Understanding signaling through the 'striatin interacting phosphatase and kinase' (STRIPAK) complex in eukaryotic development

了解真核发育中通过“条纹蛋白相互作用磷酸酶和激酶”(STRIPAK) 复合物发出的信号

• 批准号: 321675624
• 负责人: Professor Dr. Ulrich Kück
• 金额: --
• 依托单位: Lehrstuhl für Allgemeine und Molekulare Botanik
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2016
• 资助国家: 德国
• 起止时间: 2015-12-31 至 2021-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/321675624?language=en
• 关键词: Understanding; signaling; through; striatin; interacting

Striatin-interacting phosphatase and kinase (STRIPAK) complexes are supramolecular structures that are evolutionary conserved from yeast to man. Molecular genetic analysis has shown that diverse processes are STRIPAK complex dependent, such as mammalian cell proliferation, differentiation, and embryonic development, autophagosome transport in Drosophila neurons, viability of Caenorhabditis elegans embryos, and cellular fusion and organ formation in microbial systems. Further, medical studies revealed that dysregulation of the STRIPAK complex is correlated with diverse human diseases, including cancer.Recently, several reports have provided evidence that STRIPAK complexes are regulators or mediators of vital cell signaling pathways including the Hippo signaling pathway in Drosophila, the septation initiation network (SIN) in fungal systems or mitogen-activated kinases (MAPK) pathways in various eukayotes. However, the precise targets of phosphorylation and dephosphorylation by STRIPAK associated kinases and phosphatases have not been identified. Previously, we have established the filamentous fungus Sordaria macrospora as a model system for eukaryotic multicellular development. Studying mutant strains that show defects in hyphal fusion and fruiting body development, we discovered subunits of a fungal STRIPAK complex. To get a detailed insight into the regulatory mechanisms of the STRIPAK complex, we will identify specific phosphorylation sites of target proteins, which are differentially phosphorylated or dephosphorylated in developmental mutants. Included in our study are strains that lack one or two subunits of the STRIPAK complex. We have established a multi-level protocol to isolate protein extract in a reproducible manner, in particular we tested different media and growth conditions to get optimal protein yields. Further, we have tested different protease inhibitors to prevent semi-tryptic cleavage of proteins. Finally, we reduced the rate of semi-tryptic peptides by about 46%. Using a modified protocol for iTRAQ-labelling of peptides combined with a 2D LC-MS/MS in 24 experiments, we identified 228 differentially phosphorylated proteins. These will be the subjects for further functional analysis in S. macrospora. The identification of targets of the STRIPAK complex will contribute to our understanding of STRIPAK complex functions and mechanisms in the context of cell signaling.

Striatin-interacting phosphatase and kinase（STRIPAK）复合物是一种从酵母到人类进化上保守的超分子结构，分子遗传学研究表明，在哺乳动物细胞增殖、分化、胚胎发育、果蝇神经元自噬体转运、秀丽隐杆线虫胚胎发育、微生物系统中的细胞融合和器官形成等过程中，STRIPAK复合物都起着重要的作用。此外，医学研究表明，STRIPAK复合物的失调与包括癌症在内的多种人类疾病相关。最近，多份报告提供了证据，证明STRIPAK复合物是重要细胞信号通路的调节剂或介导剂，包括果蝇中的Hippo信号通路、真菌系统中的分隔起始网络（SIN）或各种真核细胞中的有丝分裂原激活激酶（MAPK）通路。然而，尚未确定STRIPAK相关激酶和磷酸酶磷酸化和去磷酸化的精确靶点。在此之前，我们已经建立了丝状真菌Sordaria macrospora作为真核多细胞发育的模型系统。研究突变株，显示缺陷的菌丝融合和子实体的发展，我们发现了真菌的STRIPAK复合体的亚基。 为了详细了解STRIPAK复合物的调控机制，我们将确定靶蛋白的特异性磷酸化位点，这些位点在发育突变体中差异磷酸化或去磷酸化。在我们的研究中包括的菌株，缺乏一个或两个亚基的STRIPAK复合物。我们已经建立了一个多层次的协议，以分离蛋白质提取物在一个可重复的方式，特别是我们测试了不同的培养基和生长条件，以获得最佳的蛋白质产量。此外，我们已经测试了不同的蛋白酶抑制剂以防止蛋白质的半胰蛋白酶切割。最后，我们将半胰蛋白酶肽的比率降低了约46%。在24个实验中，我们使用iTRAQ标记肽的改良方案与2D LC-MS/MS相结合，鉴定了228种差异磷酸化蛋白质。这些都将是进一步的功能分析的主题在S。大孢子虫。对STRIPAK复合物靶点的识别将有助于我们理解细胞信号传导背景下的STRIPAK复合物功能和机制。

项目成果

Crosstalk Between Pheromone Signaling and NADPH Oxidase Complexes Coordinates Fungal Developmental Processes

• DOI: 10.3389/fmicb.2020.01722
• 发表时间: 2020-07
• 期刊: Frontiers in Microbiology
• 影响因子: 5.2
• 作者: Sarah Schmidt;R. Märker;Barbara Ramšak;Anna M. Beier-Rosberger;I. Teichert;U. Kück

Phosphoproteomic analysis of STRIPAK mutants identifies a conserved serine phosphorylation site in PAK kinase CLA4 to be important in fungal sexual development and polarized growth

• DOI: 10.1111/mmi.14475
• 发表时间: 2020-02-16
• 期刊: MOLECULAR MICROBIOLOGY
• 影响因子: 3.6
• 作者: Maerker, Ramona;Blank-Landeshammer, Bernhard;Kueck, Ulrich
• 通讯作者: Kueck, Ulrich

Combination of Proteogenomics with Peptide De Novo Sequencing Identifies New Genes and Hidden Posttranscriptional Modifications

• DOI: 10.1128/mbio.02367-19
• 发表时间: 2019-09-01
• 期刊: MBIO
• 影响因子: 6.4
• 作者: Blank-Landeshammer, B.;Teichert, I.;Sickmann, A.
• 通讯作者: Sickmann, A.