A link between TORC1 and CK2 in S. cerevisiae via the Cdc-like kinase Knsl

Abstract

The regulation of energy utilization in response to changing intracellular and extracellular conditions is key for survival. The Target of Rapamycin Complex 1 (TORC1) plays a central role in signaling nutrient availability, stress conditions and cellular energy levels to downstream anabolic and catabolic pathways in order to promote cell growth and prevent cell death. TOR kinase activity balances the level of protein synthesis and protein synthetic capacity (i.e. ribosome biogenesis) according to the needs of the cell. Ribosome biogenesis is an energetically costly process that accounts for over 80% of the transcriptional activity in growing cells and involves the coordinated action of all three nuclear RNA polymerases (pols I-III). The regulation of ribosome and tRNA synthesis is therefore a central hub for cellular energy management. Additionally, the dysregulation of this process is highly correlated with multiple cancers, making this system attractive for the development of new anti-cancer therapies.;Previously, our laboratory identified a new role for the cyclin-like kinase Knsl and the GSK3 kinase Mckl in the regulation of ribosome and tRNA synthesis downstream of TORC1. The first part of this thesis describes the design of a chemical-genetic screen identifying likely new roles for Knsl and Mckl in the regulation of vesicle-mediated transport, the control of cell size and polarity, and a link to protein kinase CK2. The second part of this thesis focuses on understanding the connection between TORC1 and CK2. Protein kinase CK2 is a constitutively active pro-growth kinase involved in cell cycle progression, cell proliferation and transcription and which contributes to cancer in higher organisms. We present evidence of in vivo phosphoregulation of its Ckbl subunit under nutrient limitation and cellular stress, and show that Knsl mediates this phosphorylation in all tested conditions. To query CK2 regulatory mechanisms we carried out co-immunoprecipitations of its subunits and glycerol gradient fractionations of whole cell extracts and found no evidence for the dissociation of CK2 subunits under stress. Instead, chromatin immunoprecipitation assays support a model of CK2 regulation via the differential association of the holoenzyme at pol III genes, and suggest a role for Ckbl phosphorylation in this process.