Src inhibitors modulate frataxin protein levels

Abstract

Defective expression of frataxin is responsible for the inherited, progressive degenerative disease Friedreich's Ataxia (FRDA). There is currently no effective approved treatment for FRDA and patients die prematurely. Defective frataxin expression causes critical metabolic changes, including redox imbalance and ATP deficiency. As these alterations are known to regulate the tyrosine kinase Src, we investigated whether Src might in turn affect frataxin expression. We found that frataxin can be phosphorylated by Src. Phosphorylation occurs primarily on Y118 and promotes frataxin ubiquitination, a signal for degradation. Accordingly, Src inhibitors induce accumulation of frataxin but are ineffective on a non-phosphorylatable frataxin-Y118F mutant. Importantly, all the Src inhibitors tested, some of them already in the clinic, increase frataxin expression and rescue the aconitase defect in frataxin-deficient cells derived from FRDA patients. Thus, Src inhibitors emerge as a new class of drugs able to promote frataxin accumulation, suggesting their possible use as therapeutics in FRDA.

Introduction

Friedreich's Ataxia (FRDA) is an autosomal recessive disorder characterized by progressive degeneration of the central and peripheral nervous systems, cardiomyopathy and increased incidence of diabetes mellitus. FRDA is caused by homozygous hyperexpansion of GAA triplets in intron 1 of the frataxin gene on chromosome 9q21 (1). Pathological GAA expansions severely reduce transcription of the FXN gene. Frataxin is an extremely conserved mitochondrial protein synthesized as a cytosolic 210 amino acid precursor, which is then imported into mitochondria following a two-step proteolytic maturation by a mitochondrial processing peptidase (2,3). Low levels of expression of frataxin are responsible for all clinical and morphological manifestations of FRDA (4). In fact, frataxin deficiency in humans critically affects survival of large primary neurons of the dorsal root ganglia, cardiomyocytes and pancreatic β-cells. As a consequence of dysregulated mitochondrial metabolism, frataxin-defective cells have indeed reduced activity of iron sulfur cluster (ISC)-containing enzymes, a general imbalance in intracellular iron distribution, reduced ATP content and increased sensitivity to oxidative stress with increased ROS generation. Low frataxin levels and disease severity have been correlated (5). Moreover, frataxin levels are not only crucial for cell survival but also for stress handling responses (6–11). There is no current successful treatment, but considering that the frataxin coding sequence is intact in most of the patients, therapies aiming at enhancing frataxin levels are now being considered (11–15). Frataxin protein levels are controlled by the proteasome upon ubiquitination of target residue, K147, on frataxin (16). This lysine represents a crucial site for frataxin stability because a frataxin mutant lacking K147 cannot be ubiquitinated and is more stable. Therefore, preventing ubiquitination on K147 is expected to grant frataxin an increased stability and a prolonged half-life (16). Ubiquitination and phosphorylation are post-translational modifications (PTM) that often interact dictating the fate of proteins (17). In addition, PTM have emerged as powerful modulators of metabolic pathways (18) and are important regulators of mitochondrial functions (19). Moreover, Src tyrosine kinase family members such as Lyn, Fgr, Fyn and c-Src have also been reported to associate with mitochondria (19). Considering that Src tyrosine kinases can be regulated by a variety of important mitochondrial signals such as ATP levels and redox state (20), which are indeed dysregulated in FRDA, we sought to investigate whether frataxin levels could in turn be modulated by phosphorylation. In this study, we report that frataxin is phosphorylated on Y118 by Src kinase. Interestingly, non-phosphorylatable frataxin-Y118F mutant is less ubiquitinated and blocking Src activity with specific inhibitors increases frataxin levels. Accordingly, Src inhibitors are ineffective in human cells in which a frataxin-Y118F mutant was expressed. Moreover, Src inhibitors induce frataxin expression and rescue the aconitase defect in cells derived from FRDA patients. Therefore, Src inhibitors can promote frataxin accumulation in living cells, strongly supporting their potential therapeutic application.

