Browse
Submit Data
Databases
API
Help

Dataset Information

17 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Muscarinic-dependent phosphorylation of the cardiac ryanodine receptor by protein kinase G is mediated by PI3K-AKT-nNOS signaling.

ABSTRACT: Post-translational modifications of proteins involved in calcium handling in myocytes, such as the cardiac ryanodine receptor (RyR2), critically regulate cardiac contractility. Recent studies have suggested that phosphorylation of RyR2 by protein kinase G (PKG) might contribute to the cardioprotective effects of cholinergic stimulation. However, the specific mechanisms underlying these effects remain unclear. Here, using murine ventricular myocytes, immunoblotting, proximity ligation as-says, and nitric oxide imaging, we report that phosphorylation of Ser-2808 in RyR2 induced by the muscarinic receptor agonist carbachol is mediated by a signaling axis comprising phosphoinositide 3-phosphate kinase, Akt Ser/Thr kinase, nitric oxide synthase 1, nitric oxide, soluble guanylate cyclase, cyclic GMP (cGMP), and PKG. We found that this signaling pathway is compartmentalized in myocytes, as it was distinct from atrial natriuretic peptide receptor-cGMP-PKG-RyR2 Ser-2808 signaling and independent of muscarinic-induced phosphorylation of Ser-239 in vasodilator-stimulated phosphoprotein. These results provide detailed insights into muscarinic-induced PKG signaling and the mediators that regulate cardiac RyR2 phosphorylation critical for cardiovascular function.

SUBMITTER: Baine S

PROVIDER: S-EPMC7450129 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

Muscarinic Stimulation Facilitates Sarcoplasmic Reticulum Ca Release by Modulating Ryanodine Receptor 2 Phosphorylation Through Protein Kinase G and Ca/Calmodulin-Dependent Protein Kinase II.

Project description:Although the effects and the underlying mechanism of sympathetic stimulation on cardiac Ca handling are relatively well established both in health and disease, the modes of action and mechanisms of parasympathetic modulation are poorly defined. Here, we demonstrate that parasympathetic stimulation initiates a novel mode of excitation-contraction coupling that enhances the efficiency of cardiac sarcoplasmic reticulum Ca store utilization. This efficient mode of excitation-contraction coupling involves reciprocal changes in the phosphorylation of ryanodine receptor 2 at Ser-2808 and Ser-2814. Specifically, Ser-2808 phosphorylation was mediated by muscarinic receptor subtype 2 and activation of PKG (protein kinase G), whereas dephosphorylation of Ser-2814 involved activation of muscarinic receptor subtype 3 and decreased reactive oxygen species-dependent activation of CaMKII (Ca/calmodulin-dependent protein kinase II). The overall effect of these changes in phosphorylation of ryanodine receptor 2 is an increase in systolic Ca release at the low sarcoplasmic reticulum Ca content and a paradoxical reduction in aberrant Ca leak. Accordingly, cholinergic stimulation of cardiomyocytes isolated from failing hearts improved Ca cycling efficiency by restoring altered ryanodine receptor 2 phosphorylation balance.

| S-EPMC5063706 | biostudies-literature

Casein kinase 2 dependent phosphorylation of neprilysin regulates receptor tyrosine kinase signaling to Akt.

Project description:Neprilysin (NEP) is a type II membrane metalloproteinase that cleaves physiologically active peptides at the cell surface thus regulating the local concentration of these peptides available for receptor binding and signal transduction. In addition, the cytoplasmic N-terminal domain of NEP interacts with the phosphatase and tensin homologue deleted on chromosome 10 (PTEN) thereby regulating intracellular signaling via Akt. Thus, NEP serves dual functions in extracellular and intracellular signal transduction. Here, we show that NEP undergoes phosphorylation at serine residue 6 within the N-terminal cytoplasmic domain. In vitro and cell culture experiments demonstrate that Ser 6 is efficiently phosphorylated by protein kinase CK2. The phosphorylation of the cytoplasmic domain of NEP inhibits its interaction with PTEN. Interestingly, expression of a pseudophosphorylated NEP variant (Ser6Asp) abrogates the inhibitory effect of NEP on insulin/insulin-like growth factor-1 (IGF-1) stimulated activation of Akt. Thus, our data demonstrate a regulatory role of CK2 in the interaction of NEP with PTEN and insulin/IGF-1 signaling.

| S-EPMC2948513 | biostudies-literature

CaMKII-dependent phosphorylation of cardiac ryanodine receptors regulates cell death in cardiac ischemia/reperfusion injury.

