The microtubule-associated protein Tau is implicated in the pathogenesis of several neurodegenerative disorders, including Alzheimer’s disease. Increasing evidence suggests that post-translational modifications play critical roles in regulating Tau normal functions and its pathogenic properties in Tauopathies. Very little is known about how phosphorylation of tyrosine residues influences the structure, aggregation, and microtubule- and lipid-binding properties of Tau. In this work, we aimed to address this knowledge gap and determine the relative contribution of phosphorylation of one or several of the five tyrosine residues in Tau (Y18, Y29, Y197, Y310 and Y394) to the regulation of its biophysical, aggregation and functional properties. Towards this goal, we used a combination of site-specific mutagenesis and in vitro phosphorylation by c-Abl kinase to generate Tau species phosphorylated at all tyrosine residues, all tyrosine residues except Y310 or Y394 (pTau-Y310F, pTau-Y394F) and Tau phosphorylated only at Y310 or Y394 (4F\pY310 or 4F\pY394). Our results show that phosphorylation at all five tyrosine residues, multiple N-terminal tyrosine residues (Y18, Y29 and Y197) or site-specific phosphorylation at residue Y310, itself located in the microtubule-binding and aggregation-prone domain of Tau, was sufficient to abolish Tau aggregation and inhibit its microtubule- and lipid-binding properties. NMR studies demonstrated that these effects were mediated by a local decrease in β−sheet propensity of the PHF6 domain. Our findings underscore the unique role of Y310 phosphorylation in the regulation of Tau aggregation, microtubule and lipid interactions and highlight the importance of conducting further studies to elucidate its role in the regulation of Tau normal functions and its pathogenic properties.*   Abl    :   Abelson murine leukemia viral oncogene homolog 1    AD    :   Alzheimer’s disease    Arg    :   Abelson tyrosine-protein kinase 2    BPS    :   brain phosphatidylserine    CBD    :   corticobasal degeneration    CD    :   circular dichroism    CTE    :   chronic traumatic encephalopathy    EM    :   electron microscopy    fPS    :   fluorescent phospholipids    FTDP-17    :   frontotemporal dementia with parkinsonism linked to chromosome 17    GTP    :   guanosine triphosphate    HSCQS    :   heteronuclear single quantum coherence spectroscopy    LC/MS    :   liquid chromatography–mass spectrometry    MAPF    :   microtubule−associated protein fraction    MS/MS    :   tandem mass spectrometry    MT    :   microtubule    MTBD    :   microtubule-binding domain    MTBR    :   microtubule-binding region    NBD    :   nitrobenzoxadiazole    NFT    :   neurofibrillary tangle    NMR    :   nuclear magnetic resonance    PD    :   Parkinson’s disease    pS    :   phospho-serine    PSP    :   progressive supranuclear palsy    pT    :   phospho-threonines    PTM    :   post-translational modification    pY    :   phospho-tyrosine    RP−HPLC    :   reversed phase high-performance liquid chromatography    RT    :   room temperature    SD    :   standard deviation    SDS-PAGE    :   sodium dodecyl sulphate-polyacrylamide gel electrophoresis    SP    :   spectral count    ssi    :   secondary shift index    Syk    :   Spleen tyrosine kinase    ThT    :   thioflavin T    TTBK1    :   Tau tubulin kinase 1    UPLC    :   ultra performance liquid chromatography    WT    :   wild-type

Phosphorylation of the overlooked tyrosine 310 regulates the structure, aggregation, and microtubule- and lipid-binding properties of Tau