1. Introduction
EYA proteins belong to a family of evolutionarily conserved transcription factors and cofactors, referred to as the Pax-Six-Eya-Dach Network (PSEDN). This network has important roles in the development and homeostasis of various tissues and organs such as eye, kidney, nervous system, ear, and muscle [
1,
2] as well as in limb formation [
3,
4], gonadogenesis [
5], and neurogenesis [
6,
7]. Loss-of-function mutations in the eyes absent genes (
EYA) can lead to several congenital syndromes (cardiofacial syndrome [
8], bronchio-oto-renal syndrome [
9], oto-facio-cervical syndrome [
10], congenital cataract [
11], and late onset deafness [
12]). On the other hand, overexpression of
EYAs has been detected in various types of cancers such as colorectal [
13], breast [
14,
15], and epithelial ovarian cancer [
16], Wilms’ tumor [
17], lung and esophageal adenocarcinoma [
18,
19], and malignant peripheral nerve sheath tumors [
20].
EYA proteins contain specific domains responsible for transactivation [
21] and protein tyrosine phosphatase [
22,
23] activities. The EYA transcriptional co-activator function resides in the N-terminal domain (NTD), which is a region poorly conserved among vertebrates [
1] and absent in plants [
24]. The protein tyrosine phosphatase (PTP) activity is localized in the C-terminal domain and contains characteristic motifs of the haloacid dehalogenase (HAD) superfamily, which makes EYA a member of the phosphatase subgroup of HAD [
2,
22,
23]. In addition to its own tyrosine phosphatase activity, EYA has threonine phosphatase activity but only when interacting with the protein phosphatase 2A (PP2A)-B55α holoenzyme. This interaction proved to play a critical role in c-Myc stabilization and late stage metastasis in the breast cancer model [
25]. There are four human homologous EYA proteins (EYA 1 to 4), which all contain a highly conserved PTP catalytic domain, termed the Eya Domain (ED) and a variable N-terminal region. EYA homologues have been shown to be involved in various diseases. For example, EYA1′s PTP activity has been implicated in breast cancer tumor growth as well as in cellular proliferation through cyclin D1 transcriptional induction [
26]. Similarly, it has been reported that the PTP activity of EYA 1, 2, and 3 is required for transformation, migration, invasion, and metastasis in MCF-7 and MDA-MB-231 breast cancer cell lines [
14]. Despite the large number of reports implicating EYA proteins in pathological conditions, limited information is available regarding their substrates. So far, three physiological substrates for EYA’s PTP activity have been identified: histone H2A.X (phosphotyrosine-pY-142) [
27,
28], estrogen receptor β (pY36) [
29], which both have nuclear localization, and WD repeat-containing protein 1 (WDR1), which is a cytoskeletal protein [
30].
Tyrosine phosphorylation, which is one of the most important post-translational modifications, regulates diverse cellular processes such as growth, proliferation, differentiation, migration, organelle trafficking, and apoptosis [
31,
32,
33]. Dysregulation of tyrosine kinase signaling pathways is one of the leading causes of cancer progression [
34]. For example, c-Src activation has been reported to generate more than 50% of tumors in liver, colon, breast, lung, and pancreas [
35]. Recently, we have demonstrated that c-Src phosphorylates tyrosine residues of human EYA1 and EYA3 to control their nuclear and cytoskeletal localization [
30]. We have also found that EYA1 and EYA3 are capable of autodephosphorylation [
30]. These data indicate a potential implication of EYA tyrosine phosphorylation and autodephosphorylation in regulating physiological processes and contributing to pathological conditions. Thus, EYA proteins have built-in self-regulating capabilities that control their own function. Information on specific phosphorylated residues and the extent to which they are modified is still unknown. Due to the simultaneous action of tyrosine phosphorylation and autodephosphorylation, it is challenging to perform such mapping studies. In this article, we used a combination of native mass spectrometry (MS) [
36,
37] and bottom-up mass spectrometry [
38,
39,
40] to reveal tyrosine phosphorylation and dephosphorylation sites of human EYA3. High resolution native MS enabled us to evaluate the stoichiometry of phosphorylation at the level of the intact protein, whereas bottom-up mass spectrometry allowed us to determine the specific sites of phosphorylation. We show that in vitro Src selectively phosphorylates 13 tyrosine sites in EYA3. Most of them are located within the N-terminal region. Then, we evaluated the contribution of the identified phosphotyrosine residues to overall EYA3 phosphorylation. To determine the biological relevance of the EYA3 phosphorylation/dephosphorylation-cycle, we investigated the proliferation of HEK293T cells overexpressing wild-type EYA3 (EYA3 WT) or an EYA3 mutant, containing tyrosine to phenylalanine (Y → F) mutations of three residues, which we identified as phosphorylation sites (Y77, Y96, and Y237). Expression of this mutant decreased the proliferation rate of HEK293T cells, which reveals a potential role for the phosphorylated sites in cell proliferation. Cell cycle analysis revealed that these residues play a role in the modulation of the cell cycle distribution. Using nano-high performance liquid chromatography with tandem mass spectrometry (nLC-MS/MS) for mapping tyrosine phosphorylation sites, we have identified a novel phosphorylation pattern of EYA3 in HEK293T cells. Overall, our results reveal the complex nature of the balance between EYA3 phosphorylation and autodephosphorylation and are expected to lead to a better understanding of EYA’s role in pathogenesis.
3. Discussion
In this article, we report the detection and detailed analysis of Src kinase-phosphorylated tyrosine residues of EYA3 as well as EYA3′s capacity to autodephosphorylate these sites.
Initially, native MS experiments revealed 12 phosphorylated tyrosine residues on EYA3 D311N. Monophosphorylated EYA3 WT molecules were detected after a 2-h incubation period with Src, which proves the autodephosphorylation capacity of the PTP and also that autodephosphorylation exceeds Src phosphorylation on these tyrosine residues. Other examples of PTPs capable of tyrosine autodephosphorylation include RPTPα [
50], PTP1C [
51], CD45 [
52], and PTP2C (SHP-2) [
53,
54,
55]. Regarding the potential role of tyrosine phosphorylation, it has been reported that this post-translational modification produced specific effects like modulation of PTP activity [
50,
53,
54,
55], which generates binding sites for SH2-containing proteins [
50] or induces various signal transduction processes [
51,
52,
53]. From our perspective, the present work represents the first in-depth analysis of tyrosine phosphorylation and dephosphorylation sites of an EYA homologue.
