Quantitative Phosphoproteomics Reveals Pathways for Coordination of Cell Growth and Division by the Conserved Fission Yeast Kinase Pom1

Complex phosphorylation-dependent signaling networks underlie the coordination of cellular growth and division. In the fission yeast Schizosaccharomyces pombe, the Dual specificity tyrosine-(Y)-phosphorylation regulated kinase (DYRK) family protein kinase Pom1 regulates cell cycle progression through the mitotic inducer Cdr2 and controls cell polarity through unknown targets. Here, we sought to determine the phosphorylation targets of Pom1 kinase activity by SILAC-based phosphoproteomics. We defined a set of high-confidence Pom1 targets that were enriched for cytoskeletal and cell growth functions. Cdr2 was the only cell cycle target of Pom1 kinase activity that we identified in cells. Mutation of Pom1-dependent phosphorylation sites in the C terminus of Cdr2 inhibited mitotic entry but did not impair Cdr2 localization. In addition, we found that Pom1 phosphorylated multiple substrates that function in polarized cell growth, including Tea4, Mod5, Pal1, the Rho GAP Rga7, and the Arf GEF Syt22. Purified Pom1 phosphorylated these cell polarity targets in vitro, confirming that they are direct substrates of Pom1 kinase activity and likely contribute to regulation of polarized growth by Pom1. Our study demonstrates that Pom1 acts in a linear pathway to control cell cycle progression while regulating a complex network of cell growth targets.The coordination of cell growth and division represents a fundamental concept in cell biology. The mechanisms that promote polarized growth and drive cell cycle progression are complex signaling networks that operate in a wide range of cell types and organisms. Understanding these networks and their molecular connections requires large-scale approaches that define the underlying biochemical reactions. Phosphorylation drives many events in both cell polarity and cell cycle signaling, and protein kinases that act in both processes represent key players in coordinated growth and division.The fission yeast S. pombe has served as a long-standing model organism for studies on cell polarity and the cell cycle. The fission yeast protein kinase Pom1 is an intriguing candidate to function in the coordination of polarized growth and cell cycle progression. This DYRK¹ family kinase was originally identified as a polarity mutant (hence the name Pom1) in a genetic screen for misshapen cells (1). Later studies revealed an additional role for Pom1 in cell cycle progression, where it delays mitotic entry until cells reach a critical size threshold (2, 3). Thus, pom1Δ mutant cells display defects in both cell polarity and cell size at mitosis, as well as misplaced division septa (1–6). Mutations that impair Pom1 kinase activity mimic these deletion phenotypes, indicating a key role for Pom1-dependent phosphorylation. The pleiotropic phenotype of pom1 mutants might result from Pom1 phosphorylating distinct substrates for cell polarity versus mitotic entry, but the targets of Pom1 kinase activity are largely unknown. Only two Pom1 substrates have been identified to date. First, Pom1 auto-phosphorylates as part of a mechanism that promotes localization in a cortical gradient enriched at cell tips (7). Second, Pom1 phosphorylates two regions of the protein kinase Cdr2. Phosphorylation of Cdr2 C terminus is proposed to prevent mitotic entry by inhibiting Cdr2 kinase activity (8, 9), while phosphorylation near membrane-binding motifs of Cdr2 promotes medial cell division by inhibiting localization of Cdr2 at cell tips (10). It has been unclear if Cdr2 represents the only cell cycle target of Pom1 kinase activity, and no cell polarity targets of Pom1 have been identified. In order to clarify how this protein kinase controls multiple cellular processes, we have comprehensively cataloged Pom1 substrates by quantitative phosphoproteomics. Such a large-scale approach also has the potential to reveal general mechanisms that operate in the coordination of cell growth and division.Stable isotope labeling of amino acids in culture (SILAC) combined with phosphopeptide enrichment and mass spectrometry has allowed the proteome-wide analysis of protein phosphorylation from diverse experimental systems (11–15). In this approach, cells are grown separately in media containing normal (“light”) or isotope-labeled (“heavy”) arginine and lysine, treated, mixed, and processed for LC-MS/MS analysis. In combination with analog-sensitive protein kinase mutants, which can be rapidly and specifically inhibited by nonhydrolyzable ATP analogs (16, 17), SILAC presents a powerful approach to identify cellular phosphorylation events that depend on a specific protein kinase. This method is particularly well suited for studies in yeast, where analog-sensitive protein kinase mutants can be readily integrated into the genome.In this study, we have employed SILAC-based phosphoproteomics to identify Pom1 substrates in fission yeast. New Pom1 targets were verified as direct substrates in vitro, and our analysis indicates that Pom1 controls cell cycle progression through a single target while coordinating a more complex network of cell polarity targets.     

