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.

Phosphorylation of types III and IV intermediate filaments (IFs) is known to regulate their organization and function. Phosphorylation of the amino-terminal head domain sites on types III and IV IF proteins plays a key role in the assembly/disassembly of IF subunits into 10 nm filaments, and influences the phosphorylation of sites on the carboxyl-terminal tail domain. These phosphorylation events are largely under the control of second messenger-dependent protein kinases and provide the cells a mechanism to reorganize the IFs in response to the changes in second messenger levels. In mitotic cells, Cdk1, Rho kinase, PAK1 and Aurora-B kinase are believed to regulate vimentin and glial fibrillary acidic protein phosphorylation in a spatio-temporal manner. In neurons, the carboxyl-terminal tail domains of the NF-M and NF-H subunits of heteropolymeric neurofilaments (NFs) are highly phosphorylated by proline-directed protein kinases. The phosphorylation of carboxyl-terminal tail domains of NFs has been suspected to play roles in forming cross-bridges between NFs and microtubules, slowing axonal transport and promoting their integration into cytoskeleton lattice and, in doing so, to control axonal caliber and stabilize the axon. The role of IF phosphorylation in disease pathobiology is discussed.

Members of the N-methyl-D-aspartate (NMDA) class of glutamate receptors (NMDARs) are critical for development, synaptic transmission, learning and memory; they are targets of pathological disorders in the central nervous system. NMDARs are phosphorylated by both serine͞threonine and tyrosine kinases. Here, we demonstrate that cyclin dependent kinase-5 (Cdk5) associates with and phosphorylates NR2A subunits at Ser-1232 in vitro and in intact cells. Moreover, we show that roscovitine, a selective Cdk5 inhibitor, blocks both long-term potentiation induction and NMDAevoked currents in rat CA1 hippocampal neurons. These results suggest that Cdk5 plays a key role in synaptic transmission and plasticity through its up-regulation of NMDARs. The N-methyl-D-aspartate (NMDA) class of glutamate receptors (NMDAR) are essential for learning, memory, and development in the central nervous system (1-5). NMDARs are multimeric complexes formed from both NMDA receptor subunit (NR1) and modulatory NR2 subunits (6-9). A single gene encodes the NR1 subunit. Eight possible alternative RNA-splice variants provide molecular diversity of NMDARs (10). NR2A-NR2D are encoded by four separate genes (11,12). NMDARs consist of NR1͞NR2 heteromeric complexes (13-15). The activation of NMDARs and the influx of Ca NMDA-channel activity is dynamically modulated in both intracellular and extracellular sites (3). Phosphorylation sites have been identified on the NR1, NR2A, and NR2B subunits, but not on the NR2C-NR2D subunits. Protein kinase C phosphorylates Ser-890 and Ser-896, and protein kinase A phosphorylates Ser-897 within the C1 exon of the NR1 subunit (17-19). Calcium͞calmodulin protein kinase II mediates phosphorylation of NR2B, but not NR2A (20). More recently, phosphorylation at tyrosine residues of NR2A and NR2B has been described as an important determinant for NMDAR functions (19)(20). In particular, Fyn, a member of the Src family of nonreceptor protein tyrosine kinases, was shown to phosphorylate the NR2A (7, 21-23). However, no information is available concerning which kinases phosphorylate NR2A at serine͞threonine sites. Cyclindependent kinase-5 (Cdk5) is a serine͞threonine kinase that is activated by neuron-specific p35 and p39 proteins (24-27). It exists as a large, multimeric complex associated with cytoskeletal proteins in the neurons. Cdk5 has been shown to phosphorylate a wide variety of proteins, all of which have serine͞threonine sites in (K͞RT͞SPXK)-type motifs (28,29). A number of synaptic proteins have been identified as Cdk5 substrates (30-32). Cdk5 and p35, predominantly expressed in postmitotic neurons, play essential roles in neuronal migration, neurite outgrowth, and laminar configuration of the cerebral cortex (25,27,33). Cdk5 in association with p25, a truncated form of p35, hyperphosphorylates the microtubule-associated protein tau. This hyperphosphorylation is thought to disrupt the neuronal cytoskeleton and ultimately contributes to neurodegeneration in Alzheimer's disease (34).Because of its virtue of phosphoryl...

