Dephosphorylation of the C-terminal Tyrosyl Residue of the DNA Damage-related Histone H2A.X Is Mediated by the Protein Phosphatase Eyes Absent
2009; Elsevier BV; Volume: 284; Issue: 24 Linguagem: Inglês
10.1074/jbc.c900032200
ISSN1083-351X
AutoresNavasona Krishnan, Dae Gwin Jeong, Suk‐Kyeong Jung, Seong Eon Ryu, Andrew Xiao, C. David Allis, Seung Jun Kim, Nicholas K. Tonks,
Tópico(s)Cancer-related Molecular Pathways
ResumoIn mammalian cells, the DNA damage-related histone H2A variant H2A.X is characterized by a C-terminal tyrosyl residue, Tyr-142, which is phosphorylated by an atypical kinase, WSTF. The phosphorylation status of Tyr-142 in H2A.X has been shown to be an important regulator of the DNA damage response by controlling the formation of γH2A.X foci, which are platforms for recruiting molecules involved in DNA damage repair and signaling. In this work, we present evidence to support the identification of the Eyes Absent (EYA) phosphatases, protein-tyrosine phosphatases of the haloacid dehalogenase superfamily, as being responsible for dephosphorylating the C-terminal tyrosyl residue of histone H2A.X. We demonstrate that EYA2 and EYA3 displayed specificity for Tyr-142 of H2A.X in assays in vitro. Suppression of eya3 by RNA interference resulted in elevated basal phosphorylation and inhibited DNA damage-induced dephosphorylation of Tyr-142 of H2A.X in vivo. This study provides the first indication of a physiological substrate for the EYA phosphatases and suggests a novel role for these enzymes in regulation of the DNA damage response. In mammalian cells, the DNA damage-related histone H2A variant H2A.X is characterized by a C-terminal tyrosyl residue, Tyr-142, which is phosphorylated by an atypical kinase, WSTF. The phosphorylation status of Tyr-142 in H2A.X has been shown to be an important regulator of the DNA damage response by controlling the formation of γH2A.X foci, which are platforms for recruiting molecules involved in DNA damage repair and signaling. In this work, we present evidence to support the identification of the Eyes Absent (EYA) phosphatases, protein-tyrosine phosphatases of the haloacid dehalogenase superfamily, as being responsible for dephosphorylating the C-terminal tyrosyl residue of histone H2A.X. We demonstrate that EYA2 and EYA3 displayed specificity for Tyr-142 of H2A.X in assays in vitro. Suppression of eya3 by RNA interference resulted in elevated basal phosphorylation and inhibited DNA damage-induced dephosphorylation of Tyr-142 of H2A.X in vivo. This study provides the first indication of a physiological substrate for the EYA phosphatases and suggests a novel role for these enzymes in regulation of the DNA damage response. Unlike kinases, which are derived from a common ancestor, the opposing phosphatases have evolved in separate, structurally distinct families. In fact, a variety of catalytic mechanisms have been harnessed to facilitate protein dephosphorylation. The protein-Ser/Thr phosphatases, such as the PPP and PPM families, mediate dephosphorylation by using two metal ions at the active site, activating a water molecule for nucleophilic attack on the substrate phosphate in a single-step reaction (1Barford D. Das A.K. Egloff M.P. Annu. Rev. Biophys. Biomol. Struct. 1998; 27: 133-164Crossref PubMed Scopus (571) Google Scholar). The family of Cys-dependent protein-tyrosine phosphatases (PTPs) 4The abbreviations used are: PTPprotein-tyrosine phosphataseHADhaloacid dehalogenaseEDEYA domainRNAiRNA interferencesiRNAsmall interfering RNASH2Src homology 2DSBsdouble-strand breaks. utilizes a two-step catalytic mechanism involving an essential nucleophilic cysteinyl residue, which forms a Cys-phosphate intermediate (2Tonks N.K. Nat. Rev. Mol. Cell Biol. 2006; 7: 833-846Crossref PubMed Scopus (1276) Google Scholar). More recently, a third family of Asp-dependent phosphatases belonging to the haloacid dehalogenase (HAD) superfamily (3Burroughs A.M. Allen K.N. Dunaway-Mariano D. Aravind L. J. Mol. Biol. 