Glucose allocation is an important cellular process that is misregulated in the interrelated diseases obesity, diabetes and cancer. Cells have evolved critical mechanisms for regulating glucose allocation, one of which is sensory protein kinases. PAS kinase is a key sensory protein kinase that regulates glucose allocation in yeast, mice and man; and is a novel therapeutic target for the treatment of metabolic diseases such as obesity, diabetes and cancer. Despite its importance, the molecular mechanisms of PAS kinase function are largely unknown. Through large-scale protein-interaction studies, we have identified 93 novel binding partners for PAS kinase which help to expand its role in glucose allocation as well as suggest novel roles for PAS kinase including mitochondrial metabolism, cell growth/division, protein modification, stress tolerance, and gene/protein expression. From a subset of these binding partners, we identified 5 in vitro substrates of PAS kinase namely Mot3, Utr1, Zds1, Cbf1 and Pbp1. Additionally, we have further characterized Pbp1 and Cbf1 as PAS kinase substrates through both in vitro and in vivo evidence as well as phenotypic analysis. Evidence is provided for the PAS kinase-dependent phosphorylation and activation of Pbp1, which in turn inhibits cell proliferation through the sequestration of TORC1. In contract, PAS kinase-dependent phosphorylation of Cbf1 inhibits its activity, decreasing cellular respiration. This work elucidates novel molecular mechanisms behind PAS kinase function in both mitochondrial and cell growth pathways in eukaryotic cells, increasing our understanding of the regulation of central metabolism.

The second identified substrate of PAS kinase discussed is Pbp1. The human homolog of Pbp1 is ataxin-2, mutations in which are a known risk factor for amyotrophic lateral sclerosis (ALS). As diet and sex have been shown to be important factors regarding PAS kinase function, they also are strong contributing factors to ALS and are extensively reviewed herein. Pbp1 is known to be sequestered by PAS kinase under glucose depravation, and it can sequester additional proteins along with it to regulate different cellular pathways. To shed light on the pathways affected by Pbp1, we performed a yeast two-hybrid assay and mass spectrometry, identifying 32 novel interacting partners of Pbp1 (ataxin-2). We provide further analysis of the direct binding partner Ptc6, measuring mitophagy, mitochondrial content, colocalization, and respiration. This work elucidates novel molecular mechanisms behind the function of PAS kinase and yields valuable insights into the role of PAS kinase in disease.