Native MS analysis of purified EYA3 proteins, pinpointed by nLC-MS/MS experiments (
Figures S1, S2 and S7), revealed several interesting facts: lack of the start methionine, acetylation of the second serine residue, and presence of two chaperons, Skp and DnaK. The first one actually forms a complex with EYA3. The N-terminal methionine cleavage in prokaryotic proteins has been shown to be promoted by the presence of Ser, Ala, Gly, Pro, and Thr residues in the second position [
56]. On the other hand, N
α-acetylation is a rare phenomenon in
E. coli [
57,
58,
59,
60,
61]. N-terminal acetylation of recombinant mammalian proteins expressed in
E. coli has also been reported [
62]. Analysis of different purified EYA3 forms (WT full length, N-terminal and C-terminal) and mutants (D309N, D311N) showed that association with Skp is dependent on the presence of the N-terminal region of the protein (
Figure S19). Accounting for the increased disorder of the N-terminal region of EYA3 (see PrDos [
63] prediction of disordered regions in EYA3 -
Figure S20) as well as for our finding that EYA3 could not be expressed in MC 4100 ΔSkp cells, we can assume that Skp plays a role in the stabilization of the N-terminal region of EYA3. A lack of a stabilizing effect of Skp in MC 4100 ΔSkp leaves disordered regions exposed to cellular proteases, which leads to proteolysis of the expressed EYA3.
The experiment concerning the time-course of the in vitro phosphorylation shows that a total of 11 tyrosine residues were phosphorylated on EYA3 D311N (Y67, Y72, Y77, Y90, Y96, Y105, Y108, Y208, Y237, Y426, and Y532). Furthermore, most phosphorylated residues belong to the N-terminal part of the protein. In the case of EYA3 WT, four tyrosine residues remained phosphorylated (Y77, Y96, Y105, and Y237) even after 6 h, which suggests that these sites have a higher resistance to autodephosphorylation. The fact that, around the 2-h time point, EYA3 autodephosphorylation dominates Src phosphorylation is supported by our native mass spectrometry experiments, in which only EYA3 WT proteoforms with one phosphorylated residue were detected after 2 h of incubation with Src. Moreover, kinetics experiment reveals a steady state process in the case of time-course phosphorylation of EYA3 WT by Src. EYA3 tyrosine phosphorylation seems to be under the dynamic control of three essential factors: (i) time-dependent EYA3 phosphorylation by Src, (ii) EYA3 autodephosphorylation activity, and (iii) moderate increase of EYA3 tyrosine phosphatase activity upon its Src phosphorylation. The situation becomes even more complicated if we take into account that both Src phosphorylation and EYA3 autodephosphorylation are site-specific. This interplay might produce transient modifications of the phosphorylation pattern of EYA3, which is in agreement with modifications observed in our time dependence experiments described in
Figure 3. We consider that the interplay between the mentioned factors confers EYA3 a phosphorylation pattern, which can be modified according to the physiological needs of the cell.
Experiments on the dynamics of Src-dependent phosphorylation of EYA3 D311N, analyzed using nLC-MS/MS, demonstrate that, out of 11 phospho-tyrosine residues, after 4 h of incubation with ED EYA3 WT, only four tyrosine residues remained phosphorylated, which include Y77, Y96, Y237, and Y508. Notably, three of these residues are the most resistant to autodephosphorylation in the experiment described in
Figure 3c. The intensive autodephosphorylation of Y105 outlined in the in vitro phosphorylation time-course at the 2-h time point (
Figure 3c, Src active) explains its eventual dephosphorylation after 2 h of incubation of D311N with ED EYA3 WT (after Src inactivation with PP2) (
Figure 4b). In this time frame (0 to 4 h incubation with ED EYA3 WT), Y508 was shown to be a residue with high resistance to autodephosphorylation (
Figure 4b). In our previously published data [
30], it is characterized as the only autodephosphorylation residue from the ED region of EYA3, which means that, in vivo, this residue is eventually autodephosphorylated. The apparent contradiction can be explained by the fact that, under in vitro conditions, phosphorylated Y508 has been exposed for a limited time to EYA3 autodephosphorylation activity. Under in vivo conditions, this phosphosite is exposed to long-term autodephosphorylation activity, which leads to complete removal of its phosphate residue. In addition, under in vivo conditions, there are other factors (including inhibitors, activators, etc.), which may modulate Src phosphorylation and/ or EYA3 phosphatase activity.
Additional experiments illustrate the significance of Y77 and Y96 on overall EYA3 tyrosine phosphorylation (
Figure 5). Residue Y77 seems to have the highest contribution to the total phosphotyrosine signal followed by Y96. On the contrary, Y237 proved to have a very small contribution to the phosphorylation signal. Variations regarding the phosphorylation pattern of different EYA3 D309N Y→F mutants denote the complex nature of the processes (including phosphorylation) in a mammalian cell line versus in vitro. This was also proven through the results obtained in our proliferation assays and cell cycle analysis. Proliferation assays (
Figure 6d,f) showed that c-Src Y527F co-expression in HEK293T cells induced an increased proliferation that masked the contribution of EYA3 WT to this process. This effect was also illustrated by flow cytometry analysis of the cell cycle. The trend toward an increase in the S and G2/M phases of cells overexpressing EYA3 WT versus mock transfected cells was abolished when EYA3 WT was co-expressed with c-Src Y527F. On the other hand, the MTS assay and cell cycle distribution analysis revealed that, by mutating three tyrosine residues from EYA3 (Y77, Y96, and Y237) to phenylalanine, the pro-proliferative effect of EYA3 was abolished. These results request further investigations in non-transformed as well as cancer cells, where it was reported that EYA proteins promote processes related to tumor progression. It remains to be elucidated which of these three tyrosine residues, if not all of them, are implicated in cell proliferation and what is the molecular mechanism generating this effect.
