Receptor-independent modulation of cAMP-dependent protein kinase and protein phosphatase signaling in cardiac myocytes by oxidizing agents

The contraction and relaxation of the heart is controlled by stimulation of the beta 1-adrenoreceptor (AR) signaling cascade, which leads to activation of cAMP-dependent protein kinase (PKA) and subsequent cardiac protein phosphorylation. Phosphorylation is counteracted by the main cardiac protein phosphatases, PP2A and PP1. Both kinase and phosphatases are sensitive to intramolecular disulfide formation in their catalytic subunits that inhibits their activity. Additionally, intermolecular disulfide formation between PKA type I regulatory subunits (PKA-RI) has been described to enhance PKA's affinity for protein kinase A anchoring proteins, which alters its subcellular distribution. Nitroxyl donors have been shown to affect contractility and relaxation, but the mechanistic basis for this effect is unclear. The present study investigates the impact of several nitroxyl donors and the thiol-oxidizing agent diamide on cardiac myocyte protein phosphorylation and oxidation. Although all tested compounds equally induced intermolecular disulfide formation in PKA-RI, only 1-nitrosocyclohexalycetate (NCA) and diamide induced reproducible protein phosphorylation. Phosphorylation occurred independently of beta(1)-AR activation, but was abolished after pharmacological PKA inhibition and thus potentially attributable to increased PKA activity. NCA treatment of cardiac myocytes induced translocation of PKA and phosphatases to the myofilament compartment as shown by fractionation, immunofluorescence, and proximity ligation assays. Assessment of kinase and phosphatase activity within the myofilament fraction of cardiac myocytes after exposure to NCA revealed activation of PKA and inhibition of phosphatase activity thus explaining the increase in phosphorylation. The data suggest that the NCA-mediated effect on cardiac myocyte protein phosphorylation orchestrates alterations in the kinase/phosphatase balance.

Identification of genetic interactors of ypa2 in Schizosaccharomyces pombe

Manuela Moraru

In this study, the fission yeast Schizosaccharomyces pombe was used as a model system to study the cellular signaling that underlies cell cycle progression. Phosphorylation plays an important part in the regulation of this process. Kinases catalyze the transfer of a phosphate group to a substrate, which may regulate its activity. Protein phosphatases oppose the activity of protein kinases; the balance of their activities regulates signaling flux in many signaling pathways. The Septation Initiation Network (SIN) is a signaling cascade that regulates cytokinesis in fission yeast. SIN signaling is triggered by a GTPase and is transduced by three protein kinases; phosphorylation plays an important role in controlling SIN signalling. Numerous genetic screens and biochemical analyses have identified phosphatases and kinases that regulate the SIN. The protein phosphatases Calcineurin/Protein Phosphatase 2B (PP2B) and Flp1p promote SIN signaling, while PP2A is a SIN inhibitor. PP2A is conserved and is activated by the chaperone Phosphatase Two A Phosphatase Activator (PTPA). S. pombe has two PTPA-related genes, ypa1 and ypa2; the deletion mutant of the latter rescues SIN mutants which cannot undergo cytokinesis, consistent with PP2A opposing SIN signaling. The phenotype of the deletion mutant of ypa2, indicates that it is involved in the control of cell polarity, cell growth, mitotic commitment and cytokinesis. In this study, we characterized the hypomorphic allele ypa2-S2 which rescues SIN mutants, but is otherwise largely normal. ypa2-s2 was used as bait in a synthetic genetic array screen, to identify genes whose products compensate for reduced function of Ypa2p. A large number of hits were identified, of which approximately ninety percent are novel genetic interactors of ypa2. Validation studies indicated that eighty percent of the interactions seen in the high-throughput screen can be reconstructed. The identified genes regulate several biological processes, including signal transduction and protein phosphorylation. This prompted us to further characterize the roles of Ckb1p and Flp1p, in regulating cytokinesis signaling. Ckb1p is a regulatory subunit for Casein Kinase II (CK II), which was previously implicated in polar growth. The phenotype of the mutant ypa2-S2 ckb1-â indicates that Ckb1p, similar to Ypa2p, contributes to the regulation of mitotic commitment and cytokinesis signaling. Furthermore, ckb1-â showed negative genetic interaction with PP2A and SIN mutants. Flp1p is a protein phosphatase that was reported to regulate mitotic commitment and to promote SIN signaling. The double mutant between ypa2-S2 and flp1-â showed cell separation defects and phenotypic analysis of double mutants between flp1-â and PP2A mutants indicates that these phosphatases cooperate in cell separation. Overall, this work has uncovered novel facets of the regulation of cytokinesis in S. pombe, implicating CK II in controlling SIN signaling, and uncovering cooperative interactions between different phosphatases in controlling cytokinesis.