Results

Src kinase triggers frataxin phosphorylation

To assess whether frataxin could be a substrate for non-receptor tyrosine kinases, frataxin was transiently transfected in human embryo kidney (HEK) 293 cells, together with several constructs encoding different forms of Src and Abl kinases. The constitutively active Src, SrcY527F, but not its inactive kinase counterpart, SrcY527FKin−, caused retarded frataxin precursor electrophoretic migration as shown by immunoblotting (Fig. 1A). To address whether this shift migration was due to precursor phosphorylation, phosphatase assay on total lysates was performed. Following phosphatase treatment, the shifted form disappeared, indicating that the frataxin precursor is indeed phosphorylated in the cells (Fig. 1B). To further validate frataxin precursor phosphorylation, immunoprecipitation experiments were performed (Fig. 1C). Interestingly, the constitutively active mutant of c-Abl, Abl-PP, could not phosphorylate frataxin precursor suggesting that this form of frataxin is specifically phosphorylated by Src.

Figure 1.

Src kinase phosphorylates frataxin. (A) HEK293 cells were transiently transfected with frataxin (FXN), and either constitutively active Src (Y527F) or its kinase-inactive counterpart (Y527F-Kin⁻). Total protein extracts (TOT) were separated by SDS–PAGE and immunoblotted (WB) with specific antibodies against frataxin and tubulin (TUB) as loading control. Data are representative of ten independent experiments. (B) Total lysate of HEK293 transfected with frataxin and constitutively active Src (Y527F) was incubated for 50 min at 37°C with buffer alone, CIP phosphatase (PPase) in the presence or absence of phosphatase inhibitors (Inh) and analyzed after separation by SDS–PAGE by immunoblotting (WB) with specific antibody against frataxin (FXN). Data are representative of three independent experiments. (C) HEK293 cells were transiently transfected with frataxin, and either constitutively active Src (Y527F), its kinase-inactive counterpart (Y527F-Kin⁻) or constitutively active Abl (Abl-PP). The kinase activity of Abl-PP was indeed controlled (data not shown). Total protein extracts (TOT) or immunoprecipitated frataxin (IP) were separated by SDS–PAGE and immunoblotted (WB) with specific antibodies against frataxin (FXN), phosphorylated tyrosine (pY) and tubulin (TUB) as loading control. Data are representative of three independent experiments. The precursor (P), intermediate (I) and mature (M) frataxin forms are indicated. The arrows show the phosphorylated shifted precursor form.

Src kinase phosphorylates frataxin on residue Y118

To identify Src-induced tyrosine phosphorylated site(s) on frataxin, single non-phosphorylatable mutants of the eight tyrosines residues of frataxin (Y74, Y95, Y118, Y123, Y143, Y166, Y175 and Y205) were generated converting each tyrosine into phenylalanine residue. Mutants were analyzed in cotransfection assays with constitutively active SrcY527F or its inactive kinase counterpart SrcY527FKin− as described in Figure 1. Interestingly, mutation of residues Y95, Y118 and Y123 induced an electrophoretic shift migration of all the frataxin forms (Fig. 2A). Though mutations are conservative, the shift migration could be due to charges modifications because the α-helix 1 (D91 to A114) is an acidic residue-rich region and the loop 1 (D115 to Y123) is important for proper protein folding and stability (21).

Figure 2A shows three representative mutants, out of the eight analyzed, whereas the table summarizes the results for all mutants. Only mutation of Y118 abrogated tyrosine phosphorylation of frataxin precursor, indicating that Y118 is the main Src phosphorylation site. To further confirm that Y118 was the main phosphorylation site, mass spectrometry was performed. In vitro phosphorylation reaction using recombinant frataxin^1–210 and recombinant Src was performed, recovered on SDS–PAGE gel after visualization by Coomassie staining, digested with chymotrypsin and analyzed by LC–MS/MS. A phosphopeptide, corresponding to amino acids 96–123, was isolated, identifying the phosphorylated residue as Y118 (Fig. 2B).

