Genetic Analysis of Yeast Casein Kinase 1 Function

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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 ***