Identification of Substrates and Pathways Regulated by PAS Kinase
Probst, Brandon Linn
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PAS kinase, a serine/threonine protein kinase, is unique in that it comprises the only mammalian protein kinase regulated by a PAS domain. The interest of the McKnight laboratory in understanding the regulation and biological role of PAS kinase stems from knowledge that PAS domains typically function as sensors in other systems. My study primarily focused on unveiling physiological PAS kinase (PASK) substrates and interacting gene products to establish pathways regulated by PASK. First, we examined the function of PASK in the budding yeast, S. cerevisiae. An unbiased biochemical screen for putative PASK substrates from cellular extracts uncovered five polypeptides phosphorylated in a PASK-dependent manner. Two of the substrates identified are known translation factors and a third is an RNA-binding protein that was also found to be a high copy suppressor of the psk1 psk2 double mutant phenotype. We further observed PASK to phosphorylate two enzymes in the pathway for the synthesis of glycogen: UDP-glucose pyrophosphorylase and glycogen synthase. Genetic and biochemical data provide evidence that both of these enzymes are inhibited by PASK-dependent phosphorylation. We next examined the role of PASK in mammalian cells. With the exception of glycogen synthase none of the mammalian homologs of the yeast PASK substrates have been found to qualify as substrates for the mammalian PASK. To this end, a second unbiased, large-scale biochemical screen was employed using HeLa cell extracts to discover phosphorylation targets of the mammalian enzyme. This biochemical screen entailed the disruption of 150 liters of HeLa cells into a soluble extract and subsequent fractionation over seven chromatographic steps to generate roughly 1000 partially purified pools of protein. This effort led to the identification of enzyme substrates involved in protein synthesis and intermediary metabolism. Phosphorylation site mapping of these substrates identified the consensus motif, R-X-A/x-S*/T* as the optimal substrate for PASK. Although it remains unclear as to the functional role of PAS kinase-dependent phosphorylation of these substrates, there appears to be a unifying theme in both yeast and mammalian systems. PAS kinase is a PAS-domain regulated enzyme controlling translation as this energetically expensive process is coupled to cellular energy metabolism.