Comparing the phosphorylation pattern of EYA3 WT co-expressed with c-Src Y527F in HEK293T cells to its pattern when only EYA3 WT was expressed (
Table 1), we noticed that four residues were still phosphorylated in the presence of c-Src Y527F co-expression (Y77, Y108, Y208, and S157). Y77 emerged as a residue with increased resistance to autodephosphorylation. It is the only tyrosine residue detected as phosphorylated in EYA3 WT in the presence of Src, both in vitro as well as in HEK293T cells. Twelve phospho-residues are reported in PhosphoSitePlus
® [
64]: S256, S262, S264, S266, T268, T269, S271, T274, T295, S297, S438, and S472. Among all of them, S438 is the only phospho-residue also detected in our experiments made on HEK293T cells. The multitude and variety of phosphosites detected on EYA3 in different studies suggests that this protein is involved in many cellular processes.
Pertaining to the phosphotyrosine residues identified in this study, an important question could be raised as to which of these sites are functionally relevant. In principle, in vitro identification of a phosphorylation site does not necessarily mean a particular phosphosite has a functional role. On the contrary, the result might be only an experimental artefact. An argument resides in the fact that, while thousands of phosphosites have been identified, only several hundred have a clear function [
65]. Therefore, it is important to predict which potential phosphosites actually have functional roles. In this respect, there are numerous reports proving that functional phosphosites are significantly more conserved than phosphosites with unknown functions [
65]. Apparently, tyrosine residues situated in disordered regions of proteins are frequently off-target phosphorylation sites [
66], whereas it has also been shown that phosphosites are enriched in disordered regions in order to be recognized more easily by the cognate kinase [
67,
68,
69]. According to our protein disorder prediction (
Figure S20), the N-terminal region of EYA3 is a good candidate for both hypotheses. To make a more accurate prediction, we sought to determine the degree of conservation of the identified EYA3 tyrosine phosphorylation residues (i.e., fifteen sites: Y67, Y72, Y77, Y90, Y96, Y105, Y108, Y208, Y237, Y325, Y329, Y426, Y508, and Y532) assuming that functional sites should be conserved to a higher extent. We performed a variability study on a group of 721 homologues of human EYA3, selected from the Representative Proteomes RP55 database [
70] (
Figure 7). This gives us a better view of the conserved residues among the 721 sequences, but, most importantly, the possibility of estimating the degree of conservation for EYA3 tyrosine residues. The fact that, in the N-terminal and ED regions of the protein, tyrosine residues are conserved to a higher extent, means they could be more likely implicated in physiological processes. Individual contributions of these residues definitely warrant further investigation. Y72, Y77, Y90, Y105, Y108, and Y208 display high conservation. The fact that these residues were found as phosphosites in HEK293T cells suggests they have a high probability of being functionally relevant. Phosphoresidues specific to one cell line could be part of signal transduction pathways which, among other mechanisms, lead to different effects on proliferation or other cellular events. The tyrosine residues that were not detected as phosphorylated by Src kinase but have a high degree of conservation could also be functional phosphosites. Further experiments should confirm the reliability of these predictions.
The 13 tyrosine phosphosites detected on EYA3 are not uniformly distributed along its sequence, with the N-terminal part of the protein being preferred for phosphorylation. These residues differ by their time-dependent phosphorylation kinetics, resistance to autodephosphorylation, and contribution to the overall tyrosine phosphorylation of EYA3. Three of these tyrosine residues (Y77, Y96, and Y237) are implicated in EYA3-dependent proliferation events in HEK293T. As supported by our results reported in this article, EYA3 displays a specific, time-dependent phosphorylation pattern that is under the control of Src phosphorylation and EYA3 autodephosphorylation activity. Apparently, this phosphorylation pattern can influence important physiological processes.
4. Materials and Methods
4.1. Antibodies, Enzymes, Reagents, and Kits
Antibodies used in Western blotting: anti-pY (pY99, sc-7020 HRP), anti-GAPDH (FL-335) HRP (sc-25778 HRP), anti-c-Myc (9E10) HRP (sc-40 HRP), and secondary antibodies goat anti-rabbit IgG-HRP (sc-2004) and rabbit anti-mouse IgG-HRP (sc-358914) from Santa Cruz Biotechnology, Dallas, TX, USA, anti-His antibody (27-4710-01), from GE Healthcare, Chicago, IL, USA, anti-v-Src (clone 327) from Merck Millipore, Burlington, MA, USA, and anti-Actin Ab-5 (Clone C4/actin) from BD Biosciences, Bedford, MA, USA. Anti-c-Myc (9E10) (sc-40) from Santa Cruz Biotechnology and anti-myc tag (clone4A6) from Merck Millipore were used for immunoprecipitation (IP). Anti-EYA3 (21196-1-AP) from Proteintech (Rosemont, IL, USA) was used in Western blot and IP experiments. The Western Blotting Chemiluminescence Luminol Reagent (sc-2048) from Santa Cruz Biotechnology, USA was used for detecting immunoreactive bands. GST-v-Src active kinase protein (S19-10G) was purchased from SignalChem Biotech Inc. (Richmond, BC, Canada).
For mass spectrometry, we used LC-MS chromasolv solvents (water, acetonitrile–noted MeCN), ammomium bicarbonate, iodoacetamide (I6125), and dithiothreitol (DTT, 43815) from Sigma-Aldrich (St. Louis, MO, USA), formic acid, eluent additive for LC-MS (56302) from Fluka (Honeywell, Romania), sequencing grade Trypsin, Chymotrypsin, and rLysC from Promega, Madison, WI, USA, C18 bulk (Aqua 5 µm C18 200Å bulk, Phenomenex, Torrance, CA, USA), C8 material (3M™ Empore™ C8 disks), Titansphere™ Phos-TiO Kit, 1mg TiO2/ 10µL tip (5010-21309), GL-Tip SDB (7820-11200), and GL-Tip GC (7820-11201) all from GL Sciences (Tokyo, Japan) and Amicon Ultra-0.5mL Centrifugal Filter Unit with Ultracel-30 membrane, from Millipore (USA).
