Timo Eichner, Steffen Kutter, Wladimir Labeikovsky, Vanessa Buosi, Dorothee Kern

Human peptidyl-prolyl isomerase (PPIase) Pin1 plays key roles in developmental processes, cell proliferation and neuronal function. Extensive phosphorylation of the microtubule binding protein tau has been implicated in neurodegeneration and Alzheimer's disease. For the past 15years these two players have been the focus of an enormous research effort to unravel the biological relevance of their interplay in health and disease resulting in a series of proposed molecular mechanism of how Pin1 catalysis of tau results in biological phenotypes. Our results presented here refute these mechanisms of Pin1 action. Using NMR, ITC and SAXS we dissect binding and catalysis on multiple phosphorylated tau with particular emphasize towards the Alzheimer's associated AT180 tau epitope containing phosphorylated THR231 and SER235. We find that phosphorylated (p-) SER235-PRO, but not pTHR231-PRO, is exclusively catalyzed by full length Pin1 and isolated PPIase domain. Importantly, site-specific measurements of Pin1-catalysis of CDK2/CycA-phosphorylated full-length tau reveal a number of sites that are catalyzed simultaneously with different efficiencies. Furthermore we show that the turnover efficiency at pSER235 by Pin1 is independent of both the WW domain and phosphorylation on THR231. Our mechanistic results on site-specific binding and catalysis together with the lack of an increase of dephosphorylation rates by PP2A counter a series of previously published models for the role of Pin1 catalysis of tau in Alzheimer's disease. Together, our data reemphasize the complicated scenario between binding and catalysis of multiple phosphorylated tau by Pin1 and the need for directly linking biological phenotypes and residue-specific turnover in Pin1 substrates.