Project description:Ca(2+)-calmodulin kinase II (CaMKII) activation is deleterious in cardiac ischemia/reperfusion (I/R). Moreover, inhibition of CaMKII-dependent phosphorylations at the sarcoplasmic reticulum (SR) prevents CaMKII-induced I/R damage. However, the downstream targets of CaMKII at the SR level, responsible for this detrimental effect, remain unclear. In the present study we aimed to dissect the role of the two main substrates of CaMKII at the SR level, phospholamban (PLN) and ryanodine receptors (RyR2), in CaMKII-dependent I/R injury. In mouse hearts subjected to global I/R (45/120min), phosphorylation of the primary CaMKII sites, S2814 on cardiac RyR2 and of T17 on PLN, significantly increased at the onset of reperfusion whereas PKA-dependent phosphorylation of RyR2 and PLN did not change. Similar results were obtained in vivo, in mice subjected to regional myocardial I/R (1/24h). Knock-in mice with an inactivated serine 2814 phosphorylation site on RyR2 (S2814A) significantly improved post-ischemic mechanical recovery, reduced infarct size and decreased apoptosis. Conversely, knock-in mice, in which CaMKII site of RyR2 is constitutively activated (S2814D), significantly increased infarct size and exacerbated apoptosis. In S2814A and S2814D mice subjected to regional myocardial ischemia, infarct size was also decreased and increased respectively. Transgenic mice with double-mutant non-phosphorylatable PLN (S16A/T17A) in the PLN knockout background (PLNDM) also showed significantly increased post-ischemic cardiac damage. This effect cannot be attributed to PKA-dependent PLN phosphorylation and was not due to the enhanced L-type Ca(2+) current, present in these mice. Our results reveal a major role for the phosphorylation of S2814 site on RyR2 in CaMKII-dependent I/R cardiac damage. In contrast, they showed that CaMKII-dependent increase in PLN phosphorylation during reperfusion opposes rather than contributes to I/R damage.

| S-EPMC4131282 | biostudies-literature

LncRNA DANCR upregulates PI3K/AKT signaling through activating serine phosphorylation of RXRA.

Project description:AbstactConventional therapies and novel molecular targeted therapies against breast cancer have gained great advances over the past two decades. However, poor prognosis and low survival rate are far from expectation for improvement, particularly in patients with triple negative breast cancer (TNBC). Here, we found that lncRNA DANCR was significantly overregulated in TNBC tissues and cell lines compared with normal breast tissues or other type of breast cancer. Knockdown of DANCR suppressed TNBC proliferation both in vitro and in vivo. Further study of underlying mechanisms demonstrated that DANCR bound with RXRA and increased its serine 49/78 phosphorylation via GSK3?, resulting in activating PIK3CA transcription, and subsequently enhanced PI3K/AKT signaling and TNBC tumorigenesis. Taken together, Our findings identified DANCR as an pro-oncogene and uncoverd a new working pattern of lncRNA to mediate TNBC tumorigenesis, which may be a potential therapeutic target for improving treatment of TNBC.

| S-EPMC6281578 | biostudies-literature

SDF-1α/CXCR4 Signaling in Lipid Rafts Induces Platelet Aggregation via PI3 Kinase-Dependent Akt Phosphorylation.

