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Quiescent stem cells are activated in response to a mechanical or chemical injury to their tissue niche. Activated cells rapidly generate a heterogeneous progenitor population that regenerates the damaged tissues. While the transcriptional cadence that generates heterogeneity is known, the metabolic pathways influencing the transcriptional machinery to establish a heterogeneous progenitor population remains unclear. Here, we describe a novel pathway downstream of mitochondrial glutamine metabolism that confers stem cell heterogeneity and establishes differentiation competence by countering post-mitotic self-renewal machinery. We discovered that mitochondrial glutamine metabolism induces CBP/EP300-dependent acetylation of stem cell-specific kinase, PAS domain-containing kinase (PASK), resulting in its release from cytoplasmic granules and subsequent nuclear migration. In the nucleus, PASK catalytically outcompetes mitotic WDR5-anaphase-promoting complex/cyclosome (APC/C) interaction resulting in the loss of post-mitotic Pax7 expression and exit from self-renewal. In concordance with these findings, genetic or pharmacological inhibition of PASK or glutamine metabolism upregulated Pax7 expression, reduced stem cell heterogeneity, and blocked myogenesis in vitro and muscle regeneration in mice. These results explain a mechanism whereby stem cells co-opt the proliferative functions of glutamine metabolism to generate transcriptional heterogeneity and establish differentiation competence by countering the mitotic self-renewal network via nuclear PASK.

OBJECTIVES: To determine how nutrient signaling impacts stem cell functions METHODS: PASK phosphorylation: We measured in situ phosphorylation of PASK by metabolic 32P labeling of stem cells expressing WT or mutant versions of PASK. PASK Activation: PASK activation was measured using in vitro kinase assay using radio-labeled ATP. Myogenesis: Myogenesis was measured by immunohistological, and immunofluorescent analysis of differentiating muscle stem cells. Antibodies used were: Myogenin (F5D-Developmental Hybridoma), MF20 (Myosin heavy chain), Pax7 and MyoD. RESULTS: Stem cell fate in the tissue niche is intimately connected with intracellular metabolic state and the extracellular hormonal stimulations. We have identified PAS domain containing Kinase (PASK) as a stem cell enriched protein kinase that is required for establishment of the differentiation program in many stem cell paradigms. For this function, PASK phosphorylates Wdr5, a member of the COMPASS family of histone methyltransferases, to activate the epigenetic processes required for the stem cell differentiation (eLife, 2016). Here we show that a master nutrient sensor, mTOR complex 1 (mTORC1) activates PASK via multi-site phosphorylation during stem cell differentiation. This phosphorylation of PASK by mTORC1 is required for epigenetic activation of the Myogenin transcription, exit from the self-renewal and induction of the myogenesis program. Our data suggest that mTORC1-PASK signaling generates MyoG + committed myoblasts (epigenetically - an early stage of myogenesis), whereas mTORC1-S6K1 signaling is required for myoblast fusion (translationally - later stage of myogenesis). CONCLUSIONS: Our discoveries show that nutrient signaling can partition stem cell fates during different stages of the myogenesis program downstream of mTOR signaling via activation of two distinct protein kinases. FUNDING SOURCES: NIH R01 (Chintan Kikani), HHMI (Jared Rutter) SUPPORTING TABLES, IMAGES AND/OR GRAPHS

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.

SummaryIn the tissue microenvironment, stem cell functions are modulated by extrinsic signaling cues such as peptide hormones and dietary nutrients. These signaling cues maintain the balance between self-renewal and differentiation of its resident stem cells. The mechanistic Target of Rapamycin Complex 1 (mTORC1) is implicated to play an important role in regulating this balance, although its downstream effectors in stem cells have been elusive. We have recently shown that the PASK protein kinase phosphorylates Wdr5 to stimulate muscle stem cell differentiation by epigenetically activating the Myogenin promoter. Here, we show that the PASK-Wdr5 signaling pathway is a nutrient-sensitive downstream target of mTORC1 in muscle stem cells. We show that phosphorylation of PASK, and in turn of Wdr5, by mTORC1 is required for the activation of Myogenin transcription, exit from the self-renewal and induction of the myogenesis program. Thus, mTOR connects the diverse extrinsic signaling cues to a central epigenetic process to regulate the muscle stem cell fate between self-renewal and differentiation.