Other reagents and kits include: QuickChange Lightning Site-Directed Mutagenesis Kit (#210519), from Agilent Technologies (Santa Clara, CA, USA), CellTiter 96® AQueous One Solution Cell Proliferation Assay System (Promega, USA), Vybrant™ CFDA SE Cell Tracer Kit (V12883, Thermo Fisher Scientific, Dreieich, Germany), Protein G Sepharose® 4 Fast Flow (17-0618-01), GE Healthcare Life Sciences (USA) and also Rec-Protein A Sepharose®4B Conjugate (101141), Invitrogen (Carlsbad, CA, USA) - for IP, 3-(N-morpholino)propanesulfonic acid (MOPS, A2947) and phenylmethylsulfonyl fluoride (PMSF, A0999) from AppliChem (Darmstadt, Germany), ethylenediaminetetraacetic acid (EDTA, ED255), adenosine 5′-triphosphate disodium salt (Na2ATP, A3377) and polyethylenimine (PEI, 40872-7) from Sigma-Aldrich (USA), Tris-base (648311) and dimethyl sulfoxide (DMSO, 317275) from Calbiochem (San Diego, CA, USA), NP-40 (A1694) from Panreac AppliChem (Germany), protease inhibitor cocktail tablets (sc-29131), and sodium orthovanadate (sc-3540) from Santa Cruz Biotechnology, USA, iodoacetic acid (35603, Pierce™, USA), NativeMark™ Unstained Protein Standard and Spectra™ Multicolor Broad Range Protein Ladder (Thermo Fisher Scientific, Dreieich, Germany), Low Molecular Weight Marker (GE Healthcare, USA), BLUelf Prestained Protein Ladder (GeneDireX-PM008-0500, Taiwan, China), RNAse (R6513, Sigma-Aldrich, USA), propidium iodide solution (S7112, Sigma-Aldrich, USA), and sodium citrate (S4641, Sigma-Aldrich, USA).
The cell culture includes Dulbecco’s Modified Eagle Medium (DMEM) (4.5 g/L D-Glucose)-GlutaMax™ media (61965059), Fetal Bovine Serum (FBS) (16000044), 100x MEM Non-Essential Amino Acids solution (NEAA) (11140), 100 mM sodium pyruvate (11360070), and 1x phosphate-buffered saline (PBS) (18912-014), which were all from Gibco (Thermo Fisher Scientific, Dreieich, Germany).
4.2. Cell Culture, Transfection, Immunoprecipitation and Western Blotting
HEK293T cells were cultured at 37 °C in a 5% CO2 atmosphere incubator, in DMEM (4.5 g/L D-Glucose)-GlutaMAX™ media, which also contained 10% heat inactivated FBS, 1 mM sodium pyruvate, and 1x NEAA.
The cells were passaged 24 h before transfection, so they would be at 70% confluency the day of the experiment. Transient co-transfections were carried out using PEI as a transfection reagent.
Unless stated otherwise, cell harvesting and lysis were performed as described. The cells were washed two times with ice-cold PBS 1× and then harvested on ice in 50 mM Tris-HCl pH 7.4, 150 mM NaCl, 10 mM MgCl2, 1% NP-40, 10% glycerol, 1× protease inhibitor cocktail, 1 mM PMSF (lysis buffer) supplemented with 1 mM sodium orthovanadate and 5 mM iodoacetic acid and lysed by passing the cellular suspension three times through a syringe with a narrow inner diameter, vortexing and incubation for 20 min on ice. After 25 min of centrifugation at 20,000× g at 4 °C, the soluble cellular extract was collected, and the total protein concentration was determined by the bicinchoninic acid (BCA) assay. Western blot analyses of the lysates were carried out to verify the expression of each of the proteins of interest (endogenous EYA3, c-myc tagged EYA3, Src, and loading control—G-actin or GAPDH) using anti-EYA3 (21196-1-AP) with goat anti-rabbit IgG-HRP (sc-2004), anti-c-Myc-HRP (sc-40 HRP), anti-v-Src (clone 327), and anti-actin (Ab-5, Clone C4/actin) both with rabbit anti-mouse IgG-HRP (sc-358914), and anti-GAPDH-HRP (sc-25778 HRP) antibodies.
IP of c-myc tagged EYA3 or the endogenous EYA3 protein was achieved by incubating the lysates with anti-c-Myc (9E10, sc-40) or anti-EYA3 (21196-1-AP) antibody for 1 h at 4°C, with rotation, which was followed by the addition of Protein G or Protein A Sepharose beads and further incubation with a rotation for 16 h at 4 °C. The next day, beads were washed three times with 50 mM Tris-HCl pH 7.4, 150 mM NaCl, 10 mM MgCl2, 1% NP-40, and 10% glycerol (wash buffer). Proteins were eluted by adding 1x Laemmli sample buffer and incubating the beads for 5 min at 95 °C. The eluates were analysed by a Western blot using the following antibodies: anti-pY (pY99, sc-7020 HRP), then anti-c-Myc (sc-40 HRP) for c-myc tagged EYA3, respectively, and anti-EYA3 antibody (21196-1-AP) with goat anti-rabbit IgG-HRP (sc-2004), for endogenous EYA3. All statistical analyses were conducted using GraphPad Prism6.
Evaluation of the tyrosine phosphorylation of EYA3 D309N Y → F mutants. Transient transfections were performed in six well plates. The pCS2-MT vector containing the sequence for myc-tagged EYA3 D309N, its corresponding Y → F mutants or EYA3 WT, along with pSLX-c-Src Y527F, were transfected at a ratio of 5 to 1 (2 µg: 0.4 µg). The EYA3 D309N Y → F mutants were obtained by site directed mutagenesis (QuickChange Lightning Site-Directed Mutagenesis Kit). After 24 h, the cells were harvested, lysed, and Western blot analyses of the lysates were performed using anti-c-Myc (sc-40 HRP), anti-v-Src (clone 327), and anti-Actin Ab-5 (Clone C4/actin) with rabbit anti-mouse IgG-HRP (sc-358914) as a secondary antibody. Transiently expressed c-myc-EYA3 protein was immunoprecipitated using the anti-c-Myc (9E10) antibody. The resulting samples were analysed by a Western blot using anti-pY (pY99, sc-7020 HRP) and anti-c-Myc (sc-40 HRP) antibodies. Quantifications were conducted using ImageJ software [
45]. Three independent experiments were performed.
