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Transcription profiling by array of human ovarian cancer cells overexpressing PKA regulatory subunits

ABSTRACT: The regulatory subunit of cAMP-dependent protein kinase (PKA) exists in two isoforms, RI and RII, which distinguish the PKA isozymes, type I (PKA-I) and type II (PKA-II). Evidence obtained from a variety of different experimental approaches has shown that the relative levels of type I and type II PKA in cells can play a major role in determining the balance between cell growth and differentiation. RI? transfected cells exhibit hyper-proliferative growth and RII? transfected cells revert to a relatively quiescent state. Profiling by microarray revealed equally profound changes in gene expression between RI?, RII?, and parental OVCAR cells.

ORGANISM(S): Homo sapiens

SUBMITTER: chris cheadle

PROVIDER: E-GEOD-12854 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Overexpression of PKA regulatory subunits in ovarian cancer cells

Project description:The regulatory subunit of cAMP-dependent protein kinase (PKA) exists in two isoforms, RI and RII, which distinguish the PKA isozymes, type I (PKA-I) and type II (PKA-II). Evidence obtained from a variety of different experimental approaches has shown that the relative levels of type I and type II PKA in cells can play a major role in determining the balance between cell growth and differentiation. RIα transfected cells exhibit hyper-proliferative growth and RIIβ transfected cells revert to a relatively quiescent state. Profiling by microarray revealed equally profound changes in gene expression between RIα, RIIβ, and parental OVCAR cells. Keywords: genetic modification

2008-09-20 | GSE12854 | GEO

Discovery of a new Cushing’s syndrome PKAc mutant that is retained within anchored type I holoenzymes

Project description:Adrenal Cushing’s syndrome is a disease of cortisol hypersecretion often caused by mutations in protein kinase A catalytic subunit (PKAc). Using a personalized medicine screening platform, we discovered a new Cushing’s driver mutation, PKAc-W196G, in ~20% of patient samples analyzed. Proximity proteomics and photokinetic imaging reveal that PKAcW196G is distinct from other Cushing’s variants. This mutant surprisingly retains association with type I regulatory subunits (RI) and their corresponding A kinase anchoring proteins (AKAPs). Molecular dynamics simulations predict that substitution of glycine for tryptophan 196 creates a 653 Å3 cleft between the catalytic core of PKAcW196G and type II regulatory subunits (RII), but only a 395 Å3 cleft with RI. Endocrine measurements show that overexpression of RIα or redistribution of PKAcW196G via AKAP recruitment counteracts stress hormone overproduction. We conclude that a W196G mutation in the kinase catalytic core skews R subunit selectivity and biases AKAP association to drive Cushing’s syndrome.

2024-02-10 | PXD045556 | Pride

Expression data from the mushroom bodies of Drosophila-PKA activity dependent manner

Project description:Temperature preference behavior in Drosophila depends on the level of PKA signaling in the mushroom bodies. To identify new components downstream to PKA, we carried out a genome-wide screen for genes regulated by PKA signaling in the mushroom bodies. Using the Gal4-UAS system, we increased or decreased PKA activity in the mushroom bodies by expressing dominant-negative (UAS-PKADN) or constitutively active PKA (UAS-PKACA), respectively. Expression of PKA transgenes was targeted to the mushroom bodies using the mushroom body-specific MB247-Gal4 driver. PKA expression was induced for 12-16 hours in three-day-old adults by inactivating the temperature-sensitive Gal80 at the restrictive temperature. We then analyzed gene-expression profiles to identify the genes showing altered expression levels in response to the high or low PKA activity.

2014-03-25 | E-GEOD-28181 | biostudies-arrayexpress

Proteomic and phosphoproteomic analyses of protein PKA catalytic-alpha-null mpkCCD cells and PKA catalytic-beta-null mpkCCD cells.

Project description:Protein Kinase A (PKA) is a widely studied protein that has been viewed by most investigators as a single entity, although its catalytic subunits are coded in the genome by two separate genes, PKA catalytic alpha (Gene symbol: Prkaca) and PKA catalytic beta Gene symbol: Prkacb). At an amino-acid level, the two are 91.5 percent identical and the catalytic domains are virtually identical. (Footnote: A third entity PKA catalytic gamma, is not widely expressed and will not be considered here.) We have recently succeeded in using CRISPR-Cas9 to create disruptive mutations in both PKA genes (PKA double KO, or PKA dKO) in vasopressin-responsive kidney epithelial cells (mpkCCD cells). Here we carry out mass spectrometry based quantitative proteomics and phosphoproteomics separately in PKA catalytic-alpha and PKA catalytic-beta single knockouts address the issue of function difference between these two PKA catalytic subunits.

2020-06-01 | PXD015050 | Pride

H89 Phosphoproteomics in PKA-Null Cells

Project description:Protein phosphorylation plays a crucial role in propagating cellular responses to both internal and external cues. One of the most important kinases responsible for protein phosphorylation is protein kinase A (PKA). N-[2-p-bromocinnamylamino-ethyl]-5-isoquinolinesulphonamide (H89) is often used as a “PKA specific inhibitor” to study the involvement of PKA in signaling pathways. However, evidence from cell-free experiments suggested that H89 can also inhibit other kinases. In this experiment, previously-generated mouse collecting duct cell lines with and without PKA were treated with H89, followed by mass spectrometry-based phosphoproteomics to globally assess changes in phosphorylation.

