激酶信号通路与受精

受精是雌雄生殖细胞相互融合形成合子的过程 ,是生命的开端。有性生殖的生命体通过受精 ,使基因组由生殖细胞的单倍体恢复为体细胞的二倍体 ;同时 ,精子与卵子的结合激活了以母源信息形式贮存在卵子中的发育程序 ,使卵子由受精前的生命活动抑制状态转变为活跃状态 ,即活化卵子。受精涉及到两个不同个体间异源细胞的识别、融合、信号转导和分裂周期调节 ,因此久已成为发育生物学、细胞生物学和生物化学的重要研究课题。多种蛋白激酶参与对受精过程的调节。作者和其他人的研究均发现丝裂原活化蛋白激酶 (ｍｉｔｏｇｅｎａｃｔｉｖａｔｅｄｐｒ…     

Feedback Regulation of SIN by Etd1 and Rho1 in Fission Yeast

In fission yeast, the septation initiation network (SIN) is thought to promote cytokinesis by downstream activation of Rho1, a conserved GTPase that controls cell growth and division. Here we show that Etd1 and PP2A-Pab1, antagonistic regulators of SIN, are Rho1 regulators. Our genetic and biochemical studies indicate that a C-terminal region of Etd1 may activate Rho1 by directly binding it, whereas an N-terminal domain confers its ability to localize at the growing tips and the division site where Rho1 functions. In opposition to Etd1, our results indicate that PP2A-Pab1 inhibits Rho1. The SIN cascade is upstream-regulated by the Spg1 GTPase. In the absence of Etd1, activity of Spg1 drops down prematurely, thereby inactivating SIN. Interestingly, we find that ectopic activation of Rho1 restores Spg1 activity in Etd1-depleted cells. By using a cytokinesis block strategy, we show that Rho1 is essential to feedback-activate Spg1 during actomyosin ring constriction. Therefore, activation of Spg1 by Rho1, which in turn is regulated by Etd1, uncovers a novel feedback loop mechanism that ensures SIN activity while cytokinesis is progressing.     

Tyr728 in the Kinase Domain of the Murine Kinase Suppressor of RAS 1 Regulates Binding and Activation of the Mitogen-activated Protein Kinase Kinase

In metazoans, the highly conserved MAPK signaling pathway regulates cell fate decision. Aberrant activation of this pathway has been implicated in multiple human cancers and some developmental disorders. KSR1 functions as an essential scaffold that binds the individual components of the cascade and coordinates their assembly into multiprotein signaling platforms. The mechanism of KSR1 regulation is highly complex and not completely understood. In this study, we identified Tyr⁷²⁸ as a novel regulatory phosphorylation site in KSR1. We show that Tyr⁷²⁸ is phosphorylated by LCK, uncovering an additional and unexpected link between Src kinases and MAPK signaling. To understand how phosphorylation of Tyr⁷²⁸ may regulate the role of KSR1 in signal transduction, we integrated structural modeling and biochemical studies. We demonstrate that Tyr⁷²⁸ is involved in maintaining the conformation of the KSR1 kinase domain required for binding to MEK. It also affects phosphorylation and activation of MEK by RAF kinases and consequently influences cell proliferation. Moreover, our studies suggest that phosphorylation of Tyr⁷²⁸ may affect the intrinsic kinase activity of KSR1. Together, we propose that phosphorylation of Tyr⁷²⁸ may regulate the transition between the scaffolding and the catalytic function of KSR1 serving as a control point used to fine-tune cellular responses.     

Rice Mitogen-activated Protein Kinase Gene Family and Its Role in Biotic and Abiotic Stress Response

The mitogen-activated protein kinase （MAPK） cascade is an important signaling module that transduces extracellular stimuli into intracellular responses in eukaryotic organisms. An increasing body of evidence has shown that the MAPK-mediated cellular signaling is crucial to plant growth and development, as well as biotic and abiotic stress responses. To date, a total of 17 MAPK genes have been Identified from the rice genome. Expression profiling, biochemical characterization and/or functional analysis were carried out with many members of the rice MAPK gene family, especially those associated with biotic and abiotic stress responses. In this review, the phylogenetic relationship and classification of rice MAPK genes are discussed to facilitate a simple nomenclature and standard annotation of the rice MAPK gene family. Functional data relating to biotic and abiotic stress responses are reviewed for each MAPK group and show that despite overlapping in functionality, there is a certain level of functional specificity among different rice MAP kinases. The future challenges are to functionally characterize each MAPK, to identify their downstream substrates and upstream kinases, and to genetically manipulate the MAPK signaling pathway in rice crops for the Improvement of agronomically important traits.     

Rice Mitogen-activated Protein Kinase Gene Family and Its Role in Biotic and Abiotic Stress Response

The mitogen-activated protein kinase (MAPK) cascade is an important signaling module that transduces extracellu-lar stimuli into intracellular responses in eukaryotic organisms. An increasing body of evidence has shown that theMAPK-mediated cellular signaling is crucial to plant growth and development, as well as biotic and abiotic stressresponses. To date, a total of 17 MAPK genes have been identified from the rice genome. Expression profiling,biochemical characterization and/or functional analysis were carried out with many members of the rice MAPKgene family, especially those associated with biotic and abiotic stress responses. In this review, the phylogeneticrelationship and classification of rice MAPK genes are discussed to facilitate a simple nomenclature and standardannotation of the rice MAPK gene family. Functional data relating to biotic and abiotic stress responses are re-viewed for each MAPK group and show that despite overlapping in functionality, there is a certain level of functionalspecificity among different rice MAP kinases, The future challenges are to functionally characterize each MAPK, toidentify their downstream substrates and upstream kinases, and to genetically manipulate the MAPK signalingpathway in rice crops for the improvement of agronomically important traits.