The following describes the analysis of endogenous EYA3 tyrosine phosphorylation. Before transfection, cultured HEK293T cells were passaged in 25 cm2 flasks. Cells received the following treatment: transfection reagent (PEI) and transient transfection with empty vector (pCiNeo) and pSLX-c-Src Y527F, respectively. Forty hours post-transfection, the cells were harvested and lysed. Following Western blot verification, lysates were subjected to IP using anti-EYA3 antibody (21196-1-AP). The resulting samples were analyzed by a Western blot using anti-pY (pY99, sc-7020 HRP) and anti-EYA3 antibody (21196-1-AP) with goat anti-rabbit IgG-HRP (sc-2004). Two independent experiments were performed.
The following describes the inhibition of endogenous EYA3 PTP activity. Cells were cultured in the same conditions as in the previous experiment. Two flasks were transfected with vector pSLX-c-Src Y527F. Twenty-four hours after having induced transient expression of c-Src Y527F, one of the flasks was treated with 10 μM BB (solubilized in DMSO) and the other one with the corresponding volume of DMSO. In addition, 24 h after treatment, cells were harvested, lysed, and lysates were analysed by Western blotting using anti-EYA3 (21196-1-AP) with goat anti-rabbit IgG-HRP (sc-2004), anti-v-Src (clone 327) with rabbit anti-mouse IgG-HRP (sc-358914), and anti-GAPDH-HRP (sc-25778 HRP) antibodies. The IP of endogenous EYA3 and its Western blot analysis was performed in the same manner as in the previous experiment. Two independent experiments were performed.
All immunoreactive bands were detected by chemiluminescence using Western Blotting Chemiluminescence Luminol Reagent (sc-2048) from Santa Cruz Biotechnology, USA.
4.3. Expression and Purification of N-Terminal His-Tagged EYA3 Proteins
Full length proteins (6xHis-EYA3 WT, 6xHis-EYA3 D309N, and 6xHis-EYA3 D311N) as well as the N-terminal and C-terminal fragments of EYA3 protein (6xHis-ΔED EYA3 WT, 6xHis-ED EYA3 WT) were expressed in a prokaryotic system (
E. coli BL21(DE3)RIL strain) and purified following the protocols described in previous studies [
30,
71].
4.4. In Vitro Src Kinase Reactions
Kinase reactions were performed at 30 °C in 25 mM MOPS pH 7.2 with 10 mM MgCl2 and 0.25 mM DTT (kinase assay buffer). The molar ratio between the EYA3 protein and GST-v-Src was 50:1. Na2ATP was added in a 150-fold molar excess compared to the concentration of EYA3. Reactions were quenched on ice by the addition of EDTA so that the molarity in the reaction was five times that of MgCl2.
4.5. Native MS
The following describes the analysis of purified 6xHis-EYA3 WT and 6xHis-EYA3 D311N. Proteins stored at −80 °C were thawed on ice and then centrifuged at 20,000× g for 20 min at 4 °C. The soluble protein sample was buffer exchanged to ammonium acetate at a pH of 6.8. Each protein sample was subjected to native mass spectrometry analysis, ESI-MS, using the Orbitrap™ Exactive™ Plus EMR (Thermo Fisher Scientific, Germany) instrument. Interpretation of the raw data obtained was conducted using Xcalibur™ Software (Thermo Fisher Scientific) and MassLynx™ v4.1 Software (Waters Corporation, Milford, MA, USA). In vitro v-Src phosphorylated 6xHis-EYA3 WT and 6xHis-EYA3 D311N (for 5 min and 2 h) were also analyzed as described above.
The following describes the native MS analysis of direct confirmation of dephosphorylation. His-tagged EYA3 D311N in vitro tyrosine phosphorylation by GST-v-Src was performed as described earlier. Samples were taken 5 min and 2 h after the addition of v-Src. The remaining reaction was split in half with one sample being further incubated at 30 °C and the other one was incubated at the same temperature with EYA3 WT in a ratio of 5.61:1 for 2 more hours. All reactions were quenched on ice by adding EDTA to a final concentration that was five times higher than that of MgCl2. The buffer was exchanged to ammonium acetate pH 6.8 and samples were analyzed by native ESI-MS (Orbitrap™ Exactive™ Plus EMR, Thermo Fisher Scientific, Germany). Raw data were acquired and analyzed using Xcalibur™ Software (Thermo Fisher Scientific). The peaks corresponding to 6xHis-EYA3 D311N molecules carrying charge 16+ were used to quantify the evolution of tyrosine phosphorylation. The intensity of each peak was taken into consideration for quantification. The acetylated molecules were separately quantified. The data were quantified by taking into consideration the signal for EYA3 WT added in one of the samples (dephosphorylation reaction).
