Investigating the Regulation and Structural Features of the conserved Membrane-Binding F-BAR Protein Cdc15

研究保守膜结合 F-BAR 蛋白 Cdc15 的调控和结构特征

• 批准号: 9248211
• 负责人: MariaSanta Mangione
• 金额: $ 2.87万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-04-01 至 2018-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9248211
• 关键词: Actins; Affect; Alpha Cell; Aneuploidy; Animal Model; Animals; Automobile Driving; Binding; Binding Proteins; Biochemical; Biochemistry; Biological Assay; Biology; C-terminal; Cell Cycle; Cell Cycle Regulation; Cell Death; Cell division; Cell physiology; Cells; Complement; Complex; Consensus Sequence; Cytokinesis; Cytoskeleton; DNA Damage; Defect; Development; Disease; Enzymes; Eukaryota; Eukaryotic Cell; Event; Failure; Family; Family member; Fanconi' s Anemia; Fission Yeast; Foundations; Gene Deletion; Genetic; Genetic Materials; Genomics; Goals; In Vitro; Individual; Interphase; Knowledge; Lead; Light; Location; Malignant Neoplasms; Mass Spectrum Analysis; Medial; Mediating; Membrane; Microscopy; Mitosis; Modeling; Molecular; Mutagenesis; Myosin ATPase; Myosin Type II; N-terminal; Normal Cell; Organism; Pathogenesis; Pathway interactions; Phenotype; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphorylation Site; Phosphotransferases; Process; Protein Family; Protein Kinase; Protein Kinase C; Protein phosphatase; Proteins; Proteomics; Recruitment Activity; Regulation; Research; Role; SH3 Domains; Serine; Signal Pathway; Signal Transduction; Site; Structure; Testing; Threonine; Yeasts; amphiphysin; anillin; base; constriction; daughter cell; deletion library; event cycle; experimental study; forward genetics; genetic analysis; genetic manipulation; improved; insight; link protein; member; novel; novel therapeutics; physical separation; protein function; public health relevance; reverse genetics; scaffold; screening; segregation; spatiotemporal; temperature sensitive mutant; tumor; tumorigenesis; zygote

 DESCRIPTION (provided by applicant): The final step in cell replication is the physical separation of daughter cells, or cytokinesis. Animal cells divide using an actin- and myosin-based contractile ring (CR). CR assembly, constriction, and disassembly are tightly regulated to protect the genomic complement. Cytokinesis failure or inappropriate location or timing of cytokinesis can lead to cell death or aneuploidy, a state that promotes tumorigenesis. The goal of this proposal is to enhance the molecular understanding of cytokinesis using the eukaryotic fission yeast Schizosaccharomyces pombe. S. pombe is an excellent model organism to study cytokinesis because this process is evolutionarily conserved from yeast to animal cells. Furthermore, S. pombe offers many technical advantages including a gene deletion collection and easy implementation of forward and reverse genetics, biochemistry, and microscopy. Facile genetic manipulation in S. pombe has allowed for large-scale screens to identify additional cytokinetic proteins, many of which are conserved in higher eukaryotes. The proposed research investigates the regulation and molecular mechanism of an essential cytokinetic protein, Cdc15. Cdc15 is the founding member of the Pombe Cdc15 Homology (PCH) / FCH-Bin/Amphiphysin/Rvs (F-BAR) family, which comprises proteins that link membranes via F-BAR domains to actin cytoskeleton remodeling through additional functional domains. Cdc15's N-terminal F-BAR domain recruits the formin Cdc12 to the division site and binds membranes, while its non-essential C-terminal SH3 domain interacts with numerous proteins involved in robust completion of cytokinesis. Cdc15 functions are cell cycle regulated by phosphorylation of >33 serines or threonines, most of which occur in the disordered central region. The phosphorylated sites match diverse kinase consensus sequences, suggesting that multiple signaling pathways converge on Cdc15. In Aim 1, kinases and phosphatases that regulate Cdc15 will be identified by screening gene deletion or temperature-sensitive mutants. A combination of mass spectrometry, mutagenesis, genetic analysis, biochemistry and microscopy will be used to characterize how the phosphorylation status of the specific residues (de)phosphorylated by a single kinase or phosphatase affects Cdc15 activity. The second aim of this proposal employs proteomics, structure-function analysis, microscopy, biochemistry, and genetic experiments to test the hypothesis that the disordered central region binds one or more proteins that participate in cytokinesis. The combined results of these aims will identify signalin pathways that temporally and spatially regulate animal cell cytokinesis and will better characterize the molecular machinery whose integrity is essential for successful cytokinesis. This knowledge will provide foundational insight for understanding the pathogenesis of disorders resulting from cytokinetic defects.