2006; 361: 1003-1034Crossref PubMed Scopus (322) Google Scholar) has been gaining prominence. protein-tyrosine phosphatase haloacid dehalogenase EYA domain RNA interference small interfering RNA Src homology 2 double-strand breaks. Various HAD phosphatases have been linked to fundamental aspects of control of cell function. One of the best characterized is FCP1, which is an important regulator of transcription through dephosphorylation of the C-terminal domain of RNA polymerase II (4Ghosh A. Shuman S. Lima C.D. Mol. Cell. 2008; 32: 478-490Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). Chronophin has been shown to dephosphorylate Ser-3 in cofilin and thereby function as a regulator of the actin cytoskeleton (5Gohla A. Birkenfeld J. Bokoch G.M. Nat. Cell Biol. 2005; 7: 21-29Crossref PubMed Scopus (257) Google Scholar). More recently, a HAD phosphatase known as Dullard has been shown to dephosphorylate the phosphatidic acid phosphatase lipin, functioning in a phosphatase cascade that regulates nuclear membrane biogenesis (6Kim Y. Gentry M.S. Harris T.E. Wiley S.E. Lawrence Jr., J.C. Dixon J.E. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 6596-6601Crossref PubMed Scopus (117) Google Scholar). In these cases, the HADs are functioning as protein-Ser/Thr phosphatases. However, there is now also an example of a member of the HAD superfamily with the capacity to dephosphorylate phosphotyrosyl residues in substrates. Eyes Absent (EYA) was identified initially as a component of a network of transcription regulators, the retinal determination gene network that is responsible for eye development in Drosophila. It is now known to be involved in tissue and organ development in many organisms (7Jemc J. Rebay I. Annu. Rev. Biochem. 2007; 76: 513-538Crossref PubMed Scopus (110) Google Scholar). There are four EYA proteins in mammals (designated EYA1–4) that are defined by a C-terminal domain of ∼270 residues termed the EYA domain (ED) (7Jemc J. Rebay I. Annu. Rev. Biochem. 2007; 76: 513-538Crossref PubMed Scopus (110) Google Scholar). This domain is required for interaction with the homeodomain protein Sine oculis (SO) in Drosophila (known as the SIX proteins in vertebrates) and the transcriptional regulator Dachshund (DACH). EYA and SIX function as a transcription factor complex, in which SIX mediates DNA binding, and EYA uses its N-terminal segment to function in transactivation. Considerable progress has been made in characterizing the function of EYA as a transcription factor (7Jemc J. Rebay I. Annu. Rev. Biochem. 2007; 76: 513-538Crossref PubMed Scopus (110) Google Scholar). The demonstration of altered expression of the SIX-EYA-DACH network has also illustrated a potential role in cancer (8Reichenberger K.J. Coletta R.D. Schulte A.P. Varella-Garcia M. Ford H.L. Cancer Res. 2005; 65: 2668-2675Crossref PubMed Scopus (95) Google Scholar, 9Zhang L. Yang N. Huang J. Buckanovich R.J. Liang S. Barchetti A. Vezzani C. O'Brien-Jenkins A. Wang J. Ward M.R. Courreges M.C. Fracchioli S. Medina A. Katsaros D. Weber B.L. Coukos G. Cancer Res. 2005; 65: 925-932PubMed Google Scholar). However, a major breakthrough came with the observation that the ED of EYA contains signature sequences from the HAD superfamily. EYA is the first example of a transcription factor with intrinsic phosphatase activity (10Rayapureddi J.P. Kattamuri C. Steinmetz B.D. Frankfort B.J. Ostrin E.J. Mardon G. Hegde R.S. Nature. 2003; 426: 295-298Crossref PubMed Scopus (200) Google Scholar, 11Tootle T.L. Silver S.J. Davies E.L. Newman V. Latek R.R. Mills I.A. Selengut J.D. Parlikar B.E. Rebay I. Nature. 2003; 426: 299-302Crossref PubMed Scopus (218) Google Scholar, 12Li X. Oghi K.A. Zhang J. Krones A. Bush K.T. Glass C.K. Nigam S.K. Aggarwal A.K. Maas R. Rose D.W. Rosenfeld M.G. Nature. 2003; 426: 247-254Crossref PubMed Scopus (514) Google Scholar). Characterization with phosphopeptides indicated specificity for phosphotyrosyl residues (10Rayapureddi J.P. Kattamuri C. Steinmetz B.D. Frankfort B.J. Ostrin E.J. Mardon G. Hegde R.S. Nature. 