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

Abstract

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.

The multi-functional eyes absent proteins

“…This Abl -Eya signaling circuitry plays a role in the development of the Drosophila visual system. There is also some evidence that EYA3 has auto-dephosphorylation activity when over-expressed in HEK293T cells (Ionescu et al 2019). Whether tyrosine phosphorylation of EYA proteins affects its other biochemical activities (transactivation and putative threonine phosphatase) remains to be determined.…”

“…Little is known thus far about cell-cycle associated fluctuations in EYA3 and EYA4 levels, but over-expression of EYA3 increases cell proliferation and this is linked to the phosphorylation of specific tyrosine residues in EYA3 (Ionescu et al 2019).…”

“…Further, this inhibitor is EYA2-specific (does not inhibit EYA3). Benzbromarone has been used widely as a pharmacological tool in evaluating the role of EYA PTP activity both in vitro (Mentel Ionescu et al 2019) and in vivo (Zhou et al 2017;Cui et al 2020).…”

Whole Genome Sequence Data Provides Novel Insights Into the Genetic Architecture of Meat Quality Traits in Beef

“…The upregulation of EYAs is present in many tumor types, performing transcriptional cofactor and tyrosine phosphatase functions. Ionescu et al (2019) found that the upregulation of EYA3 has proliferative effects. MED12L is part of the Mediator complex, a multiprotein complex involved in transcription of RNA polymerase II-dependent genes.…”

The Double Phospho/Dephosphorylation Cycle as a Benchmark to Validate an Effective Taylor Series Method to Integrate Ordinary Differential Equations

“…Phosphorylations usually introduce conformational changes that activate/inactivate the enzymatic activity of a protein, or simply prime degradation processes, like the ones involved in the yeast cell cycle (see, e.g., in [3,4] dealing with the degradation of Sic1 and Whi5, respectively) or those involving tyrosine kinase pathways, regulating diverse cellular processes, and whose dysregulation is one of the leading causes of cancer progression [5]. In many important cases, multi-site phosphorylations are required to ensure the correct timing of activation [6,7].…”