Figure 2.

Src kinase phosphorylates frataxin on residue Y118. (A) HEK293 cells were transiently transfected with wild-type frataxin (FXN), or non-phosphorylatable frataxin mutants Y175F, Y118F, Y123F and either constitutively active Src (Y527F) or its kinase-inactive counterpart (Y527F-Kin⁻). Total protein extracts (TOT) or immunoprecipitated frataxin (IP) were separated by SDS–PAGE and immunoblotted (WB) with specific antibodies against frataxin, phosphorylated tyrosine (pY) and tubulin (TUB) as loading control. Data are representative of three independent experiments. The precursor (P), intermediate (I) and mature (M) frataxin forms are indicated. (B) The picture shows the fragment ion spectrum of the phosphopeptide (96–123), with sequence ERLAEETLDSLAEFFEDLADKPpYTFEDY and triple-charged precursor ion at m/z 1146.7, obtained after in-gel digestion of frataxin by chymotrypsin. The fragment ion series are indicated on the sequence: b and y ions, which are detected in the spectrum, are in bold dark. The upper panel shows an enlarged region of the spectrum (m/z range from 1200 to 1440): here, the distance from the peaks corresponding to b₂₂ to b₂₃ ions coincides with a phosphotyrosine (pY) locating the phosphorylation on Y118.

Non-phosphorylatable Y118F frataxin mutant is less ubiquitinated

To evaluate the impact of phosphorylation on frataxin ubiquitination, HEK-293 cells were transfected with wild-type frataxin or non-phosphorylatable frataxin mutants Y118F, Y166F and Y175F together with hemagglutinin (HA)-tagged ubiquitin (HA-Ub) in the absence or presence of proteasome inhibitor MG132. As previously reported (16), frataxin ubiquitination status was evaluated by immunoblotting with anti-frataxin monoclonal antibodies on total lysates or after immunoprecipitation of ubiquitinated forms with anti-HA antibody only in the presence of MG132. Frataxin monoubiquitinated forms can be detected as slower migrating bands above the frataxin precursor. Figure 3A illustrates that accumulation of ubiquitinated frataxin forms was reduced when non-phosphorylatable Y118F mutant, but not other tyrosine mutants such as Y166F and Y175F, was transfected. Relative ubiquitination level of frataxin was quantitated as the ratio between monoubiquitinated forms versus the non-ubiquitinated precursor forms in the MG132-treated lanes (Fig. 3B). Non-phosphorylatable Y118F frataxin mutant is >60% less ubiquitinated compared with wild-type frataxin, thus suggesting that phosphorylation on Y118 is required for ubiquitination.

Figure 3.

Phosphorylation on Y118 promotes ubiquitination. (A) HEK293 cells were transiently transfected with wild-type frataxin (FXN), or non-phosphorylatable mutants Y118F, Y166F, Y175F and hemagglutinin (HA)-tagged ubiquitin (HA-Ub). Total protein extracts (TOT) or immunoprecipitated ubiquitinated frataxin (IP α-HA) were separated by SDS–PAGE and immunoblotted (WB) with specific antibodies against frataxin (FXN) and tubulin (TUB) as loading control. Data are representative of five independent experiments. (B) The graph illustrates the relative ubiquitination level quantitated as the ratio between monoubiquitinated frataxin (Mono-Ub) level versus the frataxin precursor expression in the MG132-treated lanes. The precursor (P), intermediate (I) and mature (M) frataxin forms are indicated.

