PHOSPHOPROTEOME ANALYSIS OF SELF-FUSION REGULATION IN NEUROSPORA CRASSA

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 细胞融合是真核生物的一个重要过程。它发生在受精和组织的生物发生过程中，包括肌肉，骨骼和胎盘。融合缺陷也会导致肝脏解毒和癌症转移。在真菌中，交配细胞之间的融合已被广泛研究，而遗传上相同的细胞之间的自我融合在很大程度上仍然是未知的。丝状真菌粗糙脉孢菌（Neurospora crassa）是分子生物学的模式生物，现在用于研究自我融合。在N.自融合发生在处于相同发育状态的基因相同的细胞之间，其机制在任何系统中都未知。最近的数据表明，N。crassa细胞在两种不同的生理状态之间交替，以促进通信。保守的MAP激酶MAK 2是N. crassa，并在融合细胞之间的趋化性吸引中起直接作用。除了细胞融合，mak-2突变体显示出生长缺陷，突出了这一途径在丝状真菌中的重要性。 此外，MAK 2的直向同源物是由多种子囊菌物种引起的植物和动物发病机制的重要调节剂。 自融合的分子细节在N. crassa，包括化学引诱物分子的身份和激酶途径的任何靶标。我们构建了等位基因MAK 2 Q100 G，其激酶活性可以通过加入ATP类似物1 NM-PP 1特异性抑制。使用这个等位基因，我们表明，MAK 2激酶活性所需的MAK 2复合动力学在合作伙伴的细胞，也为SO，一个未知的分子功能的蛋白质的动态活动。拟议的合作旨在确定MAK 2的靶点，这不仅将揭示参与自我融合的蛋白质，还将揭示营养生长所需的一些蛋白质。 同源比较将揭示相关致病物种中的正磷酸激酶的靶点。 具体目标包括： (1)激酶活性/非活性MAK 2 Q100 G细胞的比较磷酸蛋白质组分析。 (2)识别MAK 2靶标上的特异性磷酸化基序。 (3)鉴定的MAK 2靶标作用的生物学表征。 这种合作有可能显着增加丝状真菌自我融合机制的知识，并可以很容易地扩展到其他多细胞真核生物，可能包括哺乳动物的融合研究。从这项研究中获得的数据也将有其他的影响，因为MAK 2同源物是几种病原体入侵所必需的。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Cell fusion is an essential process in eukaryotes. It occurs during fertilization and the biogenesis of tissues including muscle, bone and placenta. Defects in fusion also have consequences for liver detoxification and cancer metastasis. In fungi, fusion between mating cells has been extensively studied, while self-fusion between genetically identical cells remains largely uncharacterized. The filamentous fungus Neurospora crassa is a model organism for molecular biology, and is now used to study self-fusion. In N. crassa, self-fusion occurs between genetically identical cells that are in the same developmental state, the mechanisms of which are unknown in any system. Recent data has shown that N. crassa cells alternate between two different physiological states in order to facilitate communication. The conserved MAP kinase MAK2 is essential for self-fusion in N. crassa and has a direct role in chemotropic attraction between fusing cells. In addition to cell fusion, mak-2 mutants show growth defects, highlighting the importance of this pathway in filamentous fungi. Furthermore, orthologs of MAK2 are important regulators of plant and animal pathogenesis caused by a variety of ascomycete species. The molecular details of self-fusion are still unknown in N. crassa, including the identity of the chemoattractant molecule and any targets of the kinase pathway. We constructed allele MAK2Q100G, whose kinase activity can be specifically inhibited through the addition of the ATP analog 1NM-PP1. Using this allele, we showed that MAK2 kinase activity is required for MAK2 complex dynamics in the partner cell, and also for the dynamic activity of SO, a protein of unknown molecular function. The proposed collaboration aims to identify targets of MAK2, which will not only reveal proteins involved in self-fusion, but also some required for vegetative growth. Homolog comparisons will reveal targets of the orthologous kinases in related pathogenic species. Specific aims include: (1) Comparative phosphoproteome analysis of kinase active/inactive MAK2Q100G cells. (2) Identify specific phosphorylation motifs on MAK2 targets. (3) Biological characterization of the roles of identified MAK2 targets. This collaboration has the potential to significantly increase the knowledge of self-fusion mechanisms in filamentous fungi, and can be easily expanded to studies of fusion in other multicellular eukaryotes, potentially including mammals. The data obtained from this study will also have other implications, since MAK2 homologs are essential for the invasion of several pathogens.