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.
Highlights
Eyes absent (EYA) proteins belong to a family of evolutionarily conserved transcription factors and cofactors, referred to as the Pax-Six-Eya-Dach Network (PSEDN)
EYA3 was incubated with Src kinase and tyrosine phosphorylation tracked by native mass spectrometry (MS) (Figure 1c,d, Figures S3 and S4)
After 2 h, up to 12 phosphorylation sites were observed on EYA3 D311N (Figure 1b versus Figure 1d, when compared to Figure S5, the intensities of the signals corresponding to the unphosphorylated molecules decreased and those of the phosphorylated molecules increased) highlighting the importance of the Eya Domain (ED) in controlling the phosphorylation status of EYA3
Summary
EYA proteins belong to a family of evolutionarily conserved transcription factors and cofactors, referred to as the Pax-Six-Eya-Dach Network (PSEDN). The EYA transcriptional co-activator function resides in the N-terminal domain (NTD), which is a region poorly conserved among vertebrates [1] and absent in plants [24]. The protein tyrosine phosphatase (PTP) activity is localized in the C-terminal domain and contains characteristic motifs of the haloacid dehalogenase (HAD) superfamily, which makes EYA a member of the phosphatase subgroup of HAD [2,22,23]. In addition to its own tyrosine phosphatase activity, EYA has threonine phosphatase activity but only when interacting with the protein phosphatase 2A (PP2A)-B55α holoenzyme This interaction proved to play a critical role in c-Myc stabilization and late stage metastasis in the breast cancer model [25]. Three physiological substrates for EYA’s PTP activity have been identified: histone H2A.X (phosphotyrosine-pY-142) [27,28], estrogen receptor β (pY36) [29], which both have nuclear localization, and WD repeat-containing protein 1 (WDR1), which is a cytoskeletal protein [30]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