Src inhibitors increase wild-type frataxin expression but not non-phosphorylatable Y118F frataxin mutant

Considering that phosphorylation on Y118 promotes ubiquitination and that preventing ubiquitin-dependent degradation increases frataxin levels (16), we sought to investigate whether inhibiting frataxin phosphorylation by Src kinase would indeed allow enhancement of frataxin expression. Different Src inhibitors such as PP2, SU6656, Saracatinib, Bosutinib and Dasatinib were therefore used to treat HEK293^FXN cells, stably expressing a single copy of wild-type frataxin or the non-phosphorylatable Y118F frataxin mutant. As illustrated in Figure 4, all the Src inhibitors can promote frataxin accumulation within 24 h in a dose-dependent manner, although with different efficacy. Among the Src inhibitors tested, Dasatinib appeared to be the most efficient in promoting frataxin accumulation, being still active in the nanomolar range of concentrations (data not shown). Interestingly, frataxin accumulation was observed for all the different processing forms such as precursor, intermediate and mature forms. All the Src inhibitors tested could increase frataxin levels in cells overexpressing wild-type frataxin, but not in cells overexpressing the non-phosphorylatable Y118F frataxin mutant (Fig. 4), suggesting that they indeed act by inhibiting phosphorylation of Y118. In addition, these inhibitors are also effective on the levels of endogenous frataxin as shown in HEK293 cells (Fig. 5).

Figure 4.

Src inhibitors upregulate wild-type frataxin but not the non-phosphorylatable Y118F frataxin mutant. HEK293^FXN cells stably expressing single copy of wild-type frataxin (WT) or non-phosphorylatable Y118F frataxin mutant (Y118F) were treated for 24 h with 1, 3 and 10 μm of either Src inhibitor SU6656, PP2, Dasatinib, Bosutinib, Saracatinib or vehicle (−). Left panels: frataxin (FXN) and tubulin expression (TUB) was analyzed by western blot. Data are representative of three independent experiments. The precursor (P), intermediate (I) and mature (M) frataxin forms are indicated. Right panels: densitometric quantification of frataxin accumulation. Frataxin expression was normalized with tubulin and frataxin expression in non-treated cells (−) set to one. Data represent the mean ± 1 S.E.M. from three different independent experiments performed for each inhibitor. P-values were calculated with Student's t-test and were statistically significant (*P < 0.05; **P < 0.01) for each treatment compared with non-treated conditions.

Figure 5.

Src inhibitors promote endogenous frataxin accumulation in HEK293 cells. Human HEK293 cells were treated with 1, 3 and 10 μm of SU6656, PP2 and Dasatinib Src inhibitor or vehicle (−) for 24 h. Frataxin (FXN) and tubulin expression (TUB) was analyzed by western blot. Data are representative of three independent experiments. The precursor (P), intermediate (I) and mature (M) frataxin forms are indicated. Right panels: densitometric quantification of frataxin accumulation. Frataxin expression was normalized with tubulin and frataxin expression in non-treated cells (−) set to one. Data represent the mean ± 1 S.E.M. from three different independent experiments performed for each inhibitor in the left panels. P-values were calculated with Student's t-test and were statistically significant (*P < 0.05; **P < 0.01) for each treatment compared with non-treated conditions.

Src inhibitors promote frataxin accumulation in frataxin-deficient cells and rescue the aconitase defect

As blocking Src activity with tyrosine kinase inhibitors increases frataxin levels in human cells, we therefore tested their efficacy on frataxin-deficient cells such as FRDA patient-derived B cells (GM16214). Among the different Src inhibitors, SU6656, PP2, and Dasatinib seemed to be best tolerated. FRDA lymphoblasts were exposed to these inhibitors for different time periods. As illustrated in Figure 6A, the upregulation of frataxin could be detected as early as 24 h of treatment and could be further accumulated within 72 h, particularly with SU6656 and Dasatinib. Moreover, Dasatinib could upregulate frataxin in primary fibroblasts (GM04078) derived from a FRDA patient in a dose-dependent manner (Fig. 6B). Furthermore, aconitase activity was also measured in FRDA lymphoblasts exposed to SU6656, PP2 and Dasatinib inhibitors for different time periods. Interestingly, the aconitase defect caused by frataxin deficiency could be restored in 48 h and further rescued in 72 h by all the Src inhibitors tested (Fig. 7).

Figure 6.

