Human Molecular Genetics, 2015, Vol. 24, No. 15 4296–4305 doi: 10.1093/hmg/ddv162 Advance Access Publication Date: 6 May 2015 Original Article ORIGINAL ARTICLE Src inhibitors modulate frataxin protein levels Downloaded from https://academic.oup.com/hmg/article/24/15/4296/2453025 by guest on 28 September 2021 Fabio Cherubini1, Dario Serio1, Ilaria Guccini1, Silvia Fortuni1,2, Gaetano Arcuri1, Ivano Condò1, Alessandra Rufini1,2, Shadman Moiz1, Serena Camerini3, Marco Crescenzi3, Roberto Testi1,2 and Florence Malisan1,* 1Laboratory of Signal Transduction, Department of Biomedicine and Prevention, University of Rome ‘Tor Vergata’, Via Montpellier 1, 00133 Rome, Italy, 2Fratagene Therapeutics Ltd, 22 Northumberland Rd, Dublin, Ireland and 3Department of Cell Biology and Neurosciences, Italian National Institute of Health, Viale Regina Elena, 299, 00161 Rome, Italy *To whom correspondence should be addressed at: Laboratory of Signal Transduction, Department of Biomedicine and Prevention, University of Rome ‘Tor Vergata’, Via Montpellier 1, 00133 Rome, Italy. Tel: +39 0672596501; Fax: +39 0672596505; Email: [email protected] 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. of dysregulated mitochondrial metabolism, frataxin-defective Introduction cells have indeed reduced activity of iron sulfur cluster (ISC)-con- Friedreich’s Ataxia (FRDA) is an autosomal recessive disorder taining enzymes, a general imbalance in intracellular iron distri- characterized by progressive degeneration of the central and per- bution, reduced ATP content and increased sensitivity to ipheral nervous systems, cardiomyopathy and increased inci- oxidative stress with increased ROS generation. Low frataxin le- dence of diabetes mellitus. FRDA is caused by homozygous vels and disease severity have been correlated (5). Moreover, fra- hyperexpansion of GAA triplets in intron 1 of the frataxin gene taxin levels are not only crucial for cell survival but also for stress on chromosome 9q21 (1). Pathological GAA expansions severely handling responses (6–11). There is no current successful treat- reduce transcription of the FXN gene. Frataxin is an extremely ment, but considering that the frataxin coding sequence is intact conserved mitochondrial protein synthesized as a cytosolic 210 in most of the patients, therapies aiming at enhancing frataxin amino acid precursor, which is then imported into mitochondria levels are now being considered (11–15). Frataxin protein levels following a two-step proteolytic maturation by a mitochondrial are controlled by the proteasome upon ubiquitination of target processing peptidase (2,3). Low levels of expression of frataxin residue, K147, on frataxin (16). This lysine represents a crucial are responsible for all clinical and morphological manifestations site for frataxin stability because a frataxin mutant lacking of FRDA (4). In fact, frataxin deficiency in humans critically K147 cannot be ubiquitinated and is more stable. Therefore, pre- affects survival of large primary neurons of the dorsal root gan- venting ubiquitination on K147 is expected to grant frataxin an glia, cardiomyocytes and pancreatic β-cells. As a consequence increased stability and a prolonged half-life (16). Ubiquitination Received: December 30, 2014. Revised and Accepted: April 30, 2015 © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: [email protected] 4296 Human Molecular Genetics, 2015, Vol. 24, No. 15 | 4297 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 path- ways (18) and are important regulators of mitochondrial func- tions (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 dysregu- lated 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 Downloaded from https://academic.oup.com/hmg/article/24/15/4296/2453025 by guest on 28 September 2021 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 constitu- tively active Src, SrcY527F, but not its inactive kinase counterpart, SrcY527FKin−, caused retarded frataxin precursor electrophoret- ic migration as shown by immunoblotting (Fig. 1A). To address whether this shift migration was due to precursor phosphoryl- ation, phosphatase assay on total lysates was performed. Follow- ing phosphatase treatment, the shifted form disappeared, indicating that the frataxin precursor is indeed phosphorylated in the cells (Fig. 1B). To further validate frataxin precursor phos- phorylation, 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. Src kinase phosphorylates frataxin on residue Y118 Figure 1. Src kinase phosphorylates frataxin. (A) HEK293 cells were transiently To identify Src-induced tyrosine phosphorylated site(s) on fra- transfected with frataxin (FXN), and either constitutively active Src (Y527F) or its − taxin, single non-phosphorylatable mutants of the eight tyro- kinase-inactive counterpart (Y527F-Kin ). Total protein extracts (TOT) were sines residues of frataxin (Y74, Y95, Y118, Y123, Y143, Y166, separated by SDS–PAGE and immunoblotted (WB) with specificantibodiesagainst Y175 and Y205) were generated converting each tyrosine into frataxin and tubulin (TUB) as loading control. Data are representative of ten phenylalanine residue. Mutants were analyzed in cotransfection 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, assays with constitutively active SrcY527F or its inactive kinase − CIP phosphatase (PPase) in the presence or absence of phosphatase inhibitors (Inh) counterpart SrcY527FKin as described in Figure 1. Interestingly, and analyzed after separation by SDS–PAGE by immunoblotting (WB) with specific mutation of residues Y95, Y118 and Y123 induced an electrophor- antibody against frataxin (FXN). Data are representative of three independent etic shift migration of all the frataxin forms (Fig. 2A). Though mu- experiments. (C) HEK293 cells were transiently transfected with frataxin, and either − tations are conservative, the shift migration could be due to constitutively active Src (Y527F), its kinase-inactive counterpart (Y527F-Kin )or charges modifications because the α-helix 1 (D91 to A114) is an constitutively active Abl (Abl-PP). The kinase activity of Abl-PP was indeed controlled (data not shown). Total protein extracts (TOT) or immunoprecipitated acidic residue-rich region and the loop 1 (D115 to Y123) is import- frataxin (IP) were separated by SDS–PAGE and immunoblotted (WB) with specific ant for proper protein folding and stability (21). antibodies against frataxin (FXN), phosphorylated tyrosine (pY) and tubulin (TUB) Figure 2A shows three representative mutants, out of the as loading control. Data are representative of three independent experiments. The eight analyzed, whereas the table summarizes the results for precursor (P), intermediate (I) and mature (M) frataxin forms are indicated. The all mutants. Only mutation of Y118 abrogated tyrosine phosphor- arrows show the phosphorylated shifted precursor form. ylation