2003; 426: 295-298Crossref PubMed Scopus (200) Google Scholar, 11Tootle T.L. Silver S.J. Davies E.L. Newman V. Latek R.R. Mills I.A. Selengut J.D. Parlikar B.E. Rebay I. Nature. 2003; 426: 299-302Crossref PubMed Scopus (218) Google Scholar), although one group suggested that EYA could function as a dual-specificity phosphatase (12Li X. Oghi K.A. Zhang J. Krones A. Bush K.T. Glass C.K. Nigam S.K. Aggarwal A.K. Maas R. Rose D.W. Rosenfeld M.G. Nature. 2003; 426: 247-254Crossref PubMed Scopus (514) Google Scholar). The phosphatase activity of EYA is important for switching SIX1-DACH function from transcriptional repression to activation, and inactivating mutations also impair EYA function in eye development (10Rayapureddi J.P. Kattamuri C. Steinmetz B.D. Frankfort B.J. Ostrin E.J. Mardon G. Hegde R.S. Nature. 2003; 426: 295-298Crossref PubMed Scopus (200) Google Scholar, 11Tootle T.L. Silver S.J. Davies E.L. Newman V. Latek R.R. Mills I.A. Selengut J.D. Parlikar B.E. Rebay I. Nature. 2003; 426: 299-302Crossref PubMed Scopus (218) Google Scholar, 12Li X. Oghi K.A. Zhang J. Krones A. Bush K.T. Glass C.K. Nigam S.K. Aggarwal A.K. Maas R. Rose D.W. Rosenfeld M.G. Nature. 2003; 426: 247-254Crossref PubMed Scopus (514) Google Scholar). Furthermore, mutations in eya1 that are associated with branchio-oto-renal syndrome have been shown to result in loss of phosphatase activity (13Rayapureddi J.P. Hegde R.S. FEBS Lett. 2006; 580: 3853-3859Crossref PubMed Scopus (25) Google Scholar). Nevertheless, the identity of downstream physiological substrates of EYA (cytosolic and nuclear) remains a major unresolved issue. In this study, we report that a striking feature of the ED of EYA2, as revealed by the crystal structure, is the clustering of acidic residues around the active site to present a negatively charged surface. This suggests that substrates of the enzyme may have basic properties. Recently, phosphorylation of the C-terminal tyrosyl residue of the DNA damage-related histone H2A.X has been revealed as a new element in the control of the DNA damage response (14Xiao A. Li H. Shechter D. Ahn S.H. Fabrizio L.A. Erdjument-Bromage H. Ishibe-Murakami S. Wang B. Tempst P. Hofmann K. Patel D.J. Elledge S.J. Allis C.D. Nature. 2009; 457: 57-62Crossref PubMed Scopus (316) Google Scholar). Considering the known functions of EYA in the nucleus and the fact that histones are recognized as highly basic proteins, we sought to determine whether EYA could dephosphorylate histone H2A.X as a substrate. We present data from in vitro assays demonstrating the specificity of EYA for Tyr-142 in H2A.X. Furthermore, we show that suppression of EYA by RNA interference (RNAi) results in enhanced basal phosphorylation and inhibition of DNA damage-induced dephosphorylation of the C-terminal tyrosyl residue of histone H2A.X in cells. These data provide the first indications of a physiological substrate of EYA and suggest that the phosphatase may be an important component of the response to DNA damage. Recombinant human EYA2 (residues 268–538) and EYA3 (residues 255–536) were produced in Escherichia coli strain BL21(DE3) as N-terminally His-tagged proteins and purified by nickel-nitrilotriacetic acid affinity chromatography. The sequence of the C-terminal H2A.X peptide was CPSGGKKATQASQEY. Phosphate release was measured using the malachite green detection assay. The small interfering RNA (siRNA) against eya3 was a pool of three target-specific 19–25-nucleotide siRNAs (Santa Cruz Biotechnology). As a control, nonspecific non-targeting fluorescein-conjugated siRNAs were used. Cells were transfected 24 h after plating (5 × 103 cells/well in 6-well plates or 1 × 106 cells in a 10-cm culture dish) with up to 30 nm siRNA pool. Total RNA was phenol-extracted, precipitated, and reverse-transcribed to synthesize total cDNA. The resulting cDNA was then used as a template for PCR amplification using specific primers for EYA3 (5′-ATGGAAGAAGAGCAAGATTTACCAGAGC- 3′ and 5′-GAGAAAATCAAGCTCTAAAGCCTGGTG-3′) and the loading control glyceraldehyde-3-phosphate dehydrogenase. We determined the structure of the ED of EYA2 at 2.4 Å resolution by the multiple isomorphous replacement method using two heavy atom derivatives. Details of the structural analysis will be described elsewhere. 