Src inhibitors promote frataxin accumulation in frataxin-deficient cells. (A). FRDA patient-derived B cells were treated with 10 μm of either Src inhibitor SU6656, PP2, Dasatinib or vehicle (−) for the time indicated. Left panels: mature frataxin (FXN) and tubulin expression (TUB) were analyzed by western blot. Data are representative of three independent experiments. Right panels: densitometric quantification of frataxin accumulation. Frataxin expression was normalized with tubulin and frataxin expression in non-treated cells (NT) set to one. Data represent the mean ± 1 S.E.M. from three different independent experiments performed for each inhibitor in the left panels. P-values were calculated with Student's t-test and were statistically significant (*P < 0.05; **P < 0.01) for each treatment compared with non-treated conditions. (B). FRDA patient-derived primary fibroblast cells were treated with 0.1, 1 and 10 μm of Src inhibitor Dasatinib or vehicle (−) for 24 h. Left panels: mature frataxin (FXN) and tubulin expression (TUB) was analyzed by western blot. Data are representative of four independent experiments. Right panels: densitometric quantification of frataxin accumulation. Frataxin expression was normalized with tubulin and frataxin expression in non-treated cells (NT) set to one. Data represent the mean ± 1 S.E.M. from four different independent experiments performed. P-values (*P < 0.05; **P < 0.01) were calculated using Student's t-test.

Figure 7.

Src inhibitors rescue the aconitase defect in frataxin-deficient cells. FRDA patient-derived B cells were treated with 10 μm of either Src inhibitor SU6656, PP2, Dasatinib or vehicle (FRDA) for the time indicated. B cells derived from unaffected carrier parent (Healthy) were treated with the vehicle for the time indicated. Total aconitase activity was measured and normalized as described in the ‘Material and Methods’ section. Data represent the mean ± 1 S.E.M. from three different independent experiments performed for each inhibitor. P-values (*P < 0.05; **P < 0.01) were calculated using Student's t-test.

Discussion

This study shows that frataxin precursor can be phosphorylated by Src kinase on Y118, promoting its ubiquitination and degradation. Moreover, Src inhibitors increase frataxin expression in human cells. Accordingly, Src inhibitors failed to upregulate frataxin in human cells in which a frataxin-Y118F mutant was expressed. More importantly, Src inhibitors are effective in FRDA cells as they promote frataxin accumulation and rescue aconitase defect.

Reduced frataxin expression, owing to abnormal GAA triplet expansion, gives rise to the degenerative disorder FRDA, a debilitating disease that leads to a premature death of the patients. To date, there is no approved treatment for FRDA (22). Main current therapeutic approaches were based on the use of anti-oxidants or iron chelators with controversial results (23,24). Other strategies aiming at improving mitochondrial function or restoring frataxin levels are being developed (4,15). We recently provided evidence that preventing frataxin ubiquitination and degradation effectively results in frataxin accumulation in cells derived from FRDA patients (16). Modulating frataxin ubiquitination therefore represents an attractive therapeutic strategy.

