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Analysis of EYA3 phosphorylation by Src kinase

ABSTRACT: Native MS analysis of prokaryotically expressed and purified EYA3. Native MS analysis of EYA3 phosphorylation by Src and EYA3 autodephosphorylation reaction.

INSTRUMENT(S): Thermo Fisher Scientific instrument model

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Aura Elena Ionescu

LAB HEAD: Stefan Eugen Szedlacsek

PROVIDER: PXD016596 | Pride | 2019-12-17

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	ACL_20150727_AI_WTEya3_highenergy_FigS6.RAW	Raw
	ACL_20150727_AI_WTEya3_higher_150727165040_FigS6.RAW	Raw
	ACL_20150727_AI_WTEya3_lowenergy_FigS6.RAW	Raw
	ACL_20150818_AI_D311NEya3_nokinase_35000_attempt2_lowmz_rpttskp.RAW	Raw
	ACL_20150904_WT_Src_ATP_2h_50uL.RAW	Raw

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Publications

Analysis of EYA3 Phosphorylation by Src Kinase Identifies Residues Involved in Cell Proliferation.

Ionescu Aura E AE Mentel Mihaela M Munteanu Cristian V A CVA Sima Livia E LE Martin Eliza C EC Necula-Petrareanu Georgiana G Szedlacsek Stefan E SE

International journal of molecular sciences 20191213 24

Eyes absent (EYA) are non-thiol-based protein tyrosine phosphatases (PTPs) that also have transcriptional co-activator functions. Their PTP activity is involved in various pathologies. Recently, we demonstrated that Src tyrosine kinase phosphorylates human EYA3 by controlling its subcellular localization. We also found EYA3's ability to autodephosphorylate, while raising the question if the two opposing processes could be involved in maintaining a physiologically adequate level of phosphorylatio ...[more]

PMID: 31847183

Similar Datasets

Multiple functions for Eya3 in mammalian organism

Project description:The important function of Eya (eyes absent) in eye development of D.melanogaster is well known, the function of its mammalian orthologs Eya1-4 is only partially understood. Yet, no phenotype for Eya2 and Eya3 in humans or mouse have been described. In our study we analysed new Eya-deficient mouse model generated by insertional mutagenesis. Expression analysis of Eya3 by in-situ hybridizations and β-Gal-staining revealed a broad expression of the gene throughout development, e.g. in brain, eyes, branchial arches, somites and limbs. This diverse expression pattern suggests pleiotropic effects of Eya3 in mammalian organisms. In contrast to D. melanogaster, a close examination of the eye by various methods did not show any obvious ocular defects in mutant animals. A broad phenotypical characterization of homozygous mutants displayed decreased body weight and body length. Additionally, behavioral changes as a educed forward locomotor activity and altered exploratory behaviour were identified. A significant reduction of forelimb grip strength and an impairment of cardiac processes in Eya3-/- mice suggest a role for Eya3 in muscle development and heart function. Our results implicate that Eya3 plays various roles outside the eye with effects particularly on heart and muscle function in the mouse. Keywords: Eya3-deficient mice, cDNA microarrays, brain, heart, muscle

2008-12-17 | GSE9999 | GEO

Multiple functions for Eya3 in mammalian organism

Project description:The important function of Eya (eyes absent) in eye development of D.melanogaster is well known, the function of its mammalian orthologs Eya1-4 is only partially understood. Yet, no phenotype for Eya2 and Eya3 in humans or mouse have been described. In our study we analysed new Eya-deficient mouse model generated by insertional mutagenesis. Expression analysis of Eya3 by in-situ hybridizations and M-NM-2-Gal-staining revealed a broad expression of the gene throughout development, e.g. in brain, eyes, branchial arches, somites and limbs. This diverse expression pattern suggests pleiotropic effects of Eya3 in mammalian organisms. In contrast to D. melanogaster, a close examination of the eye by various methods did not show any obvious ocular defects in mutant animals. A broad phenotypical characterization of homozygous mutants displayed decreased body weight and body length. Additionally, behavioral changes as a educed forward locomotor activity and altered exploratory behaviour were identified. A significant reduction of forelimb grip strength and an impairment of cardiac processes in Eya3-/- mice suggest a role for Eya3 in muscle development and heart function. Our results implicate that Eya3 plays various roles outside the eye with effects particularly on heart and muscle function in the mouse. Keywords: Eya3-deficient mice, cDNA microarrays, brain, heart, muscle analysis of 4 Eya3 mutant mice versus a pool of 5 wildtype animals; brain, heart, and muscle; 8 hybridisations for each tissue: two technical replicates for each mutant animal including one dye-swap experiments

