Cdk5 Activator P35 Research Articles

Cloning and characterization of human IC53-2, a novel CDK5 activator binding protein.

We have identified IC53-2, a human homologue of the rat C53 gene from a human placenta cDNA library (GeneBank Accession No.AF217982). IC53-2 can bind to the CDK5 activator p35 by in vitro association assay. IC53-2 is mapped to human chromosome 17q21.31. The IC53-2 transcript is highly expressed in kidney, liver, skeletal muscle and placenta. It is abundantly expressed in SMMC-7721, C-33A, 3AO, A431 and MCF-7 cancer cell lines by RT-PCR assay. Stable transfection of IC53-2 cDNA into the hepatocellular carcinoma SMMC-7721 cell remarkably stimulates its growth in vitro. The above results indicate that IC53-2 is a novel human gene, which may be involved in the regulation of cell proliferation.

Truncation of CDK5 Activator p35 Induces Intensive Phosphorylation of Ser202/Thr205 of Human Tau

Hyperphosphorylated tau is a major component of neurofibrillary tangles, one of the hallmarks of Alzheimer's disease. CDK5 is a kinase that phosphorylates the tau protein, and its endogenous activator, p35, regulates its activity. Recently, calpain was found to digest p35 to its truncated product, p25. Several lines of evidence suggest that p25-CDK5 has much more powerful kinase activity and that it may cause abnormal hyperphosphorylation of tau. In this study, we have examined the kinetic characteristics of in vitro phosphorylation of the longest isoform of human tau by CDK5 and its activators using recombinant proteins. Although the kinase activity of CDK5 in phosphorylating tau was significantly higher in the presence of p25, the affinity of CDK5 for tau was not different. Phosphopeptide mapping revealed enhanced phosphorylation of Ser(202)/Thr(205) residues by p25-CDK5 (amino acid residues of tau are numbered according to the longest isoform of human tau). These results suggest that cleavage of p35 to p25 greatly enhances the kinase activity of CDK5 and increases the phosphorylation of Ser(202)/Thr(205). Considering the fact that phosphorylation of Ser(202)/Thr(205) antagonizes the tau-mediated nucleation of tubulin, p25-CDK5 may play a pivotal role in neuronal cell death in Alzheimer's disease.

AβPP induces cdk5-dependent tau hyperphosphorylation in transgenic mice Tg2576

Previous studies of Abeta-induced neuronal damage of hippocampal cells in culture have provided strong evidence that deregulation of the Cdk5/p35 kinase system is involved in the neurodegeneration pathway. Cdk5 inhibitors and antisense probes neuroprotected hippocampal cells against the neurotoxic action of Abeta. To further investigate the mechanisms underlying the participation of Cdk5 in neuronal degeneration, the transgenic mouse containing the Swedish mutations, Tg2576, was used as an animal model. Immunocytochemical studies using anti-Abeta(1-17) antibody evidenced the presence of labeled small-clustered core plaques in the hippocampus and cortex of 18-month-old transgenic mice brains. The loss of granular cells without a compressed appearance was detected in the vicinity of the cores in the dentate gyrus of the hippocampus. Immunostaining of Tg2576 brain sections with antibodies AT8, PHF1 and GFAP labeled punctuate dystrophic neurites in and around the amyloid core. Reactive astrogliosis around the plaques in the hippocampus was also observed. Studies at the molecular level showed differences in the expression of the truncated Cdk5 activator p25 in the transgenic animal, as compared with wild type controls. However no differences in Cdk5 levels were detected, thus corroborating previous cellular findings. Interestingly, hyperphosphorylated tau epitopes were substantially increased as assessed with the AT8 and PHF1 antibodies, in agreement with the observation of a p25 increase in the transgenic animal. These observations strongly suggest that the increased exposure of Alzheimer's type tau phosphoepitopes in the transgenic mice correlated with deregulation of Cdk5 leading to an increase in p25 levels. These studies also provide further evidence on the links between extraneuronal amyloid deposition and tau pathology.

Cleavage of the cyclin-dependent kinase 5 activator p35 to p25 does not induce tau hyperphosphorylation

Hyperphosphorylated tau protein is the primary component of neurofibrillary tangles observed in several neurodegenerative disorders. It has been hypothesized that in certain pathological conditions, the calcium activated protease, calpain, would cleave the cyclin-dependent kinase 5 (cdk5) activator p35 to a p25 fragment, which would lead to augmented cdk5 activity, and cdk5-mediated tau hyperphosphorylation. To test this hypothesis, we induced calpain-mediated p35 cleavage in rat hippocampal neuronal cultures and studied the relationship between p25 production, cdk5 activity, and tau phosphorylation. In glutamate-treated cells p35 was cleaved to p25 and this was associated with elevated cdk5 activity. However, tau phosphorylation was concomitantly decreased at multiple sites. The calpain inhibitor MDL28170 prevented the cleavage of p35 but had no effect on tau phosphorylation, suggesting that calpain-mediated processes, i.e., the cleavage of p35 to p25 and cdk5 activation, do not contribute to tau phosphorylation in these conditions. Treatment of the neuronal cultures with N-methyl- d-aspartic acid or with calcium ionophores resulted in an outcome highly similar to that of glutamate. We conclude that, in neuronal cells, the cleavage of p35 to p25 is associated with increased activity of cdk5 but not with tau hyperphosphorylation.