Project description:Stromal cell-derived factor-1α (SDF-1α)-induced platelet aggregation is mediated through its G protein-coupled receptor CXCR4 and phosphatidylinositol 3 kinase (PI3K). Here, we demonstrate that SDF-1α induces phosphorylation of Akt at Thr308 and Ser473 in human platelets. SDF-1α-induced platelet aggregation and Akt phosphorylation are inhibited by pretreatment with the CXCR4 antagonist AMD3100 or the PI3K inhibitor LY294002. SDF-1α also induces the phosphorylation of PDK1 at Ser241 (an upstream activator of Akt), GSK3β at Ser9 (a downstream substrate of Akt), and myosin light chain at Ser19 (a downstream element of the Akt signaling pathway). SDF-1α-induced platelet aggregation is inhibited by pretreatment with the Akt inhibitor MK-2206 in a dose-dependent manner. Furthermore, SDF-1α-induced platelet aggregation and Akt phosphorylation are inhibited by pretreatment with the raft-disrupting agent methyl-β-cyclodextrin. Sucrose density gradient analysis shows that 35% of CXCR4, 93% of the heterotrimeric G proteins Gαi-1, 91% of Gαi-2, 50% of Gβ and 4.0% of PI3Kβ, and 4.5% of Akt2 are localized in the detergent-resistant membrane raft fraction. These findings suggest that SDF-1α/CXCR4 signaling in lipid rafts induces platelet aggregation via PI3K-dependent Akt phosphorylation.

| S-EPMC5224795 | biostudies-literature

Nutrients suppress phosphatidylinositol 3-kinase/Akt signaling via raptor-dependent mTOR-mediated insulin receptor substrate 1 phosphorylation.

Project description:Nutritional excess and/or obesity represent well-known predisposition factors for the development of non-insulin-dependent diabetes mellitus (NIDDM). However, molecular links between obesity and NIDDM are only beginning to emerge. Here, we demonstrate that nutrients suppress phosphatidylinositol 3 (PI3)-kinase/Akt signaling via Raptor-dependent mTOR (mammalian target of rapamycin)-mediated phosphorylation of insulin receptor substrate 1 (IRS-1). Raptor directly binds to and serves as a scaffold for mTOR-mediated phosphorylation of IRS-1 on Ser636/639. These serines lie close to the Y(632)MPM motif that is implicated in the binding of p85alpha/p110alpha PI3-kinase to IRS-1 upon insulin stimulation. Phosphomimicking mutations of these serines block insulin-stimulated activation of IRS-1-associated PI3-kinase. Knockdown of Raptor as well as activators of the LKB1/AMPK pathway, such as the widely used antidiabetic compound metformin, suppress IRS-1 Ser636/639 phosphorylation and reverse mTOR-mediated inhibition on PI3-kinase/Akt signaling. Thus, diabetes-related hyperglycemia hyperactivates the mTOR pathway and may lead to insulin resistance due to suppression of IRS-1-dependent PI3-kinase/Akt signaling.

| S-EPMC1317643 | biostudies-literature

The Mechanism of ATP-Dependent Allosteric Protection of Akt Kinase Phosphorylation.

Project description:Kinases use ATP to phosphorylate substrates; recent findings underscore the additional regulatory roles of ATP. Here, we propose a mechanism for allosteric regulation of Akt1 kinase phosphorylation by ATP. Our 4.7-μs molecular dynamics simulations of Akt1 and its mutants in the ATP/ADP bound/unbound states revealed that ATP occupancy of the ATP-binding site stabilizes the closed conformation, allosterically protecting pT308 by restraining phosphatase access and key interconnected residues on the ATP→pT308 allosteric pathway. Following ATP→ADP hydrolysis, pT308 is exposed and readily dephosphorylated. Site-directed mutagenesis validated these predictions and indicated that the mutations do not impair PDK1 and PP2A phosphatase recruitment. We further probed the function of residues around pT308 at the atomic level, and predicted and experimentally confirmed that Akt1(H194R/R273H) double mutant rescues pathology-related Akt1(R273H). Analysis of classical Akt homologs suggests that this mechanism can provide a general model of allosteric kinase regulation by ATP; as such, it offers a potential avenue for allosteric drug discovery.

| S-EPMC7734571 | biostudies-literature

Muscarinic 2 receptors modulate cardiac proteasome function in a protein kinase G-dependent manner.

