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.

Cyclin-dependent protein kinase 5 (cdk5), a member of the cdk family, is active mainly in postmitotic cells and plays important roles in neuronal development and migration, neurite outgrowth, and synaptic transmission. In this study we investigated the relationship between cdk5 activity and regulation of the mitogen-activated protein (MAP) kinase pathway. We report that cdk5 phosphorylates the MAP kinase kinase-1 (MEK1) in vivo as well as the Ras-activated MEK1 in vitro. The phosphorylation of MEK1 by cdk5 resulted in inhibition of MEK1 catalytic activity and the phosphorylation of extracellular signal-regulated kinase (ERK) 1/2. In p35 (cdk5 activator) ؊/؊ mice, which lack appreciable cdk5 activity, we observed an increase in the phosphoryla- 1 is a member of the cyclin-dependent protein kinase family (cdc2, CDC28, and other generically cyclin-dependent CDKs). Although cdk5 binds to cyclin D, its activity is not regulated by cyclins and there is little evidence that cdk5 is involved in the progression of the cell cycle (for review see Ref.1; see also Refs. 2 and 3). cdk5 is active mainly in post-mitotic cells such as neurons (4, 5), retinal cells (6), and muscle cells (7), where its activators p35 (or its truncated form p25) (4, 5) and p39 (8 -11) are specifically expressed. cdk5 has been suggested to play important roles in neurite outgrowth (12, 13), neuronal migration (14 -16), dopamine signaling in the striatum (17), exocytosis (18 -21), differentiation of muscle cells (7), and organization of acetylcholine receptors at the neuromuscular junction (22). Although neuronal cytoskeletal proteins were initially identified as the major target substrates (4, 23, 24), the number of cdk5 substrates has expanded considerably (see Table I in Ref. 25). These include DARPP-32, a dopamine and cyclic AMP-regulated phosphoprotein involved in dopamine signaling (17), NUDEL (a murine homolog of the Aspergillus nidulans nuclear migration mutant NudE), a protein involved in neuronal migration and axon transport (26), and other proteins involved in cross-talk between protein kinases and phosphatases (27). cdk5 also modulates protein kinase reactions such as the small GTPase-Rac dependent phosphorylation of p21-activated kinase, which results in modification of the actin cytoskeleton (28). By virtue of phosphorylating these diverse substrates, cdk5 plays a multifunctional role in the nervous system. It has been demonstrated that the absence of cdk5 in cdk5 Ϫ/Ϫ mice results in embryonic lethality (16). Although the p35 knockout mice survive longer (14), both cdk5 Ϫ/Ϫ and p35 Ϫ/Ϫ mice exhibit similar defects in cortical neuronal migration and affect the development of the nervous system (14 -16). We observed that in cdk5 Ϫ/Ϫ mice brain stem neurons showed ballooning and hyperphosphorylation of cytoskeletal proteins as detected by the SMI31 antibody (see Fig. 1). Similar observations were obtained from p35 (Ϫ/Ϫ) mice. 2 The antibody cross-reacts with phosphorylated Lys-Ser-Pro (KSP) motifs in neurofilament proteins, tau, and MAPs ...

The extracellular aggregation of amyloid b (Ab) peptides and the intracellular hyperphosphorylation of tau at specific epitopes are pathological hallmarks of neurodegenerative diseases such as Alzheimer's disease (AD). Cdk5 phosphorylates tau at AD-specific phospho-epitopes when it associates with p25. p25 is a truncated activator, which is produced from the physiological Cdk5 activator p35 upon exposure to Ab peptides. We show that neuronal infections with Cdk5 inhibitory peptide (CIP) selectively inhibit p25/ Cdk5 activity and suppress the aberrant tau phosphorylation in cortical neurons. Furthermore, Ab 1À42 -induced apoptosis of these cortical neurons was also reduced by coinfection with CIP. Of particular importance is our finding that CIP did not inhibit endogenous or transfected p35/ Cdk5 activity, nor did it inhibit the other cyclin-dependent kinases such as Cdc2, Cdk2, Cdk4 and Cdk6. These results, therefore, provide a strategy to address, and possibly ameliorate, the pathology of neurodegenerative diseases that may be a consequence of aberrant p25 activation of Cdk5, without affecting 'normal' Cdk5 activity.

Glucocorticoids, major end effectors of the stress response, play an essential role in the homeostasis of the central nervous system and influence diverse functions of neuronal cells. We found that cyclin-dependent kinase 5 (CDK5), which plays important roles in the morphogenesis and functions of the nervous system and whose aberrant activation is associated with development of neurodegenerative disorders, interacted with the ligand-binding domain of the glucocorticoid receptor (GR) through its activator p35 or its active proteolytic fragment p25. CDK5 phosphorylated GR at multiple serines, including Ser203 and Ser211 of its N-terminal domain, and suppressed the transcriptional activity of this receptor on glucocorticoid-responsive promoters by attenuating attraction of transcriptional cofactors to DNA. In microarray analyses using rat cortical neuronal cells, the CDK5 inhibitor roscovitine differentially regulated the transcriptional activity of the GR on more than 90% of the endogenous glucocorticoid-responsive genes tested. Thus, CDK5 exerts some of its biological activities in neuronal cells through the GR, dynamically modulating GR transcriptional activity in a target promoter-dependent fashion.