Eyes Absent Proteins Research Articles

EYA protein complex is required for Wntless retrograde trafficking from endosomes to Golgi

Retrograde transport of WLS (Wntless) from endosomes to trans-Golgi network (TGN) is required for efficient Wnt secretion during development. However, the molecular players connecting endosomes to TGN during WLS trafficking are limited. Here, we identified a role for Eyes Absent (EYA) proteins during retrograde trafficking of WLS to TGN in human cell lines. By using worm, fly, and zebrafish models, we found that the EYA-secretory carrier-associated membrane protein 3 (SCAMP3) axis is evolved in vertebrates. EYAs form a complex and interact with retromer on early endosomes. Retromer-bound EYA complex recruits SCAMP3 to endosomes, which is necessary for the fusion of WLS-containing endosomes to TGN. Loss of EYA complex or SCAMP3 leads to defective transport of WLS to TGN and failed Wnt secretion. EYA mutations found in patients with hearing loss form a dysfunctional EYA-retromer complex that fails to activate Wnt signaling. These findings identify the EYA complex as a component of retrograde trafficking of WLS from the endosome to TGN.

The Eyes Absent family members EYA4 and EYA1 promote PLK1 activation and successful mitosis through tyrosine dephosphorylation

The Eyes Absent proteins (EYA1-4) are a biochemically unique group of tyrosine phosphatases known to be tumour-promoting across a range of cancer types. To date, the targets of EYA phosphatase activity remain largely uncharacterised. Here, we identify Polo-like kinase 1 (PLK1) as an interactor and phosphatase substrate of EYA4 and EYA1, with pY445 on PLK1 being the primary target site. Dephosphorylation of pY445 in the G2 phase of the cell cycle is required for centrosome maturation, PLK1 localization to centrosomes, and polo-box domain (PBD) dependent interactions between PLK1 and PLK1-activation complexes. Molecular dynamics simulations support the rationale that pY445 confers a structural impairment to PBD-substrate interactions that is relieved by EYA-mediated dephosphorylation. Depletion of EYA4 or EYA1, or chemical inhibition of EYA phosphatase activity, dramatically reduces PLK1 activation, causing mitotic defects and cell death. Overall, we have characterized a phosphotyrosine signalling network governing PLK1 and mitosis.

Stem Cell Bioengineering with Bioportides: Inhibition of Planarian Head Regeneration with Peptide Mimetics of Eyes Absent Proteins.

Djeya1 (RKLAFRYRRIKELYNSYR) is a very effective cell penetrating peptide (CPP) that mimics the α5 helix of the highly conserved Eya domain (ED) of eyes absent (Eya) proteins. The objective of this study was to bioengineer analogues of Djeya1 that, following effective translocation into planarian tissues, would reduce the ability of neoblasts (totipotent stem cells) and their progeny to regenerate the anterior pole in decapitated S. mediterranea. As a strategy to increase the propensity for helix formation, molecular bioengineering of Djeya1 was achieved by the mono-substitution of the helicogenic aminoisobutyric acid (Aib) at three species-variable sites: 10, 13, and 16. CD analyses indicated that Djeya1 is highly helical, and that Aib-substitution had subtle influences upon the secondary structures of bioengineered analogues. Aib-substituted Djeya1 analogues are highly efficient CPPs, devoid of influence upon cell viability or proliferation. All three peptides increase the migration of PC-3 cells, a prostate cancer line that expresses high concentrations of Eya. Two peptides, [Aib13]Djeya1 and [Aib16]Djeya1, are bioportides which delay planarian head regeneration. As neoblasts are the only cell population capable of division in planaria, these data indicate that bioportide technologies could be utilised to directly manipulate other stem cells in situ, thus negating any requirement for genetic manipulation.

Seasonal cues act through the circadian clock and pigment-dispersing factor to control EYES ABSENT and downstream physiological changes

Organisms adapt to seasonal changes in photoperiod and temperature to survive; however, the mechanisms by which these signals are integrated in the brain to alter seasonal biology are poorly understood. We previously reported that EYES ABSENT (EYA) shows higher levels in cold temperature or short photoperiod and promotes winter physiology in Drosophila. Nevertheless, how EYA senses seasonal cues is unclear. Pigment-dispersing factor (PDF) is a neuropeptide important for regulating circadian output rhythms. Interestingly, PDF has also been shown to regulate seasonality, suggesting that it may mediate the function of the circadian clock in modulating seasonal physiology. In this study, we investigated the role of EYA in mediating the function of PDF on seasonal biology. We observed that PDF abundance is lower on cold and short days as compared with warm and long days, contrary to what was previously observed for EYA. We observed that manipulating PDF signaling in eya+ fly brain neurons, where EYA and PDF receptor are co-expressed, modulates seasonal adaptations in daily activity rhythm and ovary development via EYA-dependent and EYA-independent mechanisms. At the molecular level, altering PDF signaling impacted EYA protein abundance. Specifically, we showed that protein kinase A (PKA), an effector of PDF signaling, phosphorylates EYA promoting its degradation, thus explaining the opposite responses of PDF and EYA abundance to changes in seasonal cues. In summary, our results support a model in which PDF signaling negatively modulates EYA levels to regulate seasonal physiology, linking the circadian clock to the modulation of seasonal adaptations.

