Cdk5 and MAPK are associated with complexes of cytoskeletal proteins in rat brain

Neurofilament proteins, the major cytoskeletal components of large myelinat ed axons, are highly phosphorylated by second messenger-dependent and -inde pendent kinases. These kinases, together with tubulins and other cytoskelet al proteins, have been shown to bind to neurofilament preparations. Cdk5 an d Erk2, proline-directed kinases in neuronal tissues, phosphorylate the Lys -Ser-Pro (KSP) repeats in tail domains of NF-H, NF-M, and other axonal prot eins such as tau and synapsin. In neurofilament and microtubule preparation s from rat brain, we demonstrated by Western blot analysis that cdk5, a neu ronal cyclin dependent kinase and Erk1/2 were associated with complexes of NF proteins, tubulins and tau. Using P13(suc1) affinity chromatography, a p rocedure known to bind cdc2-like kinases in proliferating cells with high a ffinity, we obtained a P13 complex from a rat brain extract exhibiting the same profiles of cdk5 and Erk2 bound to cytoskeletal proteins. The phosphor ylation activities of these preparations and the effect of the cdk5 inhibit or, butyrolactone, were consistent with the presence of active kinases. Fin ally, during a column fractionation and purification of Erk kinases from ra t brain extracts, fractions enriched in Erk kinase activity also exhibited co-elution of phosphorylated NF-H, tubulin, tau and cdk5. We suggest that i n mammalian brain, different kinases, their regulators and phosphatases for m multimeric complexes with cytoskeletal proteins and regulate multisite ph osphorylation from synthesis in the cell body to transport and assembly in the axon. (C) 2000 Elsevier Science B.V. All rights reserved.