EPFL2016

Phosphorylation of the nuclear receptor corepressor 1 by protein kinase B switches its corepressor targets in the liver in mice

Johan Auwerx, Adriano Maida, Dongryeol Ryu, Kristina Schoonjans, Xu Wang

Nuclear receptor corepressor 1 (NCoR1) is a transcriptional coregulator that has wide-ranging effects on gene expression patterns. In the liver, NCoR1 represses lipid synthesis in the fasting state, whereas it inhibits activation of peroxisome proliferator-activated receptor alpha (PPAR) upon feeding, thereby blunting ketogenesis. Here, we show that insulin by activation of protein kinase B induces phosphorylation of NCoR1 on serine 1460, which selectively favors its interaction with PPAR and estrogen-related receptor alpha (ERR) over liver X receptor alpha (LXR). Phosphorylation of NCoR1 on S1460 selectively derepresses LXR target genes, resulting in increased lipogenesis, whereas, at the same time, it inhibits PPAR and ERR targets, thereby attenuating oxidative metabolism in the liver. Phosphorylation-gated differential recruitment of NCoR1 to different nuclear receptors explains the apparent paradox that liver-specific deletion of NCoR1 concurrently induces both lipogenesis and oxidative metabolism owing to a global derepression of LXR, PPAR, and ERR activity. Conclusion: Phosphorylation-mediated recruitment switch of NCoR1 between nuclear receptor subsets provides a mechanism by which corepressors can selectively modulate liver energy metabolism during the fasting-feeding transition. (Hepatology 2015;62:1606-1618)

Wiley-Blackwell2015

Phosphorylation of the nuclear receptor co-repressor 1 by protein kinase B (PKB/Akt) switches its co-repressor targets in the liver

Johan Auwerx, Adriano Maida, Dongryeol Ryu, Kristina Schoonjans, Xu Wang

The nuclear receptor corepressor 1 (NCoR1) is a transcriptional co-regulator that has wide-ranging effects on gene expression patterns. In the liver, NCoR1 represses lipid synthesis in the fasting state, whereas it inhibits the activation of PPARα upon feeding, thereby blunting ketogenesis. Here, we show that insulin via the activation of PKB/Akt induces the phosphorylation of NCoR1 on serine 1460, which selectively favors its interaction with PPARα and ERRα over LXRα. Phosphorylation of NCoR1 on S1460 selectively derepresses LXRα target genes, resulting in increased lipogenesis, while at the same time it inhibits PPARα and ERRα targets, thereby attenuating oxidative metabolism in the liver. The phosphorylation-gated differential recruitment of NCoR1 to different nuclear receptors explains the apparent paradox that liver-specific deletion of NCoR1 concurrently induces both lipogenesis and oxidative metabolism, due to a global derepression of LXRα, PPARα and ERRα activity. This phosphorylation-mediated recruitment switch of NCoR1 between nuclear receptor subsets hence provides a mechanism by which corepressors can selectively modulate liver energy metabolism during the fasting-feeding transition. This article is protected by copyright. All rights reserved.

Wiley-Blackwell2015

Identification of genetic interactors of ypa2 in Schizosaccharomyces pombe

Manuela Moraru

EPFL2016