The Eyes Absent proteins in development and in developmental disorders.

The Eyes Absent (EYA) transactivator-phosphatase proteins are important contributors to cell-fate determination processes and to the development of multiple organs. The transcriptional regulatory activity as well as the protein tyrosine phosphatase activities of the EYA proteins can independently contribute to proliferation, differentiation, morphogenesis and tissue homeostasis in different contexts. Aberrant EYA levels or activity are associated with numerous syndromic and non-syndromic developmental disorders, as well as cancers. Commensurate with the multiplicity of biochemical activities carried out by the EYA proteins, they impact upon a range of cellular signaling pathways. Here, we provide a broad overview of the roles played by EYA proteins in development, and highlight the molecular signaling pathways known to be linked with EYA-associated organ development and developmental disorders.

The Eyes Absent Proteins: Unusual HAD Family Tyrosine Phosphatases

Here, we review the haloacid dehalogenase (HAD) class of protein phosphatases, with a particular emphasis on an unusual group of enzymes, the eyes absent (EYA) family. EYA proteins have the unique distinction of being structurally and mechanistically classified as HAD enzymes, yet, unlike other HAD phosphatases, they are protein tyrosine phosphatases (PTPs). Further, the EYA proteins are unique among the 107 classical PTPs in the human genome because they do not use a Cysteine residue as a nucleophile in the dephosphorylation reaction. We will provide an overview of HAD phosphatase structure-function, describe unique features of the EYA family and their tyrosine phosphatase activity, provide a brief summary of the known substrates and cellular functions of the EYA proteins, and speculate about the evolutionary origins of the EYA family of proteins.

Regulation of cancer stem cell properties by SIX1, a member of the PAX-SIX-EYA-DACH network.

The PAX-SIX-EYA-DACH network (PSEDN) is a central developmental transcriptional regulatory network from Drosophila to humans. The PSEDN is comprised of four conserved protein families; including paired box (PAX), sine oculis (SIX), eyes absent (EYA), and dachshund (DACH). Aberrant expression of PSEDN members, particularly SIX1, has been observed in multiple human cancers, where SIX1 expression correlates with increased aggressiveness and poor prognosis. In conjunction with its transcriptional activator EYA, the SIX1 transcription factor increases cancer stem cell (CSC) numbers and induces epithelial-mesenchymal transition (EMT). SIX1 promotes multiple hallmarks and enabling characteristics of cancer via regulation of cell proliferation, senescence, apoptosis, genome stability, and energy metabolism. SIX1 also influences the tumor microenvironment, enhancing recruitment of tumor-associated macrophages and stimulating angiogenesis, to promote tumor development and progression. EYA proteins are multifunctional, possessing a transcriptional activation domain and tyrosine phosphatase activity, that each contributes to cancer stem cell properties. DACH proteins function as tumor suppressors in solid cancers, opposing the actions of SIX-EYA and reducing CSC prevalence. Multiple mechanisms can lead to increased SIX1 expression, including loss of SIX1-targeting tumor suppressor microRNAs (miRs), whose expression correlates inversely with SIX1 expression in cancer patient samples. In this review, we discuss the major mechanisms by which SIX1 confers CSC and EMT features and other important cancer cell characteristics. The roles of EYA and DACH in CSCs and cancer progression are briefly highlighted. Finally, we summarize the clinical significance of SIX1 in cancer to emphasize the potential therapeutic benefits of effective strategies to disrupt PSEDN protein interactions and functions.