Project description:Proteasome function insufficiency and inadequate protein quality control are strongly implicated in a large subset of cardiovascular disease and may play an important role in their pathogenesis. Protein degradation by the ubiquitin proteasome system can be physiologically regulated. Cardiac muscarinic 2 (M2) receptors were pharmacologically interrogated in intact mice and cultured neonatal rat ventricular myocytes (NRVMs). Proteasome-mediated proteolysis was measured with a surrogate misfolded protein, proteasome peptidase assay, and by characterizing key proteasome subunits. Successful M2 receptor manipulation in cardiomyocytes was determined by measuring an endogenous protein substrate, and in mice, the cardiovascular physiological response. M2 receptor stimulation was associated with increased proteasome-mediated proteolysis and enhanced peptidase activities, while M2 receptor inhibition yielded opposing results. Additionally, M2 receptor manipulation did not alter abundance of the key proteasome subunits, Rpt6 and β5, but significantly shifted their isoelectric points. Inhibition of protein kinase G abrogated the stimulatory effects on proteasome-mediated proteolysis from M2 receptor activation. We conclude that M2 receptor stimulation enhances, whereas M2 receptor inhibition reduces, proteasome-mediated proteolysis likely through posttranslational modifications. Protein kinase G appears to be the mediator of the M2 receptors actions.

| S-EPMC3977985 | biostudies-literature

Genomewide analysis of inherited variation associated with phosphorylation of PI3K/AKT/mTOR signaling proteins.

Project description:While there exists a wealth of information about genetic influences on gene expression, less is known about how inherited variation influences the expression and post-translational modifications of proteins, especially those involved in intracellular signaling. The PI3K/AKT/mTOR signaling pathway contains several such proteins that have been implicated in a number of diseases, including a variety of cancers and some psychiatric disorders. To assess whether the activation of this pathway is influenced by genetic factors, we measured phosphorylated and total levels of three key proteins in the pathway (AKT1, p70S6K, 4E-BP1) by ELISA in 122 lymphoblastoid cell lines from 14 families. Interestingly, the phenotypes with the highest proportion of genetic influence were the ratios of phosphorylated to total protein for two of the pathway members: AKT1 and p70S6K. Genomewide linkage analysis suggested several loci of interest for these phenotypes, including a linkage peak for the AKT1 phenotype that contained the AKT1 gene on chromosome 14. Linkage peaks for the phosphorylated:total protein ratios of AKT1 and p70S6K also overlapped on chromosome 3. We selected and genotyped candidate genes from under the linkage peaks, and several statistically significant associations were found. One polymorphism in HSP90AA1 was associated with the ratio of phosphorylated to total AKT1, and polymorphisms in RAF1 and GRM7 were associated with the ratio of phosphorylated to total p70S6K. These findings, representing the first genomewide search for variants influencing human protein phosphorylation, provide useful information about the PI3K/AKT/mTOR pathway and serve as a valuable proof of concept for studies integrating human genomics and proteomics.

| S-EPMC3176272 | biostudies-literature

M3-muscarinic receptor promotes insulin release via receptor phosphorylation/arrestin-dependent activation of protein kinase D1.

Project description:The activity of G protein-coupled receptors is regulated via hyper-phosphorylation following agonist stimulation. Despite the universal nature of this regulatory process, the physiological impact of receptor phosphorylation remains poorly studied. To address this question, we have generated a knock-in mouse strain that expresses a phosphorylation-deficient mutant of the M(3)-muscarinic receptor, a prototypical G(q/11)-coupled receptor. This mutant mouse strain was used here to investigate the role of M(3)-muscarinic receptor phosphorylation in the regulation of insulin secretion from pancreatic islets. Importantly, the phosphorylation deficient receptor coupled to G(q/11)-signaling pathways but was uncoupled from phosphorylation-dependent processes, such as receptor internalization and ?-arrestin recruitment. The knock-in mice showed impaired glucose tolerance and insulin secretion, indicating that M(3)-muscarinic receptors expressed on pancreatic islets regulate glucose homeostasis via receptor phosphorylation-/arrestin-dependent signaling. The mechanism centers on the activation of protein kinase D1, which operates downstream of the recruitment of ?-arrestin to the phosphorylated M(3)-muscarinic receptor. In conclusion, our findings support the unique concept that M(3)-muscarinic receptor-mediated augmentation of sustained insulin release is largely independent of G protein-coupling but involves phosphorylation-/arrestin-dependent coupling of the receptor to protein kinase D1.

| S-EPMC3000281 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data