Activation of glycogen synthase kinase 3beta promotes the intermolecular association of tau. The use of fluorescence resonance energy transfer microscopy.
Tau is hyperphosphorylated and undergoes proteolysis in Alzheimer disease brain. Caspase-cleaved tau efficiently forms fibrillary structures in vitro and in situ. Glycogen synthase kinase 3beta (GSK3beta) phosphorylates tau and induces the aggregation of caspase-cleaved tau in situ. Given the hypothesis that increased association of tau precedes the formation of fibrillar structures, we generated a cell model to quantitate the extent of tau association in situ using fluorescence resonance energy transfer (FRET) microscopy. The cyan and yellow fluorescent proteins were attached to full-length (T4) and caspase-cleaved (T4C3) tau at either the N or C termini, and a pair of cyan and yellow fluorescent protein-tagged tau were co-transfected into human embryonic kidney cells. The FRET efficiency was examined in the presence of a constitutively active or a kinase-dead GSK3beta. Active GSK3beta significantly increased FRET efficiency with both T4 and T4C3, indicating that GSK3beta activation resulted in an increase in the self-association of both T4 and T4C3, but interestingly only T4 is efficiently phosphorylated by GSK3beta. There was no significant difference in FRET efficiency between T4 and T4C3, although only T4C3 in the presence of active GSK3beta leads to the formation of Sarkosyl-insoluble inclusions. These FRET studies demonstrate that GSK3beta facilitates the association of T4 and T4C3, and the presence of caspase-cleaved tau is necessary for the evolution of tau oligomers into Sarkosyl-insoluble inclusions even though it is not extensively phosphorylated. These data imply that increased association of tau should not be regarded as a direct indicator of the formation of insoluble tau aggregates.
Chun
Wanjoo
W
Department of Psychiatry, University of Alabama at Birmingham School of Medicine, Birmingham, Alabama 35294, USA.
Johnson
Gail V W
GV
D001426:Bacterial Proteins / Q000378:metabolism; D002460:Cell Line; D004789:Enzyme Activation; D031541:Fluorescence Resonance Energy Transfer; D038362:Glycogen Synthase Kinase 3 / Q000378:metabolism*; D000071679:Glycogen Synthase Kinase 3 beta; D049452:Green Fluorescent Proteins / Q000737:chemistry; D006801:Humans; D007668:Kidney / Q000378:metabolism; D008164:Luminescent Proteins / Q000378:metabolism; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D014162:Transfection; D016875:tau Proteins / Q000378:metabolism*
D001426:Bacterial Proteins; C485182:Cyan Fluorescent Protein; D008164:Luminescent Proteins; D016875:tau Proteins; C054562:yellow fluorescent protein, Bacteria; D049452:Green Fluorescent Proteins; C000605506:GSK3B protein, human; D000071679:Glycogen Synthase Kinase 3 beta; D038362:Glycogen Synthase Kinase 3