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Oxytocin inactivates and phosphorylates rat hepatocyte glycogen synthase.

ABSTRACT: Incubation of isolated rat hepatocytes with oxytocin produces a time- and dose-dependent inactivation of glycogen synthase. Such inactivation is associated with an increase in the phosphorylation state of the 88 kDa subunit of the enzyme, as observed after electrophoretic analysis of the 32P-labelled enzyme isolated by immunoprecipitation from cells incubated with [32P]phosphate. CNBr cleavage of the immunoprecipitated glycogen synthase showed that multiple sites were phosphorylated after exposure of the cells to the hormone. The effect of oxytocin on hepatocyte glycogen synthase activity was not observed in the absence of extracellular Ca2+. Inactivation of glycogen synthase by oxytocin was partially abolished in the presence of insulin. These results indicate that the effects of oxytocin on glycogen synthase from rat hepatocytes are similar to those observed for other Ca2+-mediated glycogenolytic hormones, such as vasopressin.

SUBMITTER: Arino J

PROVIDER: S-EPMC1138905 | biostudies-other | 1989 Aug

REPOSITORIES: biostudies-other

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Glycogen synthase kinase 3beta phosphorylates p21WAF1/CIP1 for proteasomal degradation after UV irradiation.

Project description:UV irradiation has been reported to induce p21(WAF1/CIP1) protein degradation through a ubiquitin-proteasome pathway, but the underlying biochemical mechanism remains to be elucidated. Here, we show that ser-114 phosphorylation of p21 protein by glycogen synthase kinase 3beta (GSK-3beta) is required for its degradation in response to UV irradiation and that GSK-3beta activation is a downstream event in the ATR signaling pathway triggered by UV. UV transiently increased GSK-3beta activity, and this increase could be blocked by caffeine or by ATR small interfering RNA, indicating ATR-dependent activation of GSK-3beta. ser-114, located within the putative GSK-3beta target sequence, was phosphorylated by GSK-3beta upon UV exposure. The nonphosphorylatable S114A mutant of p21 was protected from UV-induced destabilization. Degradation of p21 protein by UV irradiation was independent of p53 status and prevented by proteasome inhibitors. In contrast to the previous report, the proteasomal degradation of p21 appeared to be ubiquitination independent. These data show that GSK-3beta is activated by UV irradiation through the ATR signaling pathway and phosphorylates p21 at ser-114 for its degradation by the proteasome. To our knowledge, this is the first demonstration of GSK-3beta as the missing link between UV-induced ATR activation and p21 degradation.

| S-EPMC1899930 | biostudies-literature

The Glycogen Synthase Kinase-3 Phosphoproteome: GSK-3 Phosphorylates Multiple Splicing Factors and Regulates Alternative Splicing

Project description:Glycogen Synthase Kinase-3 (GSK-3) is a constitutively active, ubiquitously expressed protein kinase that regulates multiple signaling pathways. Over 100 putative GSK-3 substrates have been reported in diverse cell types based on in vitro kinase assays or genetic and pharmacological manipulation of GSK-3. Many more have been predicted based on a recurrent GSK-3 consensus motif, but this prediction has not been tested by analyzing the GSK-3 phosphoproteome. We used stable isotope labeling of amino acids in culture (SILAC) and mass spectrometry to analyze the repertoire of GSK-3 dependent substrates in mouse embryonic stem cells (ESCs). A comparison of wild-type and Gsk3a;Gsk3b knockout (DKO) ESCs revealed prominent GSK-3-dependent phosphorylation of multiple splicing factors and regulators of RNA biosynthesis, as well as proteins that regulate transcription, translation, and cell division. We demonstrate direct, GSK-3-dependent phosphorylation of the splicing factors RBM8A and PSF as well as the nucleolar protein NPM1. RNA sequencing to compare the transcriptomes of wild-type and Gsk3 DKO ESCs identified more than 210 genes that are alternatively spliced in a GSK-3-dependent manner, supporting a broad role for GSK-3 in regulating alternative splicing. Overall, this study provides the first unbiased analysis of the GSK-3 phosphoproteome and strong evidence for GSK-3 as a regulator of alternative splicing.

2017-08-24 | GSE90921 | GEO

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Glycogen synthase kinase 3 beta (GSK3?) phosphorylates the RNAase III enzyme Drosha at S300 and S302.

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Rapid stimulation by vasopressin, oxytocin and angiotensin II of glycogen degradation in hepatocyte suspensions.