WDR1 is a novel EYA3 substrate and its dephosphorylation induces modifications of the cellular actin cytoskeleton

Eyes absent (EYA) proteins are unusual proteins combining in a single polypeptide chain transactivation, threonine phosphatase, and tyrosine phosphatase activities. They play pivotal roles in organogenesis and are involved in a variety of physiological and pathological processes including innate immunity, DNA damage repair or cancer metastasis. The molecular targets of EYA tyrosine phosphatase activity are still elusive. Therefore, we sought to identify novel EYA substrates and also to obtain further insight into the tyrosine-dephosphorylating role of EYA proteins in various cellular processes. We show here that Src kinase phosphorylates tyrosine residues in two human EYA family members, EYA1 and EYA3. Both can autodephosphorylate these residues and their nuclear and cytoskeletal localization seems to be controlled by Src phosphorylation. Next, using a microarray of phosphotyrosine-containing peptides, we identified a phosphopeptide derived from WD-repeat-containing protein 1 (WDR1) that is dephosphorylated by EYA3. We further demonstrated that several tyrosine residues on WDR1 are phosphorylated by Src kinase, and are efficiently dephosphorylated by EYA3, but not by EYA1. The lack of phosphorylation generates major changes to the cellular actin cytoskeleton. We, therefore, conclude that WDR1 is an EYA3-specific substrate, which implies that EYA3 is a key modulator of the cytoskeletal reorganization.

The Eyes Absent Proteins in Developmental and Pathological Angiogenesis

Management of neoangiogenesis remains a high-value therapeutic goal. A recently uncovered association between the DNA damage repair pathway and pathological angiogenesis could open previously unexplored possibilities for intervention. An attractive and novel target is the Eyes absent (EYA) tyrosine phosphatase, which plays a critical role in the repair versus apoptosis decision after DNA damage. This study examines the role of EYA in the postnatal development of the retinal vasculature and under conditions of ischemia-reperfusion encountered in proliferative retinopathies. We find that the ability of the EYA proteins to promote endothelial cell (EC) migration contributes to a delay in postnatal development of the retinal vasculature when Eya3 is deleted specifically in ECs. By using genetic and chemical biology tools, we show that EYA contributes to pathological angiogenesis in a model of oxygen-induced retinopathy. Both in vivo and in vitro, loss of EYA tyrosine phosphatase activity leads to defective assembly of γ-H2AX foci and thus to DNA damage repair in ECs under oxidative stress. These data reveal the potential utility of EYA tyrosine phosphatase inhibitors as therapeutic agents in inhibiting pathological neovascularization with a range of clinical applications.

Abstract P3-02-03: The phosphatase function of the eyes absent (EYA) homolog is required for the induction of breast cancer cellular proliferation via cyclin D1

Abstract The Drosophila Eyes Absent Homologue 1 (EYA1) is a component of the retinal determination gene network (RDGN) and serves as an H2AX phosphatase. The cell fate determination gene network includes the dachshund (dac), twin-of-eyeless (toy), eye absent (eya), teashirt (tsh) and sine oculis (So). In Drosophila, mutations of the RDGN leads to failure of eye formation, whereas, forced expression induces ectopic eye formation. EYA functions as a transcriptional co-activator being recruited in the context of local chromatin, but lacking intrinsic DNA binding activity. EYA family members EYA 1-4 are defined by a 275 amino acid carboxyl-terminal motif that is conserved between species, referred to as the EYA domain (ED). The human homologs EYA 1-4 are highly conserved in their EYA domain and amino termini, with the exception of a small tyrosine rich residue region named EYA domain II. Altered expression or functional activity of the RDGN has been documented in a variety of malignancies. DACH1 expression is reduced in breast, prostate, endometrial and brain cancer. EYA2 is up regulated in ovarian cancer, promoting tumor growth. EYA1 and EYA2 enhanced survival in response to DNA damage producing agents in HEK293 cells. Eya2 was required for Six1/TGFb signals that govern a prometastatic phenotype and epithelial mesenchymal transition (EMT). Although EYA proteins are expressed in human breast cancer, the relationship to molecular genetic subtype, prognosis and the molecular mechanisms governing contact-independent growth are not known. The cyclin D1 gene encodes the regulatory subunits of a holoenzyme that phosphorylates and inactivates the pRb protein. Herein, comparison with normal breast demonstrated EYA1 is overexpressed with cyclin D1 in luminal B breast cancer subtype. EYA1 enhanced breast tumor growth in mice in vivo requiring the phosphatase domain. EYA1 enhanced cellular proliferation, inhibited apoptosis, and induced contact-independent growth and cyclin D1 abundance. The induction of cellular proliferation and cyclin D1 abundance, but not apoptosis, was dependent upon the EYA1 phosphatase domain. The EYA1-mediated transcriptional induction of cyclin D1 occurred via the AP-1 binding site at -953 and required the EYA1 phosphatase function. The AP-1 mutation did not affect SIX1-dependent activation of cyclin D1. EYA1 was recruited in the context of local chromatin to the cyclin D1 AP-1 site. The EYA1 phosphatase function determined the recruitment of CBP, RNA polymerase II and acetylation of H3K9 at the cyclin D1 gene AP-1 site regulatory region in the context of local chromatin. The EYA1 phosphatase regulates cell cycle control via transcriptional complex formation at the cyclin D1 promoter. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P3-02-03.