Ubiquitination frequently interplays with phosphorylation to regulate many important signaling processes (17,25). Both serine/threonine and tyrosine phosphorylation of protein substrates can indeed promote or inhibit ubiquitination, in different manners, leading to either proteasomal degradation or processing, or the regulation of intracellular compartmentalization. Phosphorylation can, for instance, promote ubiquitination by creating a phosphodegron, a recognition signal for some E3 ligases. Many of these short motifs are serine/threonine phosphorylation-dependent ubiquitination targets. Phosphorylated tyrosine in target proteins are also specifically recognized by SH2 domain-containing E3 ligases such as Cbl family members. Cbls are RING ubiquitin ligases that modulate receptor tyrosine kinases (RTKs) by binding to phosphotyrosine residues on activated RTK such as the epidermal growth factor receptor. This promotes ligand-dependent ubiquitination of these receptors, targeting them for degradation. In addition, the phosphorylation of a critical tyrosine residue of Cbl stimulates its E3 ligase activity (26). Furthermore, non-receptor tyrosine kinase activity can also be regulated by ubiquitination. One example is given by Cbl-mediated ubiquitination of Src kinases that leads to their degradation and results in the attenuation of antigen receptor signals, thus controlling immune responses (27). Conversely, different kinases can also phosphorylate a target E3 ligase modulating its activity: the activity of the ubiquitin ligase ITCH is negatively regulated by Src-kinase family member Fyn but positively regulated by JNK-mediated serine/threonine phosphorylation (25). Therefore, phosphorylation and ubiquitination often crosstalk to tightly tune crucial cellular processes. Here, we show that Src-mediated phosphorylation of frataxin promotes its ubiquitination. This prompted us to test whether Src inhibitors could modulate frataxin levels. We found that indeed all Src inhibitors tested promote frataxin accumulation and rescue aconitase defect in cells derived from FRDA patients. Src inhibitors might therefore have therapeutic potential in FRDA.

Src inhibitors Dasatinib and Bosutinib, reported to cross the blood–brain barrier (28,29), have been approved for therapeutic use in humans (30,31) for the treatment of Philadelphia chromosome positive (Ph+) chronic myelogenous leukemia. Similarly to Dasatinib, Bosutinib is an ATP competitive Abl and Src inhibitor (32). However, chemical proteomics approaches have identified over 45 target kinases for Bosutinib (33) as well as Dasatinib (34). Interestingly, tyrosine kinases have emerged as powerful modulators of mitochondrial functions (18,19). Moreover, Src tyrosine kinases can actually be modulated by redox status and ATP levels that are indeed altered in FRDA patients (20). Considering the conserved domain structure of the Src family kinases (SFK) (35), it would be interesting to therefore study whether other SFK members could also specifically phosphorylate frataxin, especially Lyn, Fgr and Fyn described to associate with mitochondria (19). Further structural studies addressed to analyze frataxin and SFK binding would also help to develop more effective inhibitors blocking specific interaction between frataxin and SFK in order to improve efficacy and eventually circumvent possible toxicity.

In summary, our results reveal a novel molecular mechanism directly controlling frataxin protein levels in living cells. Moreover, they provide the rationale for testing a whole class of drugs, some of them already available on the market, as therapeutics for FRDA.

Material and Methods

Cell cultures and transfections

Human embryonic kidney HEK-293 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS). HEK-293 cells were transfected with the calcium/phosphate precipitation method, using 20 μg of total DNA (5 or 10 μg of pIRES2-Frataxin, 10 μg of tyrosine-mutated non-phosphorylatable frataxin mutants, 10 μg of Src or Abl constructs and 10 μg of HA-Ub, or the corresponding empty vectors) on 10-cm dishes.

HEK-293 Flp-In cells (Life Technologies) are HEK-293 variants allowing the stable and isogenic integration and expression of a transfected gene. The HEK-293 clone stably expressing frataxin^1–210 (2) and the clone stably expressing frataxin^Y118F were generated by mutagenesis as described later. HEK-293 Flp-In cells were maintained in DMEM supplemented with 10% FBS.

Immortalized GM16214 lymphoblasts, from a clinically affected FRDA patient, and immortalized GM16215 lymphoblasts from an unaffected carrier parent were obtained from NIGMS Human Genetic Cell Repository, Coriell Institute for Medical Research (Camden, NJ, USA) and cultured in RPMI 1640 supplemented with 15% FBS. Primary fibroblasts GM04078 from a clinically affected FRDA patient were obtained from NIGMS Human Genetic Cell Repository, Coriell Institute for Medical Research and cultured in DMEM supplemented with 15% FBS.

