Eyes Absent Tyrosine Phosphatase Activity Is Not Required for Drosophila Development or Survival

Abstract

Eyes absent (Eya) is an evolutionarily conserved transcriptional coactivator and protein phosphatase that regulates multiple developmental processes throughout the metazoans. Drosophila eya is necessary for survival as well as for the formation of the adult eye. Eya contains a tyrosine phosphatase domain, and mutations altering presumptive active-site residues lead to strongly reduced activities in ectopic eye induction, in vivo genetic rescue using the Gal4-UAS system, and in vitro phosphatase assays. However, these mutations have not been analyzed during normal development with the correct levels, timing, and patterns of endogenous eya expression. To investigate whether the tyrosine phosphatase activity of Eya plays a role in Drosophila survival or normal eye formation, we generated three eya genomic rescue (eyaGR) constructs that alter key active-site residues and tested them in vivo. In striking contrast to previous studies, all eyaGR constructs fully restore eye formation as well as viability in an eya null mutant background. We conclude that the tyrosine phosphatase activity of Eya is not required for normal eye development or survival in Drosophila. Our study suggests the need for a re-evaluation of the mechanism of Eya action and underscores the importance of studying genes in their native context.

Retinal Axon Guidance Requires Integration of Eya and the Jak/Stat Pathway into Phosphotyrosine-Based Signaling Circuitries in Drosophila

“…Recent work has shown that this catalytic function of Eya is dispensable for Drosophila development (Jin et al 2013). What then might be the role of cytoplasmic Eya?…”

“…Eya also carries two different phosphatase domains, one with specificity for phosphotyrosine and a second directed toward phosphothreonine (Li et al 2003;Rayapureddi et al 2003;Tootle et al 2003;Okabe et al 2009). Although Eya's tyrosine phosphatase is not required for normal development in Drosophila (Jin et al 2013), in mammals it dephosphorylates H2AX to promote repair and survival in response to DNA damage (Cook et al 2009;Krishnan et al 2009) and aPKCz to direct lung epithelial polarity and morphogenesis (El-Hashash et al 2012). Eya's threonine phosphatase is less well characterized, but appears to act both cytoplasmically to regulate innate immunity and nuclearly to provide transactivation and regulate the activity of other transcription factors (Okabe et al 2009;Liu et al 2012;Xu et al 2014;Jin and Mardon 2016).…”

Multiple Functions of the Eya Phosphotyrosine Phosphatase

“…If so, then plants provide an opportunity to study the in vivo function and regulation of Eya tyrosine phosphatase activity without the complications associated with its other role as a transcription factor. The plant ED also stands out as having the strongest in vitro catalytic activity of all Eya family members tested (58) (40,55,58), perhaps plant Eya offers the best path toward unraveling the mysteries of this novel eukaryotic phosphatase. From a structural biology perspective, determination of whether the plant ED fold has intrinsic Six binding potential might provide insight into the evolutionary trajectory that separates plant from animal Eya proteins, and by extension, the physical mechanisms by which animal Eya proteins integrate their dual functions as transcription factors and protein tyrosine phosphatases.…”

“…A new twist emerged in 2013 when the Drosophila Eya field was turned upside down by the demonstration that Eya's tyrosine phosphatase activity is dispensable for normal development, fertility, and survival (55). Whereas earlier studies had relied on overexpression-and misexpression-based genetic assays to show compromised output from phosphatase-dead Eya (39,40,49,56,57), Percentages of amino acid identities and similarities were calculated using LALIGN and are shown above and below the diagonal white line, respectively.…”

“…Mardon and colleagues used functional genomic transgenes to express Eya at endogenous levels, times, and places (55). Shockingly, three different phosphatase-dead Eya mutants fully complemented eya null alleles and exhibited completely wild-type function across a range of in vivo assays.…”