Arabidopsis FIMBRIN5, an actin bundling factor, is required for pollen germination and pollen tube growth

Actin cables in pollen tubes serve as molecular tracks for cytoplasmic streaming and organelle movement and are formed by actin bundling factors like villins and fimbrins. However, the precise mechanisms by which actin cables are generated and maintained remain largely unknown. Fimbrins comprise a family of five members in Arabidopsis thaliana. Here, we characterized a fimbrin isoform, Arabidopsis FIMBRIN5 (FIM5). Our results show that FIM5 is required for the organization of actin cytoskeleton in pollen grains and pollen tubes, and FIM5 loss-of-function associates with a delay of pollen germination and inhibition of pollen tube growth. FIM5 decorates actin filaments throughout pollen grains and tubes. Actin filaments become redistributed in fim5 pollen grains and disorganized in fim5 pollen tubes. Specifically, actin cables protrude into the extreme tips, and their longitudinal arrangement is disrupted in the shank of fim5 pollen tubes. Consequently, the pattern and velocity of cytoplasmic streaming were altered in fim5 pollen tubes. Additionally, loss of FIM5 function rendered pollen germination and tube growth hypersensitive to the actin-depolymerizing drug latrunculin B. In vitro biochemical analyses indicated that FIM5 exhibits actin bundling activity and stabilizes actin filaments. Thus, we propose that FIM5 regulates actin dynamics and organization during pollen germination and tube growth via stabilizing actin filaments and organizing them into higher-order structures.     

Simultaneous determination of didanosine and its amino acid prodrug,valdidanosine by hydrophilic interaction chromatography coupled with electrospray ionization tandem mass spectrometry: Application to a pharmacokinetic study in rats

A rapid, sensitive and selective ultra-performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) method with hydrophilic interaction chromatography has been developed and validated for the simultaneous determination of didanosine and valdidanosine (L-valine amino acid ester prodrug of didanosine) in rat plasma. Solid-phase extraction (SPE) column was employed to extract the analytes from rat plasma, with high extraction recovery (>85%) for both didanosine and valdidanosine. The analytes were then separated by hydrophilic interaction chromatography (HILIC column) and detected by a triple-quadrupole mass spectrometry equipped with an electrospray ionization (ESI) source. The method was linear over the concentration ranges of 2–20,000 ng/mL for didanosine and 4–300 ng/mL for valdidanosine. The lower limit of quantitation (LLOQ) of didanosine and valdidanosine was 2 and 4 ng/mL, respectively. The intra-day and inter-day relative standard deviation (RSD) were less than 15% and the relative errors (RE) were all within 15%. Finally, the validated UPLC–MS/MS method was successfully applied to the pharmacokinetic study after either didanosine or valdidanosine orally administrated to the Sprague–Dawley rats.     

鼠伤寒沙门菌VI型分泌系统效应因子Rhs生物学特性分析

【背景】细菌的Ⅵ型分泌系统作为杀死真核捕食者或原核竞争对手的“武器”,其杀伤作用是通过释放有毒物质即效应因子来实现。尽管已发现一些效应因子,但大多数效应因子的功能仍然未知。【目的】研究rhs基因编码的效应因子Rhs对鼠伤寒沙门菌生物学特性的影响。【方法】利用Red同源重组的方法构建鼠伤寒沙门菌rhs基因缺失株及相应的基因回补株。通过试验分析比较亲本株与缺失株、回补株在生化特性、生物被膜形成、耐药性、细菌间竞争、抗血清补体杀菌能力、组织载菌量及感染小鼠后炎症因子IL-18、IL-1β释放量上的差异。【结果】效应因子Rhs不影响鼠伤寒沙门菌的生化代谢、生物被膜形成、耐药性及抗血清补体杀菌能力。细菌种间竞争试验中,基因缺失株CVCC541Δrhs1、CVCC541Δrhs2和CVCC541Δrhs1-2的竞争指数(competition index,CI)值分别为0.85、0.77和0.87,毒力均被轻度致弱。体内组织载菌量试验中,CVCC541Δrhs1、CVCC541Δrhs2和CVCC541Δrhs1-2基因缺失株在小鼠肝脏和脾脏中的细菌数均较亲本株明显下降(P<0.05);机体...     