The Eyes Absent proteins in development and disease

The Eyes Absent (EYA) proteins, first described in the context of fly eye development, are now implicated in processes as disparate as organ development, innate immunity, DNA damage repair, photoperiodism, angiogenesis, and cancer metastasis. These functions are associated with an unusual combination of biochemical activities: tyrosine phosphatase and threonine phosphatase activities in separate domains, and transactivation potential when associated with a DNA-binding partner. EYA mutations are linked to multiorgan developmental disorders, as well as to adult diseases ranging from dilated cardiomyopathy to late-onset sensorineural hearing loss. With the growing understanding of EYA biochemical and cellular activity, biological function, and association with disease, comes the possibility that the EYA proteins are amenable to the design of targeted therapeutics. The availability of structural information, direct links to disease states, available animal models, and the fact that they utilize unconventional reaction mechanisms that could allow specificity, suggest that EYAs are well-positioned for drug discovery efforts. This review provides a summary of EYA structure, activity, and function, as they relate to development and disease, with particular emphasis on recent findings.

The EYA-SO/SIX complex in development and disease

Eyes absent (EYA) and Sine oculis (SO/SIX) proteins function as transcriptional activation complexes and play essential roles in organogenesis during embryonic development in regulating cell proliferation and survival and coordination of particular differentiation programs. Mutations of the Eya and So/Six genes cause profound developmental defects in organisms as diverse as flies, frogs, fish, mice, and humans. EYA proteins also possess an intrinsic phosphatase activity, which is essential for normal development. Here, we review crucial roles of EYA and SO/SIX in development and disease in mice and humans.

Structure-based de novo design of Eya2 phosphatase inhibitors

Although Eyes absent protein tyrosine phosphatases proved to be involved in various human cancers by a series of persuasive experimental evidence, only a very few number of small-molecule inhibitors have been reported so far. We have been able to identify 29 novel inhibitors of Eyes absent homologue 2 (Eya2) by means of a structure-based de novo design with the two known inhibitor scaffolds that contain a proper chelating group for the active-site Mg2+ ion. Because these newly found inhibitors were screened for having desirable physicochemical properties as a drug candidate and exhibited a moderate inhibitory activity with IC50 values ranging from 6 to 50μM, they deserve consideration for further investigation to develop new anticancer medicines. Structural features relevant to the stabilization of the identified inhibitors in the active site of Eya2 phosphatase are discussed in detail.

Structure‐Based Virtual Screening Approach to the Discovery of Novel Inhibitors of Eyes Absent 2 Phosphatase with Various Metal Chelating Moieties

Despite a series of persuasive experimental evidence for the involvement of eyes absent protein tyrosine phosphatases in various human cancers, no small-molecule inhibitor has been reported so far. We have identified seven novel inhibitors of eyes absent homologue 2 (Eya2) with IC(50) values ranging from 1 to 70 μm by the virtual screening with docking simulations and enzyme inhibition assay. Atomic charges of the active-site Mg(2+) ion complex are calculated to enhance the accuracy of docking simulations. The newly discovered inhibitors are structurally diverse and have various chelating groups for the Mg(2+) ion. The interactions with the amino acid residues responsible for the stabilizations of the inhibitors in the active site of Eya2 are addressed in detail.

Eyes Absent Proteins: Characterization of Substrate Specificity and Phosphatase Activity of Mutants Associated with Branchial, Otic and Renal Anomalies

The eyes absent (Eya) genes encode a family of proteins that combine the functions of transcriptional cofactors, signal transducers and enzymes, namely protein tyrosine phosphatases. The latter activity resides in the highly conserved C-terminal Eya domain (ED). Here, we investigated the substrate specificity of the Arabidopsis thaliana homologue (AtEya) by using low-molecular-weight compounds and synthetic phosphotyrosine (pY)-containing peptides that correspond either to phosphorylation sites in proteins or to peptides that were selected through the screening of a combinatorial peptide library. AtEya displayed modest peptide substrate specificity and was sensitive to charges adjacent to pY. In general, the presence of acidic residues on the N-terminal side of the phosphorylation site was critical for catalysis, whereas basic amino acids seemed to be preferred with respect to high-affinity binding. We also detected significant acyl phosphatase activity of AtEya; this suggests that Eya proteins might have further substrates in vivo. In addition, we analysed the phosphatase activity of a number of variants of the mouse Eya1 protein that harbours single point mutations that were associated with branchio-oto-renal syndrome (BOR), branchio-oto syndrome (BO) and ocular defects, respectively, in humans. While BOR mutations led to a significantly reduced phosphatase activity, BO mutants as well as those that are associated with ocular defects only displayed activity that was similar to wild-type levels.

