Genetic Analysis of Yeast Casein Kinase 1 Function

酪蛋白激酶1家族的9601294个罗宾逊蛋白激酶广泛存在于所有真核生物中。这些酶在局部酸性环境中磷酸化丝氨酸或苏氨酸残基，这些残基可以由上游的磷酸化氨基酸组成。哺乳动物细胞中至少有四种不同的CK1亚型，其中几种是由多个基因编码的。这些异构体具有不同的细胞位置和功能。例如，一种定位于有丝分裂纺锤体的人类形态似乎是有丝分裂所必需的。在肝细胞中，另一种形式可能参与了对肾上腺素的反应。将这些蛋白质作为不同的基因产物进行研究是非常新的，对大多数形式的CK1的功能还知之甚少。酿酒酵母是一种可以进行遗传操作的芽生酵母，它含有三种CK1亚型。一种异构体影响DNA重组和修复。第二个具有尚未定义的功能，这些功能似乎与其他两个不同。第三个是由功能冗余的YCK1/YCK2基因对编码的质膜相关CK1亚型，它是第一个被证明是细胞生长和分裂所必需的CK1，也是本研究的重点。我们的目标是确定需要YCK活性的细胞通路，并确定这些激酶的生物靶标。最近的结果表明，多个细胞过程需要YCK蛋白的活性。将详细研究两个过程：形态发生和内吞作用。对突变体的分析和对条件性yck突变体的多效性表型的表征将使我们能够深入了解这些和其他需要yck活性的过程。这项拟议的研究将经典和分子遗传学技术与生化和细胞生物学分析方法相结合，以实现三个具体目标。首先是评估YCK蛋白在萌芽部位定位的要求和时间特异性，并确定YCK介导的磷酸化与该部位其他蛋白质活性的关系。这将通过免疫组织化学和体内差异磷酸盐标记条件YCK突变体的潜在靶点的分析来完成。二是阐明YCK活性与内吞过程的关系。这将通过对条件突变体的各种内吞过程进行遗传和生化分析来实现。通过消除YCK功能的突变，鉴定了四种与笼状蛋白适配器复合体的四个亚基序列相似的基因产物。将评估这些细胞在复合体中相互作用的能力以及与特定囊泡群体的联系。此外，将在功能突变缺失的情况下进行内吞作用的测试。第三个目标是确定在这些途径中起作用或受YCK磷酸化影响的其他基因产物。将使用yck突变体对需要完整水平的yck活性的突变体进行遗传筛选。这种方法将允许独立地确认YCK在内吞作用和形态发生中的活性，并可能识别下游功能以及这些蛋白激酶在其他途径中的潜在靶点。YCK激酶在功能上类似于哺乳动物的CK1亚型；因此，这些研究结果可能为CK1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 ***