Microtubule binding and clustering of human Tau-4R and Tau-P301L proteins isolated from yeast deficient in orthologues of glycogen synthase kinase-3beta or cdk5.
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-3beta and cdk5, respectively (1). We isolated recombinant Tau-4R and mutant Tau-P301L from wild type, Delta mds1 and Delta 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.
Vandebroek
Tom
T
Experimental Genetics Group, KULeuven, B-3000 Leuven, Belgium.
Terwel
Dick
D
Vanhelmont
Thomas
T
Gysemans
Maarten
M
Van Haesendonck
Chris
C
Engelborghs
Yves
Y
Winderickx
Joris
J
Van Leuven
Fred
F
D051360:Cyclin-Dependent Kinase 5 / Q000502:physiology*; D000939:Epitopes / Q000737:chemistry; D005656:Fungal Proteins / Q000737:chemistry; D038362:Glycogen Synthase Kinase 3 / Q000502:physiology*; D000071679:Glycogen Synthase Kinase 3 beta; D006801:Humans; D007700:Kinetics; D008869:Microtubule-Associated Proteins / Q000737:chemistry*; D008870:Microtubules / Q000737:chemistry*; D009474:Neurons / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry; D014404:Tubulin / Q000737:chemistry; D016875:tau Proteins / Q000737:chemistry*
D000939:Epitopes; D005656:Fungal Proteins; C054369:MAPT protein, human; D008869:Microtubule-Associated Proteins; D011994:Recombinant Proteins; D014404:Tubulin; D016875:tau Proteins; D051360:Cyclin-Dependent Kinase 5; C000605506:GSK3B protein, human; D000071679:Glycogen Synthase Kinase 3 beta; D038362:Glycogen Synthase Kinase 3