c-Abl phosphorylates α-synuclein and regulates its degradation: implication for α-synuclein clearance and contribution to the pathogenesis of Parkinson's disease

Human Molecular Genetics, 2014, Vol. 23, No. 11 doi:10.1093/hmg/ddt674 Advance Access published on January 9, 2014 2858–2879 c-Abl phosphorylates a-synuclein and regulates its degradation: implication for a-synuclein clearance and contribution to the pathogenesis of Parkinson’s disease Anne-Laure Mahul-Mellier1, Bruno Fauvet1, Amanda Gysbers3, Igor Dikiy4, Abid Oueslati1, Sandrine Georgeon2, Allan J. Lamontanara2, Alejandro Bisquertt5, David Eliezer4, Eliezer Masliah5, Glenda Halliday3, Oliver Hantschel2 and Hilal A. Lashuel1,∗ 1 Received October 17, 2013; Revised December 11, 2013; Accepted December 31, 2013 Increasing evidence suggests that the c-Abl protein tyrosine kinase could play a role in the pathogenesis of Parkinson’s disease (PD) and other neurodegenerative disorders. c-Abl has been shown to regulate the degradation of two proteins implicated in the pathogenesis of PD, parkin and a-synuclein (a-syn). The inhibition of parkin’s neuroprotective functions is regulated by c-Abl-mediated phosphorylation of parkin. However, the molecular mechanisms by which c-Abl activity regulates a-syn toxicity and clearance remain unknown. Herein, using NMR spectroscopy, mass spectrometry, in vitro enzymatic assays and cell-based studies, we established that a-syn is a bona fide substrate for c-Abl. In vitro studies demonstrate that c-Abl directly interacts with a-syn and catalyzes its phosphorylation mainly at tyrosine 39 (pY39) and to a lesser extent at tyrosine 125 (pY125). Analysis of human brain tissues showed that pY39 a-syn is detected in the brains of healthy individuals and those with PD. However, only c-Abl protein levels were found to be upregulated in PD brains. Interestingly, nilotinib, a specific inhibitor of c-Abl kinase activity, induces a-syn protein degradation via the autophagy and proteasome pathways, whereas the overexpression of a-syn in the rat midbrains enhances c-Abl expression. Together, these data suggest that changes in c-Abl expression, activation and/or c-Abl-mediated phosphorylation of Y39 play a role in regulating a-syn clearance and contribute to the pathogenesis of PD. INTRODUCTION The tyrosine kinase c-Abl is involved in regulating several cellular processes and has been implicated in the development of the central nervous system (1) by controling neurogenesis, neurite outgrowth and neuronal plasticity (2 – 7). More recently, increasing evidence from various experimental model systems has also revealed that c-Abl is activated in neurodegenerative diseases (8) such as Alzheimer’s disease (AD) (9– 11), Parkinson’s disease (PD) (12,13), Niemann – Pick type C disease (14) and tauopathies (15). However, the mechanisms by which c-Abl contributes to the initiation and/or propagation of the pathogenic events underlying these neurodegenerative diseases remain poorly understood. c-Abl is a 120 kDa protein belonging to the cytoplasmic tyrosine kinase family. Similar to Src kinases, c-Abl possesses ∗ To whom correspondence should be addressed at: Laboratory of Molecular and Chemical Biology of Neurodegeneration, Brain Mind Institute, Ecole Polytechnique Fédérale de Lausanne, 1015 Lausanne, Switzerland. Tel: +41 216939691; Fax: +41 216939665; Email: # The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: Laboratory of Molecular and Chemical Biology of Neurodegeneration, Brain Mind Institute, 2ISREC Foundation Chair in Translational Oncology, Swiss Institute for Experimental Cancer Research, Ecole Polytechnique Fédérale de Lausanne, 1015 Lausanne, Switzerland, 3Neuroscience Research Australia and the School of Medical Sciences, University of New South Wales, Randwick, Sydney, NSW 2031, Australia, 4Department of Biochemistry and Program in Structural Biology, Weill Cornell Medical College, NY 10065, USA 5Department of Neuroscience, University of California San Diego, La Jolla, CA, USA Human Molecular Genetics, 2014, Vol. 23, No. 11 accumulation (26,43), suggesting that inhibition of c-Abl might constitute a viable strategy for attenuating a-syn accumulation and protecting against a-syn toxicity in PD. This hypothesis is supported by the observation that nilotinib increases a-syn clearance via the autophagy pathway and protects against a-syn-induced loss of dopaminergic neurons in a mouse model of PD (13,26). Nevertheless, the molecular mechanisms by which changes in c-Abl expression levels and/or activation regulate a-syn clearance and toxicity in vivo remain unknown. Given our interest in the role of post-translational modifications in regulating a-syn functions in health and disease and previous observations demonstrating c-Abl-mediated neuroprotection in PD models, we sought to further explore the relationship between a-syn and c-Abl. Towards this goal, we sought to (i) determine whether a-syn is a physiological substrate of c-Abl, (ii) identify the tyrosine residues that are phosphorylated by c-Abl in vitro and validate our findings in vivo and (iii) determine the role of c-Abl-mediated phosphorylation in regulating a-syn clearance. In this study, we demonstrate, for the first time, that a-syn is a bona fide substrate of c-Abl and showed that c-Abl phosphorylates a-syn, primarily at tyrosine 39 (Y39) and to a lesser extent at tyrosine 125 (Y125) in vitro, in neuroblastoma cell lines and in primary cultures of mouse cortical neurons. We also provide direct evidence that a-syn phosphorylated at Y39 is present in normal and PD human brain tissues. The phosphorylation of a-syn at Y39 can be abolished by using specific c-Abl inhibitors (imatinib, nilotinib and GNF-2) (44) or through the downregulation of c-Abl protein levels using siRNA. Conversely, the activation of c-Abl using DPH (5-[3-(4-fluorophenyl)-1-phenyl-1H-pyrazol-4-yl]-2,4-imidazolidinedione) (45) enhances a-syn phosphorylation at Y39. Moreover, the effects of c-Abl-mediated phosphorylation on a-syn clearance were also investigated. RESULTS c-Abl preferentially phosphorylates a-syn at Y39 in an in vitro kinase assay a-syn contains four tyrosine residues (Y39, Y125, Y133 and Y136). Y125, Y133 and Y136 have previously been shown to undergo phosphorylation by several tyrosine kinases including c-Src (46), Fyn (47) and the Syk family (48). However, no kinase has been shown to phosphorylate a-syn at Y39. We first investigated whether c-Abl could be a tyrosine kinase for a-syn, using in vitro kinase assays. Purified a-syn was incubated in the presence or absence of a recombinant active c-Abl fragment comprising the SH2 and catalytic domains (residues 138–534), also known as SH2-CD c-Abl. In the presence of c-Abl, electrospray ionization mass spectrometry (ESI-MS) analysis showed the appearance of a new peak with a deconvoluted mass of 14 541 Da for WT a-syn, which is consistent with a single phosphorylation event (calculated mass: 14 540 Da) (Fig. 1A). Mutating Y39 to phenylalanine (Y39F) nearly abolished a-syn phosphorylation b (...truncated)