DNA constructs

The construct pIRES2-Frataxin^1-210 contains human frataxin cDNA cloned into the bicistronic expression vector pIRES2-EGFP (Clontech Laboratories) and was previously generated in this laboratory (6). All the tyrosine-mutant constructs were generated using the Quick-Change site-directed mutagenesis kit (Agilent Technologies) with specific primers using pIRES2-Frataxin^1–210 as template. The HA-Ub construct was generated by M. Treier in Dirk Bohmann's lab (36). Constitutively active Src (pSGTSrcY527F), its inactive kinase counterpart (pSGTSrcY527F-kin−) and constitutively active Abl (pSGTAbl-PP) have been previously described (37,38). All the constructs generated were verified by DNA sequencing.

Dephosphorylation assay

CIP dephosphorylation assay kit (New England BioLabs^®, Inc.) was used to release phosphate groups from residues of tyrosine. Fifty units of CIP (Alkaline Phosphatase, Calf Intestinal) were added to total cell extracts resuspended in NE3 buffer pH 7.9 (100 mm NaCl, 50 mm Tris–HCl, 10 mm MgCl₂ and 1 mm dithiothreitol) and incubated for 60 min at 37°C. Sodium orthovanadate 4 mm and EDTA 50 mm were used to inhibit CIP activity.

Immunoprecipitation and western blot

Total cell extracts were prepared in ice-cold modified RIPA buffer (10 mm sodium phosphate, pH 7.2, 150 mm NaCl, 1% Na deoxycholate, 0.1% SDS, 1% Igepal CA-630 and 2 mm EDTA) or IP buffer (50 mm Tris–HCl, pH 7.5, 150 mm NaCl, 1% Igepal CA-630, 5 mm EDTA and 5 mm EGTA) supplemented with complete protease inhibitor cocktail (Roche Diagnostics, Milan, Italy), sodium orthovanadate 1 mm and NaF 25 mm to inhibit phosphatases.

For in vivo detection of ubiquitin conjugates, 100 μm MG132, 50 ng/ml Ub-aldehyde and N-ethylmaleimide 2 mm (NEM; Sigma–Aldrich) were added to the lysis buffer. Cell lysates (100 μg) were resolved by SDS–PAGE and analyzed by immunoblot with specific mAb anti-frataxin clone 1G2 and STR-23 (Immunological Sciences, Rome, Italy), mAb anti-tubulin (Sigma), mAb anti-actin (Sigma), mAb anti-phosphotyrosine (Millipore), pAb anti-phospho-Src (Life Technologies), mAb anti-GFP (Takara), secondary antibody horseradish peroxidase-conjugated goat anti-mouse (Pierce), secondary antibody horseradish peroxidase-conjugated mouse anti-rabbit (Pierce), secondary antibody horseradish peroxidase-conjugated goat anti-Fc anti-mouse (Thermo Scientific) using ECL system detection (GE Healthcare Europe GmbH, Milan, Italy).

For immunoprecipitation, 5 mg of total protein extract prepared as mentioned above were incubated for 1–2 h at 4°C with specific antibodies, previously conjugated to protein G-Sepharose (GE Healthcare). Immunocomplexes were then resolved and analyzed by SDS–PAGE.

Densitometric analyses were performed using ImageLab software (Biorad).

MS/MS identification of nitrated or phosphorylated residues in frataxin

Human Recombinant Frataxin protein (GenScript Corp., New Jersey, USA) was previously treated with the Src Kinase Assay (Millipore) and then separated on a 1D-gel NuPAGE 4–12% (Novex, Invitrogen) run in morpholinepropanesulfoninic acid (MOPS) buffer and stained with the Colloidal Blue Staining kit (Invitrogen).

The stained bands were cut from the gel and destained with a solution containing 50 mm ammonium bicarbonate/acetonitrile (1:1v/v) (ACN, Merck Darmstadt, Germany). Protein bands were subsequently subjected to cysteine reduction by 10 mm DTT for 1 h at 56°C and alkylation by 50 mm iodoacetamide for 45 min at RT in the dark and then dried by acetonitrile treatment and evaporation in a SpeedVac concentrator.

