Unravelling the roles of the Mck1 kinase in metabolic reprogramming and the stress response
During the transition into stationary phase, S. cerevisiae cells acquire a set of characteristics that are typical of G0 cells, which include upregulation of the starvation- specific gene expression program, enhanced resistance to environmental stressors, the accumulation of storage carbohydrates, the thickening of the cell wall and ultimately the ability to maintain long-term survival. Previous work in the lab revealed that the Mck1 kinase, one of the four GSK3 homologues in Saccharomyces cerevisiae, is a key regulator of stationary phase entry. Further study indicated that Mck1 promotes UDPG synthesis during the transition to the stationary phase to enable cell wall thickening as well as the accumulation of storage carbohydrates. In this Project, I intended to find how the Mck1 kinase regulates metabolic reprogramming and the stress response in response to glucose depletion. In the first result chapter (chapter 3), the functional conservation between the budding S. cerevisiae Mck1 and those from the pathogenic fungi was initially demonstrated. Following-up transcriptome studies revealed that Mck1 may promote metabolic reprogramming and the starvation-induced stress response by activating two regulons: the Cat8- and Adr1-mediated gene expression program promoting the TCA cycle and gluconeogenesis, and the Msn2/4- and /Gis1-dependent stress response. Transcriptome and genetic analyses also suggested that Mck1 regulates metabolic reprogramming and the stress response independent of Mpk1/Slt2, the MAP kinase of the cell wall integrity (CWI) pathway. 5 In the second result chapter (chapter 4), Mck1-regulated proteome changes in response to glucose starvation were revealed. The proteome analysis indicated that Mck1 promotes the expression of the Cat8-/Adr1- and the Msn2/4/Gis1-dependent regulons at the protein levels, supporting that Mck1 regulates metabolic reprogramming and the stress response program mainly at the transcriptional level. The levels of a number of proteins were shown to be poorly correlated with those of their corresponding transcripts, suggesting that Mck1 may also modulate their expression post-transcriptionally. In the third result chapter (chapter 5), integrative analysis of the proteome and phosphoproteome was conducted with the aim to identify the targets of Mck1 that are implicated in metabolic reprogramming and the stress response. Many known and potential targets were revealed. Subsequent genetic and phenotypic assays indicated that Rcn1 in the calcineurin (CN) pathway may not participate in the Mck1-mediated gene expression. Bioinformatic analysis suggested a number of potential targets, including Hlr1, which may be involved in the regulation of cell separation and cell integrity. The final result chapter (chapter 6) focused on the transcription factor Msn2, whose phosphorylation levels at multiple sites were decreased in the starved mck1Δ mutants from the phosphoproteome analysis. Surprisingly, experiments using Phos-tag gel and Western blotting indicated that phosphorylation of Msn2 was independent of Mck1 but rather dependent on the PAS kinase Rim15 and casein kinase 2 (CK2). CK2-dependent phosphorylation of Msn2 at S638 was shown to be essential to Msn2 protein stability regardless of nutrient status. Based on the above findings, further analysis of the phosphoproteome data and literature review, an integrative model was proposed in the discussion chapter to suggest that Mck1 may be implicated in the stress response and metabolic reprogramming by targeting the mRNA decay pathway. Such a model, however, remains to be experimentally confirmed.