Neurofilament (NF) proteins, though synthesized in neuronal cell bodies, are normally posttranslationally phosphorylated in axons. In some neuro-degenerative disorders (e.g., ALS), however, they are abnormally hyperphosphorylated in cell bodies. We have been studying the factors regulating these topographic pattern of neurofilament (NF) phosphorylation. One kinase, cdk5, identified in our laboratory, specifically phosphorylates KSPXK motifs which constitutes 20% of the total repeats in high molecular weight NF-subunit, NF-H. The remaining 80% repeats are KSPXXXK motifs kinase(s) phosphorylating these motifs are not known. A synthetic KSPXXXK peptide, KSPAEAKSPAEAKS, which repeats 41 times with minor variations at non-KSP residues was used as a substrate to identify the kinase in rat brain that phospshorylates NF- H. On the basis of biochemical, immunochemical, pharmacological and amino acid sequence analysis, the purified kinase appeared to be MEK- activated MAP kinase. To verify this statement, we demonstrated that bacterially expressed MAP kinase phosphorylated expressed rat NF-H in addition to KSPXXXK, KSPXXK and KSPXK peptides. This study suggests that neuronal MAPK can phosphsorylate all KSP repeats in rat NF-H and may be the principal kinase in vivo that phosphorylates serine residues in KSP repeats in neurofilament tail domains. Since cdk5 specifically phosphorylates KSPXK peptides and not KSPXXXK, we investigated the structural differences of these motifs, by analyzing the conformation of four peptides with either KSPXK or KSPXXXK motifs. The KSPXXXK peptide exhibited a CD spectrum indicative of a helical conformation; under the same conditions, however, the KSPXK peptide showed a mainly extended conformation with beta-turns. These differences revealed a structural specificity for the substrate of this kinase. using two dimensional NMR methods and molecular modeling. We are also studying the mechanism of cdk5 activation. Though, cdk5 is similar to other cdc2 kinases, its regulation is completely different from mitotic cdc2 kinases; e.g., cdk5 is active only in postmitotic neurons, its activity is independent of its phosphorylation, nor is its activity regulated by cyclin binding. Instead association of neuron specific proteins, P35 and P67 are responsible for its activation. These regulator proteins share no homology with cyclins. We have begun to locate the activation domains in P35 using various mutations and truncations of P35 in in vitro assays. These studies suggest the potential residues for kinase activation reside in amino acid residues 137-167 and 291-263 of N- and C-terminal tail domains respectively of P35. Further mutational and truncational studies are in progress.