Although cyclin-dependent kinase 5 (Cdk5) is closely related to other cyclin-dependent kinases, its kinase activity is detected only in the postmitotic neurons. Cdk5 expression and kinase activity are correlated with the extent of differentiation of neuronal cells in developing brain. CdkS purified from nervous tissue phosphorylates neuronal cytoskeletal proteins including neurofilament proteins and microtubule-associated protein tau in vitro. These findings indicate that Cdk5 may have unique functions in neuronal cells, especially in the regulation of phosphorylation of cytoskeletal molecules. We report here generation of CdkS(-/-) mice through gene targeting and their phenotypic analysis.CdkS(-/-) mice exhibit unique lesions in the central nervous system associated with perinatal mortality. The brains of CdkS(-/-) mice lack cortical laminar structure and cerebellar foliation. In addition, the large neurons in the brain stem and in the spinal cord show chromatolytic changes with accumulation of neurofilament immunoreactivity. These findings indicate that Cdk5 is an important molecule for brain development and neuronal differentiation and also suggest that Cdk5 may play critical roles in neuronal cytoskeleton structure and organization.

Neuronal transmission of information requires polarized distribution of membrane proteins within axonal compartments. Membrane proteins are synthesized and packaged in membrane‐bounded organelles (MBOs) in neuronal cell bodies and later transported to axons by microtubule‐dependent motor proteins. Molecular mechanisms underlying targeted delivery of MBOs to discrete axonal subdomains (i.e. nodes of Ranvier or presynaptic terminals) are poorly understood, but regulatory pathways for microtubule motors may be an essential step. In this work, pharmacological, biochemical and in vivo experiments define a novel regulatory pathway for kinesin‐driven motility in axons. This pathway involves enzymatic activities of cyclin‐dependent kinase 5 (CDK5), protein phosphatase 1 (PP1) and glycogen synthase kinase‐3 (GSK3). Inhibition of CDK5 activity in axons leads to activation of GSK3 by PP1, phosphorylation of kinesin light chains by GSK3 and detachment of kinesin from transported cargoes. We propose that regulating the activity and localization of components in this pathway allows nerve cells to target organelle delivery to specific subcellular compartments. Implications of these findings for pathogenesis of neurodegenerative diseases such as Alzheimer's disease are discussed.

Mammalian neurofilament proteins, particularly midsized (NF-M) and heavy (NF-H) molecular weight neurofilament proteins, are highly phosphorylated in axons. Neurofilament function depends on the state of phosphorylation of the numerous serine/threonine residues in these proteins. Most phosphorylation occurs in the lys-ser-pro (KSP) repeats in the C-terminal tail domains of NF-H and NF-M. In our previous study, cyclin-dependent kinase 5 (cdk5) was shown to phosphorylate specifically the KSPXK repeats in rat NF-H. Because 80% of the repeats are of the KSPXXXK type, it was of interest to determine which kinase phosphorylates these motifs. Using a synthetic KSPXXXK peptide to screen for a specific kinase, we fractionated rat brain extracts by column chromatography and identified extracellular signal-regulated kinase (Erk2) activated by an upstream activator, the mitogen-activated protein kinase kinase MAPKK (MEK), by Western blot analysis, sequence identification, and inhibition by a specific MEK inhibitor (PD 98059). The fraction containing Erk2, as well as bacterially expressed Erk1 and Erk2, phosphorylated all types of KSP motifs in peptides (KSPXK, KSPXXK, KSPXXXK, and KSPXXXXK) derived from NF-M and NF-H. They also phosphorylated an expressed 24 KSPXXXK repeat NF-H polypeptide, an expressed NF-H as well as dephosphorylated native rat NF-H, and NF-M proteins with accompanying decreases in their respective electrophoretic mobilities. A comparative kinetic study of Erk2 and cdk5 phosphorylation of KSPXK and KSPXXXK peptides revealed that, in contrast to cdk5, which phosphorylated only the KSPXK peptide, Erk2 could phosphorylate both. The preferred substrate for Erk2 was KSPXXXK peptide. The MEK inhibitor PD 98059 also inhibited phosphorylation of NF-H, NF-M, and microtubule-associated protein (MAP) in primary rat hippocampal cells and caused a decrease in neurite outgrowth, suggesting that Erk1,2 may play an important role in neurite growth and branching. These data suggest that neuronal Erk1 and Erk2 are capable of phosphorylating serine residues in diverse KSP repeat motifs in NF-M and NF-H.