Yeast MAPK Signaling Pathways: Specificity & Regulation

描述(申请人提供)：酿酒酵母交配信息素反应途径可以说是任何真核生物中最了解的多层MAPK信号级联。然而，协调交配所需的基因表达和细胞形态的变化涉及一个复杂的连锁事件网络，而不是一个简单的线性路径。此外，存在反馈机制来调节这些信令事件的效率和持续时间，基本上在每一步。此外，这个信号通路必须在正确的刺激下引起适当的反应，但又要避免不定的激活。此外，人们现在认识到，信息素反应所需的许多成分也被用于不同的发育结果，称为膜状/侵入性生长，以响应营养限制。在任何生物体中，不同的细胞外信号如何影响同一MAPK级联，但又是如何以不同的方式被破译的，还没有完全理解。因此，酵母继续提供机会来研究MAPK信号通路的组织、特异性、保真度和调控的基本方面，包括相同的成分如何与相同细胞类型中的不同上游输入和下游反应相耦合。具体目标包括：(1)Ste5支架蛋白的鉴定，包括晶体结构测定，阐明其调控核质转运的机制，以及其环-H2结构域在Ste5齐聚、分子内和分子间泛素化以及与Gbeta/Gamma相互作用中的作用(Ste4-Ste18)。(2)基于蓝藻藻胆蛋白的荧光特性，开发了一种适用于功能基因组学的实时可视化活细胞信号蛋白动力学的新方法。(3)接头蛋白Ste50的遗传和生化特性及其与小GTP酶CDC42的相互作用，包括溶液结构的确定。(4)使用酵母RGS蛋白Sst2检验DEP结构域与Gbeta/Gamma相关的假设。(5)探讨丝状化过程中对PKA功能的要求是通过阻断MAPK磷酸酶的作用来维持MAPK信号转导的可能性。(6)研究肌醇磷脂在Bem1接头蛋白膜募集中的作用，以及信息素信号阻断芽形成和胞质分裂的机制。(7)转录调控因子Dig1和Dig2对STE12依赖基因表达的抑制和启动子特异性识别的生化特征。研究酵母MAPK信号可能会为抗癌治疗提供启示，因为在人类中，众所周知的癌蛋白(如Ras和Raf)引起的MAPK不适当的激活会导致肿瘤的形成。

DESCRIPTION (provided by applicant): The Saccharomyces cerevisiae mating pheromone response pathway is arguably the best understood multi-tiered, MAPK signaling cascade in any eukaryote. However, coordinating the changes in gene expression and cell morphology necessary for mating involves an elaborate network of interlocking events rather than a simple linear pathway. In addition, feedback mechanisms exist to modulate the efficiency and duration of these signaling events at essentially every step. Moreover, this signaling pathway must evoke an appropriate response upon the correct stimulus, yet avoid adventitious activation. Also, it is now appreciated that many components required for pheromone response are also utilized for a different developmental outcome, termed filamentous/invasive growth, in response to nutrient limitation. How different extracellular signals impinge on the same MAPK cascade, yet are deciphered differently, is not understood fully in any organism. Yeast continues, therefore, to provide opportunities to examine basic aspects of the organization, specificity, fidelity, and regulation of MAPK signaling pathways, including how the same components can be coupled to different upstream inputs and downstream responses in the same cell type. Specific aims include: (1) Characterization of Ste5 scaffold protein, including crystal structure determination, elucidating the mechanism of its regulated nucleocytoplasmic transport, and genetic and biochemical analysis of the role of its RING-H2 domain in Ste5 oligomerization, in intra- and intermolecular ubiquitinylation, and in interaction with Gbeta/gamma (Ste4-Ste18). (2) Development of a new method for visualization of signaling protein dynamics in real time in live cells based on exploitation of the fluorescent properties of cyanobacterial phycobiliproteins, also applicable to functional genomics. (3) Genetic and biochemical characterization of the adaptor protein, Ste50, and its interaction with the small GTPase, Cdc42, including solution structure determination. (4) Testing a hypothesis, using yeast RGS protein, Sst2, that DEP domains associate with Gbeta/gamma. (5) Exploring the possibility that the requirement for PKA function in filamentation is to sustain MAPK signaling by blocking MAPK phosphatase action. (6) Investigating the role of phosphoinositides in membrane recruitment of Bem1 adaptor protein and in the mechanisms by which pheromone signaling interdicts both bud formation and cytokinesis. (7) Biochemical characterization of repression and promoter-specific discrimination by transcriptional regulators, Dig1 and Dig2, in Ste12-dependent gene expression. Studying yeast MAPK signaling may provide insights for anti-cancer therapy because inappropriate MAPK activation in humans, evoked by well-known oncoproteins (e.g. Ras and Raf), leads to tumor formation.