5S.-K. Jung, et al., manuscript in preparation. The domain has a typical α/β-hydrolase fold and possesses the three conserved sequence motifs and the bound magnesium ion that are common to the active site of members of the HAD family (3Burroughs A.M. Allen K.N. Dunaway-Mariano D. Aravind L. J. Mol. Biol. 2006; 361: 1003-1034Crossref PubMed Scopus (322) Google Scholar), suggesting that EYA shares a common mechanism with other HADs. A striking feature is that with five aspartate and five glutamate residues exposed around the active site, the surface is predominantly negatively charged (Fig. 1). These residues show 100% sequence conservation among EYA homologs, suggesting the possibility that these phosphatases may recognize basic substrates. A comparison of the surface charge properties of the EYA2 ED with those of PTP1B, the prototype for the family of classical Cys-based PTPs, revealed a striking difference. Unlike the EYA2 ED, the surface of PTP1B, like that of most of the non-transmembrane Cys-based PTPs (15Barr A.J. Ugochukwu E. Lee W.H. King O.N. Filippakopoulos P. Alfano I. Savitsky P. Burgess-Brown N.A. Müller S. Knapp S. Cell. 2009; 136: 352-363Abstract Full Text Full Text PDF PubMed Scopus (353) Google Scholar), is mostly positively charged close to the active site, suggesting differences in substrate recognition by these enzymes and EYA. In light of the highly basic nature of histones and the recent discovery that phosphorylation of the C-terminal tyrosyl residue of histone H2A.X (Tyr-142 in human and mouse H2A.X) may be of regulatory importance (14Xiao A. Li H. Shechter D. Ahn S.H. Fabrizio L.A. Erdjument-Bromage H. Ishibe-Murakami S. Wang B. Tempst P. Hofmann K. Patel D.J. Elledge S.J. Allis C.D. Nature. 2009; 457: 57-62Crossref PubMed Scopus (316) Google Scholar), we decided to test first a matched series of C-terminal H2A.X peptides as substrates. Synthetic peptides with a sequence derived from the C terminus of histone H2A.X were generated in monophosphorylated (Ser-139 or Tyr-142) and dual-phosphorylated (Ser-139 and Tyr-142) forms and tested as substrates of recombinant EYA2 (Fig. 2A) and EYA3 (Fig. 2B). Both enzymes displayed classic Michaelis-Menten kinetics and dephosphorylated phospho-Tyr-142 with similar kinetic constants (EYA2, Km = 1.9 μm and kcat = 5.1 s−1; EYA3, Km = 1.8 μm and kcat = 5.2 s−1). In both cases, there was a dramatic decrease in the efficiency of dephosphorylation of phospho-Ser-139 (EYA2, Km = 80 μm and kcat = 0.9 s−1; EYA3, Km = 72 μm and kcat = 0.9 s−1). Interestingly, as illustrated for EYA3 (Fig. 2B), when the dual-phosphorylated form (phospho-Ser-139/phospho-Tyr-142) of the peptide was tested, an inhibitory effect was observed on both Km and kcat compared with the monophospho-Tyr-142 peptide (EYA2, Km = 3.8 μm and kcat = 2.9 s−1; EYA3, Km = 3.5 μm and kcat = 3.2 s−1). This indicates not only a striking preference of EYA2 and EYA3 for the dephosphorylation of phospho-Tyr-142 but also raises the possibility that the dephosphorylation of the C-terminal tyrosyl residue may be influenced by phosphorylation of the neighboring residue Ser-139 in a "cross-talk" pathway. To investigate the substrate specificity of EYA3, we tested its activity against other phosphotyrosyl synthetic peptides. The monophosphorylated synthetic peptide pYEEY is derived from a well characterized SH2 domain-binding motif from the RNA-binding protein SAM68 (16Lukong K.E. Richard S. Biochim. Biophys. Acta. 2003; 1653: 73-86Crossref PubMed Scopus (211) Google Scholar). This peptide was an efficient substrate for PTP1B, with kcat = 30 s−1. In contrast, EYA3 showed no