ABSTRACT
The microtubule-associated protein Tau is implicated in the pathogenesis of several neurodegenerative disorders, including Alzheimer’s disease. Increasing evidence suggest 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, 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 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, except Y310 or Y394 (pTau-Y310F, pTau-Y394F) and tau phosphorylated only at Y310 and 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 PHF6 domain. Our findings underscore the unique role of Y310 phosphorylation and highlight the importance of further studies to elucidate its role in tauopathies and its role in regulating Tau function in health and disease.
FOOTNOTES
- 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
- CET
- chronic traumatic encephalopathy
- EM
- electron microscopy
- ESI/MS
- electrospray ionisation mass spectrometry
- 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
- S.E.M
- standard error of measurement
- 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