Angiomotin binding-induced activation of Merlin/NF2 in the Hippo pathway

The tumor suppressor Merlin/NF2 functions upstream of the core Hippo pathway kinases Lats1/2 and Mst1/2, as well as the nuclear E3 ubiquitin ligase CRL4^DCAF1. Numerous mutations of Merlin have been identified in Neurofibromatosis type 2 and other cancer patients. Despite more than two decades of research, the upstream regulator of Merlin in the Hippo pathway remains unknown. Here we show by high-resolution crystal structures that the Lats1/2-binding site on the Merlin FERM domain is physically blocked by Merlin''s auto-inhibitory tail. Angiomotin binding releases the auto-inhibition and promotes Merlin''s binding to Lats1/2. Phosphorylation of Ser518 outside the Merlin''s auto-inhibitory tail does not obviously alter Merlin''s conformation, but instead prevents angiomotin from binding and thus inhibits Hippo pathway kinase activation. Cancer-causing mutations clustered in the angiomotin-binding domain impair angiomotin-mediated Merlin activation. Our findings reveal that angiomotin and Merlin respectively interface cortical actin filaments and core kinases in Hippo signaling, and allow construction of a complete Hippo signaling pathway.     

Synthesis and acrosin inhibitory activity of substituted 4-amino-N-(diaminomethylene) benzenesulfonamide derivatives

A series of new substituted 4-amino-N-(diaminomethylene) benzenesulfonamides were synthesized and their in vitro acrosin inhibitory activities were evaluated. Most of the compounds showed potent acrosin inhibitory activities with compounds 4o and 4p being significantly more potent than the control compound N-alpha-tosyl-l-lysyl-chloromethyl-ketone (TLCK). The compounds provide a new scaffold for the development of acrosin inhibitory agents.     

不同番茄材料对B型烟粉虱个体发育和繁殖能力的影响

以甘蓝寄主上连续繁殖多代后的B型烟粉虱为对象,对其在8种番茄材料(4个栽培番茄、3个多毛番茄和1个醋栗番茄)上的产卵量、体型大小、发育历期、存活率以及第2代成虫的产卵量和寿命等生物学参数进行观察.自然情况下(10:00-14:00)接虫,烟粉虱在多毛番茄LA2329上的平均产卵量显著低于栽培番茄9706上的产卵量(分别为11粒,164粒).羽化后,烟粉虱雌虫在多毛番茄LA1777上的寿命显著低于在栽培番茄Moneymaker上的存活寿命(分别为5d,22d);而羽化后雌虫在LA1777上的平均产卵量显著低于在栽培番茄早粉2号上的产卵量(分别为7粒/头,95粒/头).在其他参数,如体型大小、存活率、发育历期等,没有显著性的变化.结果显示,较多毛番茄而言,栽培番茄是烟粉虱的较好寄主.而且,在评价抗烟粉虱番茄材料时,平均产卵量、羽化后雌虫寿命及产卵量是3个有效的评价参数.     

我国甜菜夜蛾间歇性暴发的非均衡性循环波动

[摘要] 为进一步了解我国甜菜夜蛾Spodoptera exigua ( Hbner)间歇性暴发成灾的规律,作者应用时间序列分析技术研究了我国甜菜夜蛾间歇性暴发的时间序列波动规律。结果表明,在1949—2008年的60a中,我国甜菜夜蛾大尺度暴发总年频次为120次,各年度暴发次数(年频次)存在着非均衡性循环波动特征,并且在其波动过程中呈明显的上升趋势。按“年频次”强度,可将我国1949—2008年60a中甜菜夜蛾发生过程,划分成两个明显不同的阶段。第一阶段（1949—1984年）为平稳低发阶段,36a总年频次量为4次,年平均仅0.11次;第二阶段（1985—2008年）则为波浪式上升性高发阶段,24a总年频次为116次,年平均4.83次（为第一阶段的43.91倍））。按第二阶段24a（1985—2008）数据进行自相关系数和频谱图分析,结果表明,我国甜菜夜蛾大尺度暴发存在2.8a和11.2a2种不同长度的循环周期,其暴发趋势指数对滞后1a和滞后4a的影响为正相关,而对滞后5a和滞后6a的影响则为负相关。本文根据甜菜夜蛾暴发指数的非均衡周期性特征,建立了以时间序列为自变量的甜菜夜蛾暴发指数非均衡周期性预测模型,经回代结果检验,理论值与实测值之间无显著差异。     

Molecular cloning and functional expression of d-arabitol dehydrogenase gene from Gluconobacter oxydans in Escherichia coli

A NADP-dependent d-arabitol dehydrogenase gene was cloned from Gluconobacter oxydans CGMCC 1.110 and functionally expressed in Escherichia coli. With d-arabitol as sole carbon source, E. coli transformants grew rapidly in minimal medium, and produced d-xylulose. The enzymatic properties of the 29kDa enzyme were documented. The DNA sequence surrounding the gene suggested that it is part of an operon with several components of a sugar alcohol transporter system, and the d-arabitol dehydrogenase gene belongs to the short-chain dehydrogenase family.