PAS Kinase Research Articles

Increased Voluntary Running in PAS Kinase-knockout Mice

PAS kinase (PASK) deletion in mice leads to increased oxidative metabolism, increased ATP production, and protection from diet-induced obesity. The role that PASK plays in exercise metabolism and performance is not known. PURPOSE: To assess voluntary running capacity and muscle expression of proteins related to metabolism in PASK knockout (KO) mice. METHODS: Voluntary running of female KO and littermate wild-type mice (n=4/group) was assessed in cages equipped with computer-monitored wheels. Mice in cages without wheels served as sedentary controls. After 3 weeks of voluntary run training, heart and triceps were excised and immediately frozen. Western blot (WB) analysis was performed for cytochrome C, phospho-AMPK, and phospho-ACC. RESULTS: KO mice ran 39% more than WT mice over the course of the 3-week running period (9513 ± 144 m/day vs. 6852 ± 829 m/day). No differences in AMPK phosphorylation were observed in the heart or triceps, but ACC phosphorylation was elevated in sedentary KO hearts (27% greater than in WT). Cytochrome C levels in sedentary mice were 40% higher in KO compared to WT mice, and increased with training in both genotypes (62% increase in WT; 28% increase in KO). No significant differences in cytochrome C levels were observed in the heart. CONCLUSIONS: PASK negatively impacts voluntary running performance in mice. Elevated cytochrome C levels in the triceps of PASK-KO mice suggest that increased oxidative capacity in skeletal muscle may contribute to the increased running performance in PASK-KO mice.

Involvement of Per-Arnt-Sim Kinase and Extracellular-Regulated Kinases-1/2 in Palmitate Inhibition of Insulin Gene Expression in Pancreatic β-Cells

OBJECTIVEProlonged exposure of pancreatic β-cells to simultaneously elevated levels of fatty acids and glucose (glucolipotoxicity) impairs insulin gene transcription. However, the intracellular signaling pathways mediating these effects are mostly unknown. This study aimed to ascertain the role of extracellular-regulated kinases (ERKs)1/2, protein kinase B (PKB), and Per-Arnt-Sim kinase (PASK) in palmitate inhibition of insulin gene expression in pancreatic β-cells.RESEARCH DESIGN AND METHODSMIN6 cells and isolated rat islets were cultured in the presence of elevated glucose, with or without palmitate or ceramide. ERK1/2 phosphorylation, PKB phosphorylation, and PASK expression were examined by immunoblotting and real-time PCR. The role of these kinases in insulin gene expression was assessed using pharmacological and molecular approaches.RESULTSExposure of MIN6 cells and islets to elevated glucose induced ERK1/2 and PKB phosphorylation, which was further enhanced by palmitate. Inhibition of ERK1/2, but not of PKB, partially prevented the inhibition of insulin gene expression in the presence of palmitate or ceramide. Glucose-induced expression of PASK mRNA and protein levels was reduced in the presence of palmitate. Overexpression of wild-type PASK increased insulin and pancreatic duodenal homeobox-1 gene expression in MIN6 cells and rat islets incubated with glucose and palmitate, whereas overexpression of a kinase-dead PASK mutant in rat islets decreased expression of insulin and pancreatic duodenal homeobox-1 and increased C/EBPβ expression.CONCLUSIONSBoth the PASK and ERK1/2 signaling pathways mediate palmitate inhibition of insulin gene expression. These findings identify PASK as a novel mediator of glucolipotoxicity on the insulin gene in pancreatic β-cells.

Genomewide Screen for Negative Regulators of Sirtuin Activity inSaccharomyces cerevisiaeReveals 40 Loci and Links to Metabolism

Sirtuins are conserved proteins implicated in myriad key processes including gene control, aging, cell survival, metabolism, and DNA repair. In Saccharomyces cerevisiae, the sirtuin Silent information regulator 2 (Sir2) promotes silent chromatin formation, suppresses recombination between repeats, and inhibits senescence. We performed a genomewide screen for factors that negatively regulate Sir activity at a reporter gene placed immediately outside a silenced region. After linkage analysis, assessment of Sir dependency, and knockout tag verification, 40 loci were identified, including 20 that have not been previously described to regulate Sir. In addition to chromatin-associated factors known to prevent ectopic silencing (Bdf1, SAS-I complex, Rpd3L complex, Ku), we identified the Rtt109 DNA repair-associated histone H3 lysine 56 acetyltransferase as an anti-silencing factor. Our findings indicate that Rtt109 functions independently of its proposed effectors, the Rtt101 cullin, Mms1, and Mms22, and demonstrate unexpected interplay between H3K56 and H4K16 acetylation. The screen also identified subunits of mediator (Soh1, Srb2, and Srb5) and mRNA metabolism factors (Kem1, Ssd1), thus raising the possibility that weak silencing affects some aspect of mRNA structure. Finally, several factors connected to metabolism were identified. These include the PAS-domain metabolic sensor kinase Psk2, the mitochondrial homocysteine detoxification enzyme Lap3, and the Fe-S cluster protein maturase Isa2. We speculate that PAS kinase may integrate metabolic signals to control sirtuin activity.

