Analysis of Kinase Signaling Pathways Regulating Filamentous Growth and mRNP Granules in Filamentous Yeast

Abstract

Pseudoyphal growth is a stress response in which S. cerevisiae cells form elongated multicellular filaments, similar to processes of filamentous development required for virulence in pathogenic yeast such as C. albicans. The signaling pathways that regulate pseudohyphal growth in S. cerevisiae include TORC1, MAPK, PKA and AMPK; however, the exact mechanisms by which these pathways integrate and regulate pseudohyphal growth is largely unknown. To fill this gap in knowledge, our lab previously identified targets of kinases that regulate this process via SILAC-based quantitative phosphoproteomics. This thesis builds upon these studies by focusing on a kinase that is required for pseudohyphal growth as evidenced by genome-wide studies of regulators of pseudohyphal growth. Ksp1 is a kinase that regulates pseudohyphal growth, however the mechanism was unknown. Here, we show that Ksp1 regulates pseudohyphal growth both at the transcriptional and post-translational levels. Transcriptional profiling revealed Ksp1 kinase activity is required for wild type transcript abundance of genes related to a variety of stress responses, including pseudohyphal growth, sporulation, cell morphology, DNA damage, and autophagy, as well as amino acid metabolism. Mass spectrometry-based analysis of the Ksp1-dependent phosphoproteome identified Ksp1 regulation of a statistically overrepresented set of stress granule-localized proteins, including the p21-activated kinase Ste20 which localizes to stress granules. Deletion of KSP1 resulted in elevated abundance of stress granule marker protein Pbp1-containing foci, suggesting a function for Ksp1 in modulating stress granule abundance. In total, we identify Ksp1 as an effector of different stress responses and as one of a handful of kinases identified as a regulator of stress granules via the phosphorylation-based control of stress granule components. In addition to Ksp1, our lab performed quantitative phosphoproteomic analysis of additional kinases required for pseudohyphal growth. These analyses identified target proteins that regulate inositol polyphosphate metabolism. Through metabolite analysis by HPLC, we observe a correlation between the ratio of different inositol polyphosphate species in the cell and pseudohyphal growth phenotypes, as well as expression levels of Flo11, the master transcriptional regulator of pseudohyphal growth. Similar metabolite analysis in Candida albicans suggests inositol polyphosphate metabolism might play a role in filamentation in other fungi as well. To further understand the genes important for pseudohyphal growth in S. cerevisiae, I constructed an overexpression library which then used to screen for effects on filament formation. Using a collection of approximately 1500 plasmids from an existing yeast genomic tiling library, I created a corresponding collection of filamentous Saccharomyces cerevisiae strains that overexpresses 95% of the genome in large fragments. The resulting library library has been used by Damien Krysan’s laboratory at University of Iowa to screen for regulators of yeast-triggered macrophage pyroptosis, indicating its utility as a tool in dissecting signaling pathways mediating filamentation. Collectively, the work included in this thesis sheds light on the control of two different stress responses, namely pseudohyphal growth and translational regulation in stress granules, and suggests a link between the signaling pathways that regulate these processes.