4.6. nLC-MS/MS Detection of Phosphorylated Residues and Mass Spectrometry Data Analysis
4.6.1. Detection of Phosphorylated and Autodephosphorylated Tyrosine Residues
6xHis-EYA3 WT and, respectively, 6xHis-EYA3 D311N were incubated either with ATP or with GST-v-Src and ATP, at 30 °C. The kinase reactions were performed as previously mentioned, with 10 µM EYA3 in a 10-µL reaction volume. The reactions were quenched with EDTA, at several time points: 5 min, 30 min, 2 h, and 6 h, and then stored at −80 °C. Regarding the in-solution digestion, after thawing them on ice, the same volume from a buffer containing 8 M urea in 100 mM Tris, pH 8 was added to the samples in order to aid the digestion. Reduction was performed by incubation in 2 mM DTT final concentration for 40 min at 60 °C and alkylation was achieved by incubating in 5 mM iodoacetamide for 40 min, at 25 °C, in the dark. Before adding the protease, the samples were diluted with 100 mM Tris pH 8 to a final volume of 100 µL and the EDTA concentration was adjusted to 20 mM in order make sure Mg
2+ ions (now at 2.5 mM) were complexed, which inhibits any phosphorylation or autodephosphorylation (EYA’s cofactor is Mg
2+) from taking place. Sequencing grade chymotrysin was added in a 1:100 (
w/w) ratio with EYA3 protein and incubated for 15 h at 25 °C. The resulting samples were acidified to 10% formic acid (FA) and peptides were desalted using tips with C18 bulk plugged with C8 material, manufactured in the laboratory. After elution from the tips in 100% MeCN, peptides were dried in a vacuum concentrator to be stored at −80 °C until nLC-MS/MS analysis. In nLC-MS/MS analysis, the samples were analyzed by reversed phase nLC-MS/MS using an LTQ-Orbitrap Elite™ mass spectrometer coupled to a Proxeon EASY-nLC 1000 (nano-high performance liquid chromatograph, Thermo Fisher Scientific). Peptides were resuspended and loaded on the C18 analytical column (75 μm particles) in solvent A (0.1% (
v/v) FA in H
2O) and the elution was achieved over a 55-min gradient of solvent B (0.1% (
v/v) FA in MeCN). Precursor and fragment ion spectra were acquired in the Orbitrap analyzer. Gas phase fragmentation was obtained by applying HCD on the 10 most abundant precursor ions, rejecting those that had a charge state of 1+. Three technical replicates were performed from each sample (
n = 3). Peptides were identified using Proteome Discoverer v1.4 (Thermo Fisher Scientific) with Sequest HT as an algorithm and the precursor ions area detector node was used to determine the area of each peptide ion. The searches were performed using a modified version of the Human database from UniProtKB/ Swiss-Prot, containing the sequences of purified recombinant EYA3 proteins, and Skp and DnaK. Other settings included: chymotrypsin (full) as enzyme, with a maximum of five missed cleavages [
72] (we found that it did not cut at C-terminal of pY), precursor mass tolerance of 10 ppm, fragment mass tolerance of 0.05 Da, serine, threonine, and tyrosine phosphorylation (+79.966 Da) and also methionine oxidation (+15.995 Da) as dynamic whereas carbamidomethylation (+57.021 Da) on cysteine residues as static modification. A decoy database search having reversed protein sequences was enabled. Although validation of the identified peptide spectrum matches (PSMs) was conducted using the target decoy PSM validator node, which allows for a false discovery rate (FDR) of 1%, and the phospho RS 3.1 [
73] node was introduced in the workflow for phosphorylation site assessment, phosphorylated peptides were also manually checked. Thus, some were accepted as tyrosine phosphorylated even if they would not fit in the FDR threshold of 1% and/or would not have a value for phosphoRS Site Probabilities above 75%. For a peptide to be designated as tyrosine phosphorylated, it was imperative that the b and/or y ions from the phosphorylation site (several fragment ions before, with and after the respective phosphorylated tyrosine) and also other fragment ions that would prove the specific phosphorylation at that residue and not at other eventual serine or threonine residues, should be identified. The presence of the characteristic immonium ion of phosphotyrosine [
74,
75] was also taken into consideration when designating a peptide as being phosphorylated at a tyrosine residue (only in this experiment, because HCD fragmentation was implemented in the MS analysis). Regarding relative quantification and normalization, since we detected chymotrypsin could cut after F, Y, W, and L, but not after pY, and this generated heterogenous phosphopeptides (different length, charge, even triple-phosphorylated forms), we chose a representative peptide for every phosphotyrosine residue, which had only that residue phosphorylated. Only a single charge of the peptide was chosen for relative quantification. In the cases where peptides contained methionine (same sequence and charge), both oxidized and unoxidized forms were taken into consideration. The oxidized forms manifested the same phosphorylation trend. Relative quantification was used to characterize the evolution of phosphorylation for a certain tyrosine residue. For each technical replicate, the AUC (area under the curve) of peptides corresponding to identified phospho-tyrosine residues were normalized to the AUC of a peptide that could not be phosphorylated in these in vitro reactions and did not contain C or M residues (susceptible to modification). For normalization of EYA3 D311N in vitro phosphorylation, the peptide GVTGQTNSDAESTTL (charge 2+, belonging to EYA3 D311N) was used. For normalization of EYA3 WT in vitro phosphorylation, peptide ENAEGDRTTPSIIAY (charge 2+) from DnaK (DnaK—copurified from E. coli, low amount in sample—is not phosphorylated by Src) was used. The mean value was determined from three technical replicates. Then, for each tyrosine residue, the average values obtained (at 5 min, 30 min, 2 h, and 6 h) were normalized against the highest average value for that certain residue. The values shown in the bar graphs represent these last values obtained (percent from a maximum) with the standard deviation for the three technical replicates also calculated and shown (did not exceed 10.3297) (
n = 3).
4.6.2. In Vitro Tyrosine Phosphorylation and Dephosphorylation of 6xHis-EYA3 D311N
Purified 6xHis-EYA3 D311N was subjected to a kinase reaction using GST-v-Src. The molar concentration of EYA3 D311N in all reactions was 10 µM. All reactions were stopped with 5× Laemmli sample buffer. The first sample was taken and quenched after 5 min of incubation and the second sample was taken after 90 min of incubation. At this time point, PP2 was added so its concentration would be 1 mM and the reaction was incubated at 30 °C for another hour. The reaction was divided in four equal reactions after this hour. One was stopped with 5× Laemmli sample buffer whereas the other three were treated with purified 6xHis-ED EYA3 WT, in twice the molar concentration of EYA3 D311N, and incubated for 0, 2, and 4 h at 30 °C. The concentration of PP2 in these four reactions became 0.5 mM. The maximum concentration of DMSO (the solvent for PP2) in every reaction was 10%, in order to avoid ED EYA3 WT inactivation. Tyrosine phosphorylation of EYA3 D311N in every sample was verified by Western blot analysis using anti-pY antibody and loading with the anti-His antibody. Two control reactions were performed by incubating EYA3 D311N with and without ATP for 2 h at 30 °C in a kinase assay buffer. Two independent experiments were performed. Within in-gel digestion, samples were migrated through SDS-PAGE and stained with Coomassie Brilliant Blue. Bands containing EYA3 D311N from each time point were subjected to an in-gel digestion protocol with 10 ng/µL sequencing grade chymotrypsin, at 25 °C for 16 h, as previously described [
