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The ligand-regulated PAS domains are one of the most diverse signal-integrating domains found in proteins from prokaryotes to humans. By biochemically connecting cellular processes with their environment, PAS domains facilitate an appropriate cellular response. PAS domain-containing Kinase (PASK) is an evolutionarily conserved protein kinase that plays important signaling roles in mammalian stem cells to establish stem cell fate. We have shown that the nuclear translocation of PASK is stimulated by differentiation signaling cues in muscle stem cells. However, the mechanistic basis of the regulation of PASK nucleo-cytoplasmic translocation remains unknown. Here, we show that the PAS-A domain of PASK contains a putative monopartite nuclear localization sequence (NLS) motif. This NLS is inhibited in cells through intramolecular association with a short linear motif, termed the PAS Interacting Motif (PIM), found upstream of the kinase domain. This interaction serves to retain PASK in the cytosol in the absence of signaling cues. Consistent with that, we show that metabolic inputs induce PASK nuclear import, likely by disrupting this association. We suggest that a route for such linkage may occur through the PAS-A ligand binding cavity. We show that PIM recruitment and artificial ligand binding to the PAS-A domain occur at neighboring locations that could facilitate metabolic control of the PAS-PIM interaction. Thus, the intramolecular interaction in PASK integrates metabolic signaling cues for nuclear translocation and could be targeted to control the balance between self-renewal and differentiation in stem cells., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: C.K.K. is an inventor on a provisional U.S. patent application covering the potential uses of PIM peptides as modulators of stem cell differentiation., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Tissue regeneration is a complex molecular and biochemical symphony. Signaling pathways establish the rhythmic proliferation and differentiation cadence of participating cells to repair the damaged tissues and repopulate the tissue-resident stem cells. Sensory proteins form a critical bridge between the environment and cellular response machinery, enabling precise spatiotemporal control of stem cell fate. Of many sensory modules found in proteins from prokaryotes to mammals, Per-Arnt-Sim (PAS) domains are one of the most ancient and found in the most diverse physiological context. In metazoa, PAS domains are found in many transcription factors and ion channels; however, PAS domain-containing Kinase (PASK) is the only metazoan kinase where the PAS sensory domain is connected to a signaling kinase domain. PASK is predominantly expressed in undifferentiated, self-renewing embryonic and adult stem cells, and its expression is rapidly lost upon differentiation, resulting in its nearly complete absence from the adult mammalian tissues. Thus, PASK is expressed within a narrow but critical temporal window when stem cell fate is established. In this review, we discuss the emerging insight into the sensory and signaling functions of PASK as an integrator of metabolic and nutrient signaling information that serves to balance self-renewal and differentiation programs during mammalian tissue regeneration.

During skeletal muscle regeneration, muscle stem cells (MuSCs) respond to multiple signaling inputs that converge onto mammalian target of rapamycin complex 1 (mTORC1) signaling pathways. mTOR function is essential for establishment of the differentiation-committed progenitors (early stage of differentiation, marked by the induction of myogenin expression), myotube fusion, and, ultimately, hypertrophy (later stage of differentiation). While a major mTORC1 substrate, p70S6K, is required for myotube fusion and hypertrophy, an mTORC1 effector for the induction of myogenin expression remains unclear. Here, we identified Per-Arnt-Sim domain kinase (PASK) as a downstream phosphorylation target of mTORC1 in MuSCs during differentiation. We have recently shown that the PASK phosphorylates Wdr5 to stimulate MuSC differentiation by epigenetically activating the myogenin promoter. We show that phosphorylation of PASK by mTORC1 is required for the activation of myogenin transcription, exit from self-renewal, and induction of the myogenesis program. Our studies reveal that mTORC1-PASK signaling is required for the rise of myogenin-positive committed myoblasts (early stage of myogenesis), whereas mTORC1-S6K signaling is required for myoblast fusion (later stage of myogenesis). Thus, our discoveries allow molecular dissection of mTOR functions during different stages of the myogenesis program driven by two different substrates., Competing Interests: The authors declare no conflict of interest.

