Evidence for cdk5 as a major activity phosphorylating tau protein in porcine brain extract.
The major kinase capable of phosphorylating tau in a porcine brain extract was suggested to be a brain cdc2-like kinase, called cdk5. Tau protein components of microtubules assembled in the brain extract using ATP were phosphorylated to a higher level, and showed a slower electrophoretic mobility than those assembled with GTP. Most of this phosphorylation and electrophoretic mobility shift, that occurred in the brain extract incubated with ATP, were inhibited by butyrolactone I, a specific inhibitor of cdc2 kinase and cdk5. Further, butyrolactone I inhibited phosphorylation of tau exogenously added to the brain extract by approximately 70%. cdk5 purified from porcine brain decreased the electrophoretic mobility of dephosphorylated tau by in vitro phosphorylation of tau to the level present in microtubules polymerized with ATP. cdc2 kinase purified from starfish oocytes also phosphorylated tau and shifted its electrophoretic mobility to an extent greater than that obtained with cdk5. Western blot analysis showed that cdc2 kinase phosphorylated epitopes recognized by SMI31, 33, 34, and tau 1 antibodies in tau proteins , while cdk5 phosphorylated the site recognized by SMI33 (corresponding to phosphorylation at Ser235 in the longest human tau isoform) and partially phosphorylated the tau 1 site. Phosphorylation experiments performed on tau in brain extracts, in the presence of okadaic acid, suggested the presence of both okadaic acid-sensitive and -insensitive phosphatases acting on phosphorylated Ser235. Rat tau that was prepared immediately after decapitation showed a similar phosphorylation state to tau in microtubules polymerized with ATP, suggesting that tau is relatively phosphorylated in vivo.
Hosoi
T
T
Laboratory of Cell and Developmental Biology, Faculty of Biosciences, Tokyo Institute of Technology.
Uchiyama
M
M
Okumura
E
E
Saito
T
T
Ishiguro
K
K
Uchida
T
T
Okuyama
A
A
Kishimoto
T
T
Hisanaga
S
S
D015107:4-Butyrolactone / Q000031:analogs & derivatives / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000378:metabolism / Q000494:pharmacology; D000818:Animals; D015153:Blotting, Western; D001921:Brain / Q000201:enzymology / Q000378:metabolism*; D001923:Brain Chemistry; D016203:CDC2 Protein Kinase / Q000037:antagonists & inhibitors / Q000378:metabolism; D051360:Cyclin-Dependent Kinase 5; D018844:Cyclin-Dependent Kinases; D004586:Electrophoresis; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004988:Ethers, Cyclic / Q000494:pharmacology; D006160:Guanosine Triphosphate / Q000378:metabolism; D008870:Microtubules / Q000378:metabolism; D019319:Okadaic Acid; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D012694:Serine / Q000378:metabolism; D013552:Swine; D014020:Tissue Extracts / Q000378:metabolism; D016875:tau Proteins / Q000378:metabolism*
D004791:Enzyme Inhibitors; D004988:Ethers, Cyclic; D047428:Protein Kinase Inhibitors; D014020:Tissue Extracts; D016875:tau Proteins; D019319:Okadaic Acid; D012694:Serine; D006160:Guanosine Triphosphate; C082868:butyrolactone I; D000255:Adenosine Triphosphate; D051360:Cyclin-Dependent Kinase 5; D017346:Protein Serine-Threonine Kinases; D016203:CDC2 Protein Kinase; D018844:Cyclin-Dependent Kinases; D015107:4-Butyrolactone