76,
77]. In the nLC-MS/MS analysis, the peptides were resuspended in solvent A (0.1% FA in 2% MeCN) and then injected into an nLC (EASY nLC II, Thermo Fisher Scientific, Germany) that was coupled online with an LTQ™ - Orbitrap Velos Pro™ mass spectrometer (Thermo Fisher Scientific). First, the peptides were trapped and desalted on a C18 (2 cm × 100 µm) column (Thermo Fisher Scientific), and then separated on an analytical C18 column (10 cm × 75 µm) (Thermo Fisher Scientific) prior to MS/MS analysis. A 40-min gradient of 2% to 30% solvent B (0.1% FA in 98% MeCN) with a flow rate of 300 nL/ min was implemented for the elution of the peptides. The acquisition method is comprised of a precursor ion scan (300–1650 m/z interval, resolution of 30,000 at 400 m/z) followed by a data-dependent analysis of the five most intense peaks from the survey scan (having charges +2, +3, and higher) by CID and ETD. For the MS1 survey scan, the lock mass option was set to ON and dynamic exclusion had the following settings: repeat count = 1, repeat duration = 30 s, exclusion list size = 500, exclusion duration = 30, and exclusion relative to reference mass = 10 ppm. The mass spectra of the precursor ions were acquired in the profile mode, in the Orbitrap, whereas the fragment ions spectra were obtained in centroid mode, in the linear ion trap. Data analysis and interpretation were performed as described in the previous experiment, with the following modifications: the sequences for 6xHis-EYA3 WT and D311N without the start methionine, Skp without the first 20 amino acids and DnaK, without the start methionine were introduced in the Human database from UniProtKB/ Swiss-Prot, the fragment mass tolerance was set to 0.6 Da, and a maximum of six missed cleavages were set for chymotrypsin (full). For each technical replicate, a normalization of the AUC of peptides corresponding to each phosphorylated tyrosine residue identified was achieved against the AUC of a peptide from EYA3, GVTGQTNSDAESTTL (charge 2+). This peptide does not contain any tyrosine residues so it could not be phosphorylated by Src kinase in the in vitro experiment described and does not contain C and M, which are residues susceptible to modification. The values resulted after normalization of the two technical replicates were averaged. Then, for each residue, the average values obtained at every time point of the experiment were normalized against the highest average value for that certain residue (noted ”percent from a maximum”). The bar chart shows the mean ± SEM corresponding to two independent experiments (each having two technical replicates,
n = 4).
4.6.3. Detection of Phosphorylated Residues of Transiently Expressed EYA3
HEK293T cells were passaged 24 h before transfection. In the case where only a pCS2-MT vector encoding for EYA3 WT, EYA3 D309N, or EYA3 D309N Y77F was transfected, 22 µg of DNA were used. When pCS2-MT EYA3 vector was co-transfected with the pSLX-c-Src Y527F vector, a ratio of 1:1 between the two vectors was used, with 18 µg total DNA quantity. The cells were grown for 40 h, (changing the media 24 h post-transfection) and then harvested and lysed. The total protein concentration of the soluble protein extract was measured. The expression for each of the proteins of interest (c-myc-EYA3, c-Src Y527F) and for the loading control (GAPDH) was determined by Western blot using anti-c-Myc, anti-v-Src, and anti-GAPDH antibodies. IP of c-myc tagged EYA3 protein was performed using an anti-c-Myc (9E10, sc-40) antibody, as previously described. The resulting samples were first analysed by Western blot using anti-c-Myc and anti-pY antibodies. In-gel digestion was performed as described earlier. The phosphopeptide enrichment for every peptide sample was performed using Titansphere™ Phos-TiO Kit with 1 mg of TiO
2/ 10 µL tip (GL Sciences, Tokyo, Japan), according to the manufacturer’s specifications (lactic acid to inhibit non-specific adsorbtion of peptides and elution made first with aqueous ammonia solution followed by pyrrolidine solution) [
78,
79]. The enriched phosphopeptides were further cleaned-up and desalted using reversed solid phase extraction materials such as styrene divinylbenzene (GL-Tip SDB, GL Sciences) and graphite carbon (GL-Tip GC, GL Sciences) using the protocol described by the manufacturer. The eluates were dried in a vacuum concentrator and stored at −20 °C. nLC-MS/MS analysis was performed as previously described (
Section 4.6.2 In vitro tyrosine phosphorylation and dephosphorylation of 6xHis-EYA3 D311N). Two technical replicates were performed from each sample. Data analysis and interpretation were performed as described in the first experiment, with some observations. Regarding ETD, the Non-fragment Filter processing node was introduced in the workflow, having set the removal of precursor peaks within a 4 Da mass window. The removal of charge reduced precursors within a 2 Da mass window and the removal of neutral losses reduced charge-reduced species within a 2 Da mass window (maximum neutral loss mass set to 130 Da). The peaks generated from the two activation types (CID and ETD) were searched using Sequest HT, against the UniProtKB/ Swiss-Prot database in which the sequences for several c-Myc tagged EYA3 proteins (WT, D309N, D309N Y77F) were introduced. The searches were performed with a set of three missed cleavages for chymotrypsin (full) and the fragment mass tolerance at 0.6 Da.
4.7. In Vitro PTP Assay
HEK293T cells were transiently transfected with vectors encoding for myc-tagged EYA3-WT, D309N, WT T2, and WT T9. Cells were harvested 24 h post-transfection and then lysed. The total protein concentration of soluble extract was measured. Myc-tagged EYA3 as well as anti-actin Western blot experiments verified EYA3 expression in the lysates. EYA3 IP followed, by incubating the soluble protein extract with anti-c-Myc antibody for 1 h at 4 °C and then adding 10 µL of Protein G Sepharose
® and overnight incubation on a rotator at 4 °C. The next day, the PTP assay was performed, according to the protocol described by Lorenz U [
80]. Beads were washed (10 min, rotator, 4 °C) three times with 20 mM HEPES pH 7 buffer containing 10 mM MgCl
2 and two times with phosphatase assay buffer (20 mM MES pH 6.5, 100 mM NaCl, 10 mM MgCl
2, and 5 mM DTT). Each sample was incubated with 10 mM pNPP in a final volume of 100 µL phosphatase assay buffer. After 30 min at 37 °C with mixing, 500 µL of 3N NaOH were added to stop the reactions and the samples were kept for 5 min at RT and then centrifuged 1 min at 13,000 rpm. The absorbance of the supernatant was measured in triplicate, at 405 nm, in a 96-well microplate using FLUOstar Omega (BMG Labtech, Ortenberg, Germany) microplate reader.
