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Open AccessArticle

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

1
Department of Enzymology, Institute of Biochemistry of the Romanian Academy, Splaiul Independentei 296, 060031 Bucharest, Romania
2
Department of Bioinformatics and Structural Biochemistry, Institute of Biochemistry of the Romanian Academy, Splaiul Independentei 296, 060031 Bucharest, Romania
3
Department of Molecular Cell Biology, Institute of Biochemistry of the Romanian Academy, Splaiul Independentei 296, 060031 Bucharest, Romania
*
Author to whom correspondence should be addressed.
Present address: TRANSCEND Research Center, Regional Institute of Oncology, 700483 Iasi, Romania.
Int. J. Mol. Sci. 2019, 20(24), 6307; https://doi.org/10.3390/ijms20246307
Received: 1 November 2019 / Revised: 2 December 2019 / Accepted: 9 December 2019 / Published: 13 December 2019
(This article belongs to the Section Biochemistry)
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. View Full-Text
Keywords: eyes absent 3; Src kinase; mass spectrometry; protein phosphorylation; cell proliferation eyes absent 3; Src kinase; mass spectrometry; protein phosphorylation; cell proliferation
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Ionescu, A.E.; Mentel, M.; Munteanu, C.V.; Sima, L.E.; Martin, E.C.; Necula-Petrareanu, G.; Szedlacsek, S.E. Analysis of EYA3 Phosphorylation by Src Kinase Identifies Residues Involved in Cell Proliferation. Int. J. Mol. Sci. 2019, 20, 6307.

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