Phosphorylation of microtubule-associated protein tau by Ca2+/calmodulin-dependent protein kinase II in its tubulin binding sites.
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.
Yamamoto
Hideyuki
H
Department of Pharmacology, Kumamoto University School of Medicine, Kumamoto, Japan. hideyuki@gpo.kumamoto-u.ac.jp
Yamauchi
Emiko
E
Taniguchi
Hisaaki
H
Ono
Tsunehiko
T
Miyamoto
Eishichi
E
D000595:Amino Acid Sequence; D001665:Binding Sites; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D002853:Chromatography, Liquid / Q000379:methods; D006801:Humans; D008870:Microtubules / Q000187:drug effects / Q000378:metabolism; D008969:Molecular Sequence Data; D017239:Paclitaxel / Q000378:metabolism / Q000494:pharmacology; D010446:Peptide Fragments / Q000378:metabolism; D010766:Phosphorylation; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012697:Serine Endopeptidases / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D014404:Tubulin / Q000378:metabolism*; D016875:tau Proteins / Q000235:genetics / Q000378:metabolism*
D010446:Peptide Fragments; D011994:Recombinant Proteins; D014404:Tubulin; D016875:tau Proteins; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D012697:Serine Endopeptidases; C036813:lysyl endopeptidase; D017239:Paclitaxel