Genetic Analysis of Yeast Casein Kinase 1 Function

9601294罗宾逊 酪蛋白激酶1（CK 1）家族的蛋白激酶存在于所有真核生物中。 这些激酶在局部酸性环境中磷酸化丝氨酸或苏氨酸残基，其可由上游磷酸化氨基酸组成。 在哺乳动物细胞中至少有四种不同的CK 1亚型，其中几种由多个基因编码。 这些同种型具有不同的细胞位置和功能。 例如，一个定位于有丝分裂纺锤体的人类形体似乎是有丝分裂所必需的。 另一种形式，在肝细胞中，可能参与对肾上腺素的反应。 这些蛋白质作为不同的基因产物的研究是最近的，并且对大多数形式的CK 1的功能知之甚少。 芽殖酵母，酿酒酵母，这是适合遗传操作，含有三个CK 1亚型。 一种亚型影响DNA重组和修复。 第二个有尚未定义的功能，这似乎是不同于其他两个。 第三，质膜相关的CK 1亚型编码的功能冗余的YCK 1/YCK 2基因对，是第一个CK 1被证明是必不可少的细胞生长和分裂，是本研究的重点。 目的是确定需要Yck活性的细胞途径，并确定这些激酶的生物学靶点。 最近的研究结果表明，多种细胞过程需要Yck蛋白的活性。 两个过程将被详细研究，形态发生和内吞作用。 突变体的分析和条件性yck突变体的多效性表型的表征将允许洞察这些和其他需要Yck活性的过程。 拟议的研究将经典和分子遗传技术与生物化学和细胞生物学分析方法相结合，以实现三个具体目标。 第一个是评估的要求和时间特异性本地化的YCK蛋白的网站出现的芽，并确定YCK介导的磷酸化的其他蛋白质在这个网站的活动的关系。 这将通过免疫组织化学和条件性YCK突变体中潜在靶点的差异体内磷酸盐标记试验来完成。 二是阐明Yck活性与内吞过程的关系。 这将通过条件突变体中各种内吞过程的遗传和生物化学测定来完成。 通过消除对Yck功能的要求的突变鉴定了与网格蛋白衔接子复合物的四个亚基具有序列相似性的四种基因产物。 将评估它们在复合物中相互作用的能力以及与特定囊泡群体的关联。 此外，还将对功能缺失突变体进行内吞作用试验。 第三个目的是鉴定在这些通路中起作用或受Yck磷酸化影响的其他基因产物。 将使用yck突变体进行需要全水平Yck活性的突变体的遗传筛选。 这种方法将允许独立确认Yck活性的内吞作用和形态发生，并可能允许识别的下游功能，以及这些蛋白激酶在其他途径的潜在目标。Yck激酶在功能上类似于哺乳动物CK 1亚型;因此，这些研究的结果可以为CK 1在高等细胞中的功能提供线索 *

9601294 Robinson Protein kinases of the casein kinase 1 (CK1) family are found in all eukaryotes. These kinases phosphorylate serine or threonine residues in an local acidic context, which can be comprised of an upstream phosphorylated amino acid. There are at least four distinct CK1 isoforms in mammalian cells, several of which are encoded by multiple genes. These isoforms have distinct cellular locations and functions. For example, one human form that localizes to the mitotic spindle appears to be required for mitosis. Another form, in liver cells, may participate in the response to epinephrine. The study of these proteins as distinct gene products is very recent, and little is yet known about the functions of most forms of CK1. The budding yeast, Saccharomyces cerevisiae, which is amenable to genetic manipulation, contains three CK1 isoforms. One isoform affects DNA recombination and repair. The second has as yet undefined functions, which appear to be distinct from those of the other two. The third, a plasma membrane-associated CK1 isoform encoded by the functionally redundant YCK1 / YCK2 gene pair, is the first CK1 demonstrated to be essential for cell growth and division and is the focus of this study. The goal is to identify the cellular pathways that require Yck activity and to identify the biological targets of these kinases. Recent results suggest that multiple cellular processes require the activity of the Yck proteins. Two processes will be studied in detail, morphogenesis and endocytosis. The analysis of mutants and the characterization of the pleiotropic phenotype of the conditional yck mutant will allow insight into these and other processes that require Yck activity. The proposed research combines classical and molecular genetic techniques with biochemical and cell biological methods of analysis to achieve three specific goals. The first is to assess the requirement and temporal specificity for localization of the Yck proteins to the site of the emerging bud, and to determine the relationship of Yck-mediated phosphorylation to the activities of other proteins at this site. This will be accomplished by immunohistochemistry and by assay of differential in vivo phosphate labeling of potential targets in a conditional YCK mutant. The second is to clarify the relationship of Yck activity with endocytic processes. This will be accomplished by genetic and biochemical assays of various endocytic processes in a conditional mutant. Four gene products with sequence similarity to the four subunits of clathrin adaptor complexes were identified by mutations that eliminate the requirement for Yck function. These will be assessed for their ability to interact in a complex and for association with specific vesicle populations. Also, tests for a role in endocytosis will be performed with loss of function mutants. The third aim is to identify other gene products that either act in these pathways or that are affected by Yck phosphorylation. A genetic screen for mutants requiring full levels of Yck activity will be carried out using yck mutants. This approach will allow independent confirmation of Yck activity in endocytosis and morphogenesis, and may allow identification of downstream functions as well as potential targets for these protein kinases in other pathways. The Yck kinases are functionally similar to a mammalian CK1 isoform; therefore the results of these studies could provide clues for CK1 functions in higher cells ***