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J. Biol. Chem., Vol. 281, Issue 35, 25388-25397, September 1, 2006

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Microtubule Binding and Clustering of Human Tau-4R and Tau-P301L Proteins Isolated from Yeast Deficient in Orthologues of Glycogen Synthase Kinase-3 or cdk5^*

Tom Vandebroek¹, Dick Terwel¹, Thomas Vanhelmont, Maarten Gysemans^¶, Chris Van Haesendonck^¶, Yves Engelborghs^||, Joris Winderickx, and Fred Van Leuven²

From the Experimental Genetics Group, KULeuven, B-3000 Leuven, the Laboratory of Functional Biology, the ^¶Laboratory of Solid State Physics and Magnetism, and the ^||Laboratory of Biomolecular Dynamics, KULeuven, B-3001 Leuven, Belgium

Phosphorylation of Tau protein and binding to microtubules is complex in neurons and was therefore studied in the less complicated model of humanized yeast. Human Tau was readily phosphorylated at pathological epitopes, but in opposite directions regulated by kinases Mds1 and Pho85, orthologues of glycogen synthase kinase-3 and cdk5, respectively (1). We isolated recombinant Tau-4R and mutant Tau-P301L from wild type, mds1 and pho85 yeast strains and measured binding to Taxol-stabilized mammalian microtubules in relation to their phosphorylation patterns. Tau-4R isolated from yeast lacking mds1 was less phosphorylated and bound more to microtubules than Tau-4R isolated from wild type yeast. Paradoxically, phosphorylation of Tau-4R isolated from kinase Pho85-deficient yeast was dramatically increased resulting in very poor binding to microtubules. Dephosphorylation promoted binding to microtubules to uniform high levels, excluding other modifications. Isolated hyperphosphorylated, conformationally altered Tau-4R completely failed to bind microtubules. In parallel to Tau-4R, we expressed, isolated, and analyzed mutant Tau-P301L. Total dephosphorylated Tau-4R and Tau-P301L bound to microtubules very similarly. Surprisingly, Tau-P301L isolated from all yeast strains bound to microtubules more extensively than Tau-4R. Atomic force microscopy demonstrated, however, that the high apparent binding of Tau-P301L was due to aggregation on the microtubules, causing their deformation and bundling. Our data explain the pathological presence of granular Tau aggregates in neuronal processes in tauopathies.

Received for publication, March 24, 2006 , and in revised form, June 26, 2006.

^* This work was supported by the Fonds voor Wetenschappelijk Onderzoek-Vlaanderen (FWO-Vlaanderen), KULeuven Special Research Fund (KULeu-ven-BOF), Instituut voor Wetenschappelijk en Technisch Onderzoek (IWT), KULeuven R&D, and the Roomsfund. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Both authors contributed equally to this work.

² To whom correspondence should be addressed: KULeuven Campus Gasthuisberg ON1-06.602, B-3000 Leuven, Belgium. Tel.: 32-16-34-58-88; Fax: 32-16-34-58-71; E-mail: fredvl{at}med.kuleuven.be.

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