Porcine brain neurofilament-H tail domain kinase: its identification as cdk5/p26 complex and comparison with cdc2/cyclin B kinase.
Using dephosphorylated neurofilament (NF) proteins as substrates, the kinase with a higher activity for the dephosphorylated NF-H than the phosphorylated form of NF-H was searched for in the porcine brain extract. Most NF-H kinase activity in the brain extract pelleted with microtubules. The NF-H kinase purified from a high salt extract of the microtubule pellets was composed of cdk5 and a 26 kDa protein, a fragment of the 35 kDa regulatory subunit of cdk5. In contrast to the association of the active kinase with microtubules, each of uncomplexed cdk5 and the 35 kDa regulatory subunit was differently distributed in the supernatant fraction and the pellet, respectively, by ultracentrifugation of the brain extract. Dephosphorylated forms of NF-H and NF-M became reactive to antibodies recognizing in vivo phosphorylation sites (SMI31, 34, and 36, JJ31 and 51) by phosphorylation with cdk5/p26. cdk5/p26 showed similar enzymatic properties to p34cdc2/cyclin B kinase; the substrate specificity and inhibition by a p34cdc2 kinase specific inhibitor, butyrolactone I. However, p34cdc2/cyclin B kinase was distinguished from cdk5/p26 by its binding to p13suc1 protein and by its reactivity to anti-p34cdc2 antibodies. In spite of similar enzymatic properties of cdk5/p26 and p34cdc2/cyclin B kinase, cdk5/p26 did not display M-phase promoting activity when assayed with a cell-free system of Xenopus egg extract.
Hisanaga
S
S
Laboratory of Cell and Developmental Biology, Faculty of Biosciences, Tokyo Institute of Technology, Yokohama, Japan.
Uchiyama
M
M
Hosoi
T
T
Yamada
K
K
Honma
N
N
Ishiguro
K
K
Uchida
T
T
Dahl
D
D
Ohsumi
K
K
Kishimoto
T
T
D015107:4-Butyrolactone / Q000031:analogs & derivatives / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000201:enzymology*; D016203:CDC2 Protein Kinase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002458:Cell Fractionation; D051360:Cyclin-Dependent Kinase 5; D018844:Cyclin-Dependent Kinases; D016213:Cyclins / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D016200:Maturation-Promoting Factor / Q000378:metabolism; D008870:Microtubules / Q000187:drug effects / Q000201:enzymology; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000302:isolation & purification / Q000378:metabolism*; D016900:Neurofilament Proteins / Q000302:isolation & purification / Q000378:metabolism*; D017239:Paclitaxel / Q000494:pharmacology; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000302:isolation & purification / Q000378:metabolism*; D013379:Substrate Specificity; D013552:Swine
D016213:Cyclins; D004791:Enzyme Inhibitors; D009419:Nerve Tissue Proteins; D016900:Neurofilament Proteins; C089495:neuronal Cdk5 activator (p25-p35); C051774:neurofilament protein H; C082868:butyrolactone I; D051360:Cyclin-Dependent Kinase 5; D017346:Protein Serine-Threonine Kinases; D016203:CDC2 Protein Kinase; D018844:Cyclin-Dependent Kinases; D016200:Maturation-Promoting Factor; D015107:4-Butyrolactone; D017239:Paclitaxel