p34cdc2 kinase mRNA levels during the terminal differentiation of neurons (13), demonstration ofNF-H phosphorylation by p34Cdc2 kinase from nonneuronal sources, while intriguing, has uncertain physiologic relevance. The presence of consensus sequences of substrate for p34cdc2 kinase on vertebrate NF-H suggests the possibility that there may be cdc2-related kinases in the nervous system that phosphorylate these motifs.In this report, we present the purification, biochemical, and immunologic characterization of a p34cdc2-like kinase isolated from rat spinal cords. We demonstrate that dephosphorylated NF-H and NF-M, and synthetic peptides containing KSPXK sequences are highly effective substrates and suggest that NF protein may be one of the endogenous substrates for this cdc2-like kinase.MATERIALS AND METHODS Materials. Synthetic peptide analogs of KSP sequences were custom-synthesized by Peptide Technologies (Washington, DC). Peptide substrates of protein kinase A (kemptide) and S6 kinase (S6 substrate) were obtained from Peninsula Laboratories, protein kinase C, acetyl-myelin basic protein-(4-14), tyrosine kinase (RR-SRC), p34cdc2 kinase [21-mer peptide analog, simian virus 40 (SV40) large tumor (T) antigen], and microcystin LR were obtained from GIBCO/BRL. a-Casein and dephosphorylated casein were obtained from Sigma; histone (Hi), alkaline phosphatase (calf intestine), and phosphatase-labeled goat anti-rabbit IgG were obtained from Boehringer Mannheim. Antibodies directed against C-terminal (mouse) and PSTAIRE regions (human) of p34cdc2 kinase were purchased from Upstate Biotechnology (Lake Placid, NY).Extraction. Rat spinal cords (120 g, obtained from PelFreez Biologicals) were suspended in isotonic low-salt buffer [20 mM Tris-HCl/l mM EDTA/1 mM EGTA/1 mM dithiothreitol (DTT)/leupeptin (5 ,g/ml)/0.3 M sucrose at pH 7.5] maintained at 4°C. After removing the blood contaminants and meninges, the tissues were diced and homogenized, 1:5 (wt/vol), in high-salt extraction buffer (where 0.8 M KCl was substituted for sucrose) with a Polytron homogenizer (Brinkmann). During homogenization, 0.5 M phenylmethylsulfonyl fluoride in ethanol was added to a final concentration of 2.5 mM, and the preparation was stirred for 3 hr at 4°C. The homogenate was centrifuged (100,000 x g for 60 min), and the supernate was dialyzed against column-equilibrating buffer [CEB = 20 mM Tris HCl/l mM EDTA/1 mM EGTA/1 mM DTT/5% (vol/vol) glycerol, pH 7.5]. The dialysate was centrifuged 50,000 x g for 30 min, and the resulting supemate was used for further purification.Purification. The dialyzed supernate was applied to a P-il phosphocellulose column, equilibrated in CEB, and washed Abbreviations: SV40, simian virus 40; T, tumor; DTT, dithiothreitol; NF, neurofilament.

ABSTRACTproposed to regulate the progression from G, to S phase. In this work, we have cloned and structurally characterized a third member ofthe cdc2 kinase family with 58% amino acid sequence identity to mouse cdkl and 61% identity to human cdk2. We call this kinase neuronal cdc2-like kinase (ndk) because, in contrast to either cdkl or cdk2, nclk is expressed at high levels in terminally differentiated neurons no longer in the cell cycle.

The intermediate filament protein nestin is characterized by its specific expression during the development of neuronal and myogenic tissues. We identify nestin as a novel in vivo target for cdk5 and p35 kinase, a critical signaling determinant in development. Two cdk5-specific phosphorylation sites on nestin, Thr-1495 and Thr-316, were established, the latter of which was used as a marker for cdk5-specific phosphorylation in vivo. Ectopic expression of cdk5 and p35 in central nervous system progenitor cells and in myogenic precursor cells induced elevated phosphorylation and reorganization of nestin. The kinetics of nestin expression corresponded to elevated expression and activation of cdk5 during differentiation of myoblast cell cultures and during regeneration of skeletal muscle. In the myoblasts, a disassembly-linked phosphorylation of Thr-316 indicated active phosphorylation of nestin by cdk5. Moreover, cdk5 occurred in physical association with nestin. Inhibition of cdk5 activity-either by transfection with dominant-negative cdk5 or by using a specific cdk5 inhibitorblocked myoblast differentiation and phosphorylation of nestin at Thr-316, and this inhibition markedly disturbed the organization of nestin. Interestingly, the interaction between p35, the cdk5 activator, and nestin appeared to be regulated by cdk5. In differentiating myoblasts, p35 was not complexed with nestin phosphorylated at Thr-316, and inhibition of cdk5 activity during differentiation induced a marked association of p35 with nestin. These results demonstrate that there is a continuous turnover of cdk5 and p35 activity on a scaffold formed by nestin. This association is likely to affect the organization and operation of both cdk5 and nestin during development.