In-gel digestion was performed by incubating gel slices with a solution containing 12.5 ng/ml chymotrypsin (Promega, Madison, WI, USA) in 25 mm ammonium bicarbonate at 37°C overnight under stirring.

To recognize phosphorylated residues, the peptide mixture was analyzed by nanoflow-reversed-phase liquid chromatography tandem mass spectrometry (RP-LC–MS/MS) using an HPLC Ultimate 3000 (Dionex, Sunnyvale, CA, USA) connected on line with a linear Ion Trap (LTQ, ThermoElectron, San Jose, CA, USA). Peptide mixtures were desalted in a trap-column (AcclaimPepMap100 C18, LC Packings, Dionex) and then separated in a reverse phase column, a 10-cm-long fused silica capillary (SilicaTipsFS 360-75-8, New Objective, Woburn, MA, USA), slurry-packed in-house with 5 μm and 200 Å pore size C18 resin (Michrom BioResources, CA, USA). Peptides were eluted using a 30-min linear gradient from 96% aqueous phase (H₂O with 5% ACN, 0.1% formic acid) to 60% organic buffer (ACN with 5% H₂O with 0.1% formic acid) at 300 nl/min flow rate. Analyses were performed in positive ion mode, and the HV potential was set up around 1.5–1.8 kV. The LTQ mass spectrometer operated in a data-dependent mode: each full MS scan was followed by collision-induced dissociation (CID) fragmentation of the five most abundant molecular ions, using a normalized collision energy of 35%. Tandem mass spectra were matched against the UniProtKB/Swiss-Prot protein database with the SEQUEST algorithm (39) incorporated in the Bioworks software (version3.3, Thermo Electron), using no enzyme constraint, static cysteine alkylation by iodoacetamide, dynamic modification by oxidation on methionine and phosphorylation on tyrosine residues (Δm: +80 Da). A peptide was considered reliably identified when it achieved cross-correlation scores of 1.8, 2.5 and 3 for single-, double- and triple-charged ions, respectively, and a probability cutoff for randomized identification of P < 0.001.

Reagents and treatments

The proteasome inhibitor MG132 (Sigma–Aldrich) and the deubiquitinase inhibitor Ub-aldehyde (Biomol) were added to the cell culture at the final concentration of 10 μm and 50 ng/ml respectively. Src Inhibitors: PP2, SU6656 (EMD Millipore), Bosutinib, Dasatinib and Saracatinib (Selleckchem) were added to cells at the minimum concentration of 10 nm to a maximum concentration of 10 μm. For treatment longer than 24 h, inhibitors were added every 2 days and each time lymphoblasts were centrifuged and resuspended at 400.000 cells/ml with the indicated concentration of inhibitor.

Evaluation of aconitase activity

Whole-cell extracts from immortalized lymphoblasts were prepared in ice-cold CelLytic M buffer (Sigma–Aldrich) supplemented with 2 mm sodium citrate, 1 mm sodium orthovanadate, 25 mm NaF and Complete EDTA-free protease inhibitor cocktail (Roche Diagnostic). Spectrophotometric aconitase assays were performed at 25°C with 150 μg of cell extracts using the BIOXYTECH Aconitase-340^TM Assay (OxisResearch^TM 21041). Spectrophotometric citrate synthase activities were assessed at 25°C with 15 μg of cell extracts using the Citrate Synthase Assay Kit (Sigma–Aldrich CS0720). Aconitase activities were referred to the specific activity of citrate synthase to correct for mitochondrial content. For the calculation of the activities, one unit of enzyme was expressed as the amount of protein that converted 1 μmol of substrate per minute at 25°C.

Funding

This work was supported by European Research Council (Advanced Grant #293699, FAST), Friedreich Ataxia Research Alliance USA and Telethon Italy (GGP11102).

Acknowledgements

We thank all colleagues in our laboratory, especially Dr. Simona Bagedda. We also thank Allegra Via for bioinformatics support and Dr. Daniela Barilà for helpful discussion and careful reading of the manuscript. Conflict of Interest statement. None declared.