RESUMO
The paired helical filaments (PHF) found in Alzheimer's disease (AD) brain are composed mainly of the hyperphosphorylated form of microtubule-associated protein tau (PHF-tau). It is well known that tau is a good in vitro substrate for Ca(2+)/calmodulin-dependent protein kinase II (CaM kinase II). To establish the phosphorylation sites, the longest human tau (hTau40) was bacterially expressed and phosphorylated by CaM kinase II, followed by digestion with lysyl endoprotease. The digests were subjected to liquid chromatography/mass spectrometry. We found that 5 of 22 identified peptides were phosphorylated. From the tandem mass spectrometry, two phosphorylation sites (serines 262 and 356) were identified in the tubulin binding sites. When tau was phosphorylated by CaM kinase II, the binding of tau to taxol-stabilized microtubules was remarkably impaired. As both serines 262 and 356 are reportedly phosphorylated in PHF-tau, CaM kinase II may be involved in hyperphosphorylation of tau in AD brain.
Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Tubulina (Proteína)/metabolismo , Proteínas tau/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina , Cromatografia Líquida/métodos , Humanos , Microtúbulos/efeitos dos fármacos , Microtúbulos/metabolismo , Dados de Sequência Molecular , Paclitaxel/metabolismo , Paclitaxel/farmacologia , Fragmentos de Peptídeos/metabolismo , Fosforilação , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Serina Endopeptidases/metabolismo , Espectrometria de Massas por Ionização por Electrospray/métodos , Proteínas tau/genéticaRESUMO
Inclusions, such as corpora amylacea, axonal spheroids and ubiquitin-positive granular structures, are present in aged brains. We found a phosphorylated tau-positive inclusion in brain tissues obtained from 13 non-demented subjects and five patients with Alzheimer's disease. This inclusion was spherical and 3-20 microm in size. It was most frequently detected in the hippocampal CA1 region and in the prosubiculum but was not present in the white matter. The density of this inclusion increased significantly with aging and decreased after the occurrence of neurofibrillary tangles. The presence of the inclusion was confirmed using immunoelectron microscopy. These findings show a possibility that the inclusion is a novel aging-related structure in the human brain.