Project description:1. The hormonal control of glycogen breakdown was studied in hepatocytes isolated from livers of fed rats. 2. Glucose release was stimulated by [8-arginine]vasopressin (10pm-10nm), oxytocin (1nm-1mum), and angiotensin II (1nm-0.1mum). These responses are all at least as sensitive to hormone as is glucose output in the perfused rat liver. 3. The effect of these three hormones on glucose release was critically dependent on extracellular Ca(2+), unlike that of glucagon. Half-maximal restoration of the vasopressin response occurred if 0.3mm-Ca(2+) was added back to the incubation medium. 4. Glycogen breakdown was more than sufficient to account for the glucose released into the medium, in the absence or presence of hormones. Lactate release by hepatocytes was not affected by vasopressin, but was inhibited by glucagon. 5. If Ca(2+) was omitted from the extracellular medium, vasopressin stimulated glycogenolysis, but not glucose release. 6. The phosphorylase a content of hepatocytes was increased by vasopressin, oxytocin and angiotensin II; minimum effective concentrations were 0.1pm, 0.1nm and 10pm respectively. This response was also dependent on Ca(2+). 7. These results demonstrate that hepatocytes can respond to low concentrations of vasopressin and angiotensin II, i.e. these effects are likely to be relevant in the intact animal. The role of extracellular Ca(2+) in the effects of these hormones on hepatic glycogenolysis and glucose release is discussed.

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Phosphoproteomics reveals that glycogen synthase kinase-3 phosphorylates multiple splicing factors and is associated with alternative splicing.

Project description:Glycogen synthase kinase-3 (GSK-3) is a constitutively active, ubiquitously expressed protein kinase that regulates multiple signaling pathways. In vitro kinase assays and genetic and pharmacological manipulations of GSK-3 have identified more than 100 putative GSK-3 substrates in diverse cell types. Many more have been predicted on the basis of a recurrent GSK-3 consensus motif ((pS/pT)XXX(S/T)), but this prediction has not been tested by analyzing the GSK-3 phosphoproteome. Using stable isotope labeling of amino acids in culture (SILAC) and MS techniques to analyze the repertoire of GSK-3-dependent phosphorylation in mouse embryonic stem cells (ESCs), we found that ?2.4% of (pS/pT)XXX(S/T) sites are phosphorylated in a GSK-3-dependent manner. A comparison of WT and Gsk3a;Gsk3b knock-out (Gsk3 DKO) ESCs revealed prominent GSK-3-dependent phosphorylation of multiple splicing factors and regulators of RNA biosynthesis as well as proteins that regulate transcription, translation, and cell division. Gsk3 DKO reduced phosphorylation of the splicing factors RBM8A, SRSF9, and PSF as well as the nucleolar proteins NPM1 and PHF6, and recombinant GSK-3? phosphorylated these proteins in vitro RNA-Seq of WT and Gsk3 DKO ESCs identified ?190 genes that are alternatively spliced in a GSK-3-dependent manner, supporting a broad role for GSK-3 in regulating alternative splicing. The MS data also identified posttranscriptional regulation of protein abundance by GSK-3, with ?47 proteins (1.4%) whose levels increased and ?78 (2.4%) whose levels decreased in the absence of GSK-3. This study provides the first unbiased analysis of the GSK-3 phosphoproteome and strong evidence that GSK-3 broadly regulates alternative splicing.

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Project description:Nuclear myosin 1c (NM1) mediates RNA polymerase I (pol I) transcription activation and cell cycle progression by facilitating PCAF-mediated H3K9 acetylation, but the molecular mechanism by which NM1 is regulated remains unclear. Here, we report that at early G1 the glycogen synthase kinase (GSK) 3β phosphorylates and stabilizes NM1, allowing for NM1 association with the chromatin. Genomic analysis by ChIP-Seq showed that this mechanism occurs on the rDNA as active GSK3β selectively occupies the gene. ChIP assays and transmission electron microscopy in GSK3β-/- mouse embryonic fibroblasts indicated that at G1 rRNA synthesis is suppressed due to decreased H3K9 acetylation leading to a chromatin state incompatible with transcription. We found that GSK3β directly phosphorylates the endogenous NM1 on a single serine residue (Ser-1020) located within the NM1 C-terminus. In G1 this phosphorylation event stabilizes NM1 and prevents NM1 polyubiquitination by the E3 ligase UBR5 and proteasome-mediated degradation. We conclude that GSK3β-mediated phosphorylation of NM1 is required for pol I transcription activation. Examination of GSK3beta with the genome in mouse embryonic fibroblasts

2014-05-02 | E-GEOD-57153 | biostudies-arrayexpress

Glycogen synthase kinase (GSK) 3β phosphorylates and protects nuclear myosin 1c from proteasome-mediated degradation to activate rDNA transcription in early G1 cells

2014-05-02 | GSE57153 | GEO

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