Dephosphorylation of Six2Y129 protects tyrosine hydroxylase-positive cells in SNpc by regulating TEA domain 1 expression

•Six2Y129 site was dephosphorylated by phosphatase Eya1 in injured DA cells•Dephosphorylated Six2 translocated from cytoplasm to nucleus•Dephosphorylated protected cells down-regulating Tead1 expression Parkinson’s disease (PD) is a neurodegenerative characterized selective loss of dopaminergic (DA) neurons the substantia nigra pars compacta (SNpc). We recently reported that could reverse degeneration dephosphorylation state. Here we further identified dephosphorylate tyrosine 129 (Y129) forming complex with damaged cells. Dephosphorylated then translocates nucleus. Using ChIP-qPCR and dual luciferase assay, found down-regulates TEA domain1 (Tead1) expression, thus inhibiting 6-hydroxydopamine (6-OHDA)-induced apoptosis Furthermore, showed Six2Y129F/Tead1 signaling protect against SNpc hydroxylase-positive (TH+) improve motor function PD model rats. 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IB analysis compared especially h after treatment. serine/threonine/tyrosine (S/T/Y) treatment (Figure 1A). changing trend p-Six2 similar more evident 1B). has shown overexpression reduce cells.12Gao assess Six2, first evaluated possibility interaction two proteins. IF both were co-expressed 2A). Co-IP using extracts groups 2B); however, weaker detected 2C). And GST-pull-down 293T transfected GST-Eya1/Six2 confirmed form (Figures 2D 2E). Next, tested enzyme-substrate relationship. Eya1/Six2 Eya1D327A/Six2 because 327D phosphorylase Results Eya1, Eya1D327A, overexpressed than 2F 2H). Moreover, dramatically upregulated inactive mutant Eya1D327A group wild-type 2I). Because current evidence suggests therosine-specific phosphatase, functions clearly. contrast, phosphotherosine had no change 1h 2J). Taken together, 6-OHDA. next sought identify precisely residue(s) phosphorylated. Mutagenesis each residues phenylalanine (F) Y67F, Y92F, Y109F, Y129F, Y143F, Y148F, expressed 3A). revealed mutation blocked 3B), indicating Y129 phosphorylated tyrosine. mutants aspartic (D) (Six2Y129D) 3C). 6-OHDA, displayed Six2Y129F levels, whereas Six2Y129D did not affect 3D). These indicate view vitro analysis. immunopurified His-fusion mixed Six2-pY129 peptides, effectively removed phosphate phosphatase-inactive little 3E), used known substrate positive 3F). data establish ability directly. above results, 2B). explore reason why binding becomes weaker, laser confocal primarily distributed treatment, located mainly 4A). maybe state affects localization Laser localized nucleus, even 4B). Then, indicated untreated cytoplasm, 4C). suggest Six2Y129 primary factors expression. translocate cytoplasm. For unbiased assessment plausible performed genome-wide ChIP-seq presented differentially groups. Of Six2-binding sites, 14.63% region 2000 bp upstream start differential 5A). Gene Ontology (GO) participated biological processes 5B). Meanwhile, Kyoto Encyclopedia Genes Genomes (KEGG) analyses Hippo pathways differed 5C). de novo motif discovery specific sequences bound 5D). involved qRT-PCR. mRNA downregulated 5E). Double reporter enhanced 5F). promoter group, 5G). negatively promoter. vitro. CCK8 rescued viability cells; protective When 6A). induced upregulation bax downregulation bcl-2. At same time, reversed bcl-2 6B). TUNEL analysis, apoptosis, abolished anti-apoptosis 6C). Together, inhibit downregulating strongly supported wondered prevents TH+ vivo. stereotactic injection rats 7A). apomorphine-induced rotation test abilities improved 7B). posture asymmetry experiment frequency head turning side rescue, 7C). verify numbers counted. reduced co-treatment 7D). examined TH midbrain expresion TH, 7E). protects negative regulation illness neurons. 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