Eyes absent represents a class of protein tyrosine phosphatases

Abstract

The Eyes absent proteins are members of a conserved regulatory network implicated in the development of the eye, muscle, kidney and ear1,2,3,4,5,6,7. Mutations in the Eyes absent genes have been associated with several congenital disorders including the multi-organ disease bronchio-oto-renal syndrome8, congenital cataracts9 and late-onset deafness10. On the basis of previous analyses it has been shown that Eyes absent is a nuclear transcription factor, acting through interaction with homeodomain-containing Sine oculis (also known as Six) proteins11. Here we show that Eyes absent is also a protein tyrosine phosphatase. It does not resemble the classical tyrosine phosphatases that use cysteine as a nucleophile and proceed by means of a thiol-phosphate intermediate12. Rather, Eyes absent is the prototype for a class of protein tyrosine phosphatases that use a nucleophilic aspartic acid in a metal-dependent reaction. Furthermore, the phosphatase activity of Eyes absent contributes to its ability to induce eye formation in Drosophila.

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Figure 1: Eyes absent is a phosphatase.
Figure 2: Eya3(ED) can dephosphorylate proteins in a dose- and metal-dependent fashion.
Figure 3: The effect of inhibitors on the phosphatase activity of Eya3(ED).
Figure 4: Loss of phosphatase activity compromises Eya function in vivo.

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Acknowledgements

We thank R. Lang and J. Schlessinger for discussions and comments on the manuscript. Mouse Eya1 cDNA was provided by T. Glaser and Drosophila eya cDNA was provided by N. Bonini.

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Correspondence to Rashmi S. Hegde.

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The authors declare that they have no competing financial interests.

Supplementary information

Supplementary Figure 1: Saturation kinetics for the ED domains of mouse Eya2 (d), Eya1 (e), Drosophila Eya (f) and full-length Arabidopsis Thaliana EYA (g). Rate of hydrolysis of substrate (pNPP) (assay buffer 20 mM MES pH 6, 2 mM MgCl2) is plotted against increasing substrate concentration. Data were fitted to the Michaelis-Menten equation, and the resulting kinetic parameters are indicated. (JPG 61 kb)

Supplementary Figure 2: Proposed mechanism for the phosphatase activity of Eyes Absent, by analogy with the HAD enzymes. Residues indicated in red belong to motif I, residues in green are from motif II, and residues in blue are form motif III. The substrate and cofactor are in black. The residue numbers refer to Eya3. (PDF 35 kb)

Supplementary Table 1: Mutational Analysis of Eyes Absent proteins (DOC 25 kb)

Supplementary Table 1: Activity of Eyes Absent proteins and control PTPs towards phosphorylated MyBP and a pY-containing peptide (DOC 20 kb)

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Rayapureddi, J., Kattamuri, C., Steinmetz, B. et al. Eyes absent represents a class of protein tyrosine phosphatases. Nature 426, 295–298 (2003). https://doi.org/10.1038/nature02093

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