The role of PAS kinase in regulating energy metabolism

Metabolic disorders, such as diabetes and obesity, are fundamentally caused by cellular energy imbalance and dysregulation. Therefore, understanding the regulation of cellular fuel and energy metabolism is of great importance to develop effective therapies for metabolic disease. The cellular nutrient and energy sensors, AMPK and TOR, play a key role in maintaining cellular energy homeostasis. Like AMPK and TOR, PAS kinase (PASK) is also a nutrient responsive protein kinase. In yeast, PAS kinase phosphorylates the enzyme Ugp1 and thereby shifts glucose partitioning toward cell wall glucan synthesis at the expense of glycogen synthesis. Consistent with this function, yeast PAS kinase is activated by both cell integrity stress and growth in non-fermentative carbon sources. PASK is also important for proper regulation of glucose metabolism in mammals at both the hormonal and cellular level. In cultured pancreatic beta-cells, PASK is activated by elevated glucose concentrations and is required for glucose-stimulated transcription of the insulin gene. PASK knockdown in cultured myoblasts causes increased glucose oxidation and elevated cellular ATP levels. Mice lacking PASK exhibit increased metabolic rate and resistance to diet-induced obesity. Interestingly, PGC-1 expression and AMPK and TOR activity were not affected in PASK deficient mice, suggesting PASK may exert its metabolic effects through a new mechanism. We propose that PASK plays a significant role in nutrient sensing, metabolic regulation, and energy homeostasis, and is a potential therapeutic target for metabolic disease.

PAS Kinase Mediates Palmitate Inhibition of Insulin Gene Expression in Pancreatic Beta-Cells

PAS Kinase Mediates Palmitate Inhibition of Insulin Gene Expression in Pancreatic Beta-Cells

PAS kinase is required for normal cellular energy balance

The metabolic syndrome, a complex set of phenotypes typically associated with obesity and diabetes, is an increasing threat to global public health. Fundamentally, the metabolic syndrome is caused by a failure to properly sense and respond to cellular metabolic cues. We studied the role of the cellular metabolic sensor PAS kinase (PASK) in the pathogenesis of metabolic disease by using PASK(-/-) mice. We identified tissue-specific metabolic phenotypes caused by PASK deletion consistent with its role as a metabolic sensor. Specifically, PASK(-/-) mice exhibited impaired glucose-stimulated insulin secretion in pancreatic beta-cells, altered triglyceride storage in liver, and increased metabolic rate in skeletal muscle. Further, PASK deletion caused nearly complete protection from the deleterious effects of a high-fat diet including obesity and insulin resistance. We also demonstrate that these cellular effects, increased rate of oxidative metabolism and ATP production, occur in cultured cells. We therefore hypothesize that PASK acts in a cell-autonomous manner to maintain cellular energy homeostasis and is a potential therapeutic target for metabolic disease.

Shore Up That Wall!

Single-celled and multicellular organisms must control metabolic flux in response to changing cellular and organismal needs. For example, will glucose be used to create ATP, to synthesize glycogen, to generate NADPH, or to glycosylate lipids and proteins? Disruption of these processes underlies diseases such as diabetes and obesity. Smith and Rutter found a pathway in Saccharomyces cerevisiae controlling glucose partitioning from cell wall synthesis to glycogen storage. The kinases Psk1 and Psk2 (Psk1/2) phosphorylate UDP-glucose pyrophosphorylase (Ugp1), which is a key enzyme in producing UDP-glucose for either glycogen (energy storage) or glucan (cell wall synthesis). Yeast deficient in both Psk1 and Psk2 activity or expressing a mutant version of Ugp1 in which the phosphorylated serine is replaced by alanine (Ugp1-S11A) exhibit defective growth on galactose, increased glycogen content per cell, and decreased sensitivity to a toxin that binds to β-(1,6)-glucan. Although phosphorylation did not alter the catalytic activity of Ugp1, lack of Psk1/2 activity or expression of the Ugp1-S11A mutant resulted in the loss of one population of Ugp1. In wild-type yeast, there are two populations of Ugp1, corresponding to the phosphorylated (isoform 1) and the nonphosphorylated (isoform 2) forms that were readily separated by anion exchange chromatography, and these two forms exhibit differences in conformation based on their differences in sensitivity to proteases and different proteolysis profiles. Yeast that did not produce isoform 2 showed altered sensitivity to various chemicals that cause cell wall stress. The authors propose that phosphorylation alters the cellular distribution of Ugp1 from the cytosol (for glycogen synthesis) to the cell membrane (for cell wall synthesis). Forced localization of Ugp1 to the plasma membrane rescued the Psk1/2-deficient cells from inability to grow on galactose and reduced glycogen content and restored β-(1,6)-glucan content per cell to levels similar to those in wild-type yeast. T. L. Smith, J. Rutter, Regulation of glucose partitioning by PAS kinase and Ugp1 phosphorylation. Mol. Cell 26 , 491-499 (2007). [PubMed]

PAS kinase and the maintenance of energy homeostasis

PAS kinase and the maintenance of energy homeostasis

Glucose-Stimulated Insulin Production in Mice Deficient for the PAS Kinase PASKIN