PAS domain containing protein kinase (Pask) is an evolutionarily conserved protein kinase implicated in energy homeostasis and metabolic regulation across eukaryotic species. We now describe an unexpected role of Pask in promoting the differentiation of myogenic progenitor cells, embryonic stem cells and adipogenic progenitor cells. This function of Pask is dependent upon its ability to phosphorylate Wdr5, a member of several protein complexes including those that catalyze histone H3 Lysine 4 trimethylation (H3K4me3) during transcriptional activation. Our findings suggest that, during myoblast differentiation, Pask stimulates the conversion of repressive H3K4me1 to activating H3K4me3 marks on the promoter of the differentiation gene myogenin ( Myog ) via Wdr5 phosphorylation. This enhances accessibility of the MyoD transcription factor and enables transcriptional activation of the Myog promoter to initiate muscle differentiation. Thus, as an upstream kinase of Wdr5, Pask integrates signaling cues with the transcriptional network to regulate the differentiation of progenitor cells., Competing Interests: The authors declare that no competing interests exist.

Elevated hepatic synthesis of fatty acids and triglycerides, driven by hyperactivation of the SREBP-1c transcription factor, has been implicated as a causal feature of metabolic syndrome. SREBP-1c activation requires the proteolytic maturation of the endoplasmic-reticulum-bound precursor to the active, nuclear transcription factor, which is stimulated by feeding and insulin signaling. Here, we show that feeding and insulin stimulate the hepatic expression of PASK. We also demonstrate, using genetic and pharmacological approaches, that PASK is required for the proteolytic maturation of SREBP-1c in cultured cells and in the mouse and rat liver. Inhibition of PASK improves lipid and glucose metabolism in dietary animal models of obesity and dyslipidemia. Administration of a PASK inhibitor decreases hepatic expression of lipogenic SREBP-1c target genes, decreases serum triglycerides, and partially reverses insulin resistance. While the signaling network that controls SREBP-1c activation is complex, we propose that PASK is an important component with therapeutic potential., (Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.)

Enhanced activation of phosphoinositide 3-kinase (PI3K) is a hallmark of many human tumors because it promotes cell proliferation and survival through several mechanisms. One of these mechanisms is the phosphorylation of the serine and threonine kinase Akt at the cytosolic side of the plasma membrane by phosphoinositide-dependent protein kinase 1 (PDK1), which is recruited and activated by binding to the phosphoinositides produced by PI3K. We previously demonstrated increased nuclear accumulation of PDK1 in cells with enhanced PI3K activity. We report that nuclear PDK1 promoted cell proliferation by suppressing FOXO3A-dependent transcription of the gene encoding p27Kip1 (an inhibitor of cell cycle progression), whereas it enhanced cell survival by inhibiting the activation of c-Jun amino-terminal kinase. Cells with nuclear-localized PDK1 showed anchorage-independent growth, and when injected into mice, these cells induced the formation of solid tumors. In human prostate tumors, cytoplasmic localization of PDK1 correlated only with early-stage, low-risk tumors, whereas nuclear PDK1 localization correlated with high-risk tumors. Together, our findings suggest a role for nuclear-translocated PDK1 in oncogenic cellular transformation and tumor progression in mice and humans.

Per-Arnt-Sim (PAS) domain-containing protein kinase (PASK) is an evolutionary conserved protein kinase that coordinates cellular metabolism with metabolic demand in yeast and mammals. The molecular mechanisms underlying PASK regulation, however, remain unknown. Herein, we describe a crystal structure of the kinase domain of human PASK, which provides insights into the regulatory mechanisms governing catalysis. We show that the kinase domain adopts an active conformation and has catalytic activity in vivo and in vitro in the absence of activation loop phosphorylation. Using site-directed mutagenesis and structural comparison with active and inactive kinases, we identified several key structural features in PASK that enable activation loop phosphorylation-independent activity. Finally, we used combinatorial peptide library screening to determine that PASK prefers basic residues at the P-3 and P-5 positions in substrate peptides. Our results describe the key features of the PASK structure and how those features are important for PASK activity and substrate selection.

