Dataset Information

Phosphoproteomics of DIA1-overexpressing HEK cells and protemics of probable DIA1 interactors

ABSTRACT: Many kinases are still “orphans”, i.e. knowledge about their substrates, and often also about the processes they regulate, is lacking. Here, DIA1/C3orf58, a member of a novel predicted kinase-like family is subjected to phosphoproteomics analysis in order to cast light on its signalling pathways. LC MS/MS proteomics approach with Ti phosphopeptide enrichment is applied to membrane fractions of DIA1-overexpressing and control HEK cells, and phosphosites dependent on DIA1 presence are elucidated. Most of The these phosphosites belonged to CK2- and proline-directed kinase types. In parallel, proteomics of proteins immunoprecipitated with DIA1 provided its probable interactors. Intersection of the DIA1-affected phosphoproteins and interactors provides putative DIA1 substrates, including calnexin . This pilot study data, mapped on literature, provides basis for deeper studies of DIA1 signalling.

INSTRUMENT(S): LTQ Orbitrap Elite

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: Michał Kistowski

LAB HEAD: Michał Dadlez

PROVIDER: PXD006831 | Pride | 2018-03-26

REPOSITORIES: Pride

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Publications

Phosphoproteomic insights into processes influenced by the kinase-like protein DIA1/C3orf58.

Hareza Agnieszka A Bakun Magda M Świderska Bianka B Dudkiewicz Małgorzata M Koscielny Alicja A Bajur Anna A Jaworski Jacek J Dadlez Michał M Pawłowski Krzysztof K

PeerJ 20180409

Many kinases are still 'orphans,' which means knowledge about their substrates, and often also about the processes they regulate, is lacking. Here, DIA1/C3orf58, a member of a novel predicted kinase-like family, is shown to be present in the endoplasmic reticulum and to influence trafficking via the secretory pathway. Subsequently, DIA1 is subjected to phosphoproteomics analysis to cast light on its signalling pathways. A liquid chromatography-tandem mass spectrometry proteomic approach with pho ...[more]

PMID: 29666759

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Project description:Thyroid hormones (THs) play a critical role in development and throughout adulthood. THs act through the thyroid receptor (TR), which binds to the TH response element (TRE) to regulate the expression of target genes. Although TH action has been studied for decades, surprisingly few TREs have been well validated and characterized. In this study we used chromatin immunoprecipitation followed by microarray analysis (ChIP-chip) to identify TR-binding sites in juvenile (postnatal day 15) mice liver. Microarray analysis revealed twelve TR-binding sites consistent between all analyzed samples. In silico analysis was carried out to search for moderately conserved classic TRE sequences within these novel binding regions, which led to the identification of six candidate TREs within three binding regions. Luciferase reporter assays confirmed the presence of a TRE in the promoter region of DEAD (Asp-Glu-Ala-Asp) box polypeptide 54 (Ddx54) and thyroid hormone responsive SPOT14 (Thrsp). The TR/retinoid X receptor (RXR) heterodimer and RXR homodiner were shown to bind the promoter region of Ddx54 and drive gene expression in the presence of 9-cis-retinoic acid (9cRA). The promoter region of Thrsp was shown to allow binding of the TR/RXR heterodimer, and both T3 and 9cRA were able to significantly increase luciferase activity. The RXR homodimer was also able to bind the response element in the promoter region of Thrsp and increase luciferase activity. Overall, ChIP-chip analysis revealed a relatively limited number of TR-binding sites in juvenile mouse liver despite previous studies showing that numerous genes can be affected by TH disruption at that developmental stage, suggesting that TH action may also be mediated through other intermediates. Collectively the results provide an important step towards characterizing TR-binding sites and identifying the underlying drivers of TR-gene regulation. Three samples were analyzed (total input and immunoprecipitated for each samples).

Dataset Information

Phosphoproteomics of DIA1-overexpressing HEK cells and protemics of probable DIA1 interactors

Publications

Phosphoproteomic insights into processes influenced by the kinase-like protein DIA1/C3orf58.

Similar Datasets

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