This potentially can lead to a stable state with both epitopes phosphorylated, as is found in AD neurons. An additional level of regulation has been described for the phospho-dependent prolylcis/trans isomerase Pin1. Here, we confirm the stimulatory effect of Pin1 on the PP2AT55a catalyzed dephosphorylation of wild type phospho-Tau and its absence in the Tau T231A mutant. Stimulation was most striking for the pS202 site, switching from no dephosphorylation with one unit of PP2AT55a in the absence of Pin1 to complete dephosphorylation in its presence. The effect is however not homogeneous, with at best a weak activity of PP2AT55a towards pT231 despite the presence of Pin1. Moreover, the stimulatory effect directly depends on the PR55/Ba subunit, as Pin1 rather hinders than stimulates the activity of the dimeric core enzyme. Pin1 hence counterbalances the negative regulation of PP2AT55a activity towards the AT8 site following phosphorylation of the AT80 pT231 site, in agreement with the inverse correlation between Pin1 expression and actual neurofibrillary degeneration in AD. Because PP2AT55a has been shown to regulate the phosphorylation status of Tau in vivo, a potential trigger for PHF formation might be a shift of the balance between neuronal kinases and phosphatases. Our results show that phosphorylation of T231 directly influences the dephosphorylation rate of pS202, without necessitating an alteration in the PP2AT55a pool. We have reconstituted here the complex system of phosphorylated Tau and the trimeric PP2AT55a phosphatase in the NMR tube with several recombinant proteins, and thereby unravelled a subtle regulatory Nestorone mechanism. An additional level of regulation has been shown by the prolyl cis/trans isomerase Pin1. The phosphorylation/dephosphorylation reactions in vivo might even be more complex, because of the presence of tubulin and/or other regulatory factors, but even these can be added in the NMR tube. Whereas we previously showed that NMR observation of Tau in live Xenopus ovocytes is feasible, we L-742001 hydrochloride believe that both approaches will bring novel insights in the complex regulation of multi-site phosphorylation.