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

Aura E Ionescu1, Mihaela Mentel1, Cristian V A Munteanu2

  • 1Department of Enzymology, Institute of Biochemistry of the Romanian Academy, Splaiul Independentei 296, 060031 Bucharest, Romania.

International Journal of Molecular Sciences
|December 19, 2019
PubMed

Insights

Eyes absent homolog 3 (EYA3) protein phosphorylation by Src kinase and autodephosphorylation regulates cell proliferation. Specific tyrosine sites on EYA3 impact its function and are critical for HEK293T cell growth.

Area of Science:

  • Molecular Biology
  • Cell Biology
  • Biochemistry

Background:

  • Eyes absent (EYA) proteins are non-thiol tyrosine phosphatases with transcriptional co-activator roles.
  • EYA protein tyrosine phosphatase (PTP) activity is implicated in various pathologies.
  • Src tyrosine kinase regulates human EYA3 localization, and EYA3 possesses autodephosphorylation activity.

Purpose of the Study:

  • To map and characterize Src-mediated phosphorylation sites on human EYA3.
  • To investigate the interplay between Src phosphorylation and EYA3 autodephosphorylation.
  • To determine the role of specific EYA3 phosphorylation sites in cell proliferation.

Main Methods:

  • Native and bottom-up mass spectrometry for phosphosite mapping.
  • Site-directed mutagenesis to alter tyrosine residues.
  • Cell cycle analysis to assess proliferation effects.

Main Results:

  • Thirteen tyrosine residues on EYA3 were identified with varying phosphorylation and autodephosphorylation kinetics.
  • Tyrosine residues Y77, Y96, Y237, and Y508 showed resistance to autodephosphorylation.
  • Y77 and Y96 significantly influenced overall EYA3 phosphorylation.
  • Mutations in Y77, Y96, and Y237 abolished EYA3's pro-proliferative effect on HEK293T cells.
  • A Src-induced phosphorylation pattern for EYA3 was identified.

Conclusions:

  • EYA3 tyrosine phosphorylation sites are non-equivalent and differentially regulated.
  • Phosphorylation levels of EYA3 are controlled by both Src kinase activity and EYA3's intrinsic autodephosphorylation.
  • Specific EYA3 phosphorylation sites (Y77, Y96, Y237) are crucial for regulating HEK293T cell proliferation.
Keywords:
Src kinasecell proliferationeyes absent 3mass spectrometryprotein phosphorylation

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