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 p34(cdc2)/cyclin B kinase; the substrate specificity and inhibition by a p34(cdc2) kinase specific inhibitor, butyrolactone I. However, p34(cdc2)/cyclin B kinase was distinguished from cdk5/p26 by its binding to p13(suc1) protein and by its reactivity to anti-p34(cdc2) antibodies. In spite of similar enzymatic properties of cdk5/p26 and p34(cdc2)/cyclin B kinase, cdk5/p26 did not display M-phase promoting activity when assayed with a cell-free system of Xenopus egg extract. (C) 1995 Wiley-Liss, Inc.