Chromatin recruitment of activated AMPK drives fasting response genes co-controlled by GR and PPARα

Adaptation to fasting involves both Glucocorticoid Receptor (GRα) and Peroxisome Proliferator-Activated Receptor α (PPARα) activation. Given both receptors can physically interact we investigated the possibility of a genome-wide cross-talk between activated GR and PPARα, using ChIP- and RNA-seq in primary hepatocytes. Our data reveal extensive chromatin co-localization of both factors with cooperative induction of genes controlling lipid/glucose metabolism. Key GR/PPAR co-controlled genes switched from transcriptional antagonism to cooperativity when moving from short to prolonged hepatocyte fasting, a phenomenon coinciding with gene promoter recruitment of phosphorylated AMP-activated protein kinase (AMPK) and blocked by its pharmacological inhibition. In vitro interaction studies support trimeric complex formation between GR, PPARα and phospho-AMPK. Long-term fasting in mice showed enhanced phosphorylation of liver AMPK and GRα Ser211. Phospho-AMPK chromatin recruitment at liver target genes, observed upon prolonged fasting in mice, is dampened by refeeding. Taken together, our results identify phospho-AMPK as a molecular switch able to cooperate with nuclear receptors at the chromatin level and reveal a novel adaptation mechanism to prolonged fasting

Identification of autophosphorylation sites in eukaryotic elongation factor-2 kinase

eEF2K [eEF2 (eukaryotic elongation factor 2) kinase] phosphorylates and inactivates the translation elongation factor eEF2. eEF2K is not a member of the main eukaryotic protein kinase superfamily, but instead belongs to a small group of so-called α-kinases. The activity of eEF2K is normally dependent upon Ca2+ and calmodulin. eEF2K has previously been shown to undergo autophosphorylation, the stoichiometry of which suggested the existence of multiple sites. In the present study we have identified several autophosphorylation sites, including Thr348, Thr353, Ser366 and Ser445, all of which are highly conserved among vertebrate eEF2Ks. We also identified a number of other sites, including Ser78, a known site of phosphorylation, and others, some of which are less well conserved. None of the sites lies in the catalytic domain, but three affect eEF2K activity. Mutation of Ser78, Thr348 and Ser366 to a non-phosphorylatable alanine residue decreased eEF2K activity. Phosphorylation of Thr348 was detected by immunoblotting after transfecting wild-type eEF2K into HEK (human embryonic kidney)-293 cells, but not after transfection with a kinase-inactive construct, confirming that this is indeed a site of autophosphorylation. Thr348 appears to be constitutively autophosphorylated in vitro. Interestingly, other recent data suggest that the corresponding residue in other α-kinases is also autophosphorylated and contributes to the activation of these enzymes [Crawley, Gharaei, Ye, Yang, Raveh, London, Schueler-Furman, Jia and Cote (2011) J. Biol. Chem. 286, 2607–2616]. Ser366 phosphorylation was also detected in intact cells, but was still observed in the kinase-inactive construct, demonstrating that this site is phosphorylated not only autocatalytically but also in trans by other kinases

Polymorphisms in NF-κB Inhibitors and Risk of Epithelial Ovarian Cancer

<p>Abstract</p> <p>Background</p> <p>The nuclear factor-κB (NF-κB) family is a set of transcription factors with key roles in the induction of the inflammatory response and may be the link between inflammation and cancer development. This pathway has been shown to influence ovarian epithelial tissue repair. Inhibitors of κB (IκB) prevent NF-κB activation by sequestering NF-κB proteins in the cytoplasm until IκB proteins are phosphorylated and degraded.</p> <p>Methods</p> <p>We used a case-control study to evaluate the association between single nucleotide polymorphisms (SNPs) in <it>NFKBIA </it>and <it>NFKBIB </it>(the genes encoding IκBα and IκBβ, respectively) and risk of epithelial ovarian cancer. We queried 19 tagSNPs and putative-functional SNPs among 930 epithelial ovarian cancer cases and 1,037 controls from two studies.</p> <p>Results</p> <p>The minor allele for one synonymous SNP in <it>NFKBIA</it>, rs1957106, was associated with decreased risk (p = 0.03).</p> <p>Conclusion</p> <p>Considering the number of single-SNP tests performed and null gene-level results, we conclude that <it>NFKBIA </it>and <it>NFKBIB </it>are not likely to harbor ovarian cancer risk alleles. Due to its biological significance in ovarian cancer, additional genes encoding NF-κB subunits, activating and inhibiting molecules, and signaling molecules warrant interrogation.</p

Isozymes of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase : structure, catalysis and regulation