2019-03-28 | PXD011167 | Pride

Phosphorylation of the compartmentalized PKA substrate TAF15 regulates RNA-Protein interactions

Project description:Spatiotemporal-controlled second messengers alter molecular interactions of central signaling nodes for ensuring physiological signal transmission. One prototypical second messenger molecule which modulates kinase signal transmission is the cyclic-adenosine monophosphate (cAMP). The main proteinogenic cellular effectors of cAMP are compartmentalized protein kinase A (PKA) complexes. Their cell-type specific compositions precisely coordinate substrate phosphorylation and proper signal propagation which is indispensable for numerous cell-type specific functions. Here we present evidence that TAF15, which is implicated in the etiology of amyotrophic lateral sclerosis, represents a novel nuclear PKA substrate. In cross-linking and immunoprecipitation experiments (iCLIP) we showed that TAF15 phosphorylation alters the binding to target transcripts related to mRNA maturation, splicing and protein-binding related functions. TAF15 appears to be one of multiple PKA substrates that undergo RNA-binding dynamics upon phosphorylation. We observed that the activation of the cAMP-PKA signaling axis caused a change in the composition of a collection of RNA species that interact with TAF15. This observation appears to be a broader principle in the regulation of molecular interactions, as we identified a significant enrichment of RNA-binding proteins within endogenous PKA complexes. We assume that phosphorylation of RNA-binding domains adds another layer of regulation to binary protein-RNAs interactions with consequences to RNA features including binding specificities, localization, abundance and composition.

2024-04-04 | PXD048869 | Pride

PKA C3 immunoprecipitation mass spectrometry

Project description:Toxoplasma gondii possesses three protein kinase A (PKA) orthologs. PKA C3 has been implicated as a negative regulator of transformation from the acute stage tachyzoites to the chronic stage bradyzoites. We performed immunoprecipitation of PKA C3 and identified interaction partners by mass spectrometry. The apicomplexan-specific AGC kinase SPARK emerged as a strong interaction partner of PKA C3.

2024-07-12 | PXD039896 | Pride

PKA regulated genes in HEK293 cells

Project description:When exploring the role of Gαs and β-arrestin in nuclear transcriptional programs downstream from β2-adrenergic receptor (β2AR), we noticed a dearth of information on PKA-regulated genes sets. Thus, we first used the tetracycline-regulated expression of wild type PKA Cα subunit in HEK293 cells to develop a PKA signature. This approach revealed multiple PKA-regulated genes, including well known PKA downstream transcriptional targets, such as PCK1 and FOS, and many new PKA transcriptional targets whose underlying mechanism can now be explored.

2023-10-21 | GSE245789 | GEO

Human MyBP-C PKA-treated C0-C2 LC-MSMS

Project description:Cardiac myosin-binding protein C (cMyBP-C) is a thick filament–associated protein that influences actin–myosin interactions. cMyBP-C alters myofilament structure and contractile properties in a protein kinase A (PKA) phosphorylation–dependent manner. To determine the effects of cMyBP-C and its phosphorylation on the microsecond rotational dynamics of actin filaments, we attached a phosphorescent probe to F-actin at Cys-374 and performed transient phosphorescence anisotropy (TPA) experiments. Binding of cMyBP-C N-terminal domains (C0-C2) to labeled F-actin reduced rotational flexibility by 20–25º, indicated by increased final anisotropy of the TPA decay. The effects of C0-C2 on actin TPA were highly cooperative (n ~ 8), suggesting that the cMyBP-C N terminus impacts the rotational dynamics of actin spanning seven monomers (i.e. the length of tropomyosin). PKA-mediated phosphorylation of C0-C2 eliminated the cooperative effects on actin flexibility and modestly increased actin rotational rates. Effects of Ser-to-Asp phosphomimetic substitutions in the M-domain of C0-C2 on actin dynamics only partially recapitulated the phosphorylation effects. C0-C1 (lacking M-domain/C2) similarly exhibited reduced cooperativity, but not as reduced as by phosphorylated C0-C2. These results suggest an important regulatory role of the M-domain in cMyBP-C effects on actin structural dynamics. In contrast, phosphomimetic substitution of the glycogen synthase kinase (GSK3beta) site in the Pro/Ala-rich linker of C0-C2 did not significantly affect the TPA results. We conclude that cMyBP-C binding and PKA-mediated phosphorylation can modulate actin dynamics. We propose that these N-terminal cMyBP-C–induced changes in actin dynamics help explain the functional effects of cMyBP-C phosphorylation on actin–myosin interactions.

2019-09-26 | PXD015391 | Pride

PKA C3 depletion phosphoproteomics

Project description:Toxoplasma gondii is a ubiquitous parasite of warm-blooded animals. Parasites can switch into the chronic infection following exposure to stress. PKA C3 is a protein kinase involved in the differentiation process. Here, we determine the phosphoproteome of parasites conditionally depleted of PKA C3.

2024-07-12 | PXD042989 | Pride

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