Structure–function analysis of eyes absent protein, aspartate-dependent protein tyrosine phosphatase

Structure–function analysis of eyes absent protein, aspartate-dependent protein tyrosine phosphatase

Characterization of a Plant, Tyrosine-Specific Phosphatase of the Aspartyl Class

The Arabidopsis thaliana homologue of the Eyes Absent genes (AtEYA) encodes a protein corresponding to the C-terminal conserved domain of the animal Eyes Absent proteins. We show here that AtEYA is a tyrosine-specific phosphatase that hydrolyzes its substrates in a metal-dependent reaction analogous to the phosphoserine phosphatases of the haloacid dehalogenase (HAD) family. The animal Eyes Absent proteins are a novel family of dual-function enzymes: they are transcription factors as well as phosphatases. They also represent a new mechanistic class of tyrosine phosphatases (PTPs) that do not have the Cys-containing signature motif. In contrast, AtEYA is only a tyrosine phosphatase and has no transactivation domain. Using the reaction mechanism of other HAD family enzymes as a model, we have conducted mutational analyses on AtEYA to query the roles of conserved residues. This analysis confirms the importance of the putative nucleophile, the general acid, and the metal-binding residues. Additionally, an inhibitory profile that is diagnostic of this new class of protein tyrosine phosphatases is described. The results of these studies on AtEYA reveal that while the animal and plant Eyes Absent proteins catalyze the same dephosphorylation reaction, the details of their specificity and active site environment, as well as their biological roles, are distinct.

Eyes absent represents a class of protein tyrosine phosphatases.

The Eyes absent proteins are members of a conserved regulatory network implicated in the development of the eye, muscle, kidney and ear. Mutations in the Eyes absent genes have been associated with several congenital disorders including the multi-organ disease bronchio-oto-renal syndrome, congenital cataracts and late-onset deafness. 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) proteins. 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 intermediate. 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.

Recruitment and the Role of Nuclear Localization in Polyglutamine-mediated Aggregation

The inherited neurodegenerative diseases caused by an expanded glutamine repeat share the pathologic feature of intranuclear aggregates or inclusions (NI). Here in cell-based studies of the spinocerebellar ataxia type-3 disease protein, ataxin-3, we address two issues central to aggregation: the role of polyglutamine in recruiting proteins into NI and the role of nuclear localization in promoting aggregation. We demonstrate that full-length ataxin-3 is readily recruited from the cytoplasm into NI seeded either by a pathologic ataxin-3 fragment or by a second unrelated glutamine-repeat disease protein, ataxin-1. Experiments with green fluorescence protein/polyglutamine fusion proteins show that a glutamine repeat is sufficient to recruit an otherwise irrelevant protein into NI, and studies of human disease tissue and a Drosophila transgenic model provide evidence that specific glutamine-repeat-containing proteins, including TATA-binding protein and Eyes Absent protein, are recruited into NI in vivo. Finally, we show that nuclear localization promotes aggregation: an ataxin-3 fragment containing a nonpathologic repeat of 27 glutamines forms inclusions only when targeted to the nucleus. Our findings establish the importance of the polyglutamine domain in mediating recruitment and suggest that pathogenesis may be linked in part to the sequestering of glutamine-containing cellular proteins. In addition, we demonstrate that the nuclear environment may be critical for seeding polyglutamine aggregates.

Dachshund and Eyes Absent Proteins Form a Complex and Function Synergistically to Induce Ectopic Eye Development in Drosophila

The eyeless, dachshund, and eyes absent genes encode conserved, nuclear proteins that are essential for eye development in Drosophila. Misexpression of eyeless or dachshund is also sufficient to induce the formation of ectopic compound eyes. Here we show that the dachshund and eyes absent genes act synergistically to induce ectopic retinal development and positively regulate the expression of each other. Moreover, we show that the Dachshund and Eyes Absent proteins can physically interact through conserved domains, suggesting a molecular basis for the genetic synergy observed and that a similar complex may function in mammals. We propose that a conserved regulatory network, rather than a linear hierarchy, controls retinal specification and involves multiple protein complexes that function during distinct steps of eye development.