Since the discovery of Fru-2.6-P2 more than ten years ago, a great deal has been learned about its role in the control of glycolysis in mammalian tissues and lower organisms, such as yeast. This in turn led to the recognition of the bifunctional enzyme PFK2/FBPase-2 and an understanding of its regulation at the molecular level. My own interest in the field started with the study of the role of Fru-2.6-P2 concentration in heart, PFK2/FBPase-2 was purified from bovine heart and its properties were compared with those of the rat liver bifunctional enzyme. On the basis of differences in kinetic properties and regulation by protein kinases, it became apparent that rat liver and bovine heart PFK2/FBPase-2 were distinct isozymes. Other PFK2/FBPase-2 isozymes have since been found in skeletal muscle, testis and brain. Over the last years the work has focused on the understanding of the catalytic mechanisms and regulation of PFK-2 and FBPase-2 at the molecular level, using the techniques of chemical modification and site-directed mutagenesis. One of our goals is to solve the three-dimensional structures of the PFK2/FBPase-2 isozymes by X-ray crystallography. This should lead to a detailed understanding of the catalysis and regulation of the bifunctional enzyme. The sequence similarities between the nbf in the PFK-2 domain and other nucleotide binding proteins, which have been crystallized, together with the homology between FBPase-2 and yeast PGM, whose three-dimensional structure is know, will help to fit the X-ray coordinates of PFK2/FBPase-2 into its crystal structure. Molecular biology has played an important part in our understanding of the molecular enzymology of PFK2/FBPase-2, firstly by providing amino acid sequences of the PFK2/FBPase-2 isozymes from their cDNAs. This in turn has yielded information on the evolution of the bifunctional enzyme. Secondly molecular biology has been used to identify and characterize the genes for PFK2/FBPase-2 and to study the long-term regulation of the PFK2/FBPase-2 isozymes. Thirdly, it is also the tool used in site-directed mutagenesis, which has led to an understanding of catalysis at the PFK2 and FBPase-2 active sites. The study of the physico-chemical properties and regulation of PFK2/FBPase-2 has provided information on its structure. Once the three-dimensional structure of PFK2/FBPase-2 has been solved, it will hopefully give information on the proximity of amino acid side chains ti the substrates in the PFK2 and FBPase-2 active sites. With this information, one can then go back to molecular biology and do site-directed mutagenesis experiments on the active site residues. At the end of the day, organic chemistry will have to be used to provide the molecular mechanisms of the PFK2 and FBPase-2 reactions. The three-dimensional structure of PFK2/FBPase-2 will provide a model of the folding of the PFK-2 and FBPase-2 domains, which, when compared with the topology of proteins, which share sequence similarity with PFK2 and FBPase-2, could yield important information on the evolution of the bifunctional enzyme. Finally, using the techniques of molecular biology, the PFK2/FBPase-2 isozymes could be expressed in different cell lines to study the role of Fru-2.6-P2 in controlling glycolysis. By expressing, in eukaryotic cells, PFK2/FBPase-2 carrying mutations in the PFK2 or FBPase-2 active sites, one will be able to carry out quantitative studies ti measure the degree of control exerted of whether elevated Fru-2.6-P2 concentrations are necessary for the proliferation of cancer cells. Clearly there is much work still to be done! Biochemistry, the chemistry of life, has taken us from the animal down to the molecule and back to the intact cell. The trip is fascinating, and, fortunately, is still going onThèse d'agrégation de l'enseignement supérieur (faculté de médecine) -- UCL, 199

The ubiquitin-associated domain of AMPK-related protein kinases allows LKB1-induced phosphorylation and activation

The AMPK (AMP-activated protein kinase)-related protein kinase subfamily of the human kinome comprises 12 members closely related to the catalytic α1/α2 subunits of AMPK. The precise role of the AMPK-related kinases and their in vivo substrates is rather unclear at present, but some are involved in regulating cell polarity, whereas others appear to control cellular differentiation. Of the 12 human AMPK-related protein kinase family members, 11 can be activated following phosphorylation of their T-loop threonine residue by the LKB1 complex. Nine of these AMPK-related kinases activated by LKB1 contain an UBA (ubiquitin-associated) domain immediately C-terminal to the kinase catalytic domain. In this issue of the Biochemical Journal, Jaleel et al. show that the presence of an UBA domain in AMP-related kinases allows LKB1-induced phosphorylation and activation. The findings have implications for understanding the molecular mechanisms of activation of this fascinating family of protein kinases. Also, mutations in the UBA domains of the AMP-related kinase genes might be present in families with Peutz–Jehgers syndrome and in other cancer patients