Mitochondrial Oxidative Metabolism is a critical determinant of Muscle Satellite Cell fate & function

PAS domains are evolutionarily conserved sensory modules present across all domains of life, integral to a wide array of cellular processes. In metazoans, these domains are found in key regulatory proteins, including basic Helix-Loop-Helix (bHLH) transcription factors and voltage-gated potassium channels, and their deregulation is implicated in oncogenesis, metastasis, circadian rhythms, and osmolarity. Unlike bHLH-PAS transcription factors, the PAS domain-containing protein kinase (PASK) is the sole signaling protein kinase in metazoa that combines PAS sensory domains with a serine/threonine kinase domain. PASK is a stem cell-specific protein kinase that plays important roles in the regulation of cellular metabolism, stem cell self-renewal, and differentiation, and has been implicated in aging and the development of insulin resistance in muscle and liver tissues. Despite its significant clinical relevance, the regulatory mechanisms governing PASK’s activity, subcellular localization, and the identity of its downstream targets and interacting partners remain incompletely understood. In this research project, we aim to bridge this knowledge gap by leveraging the extensive experimental toolkit developed in our laboratory. This includes the precise monitoring of PASK activity and quaternary structure, the use of a novel epitope-tagged fluorescence reporter mouse line to track PASK expression and subcellular distribution, while simultaneously facilitating the mapping of its in vivo interactome. Using these tools, we found that PASK is catalytically activated through mTOR-dependent phosphorylation, with subsequent nuclear translocation driven by glutamine-stimulated, p300-dependent acetylation. Uniquely, these post-translational modifications induce a reorganization of PASK’s quaternary structure through intra-domain interactions involving PAS domains and the kinase domain, ultimately leading to catalytic activation. Building on these findings, we will elucidate how sensory information from the three PAS domains is transmitted to the kinase domain by precisely mapping inter- and intra-domain amino acid contact sites, and how these contacts are remodeled in response to signaling cues (Aim 1). In addition to catalytic regulation, we have determined that PASK protein levels oscillate throughout the cell cycle in stem cells. In Aim 2, we will characterize the acetylation- ubiquitylation switch that regulates PASK stability under the control of cellular metabolism and its impact on self- renewal vs differentiation decisions. Finally, we have uncovered novel interacting partners and substrates of PASK in proliferating primary myoblasts. In Aim 3, we will characterize these kinase-substrate relationships and determine their functional relevance in stem cells. By integrating structural biology, biochemistry, and stem cell biology, we will develop a detailed mechanistic understanding of PASK''s role in stem cell function and metabolism. The insights gained from this work have the potential to inform novel therapeutic strategies for metabolic diseases, aging, and disorders of stem cell regulation, thereby achieving a transformational impact in the biomedical sciences.