The Per-ARNT-Sim (PAS) domain serine/threonine kinase PASKIN, or PAS kinase, links energy flux and protein synthesis in yeast and regulates glycogen synthase in mammals. A recent report suggested that PASKIN mRNA, protein, and kinase activity are increased in pancreatic islet beta-cells under hyperglycemic conditions and that PASKIN is necessary for insulin gene expression. We previously generated Paskin knockout mice by targeted replacement of the kinase domain with the beta-geo fusion gene encoding beta-galactosidase reporter activity. Here we show that no 5-bromo-4-chloro-3-indolyl-ss-d-galactopyranoside (X-gal) staining was observed in islet beta-cells derived from Paskin knockout mice, irrespective of the ambient glucose concentration, whereas adenoviral expression of the lacZ gene in beta-cells showed strong X-gal staining. No induction of PASKIN mRNA could be detected in insulinoma cell lines or in islet beta-cells. Increasing glucose concentrations resulted in PASKIN-independent induction of insulin mRNA levels and insulin release. PASKIN mRNA levels were high in testes but undetectable in pancreas and in islet beta-cells. Finally, blood glucose levels and glucose tolerance after intraperitoneal glucose injection were indistinguishable between Paskin wild-type and knockout mice. These results suggest that Paskin gene expression is not induced by glucose in pancreatic beta-cells and that glucose-stimulated insulin production is independent of PASKIN.

Control of mammalian glycogen synthase by PAS kinase

The regulation of glycogen metabolism is critical for the maintenance of glucose and energy homeostasis in mammals. Glycogen synthase, the enzyme responsible for glycogen production, is regulated by multisite phosphorylation in yeast and mammals. We have previously identified PAS kinase as a physiological regulator of glycogen synthase in Saccharomyces cerevisiae. We provide evidence here that PAS kinase is an important regulator of mammalian glycogen synthase. Glycogen synthase is efficiently phosphorylated by PAS kinase in vitro at Ser-640, a known regulatory phosphosite. Efficient phosphorylation requires a region of PAS kinase outside the catalytic domain. This region appears to mediate a direct interaction between glycogen synthase and PAS kinase, thereby targeting kinase activity to this substrate specifically. This interaction is regulated by the PAS kinase PAS domain, raising the possibility that this interaction (and phosphorylation event) is modulated by the cellular metabolic state. This mode of regulation provides a mechanism for metabolic status to impinge directly on the cellular decision of whether to store or use available energy.

Involvement of Per-Arnt-Sim (PAS) kinase in the stimulation of preproinsulin and pancreatic duodenum homeobox 1 gene expression by glucose.

Per-Arnt-Sim (PAS) domain-containing kinases are common in prokaryotes, but a mammalian counterpart has only recently been described. Although the PAS domain of the mammalian PAS kinase (PASK) is closely related to the bacterial oxygen sensor FixL, it is unclear whether PASK activity is changed in mammalian cells in response to nutrients and might therefore contribute to signal transduction by these or other stimuli. Here, we show that elevated glucose concentrations rapidly increase PASK activity in pancreatic islet beta cells, an event followed by the accumulation of both PASK mRNA and protein. Demonstrating a physiological role for PASK activation, comicroinjection into clonal beta cells of cDNA encoding wild-type PASK, or PASK protein itself, mimics the induction of preproinsulin promoter activity by high glucose concentrations. Conversely, anti-PASK antibodies block promoter activation by the sugar, and the silencing of PASK expression by RNA interference suppresses the up-regulation by glucose of preproinsulin and pancreatic duodenum homeobox 1 gene expression, without affecting glucose-induced changes in the levels of mRNAs encoding glucokinase or uncoupling protein 2. We conclude that PASK is an important metabolic sensor in nutrient-sensitive mammalian cells and plays an unexpected role in the regulation of key genes involved in maintaining the differentiated phenotype of pancreatic beta cells.

PAS kinase: an evolutionarily conserved PAS domain-regulated serine/threonine kinase.

PAS domains regulate the function of many intracellular signaling pathways in response to both extrinsic and intrinsic stimuli. PAS domain-regulated histidine kinases are common in prokaryotes and control a wide range of fundamental physiological processes. Similarly regulated kinases are rare in eukaryotes and are to date completely absent in mammals. PAS kinase (PASK) is an evolutionarily conserved gene product present in yeast, flies, and mammals. The amino acid sequence of PASK specifies two PAS domains followed by a canonical serine/threonine kinase domain, indicating that it might represent the first mammalian PAS-regulated protein kinase. We present evidence that the activity of PASK is regulated by two mechanisms. Autophosphorylation at two threonine residues located within the activation loop significantly increases catalytic activity. We further demonstrate that the N-terminal PAS domain is a cis regulator of PASK catalytic activity. When the PAS domain-containing region is removed, enzyme activity is significantly increased, and supplementation of the purified PAS-A domain in trans selectively inhibits PASK catalytic activity. These studies define a eukaryotic signaling pathway suitable for studies of PAS domains in a purified in vitro setting.