 描述（通过应用程序提供）：细胞复制的最后一步是子细胞或细胞因子的物理分离。动物细胞使用基于肌动蛋白和肌球蛋白的收缩环（CR）分裂。 CR组装，收缩和拆卸受到严格的调节，以保护基因组完成。细胞因子衰竭或不适当的位置或细胞因子的时间可能导致细胞死亡或非整倍性，该状态促进肿瘤发生。该提案的目的是使用真核裂变酵母菌酸糖果肉类增强对细胞因子的分子理解。 S. pombe是研究细胞因子的出色模型生物，因为此过程从酵母菌到动物细胞的进化保守。此外，S。Pombe提供了许多技术优势，包括基因缺失收集以及远期和反向遗传学，生物化学和显微镜的轻松实施。 S. pombe中的便捷遗传操纵允许大规模筛选鉴定出其他细胞动物蛋白，其中许多蛋白在较高的真核生物中保守。拟议的研究研究了必需细胞动力学蛋白CDC15的调节和分子机制。 CDC15是POMBE CDC15同源性（PCH）/FCH-BIN/AMPHIPHYSIN/RVS（F-BAR）家族的创始成员，该家族包括通过F-BAR结构域将机制连接到肌动蛋白细胞骨骨骼通过其他功能域通过其他功能域连接起来的蛋白质。 CDC15的N末端F-BAR结构域将formin cdc12募集到分裂位点并结合膜，而其非必需的C末端SH3结构域与涉及鲁棒性细胞因子的众多蛋白质相互作用。 CDC15功能是通过> 33丝丝或苏氨酸的磷酸化调节的细胞周期，其中大多数发生在无序中心区域。磷酸化的位点与多种激酶共有序列匹配，表明多个信号通路在Cdc15上汇聚。在AIM 1中，通过筛选基因缺失或温度敏感突变体来鉴定调节CDC15的激酶和磷酸酶。质谱，诱变，遗传分析，生物化学和显微镜的结合将用于表征由单个激酶或磷酸酶磷酸化的特定残留物（DE）的磷酸化状态如何影响CDC15活性。该提案的第二个目的是采用蛋白质组学，结构功能分析，显微镜，生物化学和遗传实验来检验该无序中心区域结合一种参与细胞因子的一种或多种蛋白质的假设。这些目标的综合结果将鉴定信号途径暂时和空间调节动物细胞因子，并更好地表征其完整性对于成功的细胞因子至关重要的分子机器。这些知识将为理解细胞动力学缺陷引起的疾病的发病机理提供基本的见解。

项目成果

Cdk1 phosphorylation of fission yeast paxillin inhibits its cytokinetic ring localization.

• DOI: 10.1091/mbc.e20-12-0807
• 发表时间: 2021-08-15
• 期刊: Molecular biology of the cell
• 影响因子: 3.3
• 作者: Mangione MC;Chen JS;Gould KL
• 通讯作者: Gould KL

Multiple polarity kinases inhibit phase separation of F-BAR protein Cdc15 and antagonize cytokinetic ring assembly in fission yeast.

• DOI: 10.7554/elife.83062
• 发表时间: 2023-02-07
• 期刊: eLife
• 影响因子: 7.7
• 作者: Bhattacharjee R;Hall AR;Mangione MC;Igarashi MG;Roberts-Galbraith RH;Chen JS;Vavylonis D;Gould KL
• 通讯作者: Gould KL