2008-12-17 | E-GEOD-9999 | biostudies-arrayexpress

Analysis of EYA3 phosphorylation by Src kinase

Project description:Data-dependent nLC-MS/MS analysis of EYA3 phosphorylation by Src protein tyrosine kinase

2019-12-17 | PXD016518 | Pride

Analysis of EYA3 phosphorylation by Src kinase

Project description:Data-dependent nLC-MS/MS analysis of EYA3 phosphorylation by Src protein tyrosine kinase

2019-12-17 | PXD016532 | Pride

Analysis of EYA3 phosphorylation by Src kinase

Project description:Data-dependent nLC-MS/MS analysis of EYA3 phosphorylation by Src protein tyrosine kinase

2019-12-17 | PXD016520 | Pride

Analysis of EYA3 phosphorylation by Src kinase

Project description:Data-dependent nLC-MS/MS analysis of EYA3 phosphorylation by Src protein tyrosine kinase

2019-12-17 | PXD016526 | Pride

Proteomic analysis of Eya3 binding partners

Project description:Eyes absent homolog 3 (EYA3) is a transcriptional cofactor that is known to partner with the sine oculis homeobox (SIX) family to regulate gene expression. In this study, we characterized a novel splice isoform of EYA3, including the alternative cassette exon 7 (EYA3+ex7), that becomes more highly expressed compared to the isoform lacking exon 7 (EYA3Δex7) during muscle cell differentiation and muscle development. This developmentally-regulated splicing pattern was evolutionarily conserved in humans, mice, rats, chickens, and zebrafish. We found that EYA3 isoforms are vital to proliferation and differentiation of myoblasts in a cell culture model of myogenesis. To understand the underlying mechanism, we defined the transcriptional networks controlled by EYA3 through RNA-sequencing of EYA3 depleted cells. We then hypothesized that binding partners of EYA3+ex7 and EYA3Δex7 might contribute to the mechanism by which they regulate transcription. An affinity-purification mass spectrometry approach was used to identify binding partners of EYA3 isoforms by heterologous expression of hemagglutinin (HA) tagged isoforms in HEK293T cells. 26 binding partners (defined as those with log2(FC) > 1 and p < 0.05 compared to HA epitope tag control) were shared among the EYA3 isoforms, including five transcription factors: zinc finger and BTB domain containing 1 (ZBTB1), tumor protein p53 (TP53), SIX5, SIX2, and SIX4. We next demonstrated that ZBTB1 and SIX4 share overlapping transcriptional targets with EYA3, indicating that EYA3 partners with ZBTB1 or SIX4 to regulate gene expression. Three and thirteen unique binding partners were discovered for EYA3Δex7 and EYA3+ex7, respectively. Therefore, inclusion of Eya3 exon 7 may allow for unique interactions and contribute to functions of EYA3+ex7 that are distinct from EYA3Δex7. This dataset includes affinity purified data for Eya3 isoforms in HEK293T cells.

2023-10-13 | PXD037572 | Pride

RNA-sequencing of differentiated mouse skeletal muscle cells following the depletion of EYA3

Project description:Mouse skeletal muscle cells were subjected to treatment with siRNAs targeting EYA3 or a non-targeting siRNA (si-Ctrl). Differential gene expression analysis was performed on the resulting data.

2023-10-06 | GSE212436 | GEO

Analysis of EYA3 Phosphorylation by Src Kinase Identifies Residues Involved in Cell Proliferation.

Project description:Eyes absent (EYA) are non-thiol-based protein tyrosine phosphatases (PTPs) that also have transcriptional co-activator functions. Their PTP activity is involved in various pathologies. Recently, we demonstrated that Src tyrosine kinase phosphorylates human EYA3 by controlling its subcellular localization. We also found EYA3's ability to autodephosphorylate, while raising the question if the two opposing processes could be involved in maintaining a physiologically adequate level of phosphorylation. Using native and bottom-up mass spectrometry, we performed detailed mapping and characterization of human EYA3 Src-phosphorylation sites. Thirteen tyrosine residues with different phosphorylation and autodephosphorylation kinetics were detected. Among these, Y77, 96, 237, and 508 displayed an increased resistance to autodephosphorylation. Y77 and Y96 were found to have the highest impact on the overall EYA3 phosphorylation. Using cell cycle analysis, we showed that Y77, Y96, and Y237 are involved in HEK293T proliferation. Mutation of the three tyrosine residues abolished the pro-proliferative effect of EYA3 overexpression. We have also identified a Src-induced phosphorylation pattern of EYA3 in these cells. These findings suggest that EYA3's tyrosine phosphorylation sites are non-equivalent with their phosphorylation levels being under the control of Src-kinase activity and of EYA3's autodephosphorylation.

| S-EPMC6940942 | biostudies-literature