CHARACTERIZATION OF BUDDING YEAST MOB1

我们的长期目标是了解控制有丝分裂退出的保守的细胞周期调节，并调节中心体复制。这两个过程都知之甚少，但对于确保分裂细胞的活力和基因组稳定性至关重要。基因组稳定性缺陷在癌细胞中很常见。这一建议的目的是通过阐明MOB1的细胞周期功能来洞察这些过程，MOB1是酿酒酵母有丝分裂退出和维持倍性所必需的、保守的基因。MOB1编码一种蛋白(Mob1p)，与几种蛋白激酶相互作用，包括Mps1p，一种SPB复制和有丝分裂检查点调节所需的蛋白激酶，以及Dbf2p/Dbf20p，一对有丝分裂退出所需的冗余蛋白激酶。Mob1p在整个细胞周期中被差异磷酸化，并在有丝分裂晚期暂时定位于纺锤体极体(SPBS，相当于中心体的功能)和胞质质环。为了阐明MOB1的S细胞周期功能(S)，提出了三个目标。第一个目标是确定Mob1p和Mps1p、Dbf2p和Dbf20p蛋白激酶之间的功能关系。实验的目的是确定1)Mob1p是否以依赖于细胞周期的方式与每个蛋白激酶相互作用，2)Mob1p是否作为每个蛋白激酶的底物或效应者，以及3)Mob1p是否调节每个蛋白激酶的亚细胞分布。第二个目的是通过分析突变的Mob1p的结合特性、亚细胞分布和功能来确定Mob1p的功能结构域。这些方法应该揭示Mob1p相互作用和亚细胞定位的功能意义。第三个目标是通过研究MOB1的S在维持倍性中的作用的遗传学方法和用于鉴定Mob1p结合蛋白的生化方法，通过鉴定其调控途径(S)中的其他成分来确定MOB1的功能。这两种方法对于任何模型的Mob1p函数都是公正的。总之，所提出的方法将揭示Mob1p功能的分子机制。鉴于MOB1在真核生物中的保守性，这些方法的数据很可能与理解保守的调控有关，这些保守的调控对于维持基因组的稳定性和细胞分裂的活性至关重要。

Our long-term objectives are to understand the conserved cell cycle regulations that control mitotic exit, and regulate centrosome duplication. Both processes are poorly understood, yet are critical for ensuring the viability and genomic stability of dividing cells. Defects in genomic stability are common among cancerous cells. The goals of this proposal are to gain insight to these processes by elucidating the cell cycle functions of MOB1, an essential and conserved the Saccharomyces cerevisiae gene required for mitotic exit and maintenance of ploidy. MOB1 encodes a protein (Mob1p) that interacts with several protein kinases, including Mps1p, a protein kinase required for SPB duplication and mitotic checkpoint regulation, and Dbf2p/Dbf20p, a pair of redundant protein kinases required for mitotic exit. Mob1p is differentially phosphorylated throughout the cell cycle and transiently localizes to spindle pole bodies (SPBs, the functional equivalent of centrosomes) and the cytokinesis ring in late mitosis. Three objectives are proposed to elucidate MOB1's cell cycle function(s). The first objective is to determine the functional relationship between Mob1p and Mps1p, Dbf2p and Dbf20p protein kinases. Experiments are proposed to determine 1) whether Mob1p interacts with each kinase in a cell cycle dependent fashion 2) whether Mob1p functions as substrate or effector of each protein kinase and 3) whether Mob1p regulates the subcellular distribution of each protein kinase. The second objective is to identify the functional domains of Mob1p by assaying the binding properties, subcellular distribution and function of mutant Mob1p. These approaches should reveal the functional significance of Mob1p's interactions and subcellular localizations. The third objective is to determine MOB1 function by identifying additional components in its regulatory pathway(s) via a genetic approach designed to investigate MOB1's role for the maintenance of ploidy and a biochemical approach designed to identify Mob1p binding proteins. Both approaches are unbiased with respect to any model of Mob1p function. Collectively, the proposed approaches will reveal insight to the molecular mechanisms of Mob1p function. Given the conservation of MOB1 among eukaryotes, it is likely that data from these approaches will be relevant for understanding the conserved regulations that are essential for maintaining the genomic stability and viability of dividing cells.