4.8. Kinetics of EYA3 WT
First, the bacterially expressed and purified 6xHis-EYA3 WT was incubated either alone, with ATP, with Src kinase, or with ATP and Src, for 2 h at 37 °C. All reactions were performed in a buffer containing 20 mM MES pH 6.5, 100 mM NaCl, 10 mM MgCl2, and 5 mM DTT. In the kinase reactions, the ratio between EYA3 and GST-v-Src was 25:1 (w/w) (4 µg of 6xHis-EYA3 WT and 0.16 µg GST-v-Src). ATP concentration was 100 µM. The total reaction volume was set at 100 µL.
Next, for the study of Src-phosphorylated or unphosphorylated EYA3 WT activity, pNPP was used as a substrate. Briefly, 20 µL from the previous reaction were incubated with 10 mM pNPP in a final volume of 100 µL. The same buffer as in the first reaction was used. The product formed, para-nitrophenol, was measured at 405 nm for 1 h at 37 °C using a spectrophotometer (FLUOstar Omega Microplate reader, BMG Labtech, Germany). Three technical replicates were made (n = 3).
4.9. MTS Assay
The proliferation assay was performed using a colorimetric method, the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) reagent, and an electron coupling reagent, phenazine ethosulfate—PES (CellTiter 96® AQueous One Solution Cell Proliferation Assay). The cells (in six well plates) were transiently transfected with the pCS2-MT empty vector, pCS2-MT EYA3 WT, or pCS2-MT EYA3 WT T2 either alone (3 µg of DNA/ well) or with pSLX c-Src Y527F vector—the DNA ratio between pCS2-MT (EYA3) and pSLX c-Src Y527F was 5:1 (2 µg:0.4 µg). After 24 h, cells were detached, counted, and plated at 10,000 cells/ 100 µL cell growth media, in a 96-well plate. The plate was incubated for 48 h at 37 °C in a humidified, 5% CO2 atmosphere. The rest of the cells were analysed by Western blot in order to verify that the proteins of interest (myc-tagged EYA3, c-Src Y527F) were expressed. After the incubation period, 20 µL of CellTiter 96® AQueous One Solution Reagent were added and the plate was incubated in the same conditions for up to 4 h. The absorbance at 490 nm was measured in a 96-well plate reader—FLUOstar Omega, BMG Labtech—with the following absorbance spectrometer-based technical specifications: OD range of 0 to 4 OD, accuracy <1% at 2 OD, precision <0.5% at 1 OD, and <0.8% at 2 OD. The values introduced in the calculations were obtained after 2 h of incubation with the MTS reagent. The coefficient of determination (R2) was verified 48 h after plating from 2500 up to 12,500 cells/well. Even after 2 h and 30 min of incubation with the MTS reagent, the R2 value was 0.9912.
4.10. Cell Cycle Analysis
HEK293T were transfected either with an empty vector (pCS2-MT), a vector encoding for c-myc tagged EYA3 WT or EYA3 WT T2, alone or with a pSLX c-Src Y527F or pSLX c-Src Y527F K295R vector. Twenty-four hours post-transfection, cells were harvested with trypsin, counted, and 2 × 105 cells were used for flow cytometry, with the rest being lysed for Western blot analysis using anti-c-myc, anti-v-Src, and anti-GAPDH antibodies. For flow cytometry, cells were washed with 1 mL of FACS buffer (2% FBS in PBS) and fixed in cold absolute ethanol while vortexing. Cells were stored at −20 °C until staining. Before analysis, they were washed two times with PBS and then stained with propidium iodide (0.05 µg/µL) in a solution containing RNAse A (0.5 ng/µL) and 38 mM citrate buffer. Cells were kept for 30 min at RT in the dark before acquisition on the FACSVerse™ instrument (BD Biosciences, USA). The gating strategy excluded cell debris and doublets. The G0/G1, S, and G2/M phases of the cell cycle were manually assessed. The gating strategy and data processing were performed using the FACSuite software (BD Biosciences).
4.11. CFSE Assay
The cultured HEK293T cells were harvested with trypsin, counted, then centrifuged at 300× g for 5 min at 25 °C, and washed once with 0.1% BSA in PBS. After resuspending them in prewarmed 0.1% BSA in PBS, approximatively 2 × 105 cells were taken to be measured at FACSVerse™ instrument (BD Biosciences) as a negative control for the CFDA SE (CFSE) staining. The rest of the cells were subjected to staining with 10 µM CFDA SE (Invitrogen), according to the manufacturer recommendations. Lastly, cells were resuspended in prewarmed complete growth media and plated in six well plates, in order to reach approximately 70% confluency. An aliquot was immediately acquired as day 0 reference. After 4 h, cells were transfected either with an empty vector (pCS2-MT), a vector encoding for c-myc tagged EYA3 WT or EYA3 WT T2, alone or with pSLX c-Src Y527F or the pSLX c-Src Y527F K295R vector. Forty-eight hours post-transfection cells were harvested with trypsin, centrifuged at 300× g for 5 min, at 4 °C, washed with 1 mL of FACS buffer (2% FBS in PBS), and then resuspended in 1 mL of the same type of buffer, to be measured on the flow cytometer. Gating strategy excluded cell debris and doublets. CFSE dilution in the proliferating cell pool was assessed as a decrease in median fluorescence intensity (MFI). The remaining cells were lysed for Western blot analysis using anti-c-myc, anti-v-Src, and anti-GAPDH antibodies. FACS analysis was performed using the FACSuite software (BD Biosciences).
4.12. Bioinformatic Sequence Analysis
The variability of tyrosine residues in EYA3 homologues was assessed starting from a set of 721 homologues identified in Reference Proteomes database RP55 [
70] using the Jackhmmer algorithm from the HMMER webserver [
81]. A number of three iterations were enough for the search to converge at a 10
−4 e-value threshold using a BLOSUM62 matrix, while the rest of the parameters were kept at the default values.
The set of 721 homologous proteins identified were realigned with MAFFT [
82] using Unipro UGENE v1.22.0 suite [
83], with the default and recommended parameters. A sequence conservation logo of the relative entropy of each amino acid position in the alignment was computed using the Schneider and Stephens approach of defining sequence variability [
84] as implemented in the WebLogo suite [
85].
4.13. Mass Spectrometry Data Deposition
The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE [
86] partner repository with the dataset identifiers PXD016518, PXD016520, PXD016526, PXD016532, and PXD016596.