measurable activity against the pYEEY peptide (Fig. 2C). Similarly, another monophosphorylated synthetic peptide with the sequence pYEEI, which is a ligand for the Fyn SH2 domain (17Songyang Z. Shoelson S.E. Chaudhuri M. Gish G. Pawson T. Haser W.G. King F. Roberts T. Ratnofsky S. Lechleider R.J. Neel B.G. Birge R.B. Fajardo J.E. Chou M.M. Hanafusa H. Schaffhausen B. Cantley L.C. Cell. 1993; 72: 767-778Abstract Full Text PDF PubMed Scopus (2391) Google Scholar), was readily dephosphorylated by PTP1B (kcat = 29 s−1); however, there was no measurable phosphatase activity when the peptide was incubated with EYA3. Similar results were obtained with EYA2. In the converse experiment, PTP1B was tested against the monophosphorylated synthetic peptide (phospho-Tyr-142) derived from histone H2A.X (Fig. 2D). Although PTP1B has been shown to dephosphorylate a wide variety of synthetic peptides in vitro (18Sarmiento M. Puius Y.A. Vetter S.W. Keng Y.F. Wu L. Zhao Y. Lawrence D.S. Almo S.C. Zhang Z.Y. Biochemistry. 2000; 39: 8171-8179Crossref PubMed Scopus (112) Google Scholar), it displayed no measurable activity against the H2A.X phospho-Tyr-142 peptide. Overall, these data suggest that EYA2 and EYA3 have the potential to exhibit selectivity in the dephosphorylation of phospho-Tyr-142 of histone H2AX. In light of the specificity for the H2A.X phospho-Tyr-142 peptide in vitro, we tested the effects of suppressing EYA expression in cells by RNAi. We transfected U2OS cells with siRNA pools (up to 30 nm) against eya3. Analysis by reverse transcription-PCR indicated that transfection of the siRNA pools reduced mRNA expression by ∼80% for eya3 (Fig. 3A). Under these conditions, we tested the phosphorylation status of Tyr-142 of histone H2A.X using a phospho-specific antibody in immunoblots of a total histone preparation (14Xiao A. Li H. Shechter D. Ahn S.H. Fabrizio L.A. Erdjument-Bromage H. Ishibe-Murakami S. Wang B. Tempst P. Hofmann K. Patel D.J. Elledge S.J. Allis C.D. Nature. 2009; 457: 57-62Crossref PubMed Scopus (316) Google Scholar). Interestingly, following suppression of EYA3, the level of H2A.X Tyr-142 phosphorylation was elevated relative to the control, consistent with a link between EYA3 and dephosphorylation of the C terminus of histone H2A.X (Fig. 3B). Furthermore, we tested the effects of DNA damage treatment on the phosphorylation status of H2A.X in the presence and absence of EYA3. In control cells, as expected, we observed that DNA damage induced dephosphorylation of Tyr-142 in H2A.X. In contrast, following suppression of eya3, this dephosphorylation was abrogated (Fig. 3C). Finally, treatment with the eya3 siRNA pools, but not the control siRNA, induced the majority of the cell population to round up and lift off the plate, consistent with cell death. Double-strand breaks (DSBs) are an extremely deleterious form of DNA damage that can lead to losses of large pieces of DNA during cell division. Incorrect repair may generate chromosomal rearrangements and mutations with the potential for cell death and carcinogenesis. Therefore, tight control over the repair process is required. The reversible phosphorylation of histone H2A.X is an important component of the cellular response to DSBs, natural or imposed, in what is beginning to be described as an "epigenetic landscape" for DNA damage and repair (19Kinner A. Wu W. Staudt C. Iliakis G. Nucleic Acids Res. 2008; 36: 5678-5694Crossref PubMed Scopus (835) Google Scholar, 20Downs J.A. Nussenzweig M.C. Nussenzweig A. Nature. 