3-Phosphoinositide-dependent protein kinase-1 (PDK1) mediates phosphorylation and activation of members of the AGC protein kinase family and plays an essential role in insulin signaling and action. However, whether and how PDK1 activity is regulated in cells remains largely uncharacterized. In the present study, we show that PDK1 undergoes insulin-stimulated and phosphatidylinositol 3-kinase-dependent phosphorylation at Ser244 in the activation loop and at a novel site: Ser163 in the hinge region between the two lobes of the kinase domain. Sequence alignment studies revealed that the residue corresponding to Ser163 of PDK1 in all other AGC kinases is glutamate, suggesting that a negative charge at this site may be important for PDK1 function. Replacing Ser163 with a negatively charged residue, glutamate, led to a 2-fold increase in PDK1 activity. Molecular modeling studies suggested that phosphorylated Ser163 may form additional hydrogen bonds with Tyr149 and Gln223. In support of this, mutation of Tyr149 to Ala is sufficient to reduce PDK1 activity. Taken together, our results suggest that PDK1 phosphorylation of Ser163 may provide a mechanism to fine-tune PDK1 activity and function in cells.

Activation of cytosolic phosphoinositide-3 kinase (PI-3K) signaling pathway has been well established to regulate gene expression, cell cycle, and survival by feeding signals to the nucleus. In addition, strong evidences accumulated over the past few years indicate the presence of an autonomous inositol lipid metabolism and PI-3K signaling within the nucleus. Much less, however, is known about the role and regulation of this nuclear PI-3K pathway. Components of the PI-3K signaling pathway, including PI 3-kinase and its downstream kinase Akt, have been identified at the nuclear level. Consistent with the presence of a complete PI-3K signaling pathway in the nucleus, we have recently found that phosphoinositide-dependent kinase 1 (PDK1), a kinase functioning downstream of PI-3K and upstream of Akt, is a nucleo-cytoplasmic shuttling protein. In the present review, we update our current knowledge on the regulatory mechanisms and the functional roles of PDK1 nuclear translocation. We also summarize some of the kinase-independent activities of PDK1 in cell signaling., (Copyright 2005 Wiley-Liss, Inc.)

3'-Phosphoinositide-dependent protein kinase 1 (PDK-1) phosphorylates and activates members of the AGC protein kinase family and plays an important role in the regulation of cell survival, differentiation, and proliferation. However, how PDK-1 is regulated in cells remains elusive. In this study, we demonstrated that PDK-1 can shuttle between the cytoplasm and nucleus. Treatment of cells with leptomycin B, a nuclear export inhibitor, results in a nuclear accumulation of PDK-1. PDK-1 nuclear localization is increased by insulin, and this process is inhibited by pretreatment of cells with phosphatidylinositol 3-kinase (PI3-kinase) inhibitors. Consistent with the idea that PDK-1 nuclear translocation is regulated by the PI3-kinase signaling pathway, PDK-1 nuclear localization is increased in cells deficient of PTEN (phosphatase and tensin homologue deleted on chromosome 10). Deletion mapping and mutagenesis studies unveiled that presence of a functional nuclear export signal (NES) in mouse PDK-1 located at amino acid residues 382 to 391. Overexpression of constitutively nuclear PDK-1, which retained autophosphorylation at Ser-244 in the activation loop in cells and its kinase activity in vitro, led to increased phosphorylation of the predominantly nuclear PDK-1 substrate p70 S6KbetaI. However, the ability of constitutively nuclear PDK-1 to induce anchorage-independent growth and to protect against UV-induced apoptosis is greatly diminished compared with the wild-type enzyme. Taken together, these findings suggest that nuclear translocation may be a mechanism to sequestrate PDK-1 from activation of the cytosolic signaling pathways and that this process may play an important role in regulating PDK-1-mediated cell signaling and function.