In pancreatic β-cells, glucose induces the binding of the transcription factor pancreatic duodenal homeobox-1 (PDX-1) to the insulin gene promoter to activate insulin gene transcription. At low glucose levels, glycogen synthase kinase 3β (GSK3β) is known to phosphorylate PDX-1 on C-terminal serine residues, which triggers PDX-1 proteasomal degradation. We previously showed that the serine/threonine Per-Arnt-Sim domain-containing kinase (PASK) regulates insulin gene transcription via PDX-1. However, the mechanisms underlying this regulation are unknown. In this study, we aimed to identify the role of PASK in the regulation of PDX-1 phosphorylation, protein expression, and stability in insulin-secreting cells and isolated rodent islets of Langerhans. We observed that glucose induces a decrease in overall PDX-1 serine phosphorylation and that overexpression of WT PASK mimics this effect. In vitro, PASK directly phosphorylates GSK3β on its inactivating phosphorylation site Ser(9). Overexpression of a kinase-dead (KD), dominant negative version of PASK blocks glucose-induced Ser(9) phosphorylation of GSK3β. Accordingly, GSK3β Ser(9) phosphorylation is reduced in islets from pask-null mice. Overexpression of WT PASK or KD GSK3β protects PDX-1 from degradation and results in increased PDX-1 protein abundance. Conversely, overexpression of KD PASK blocks glucose-induction of PDX-1 protein. We conclude that PASK phosphorylates and inactivates GSK3β, thereby preventing PDX-1 serine phosphorylation and alleviating GSK3β-mediated PDX-1 protein degradation in pancreatic β-cells.

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. DOI: http://dx.doi.org/10.7554/eLife.17985.001

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.

APPL1 is a newly identified adiponectin receptor-binding protein that positively mediates adiponectin signaling in cells. Here we report that APPL2, an isoform of APPL1 that forms a dimer with APPL1, can interacts with both AdipoR1 and AdipoR2 and acts as a negative regulator of adiponectin signaling in muscle cells. Overexpression of APPL2 inhibits the interaction between APPL1 and AdipoR1, leading to down-regulation of adiponectin signaling in C2C12 myotubes. In contrast, suppressing APPL2 expression by RNAi significantly enhances adiponectin-stimulated glucose uptake and fatty acid oxidation. In addition to targeting directly to and competing with APPL1 in binding with the adiponectin receptors, APPL2 also suppresses adiponectin and insulin signaling by sequestrating APPL1 from these two pathways. In addition to adiponectin, metformin also induces APPL1-APPL2 dissociation. Taken together, our results reveal that APPL isoforms function as an integrated Yin-Yang regulator of adiponectin signaling and mediate the cross-talk between adiponectin and insulin signaling pathways in muscle cells.

Grb10 is a pleckstrin homology and Src homology 2 domain-containing protein that interacts with a number of phosphorylated receptor tyrosine kinases, including the insulin receptor. In mice, Grb10 gene expression is imprinted with maternal expression in all tissues except the brain. While the interaction between Grb10 and the insulin receptor has been extensively investigated in cultured cells, whether this adaptor protein plays a positive or negative role in insulin signaling and action remains controversial. In order to investigate the in vivo role of Grb10 in insulin signaling and action in the periphery, we generated Grb10 knockout mice by the gene trap technique and analyzed mice with maternal inheritance of the knockout allele. Disruption of Grb10 gene expression in peripheral tissues had no significant effect on fasting glucose and insulin levels. On the other hand, peripheral-tissue-specific knockout of Grb10 led to significant overgrowth of the mice, consistent with a role for endogenous Grb10 as a growth suppressor. Loss of Grb10 expression in insulin target tissues, such as skeletal muscle and fat, resulted in enhanced insulin-stimulated Akt and mitogen-activated protein kinase phosphorylation. Hyperinsulinemic-euglycemic clamp studies revealed that disruption of Grb10 gene expression in peripheral tissues led to increased insulin sensitivity. Taken together, our results provide strong evidence that Grb10 is a negative regulator of insulin signaling and action in vivo.

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.

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 p27(Kip1) (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.

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.

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 S6KβI. 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.