2007; 447: 951-958Crossref PubMed Scopus (247) Google Scholar). A histone octamer, which is composed of two molecules each of histones H2A, H2B, H3, and H4, forms the core of the nucleosome, the basic unit of chromatin. The variant H2A.X comprises ∼10% of the H2A pool in mammalian cells and contains a unique C-terminal segment with sites of regulatory phosphorylation. Within minutes of genotoxic stress, such as ionizing radiation, histone H2A.X becomes phosphorylated at Ser-139 in its C-terminal segment by members of the PIKK family of Ser/Thr kinases, including ATM. Phosphorylated H2A.X decorates a broad region of ∼1 megabase flanking the DNA break, which serves as a platform for recruiting molecules involved in damage repair and signaling. These regions are referred to as γH2A.X foci (19Kinner A. Wu W. Staudt C. Iliakis G. Nucleic Acids Res. 2008; 36: 5678-5694Crossref PubMed Scopus (835) Google Scholar). There is evidence to suggest that this Ser/Thr phosphorylation of H2A.X is reversible and that the histone is dephosphorylated after repair is complete (21Chowdhury D. Keogh M.C. Ishii H. Peterson C.L. Buratowski S. Lieberman J. Mol. Cell. 2005; 20: 801-809Abstract Full Text Full Text PDF PubMed Scopus (424) Google Scholar, 22Kimura H. Takizawa N. Allemand E. Hori T. Iborra F.J. Nozaki N. Muraki M. Hagiwara M. Krainer A.R. Fukagawa T. Okawa K. J. Cell Biol. 2006; 175: 389-400Crossref PubMed Scopus (64) Google Scholar, 23Nakada S. Chen G.I. Gingras A.C. Durocher D. EMBO Rep. 2008; 9: 1019-1026Crossref PubMed Scopus (163) Google Scholar), emphasizing the importance of reversible Ser phosphorylation in the recovery from DNA damage. However, recently a new tier of control over the response to DNA damage has been identified, which involves reversible tyrosine phosphorylation of histone H2A.X (14Xiao A. Li H. Shechter D. Ahn S.H. Fabrizio L.A. Erdjument-Bromage H. Ishibe-Murakami S. Wang B. Tempst P. Hofmann K. Patel D.J. Elledge S.J. Allis C.D. Nature. 2009; 457: 57-62Crossref PubMed Scopus (316) Google Scholar). In mammalian cells, histone H2A.X possesses a tyrosyl residue at its C terminus (Tyr-142), which is constitutively phosphorylated under normal growth conditions (14Xiao A. Li H. Shechter D. Ahn S.H. Fabrizio L.A. Erdjument-Bromage H. Ishibe-Murakami S. Wang B. Tempst P. Hofmann K. Patel D.J. Elledge S.J. Allis C.D. Nature. 2009; 457: 57-62Crossref PubMed Scopus (316) Google Scholar). Following DNA damage, such as in response to ionizing radiation, this residue becomes dephosphorylated, while γH2A.X, the Ser-139-phosphorylated form, appears. Phosphorylation of Tyr-142 is catalyzed by WSTF, a BAZ/WAL chromatin-remodeling factor that has now been shown to possess intrinsic protein kinase activity. WSTF together with SNF2H (an ISWI ATPase) constitutes the WICH complex (WSTF-ISWI ATPase-dependent chromatin-remodeling complex), which binds to histone H2A.X under normal growth conditions and has been implicated in DNA replication. Suppression of WSTF expression by RNAi led to a decrease in the phosphorylation of Tyr-142 in H2A.X, resulting in a rapid decline in the level of γH2A.X after ionizing radiation, unlike in control cells, in which the levels were sustained for hours. The recruitment of proteins critical for formation of γH2A.X, such as MDC1 and ATM, was defective, and the normal progression from small to large γH2A.X foci was not observed in the absence of WSTF. Phosphorylation of Ser-139 and formation of γH2A.X foci were also reduced in cells expressing H2A.X in which Tyr-142 was mutated to Phe or Leu, residues that cannot be phosphorylated, suggesting that the phosphorylation/dephosphorylation of these sites may be coordinated in a poorly understood "cross-talk" pathway. Clearly, the identity of the phosphatase(s) that dephosphorylates Tyr-142 is a critical component of this novel aspect of the DNA damage response, as it would be part of the enzyme system responsible for governing the steady-state balance of this regulatory phosphorylation mark. Our data indicate that EYA3 may function as a PTP in the dephosphorylation of Tyr-142 in histone H2A.X. Whether the other EYA family members (EYA1, EYA2, and EYA4) can also serve this function remains to be established. Our data reveal specificity in the effects of EYA3 on H2A.X. The kcat for dephosphorylation of the monophosphorylated H2A.X phospho-Tyr-142 synthetic peptide was ∼600-fold faster than that for the general phosphatase substrate p-nitrophenyl phosphate. In addition, whereas there was a robust activity against the H2A.X phospho-Tyr-142 peptide, EYA2 and EYA3 did not dephosphorylate other phospho-Tyr peptides modeled on sequences that are known to engage signaling molecules. In contrast, a classical Cys-dependent PTP, PTP1B, readily dephosphorylated these SH2 domain-containing peptide ligands but was inactive against the H2A.X phospho-Tyr-142 peptide. It will be interesting to explore further whether the C-terminal location of the phosphorylation site favors recognition by EYA. Our data also provide further support for specificity of the EYA phosphatases for tyrosyl residues in proteins. In contrast to the phospho-Tyr-142 peptide, EYA displayed substantially lower activity for the same peptide that was phosphorylated at the residue equivalent to Ser-139 of H2A.X. Furthermore, both affinity and turnover were impaired by the presence of phosphate at the seryl residue in the dual-phosphorylated peptide. This raises the possibility that dual phosphorylation of these sites in histone H2A.X may impair the ability of EYA to dephosphorylate the C-terminal tyrosyl residue, perhaps due to repulsive charge interactions between the second phosphorylation site and the acidic surface surrounding the active center of the enzyme. We noted that suppression of eya3 led to increased basal phosphorylation and abrogated DNA damage-induced dephosphorylation of Tyr-142 in histone H2A.X, consistent with a role for EYA3 as a physiological H2A.X phosphatase. We observed that suppression of eya3 by RNAi was accompanied by a pronounced increase in cell death, which has also been reported to accompany suppression of EYA family members in other systems (24Kriebel M. Müller F. Hollemann T. Dev. Dyn. 2007; 236: 1526-1534Crossref PubMed Scopus (21) Google Scholar, 25Xu P.X. Adams J. Peters H. Brown M.C. Heaney S. Maas R. Nat. Genet. 1999; 23: 113-117Crossref PubMed Scopus (570) Google Scholar). Most often, γ-phosphorylation of H2A.X at Ser-139 is equated with "sensing" DSBs, leading to DNA repair in the context of a chromatin template. It is interesting to note, however, that histone H2A.X has not only been shown to function as a "caregiver" of the genome in DNA repair but has also been implicated in the control of apoptosis in a poorly understood response (26Bonner W.M. Redon C.E. Dickey J.S. Nakamura A.J. Sedelnikova O.A. Solier S. Pommier Y. Nat. Rev. Cancer. 2008; 8: 957-967Crossref PubMed Scopus (1273) Google Scholar). Thus, it is possible that the interaction of EYA and H2A.X may play a critical role in directing a cell's decision either to undergo apoptosis or the process of DNA repair. Dephosphorylation of Tyr-142 is important for the formation of γH2A.X foci and the protein-protein interactions that support the process of DNA repair (14Xiao A. Li H. Shechter D. Ahn S.H. Fabrizio L.A. Erdjument-Bromage H. Ishibe-Murakami S. Wang B. Tempst P. Hofmann K. Patel D.J. Elledge S.J. Allis C.D. Nature. 2009; 457: 57-62Crossref PubMed Scopus (316) Google Scholar). It will be important to examine how the activity of EYA is regulated and how the phosphorylation status of Tyr-142 influences the protein-protein interactions at the C terminus of H2A.X that may direct a choice between death and DNA repair. It has been shown that the gene for H2A.X maps to a region of the genome that is frequently mutated or deleted in a variety of cancers, with suggestions of a role as a tumor suppressor (26Bonner W.M. Redon C.E. Dickey J.S. Nakamura A.J. Sedelnikova O.A. Solier S. Pommier Y. Nat. Rev. Cancer. 2008; 8: 957-967Crossref PubMed Scopus (1273) Google Scholar). It would be anticipated that further characterization of the functional interaction between EYA and H2A.X will yield new insights into links between DNA damage, repair, apoptosis, and carcinogenesis. We thank Todd Miller (Stony Brook University) for the gift of pYEEY and pYEEI phosphopeptides.
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