A survey of Cdk5 activator p35 and p25 levels in Alzheimer’s disease brains

P25, a calpain cleavage product of the cyclin-dependent kinase 5 (Cdk5) activator p35, causes prolonged activation of Cdk5. Although p25 has been shown to accumulate in brains of patients with Alzheimer’s disease (AD), it is not known whether p25 accumulation in AD is brain region-specific. We analyzed the amounts of p25 and p35 in human autopsy samples from multiple brain regions including frontal cortex, inferior parietal cortex and hippocampus using immunoblotting assays. Our results show that the p25–p35 indices are higher in AD than in the control groups in all three brain regions. The most significant difference in p25–p35 indices between AD and control groups is in the frontal cortex. No significant difference in calpain activity between AD and control groups is observed, indicating that postmortem calpain activation cannot account for the elevation of p25/p35 ratios in AD brains. Our results support the notion that p25 accumulation deregulates Cdk5 activity in AD brains, and the deregulated Cdk5 activity may contribute to the pathogenesis of AD.

Identification of a Neuronal Cdk5 Activator-binding Protein as Cdk5 Inhibitor

Neuronal Cdc2-like kinase (Nclk) plays an important role in a variety of cellular processes, including neuronal cell differentiation, apoptosis, neuron migration, and formation of neuromuscular junction. The active kinase consists of a catalytic subunit, Cdk5, and an essential regulatory subunit, neuronal Cdk5 activator (p35(nck5a) or p25(nck5a)), which is expressed primarily in neurons of central nervous tissue. In our previous study using the yeast two-hybrid screening method, three novel p35(nck5a)-associated proteins were isolated. Here we show that one of these proteins, called C42, specifically inhibits the activation of Cdk5 by Nck5a. Co-immunoprecipitation data suggested that C42 and p35(nck5a) could form a complex within cultured mammalian cells. Deletion analysis has mapped the inhibitory domain of C42 to a region of 135 amino acids, which is conserved in Pho81, a yeast protein that inhibits the yeast cyclin-dependent protein kinase Pho85. The Pho85.Pho80 kinase complex has been shown to be the yeast functional homologue of the mammalian Cdk5/p35(nck5a) kinase.

GC box-binding transcription factors control the neuronal specific transcription of the cyclin-dependent kinase 5 regulator p35.

Cyclin-dependent kinase 5 (cdk5)/p35 kinase activity is highest in post-mitotic neurons of the central nervous system and is critical for development and function of the brain. The neuronal specific activity of the cdk5/p35 kinase is achieved through the regulated expression of p35 mRNA. We have identified a small 200-bp fragment of the p35 promoter that is sufficient for high levels of neuronal specific expression. Mutational analysis of this TATA-less promoter has identified a 17-bp GC-rich element, present twice, that is both required for promoter activity and sufficient for neuronal specific transcription. A GC box within the 17-bp element is critical for both promoter activity and protein-DNA complex formation. The related transcription factors Sp1, Sp3, and Sp4 constitute most of the GC box DNA binding activity in neurons. We have found that both the relative contribution of the Sp family proteins to GC box binding and the transcriptional activity of these proteins is regulated during neuronal differentiation. Thus, our data show that the GC box-binding Sp proteins contribute to the regulation of p35 expression in neurons, suggesting changes in the Sp transcription factors level and activity may contribute to cell type-specific expression of many genes in the central nervous system.

The Levels of cdk5 and p35 Proteins and Tau Phosphorylation Are Reduced during Neuronal Apoptosis

Cyclin-dependent kinase 5 (cdk5) is believed to be involved in the phosphorylation of tau protein. We studied the expression of the protein levels of cdk5 and the neuron-specific cdk5 activator p35 as well as cdk5 activity and tau phosphorylation during apoptosis in rat hippocampal neuronal cultures. We observed that in cells treated with etoposide, cyclosporin A, 4-hydroxynonenal (HNE), or okadaic acid, there was an early reduction in the protein levels of p35, and later also in cdk5 with all treatments except etoposide. The level of p25, a calpain cleavage product of p35 suggested to have increased ability to activate cdk5, was reduced paralleling the amount of p35. The changes in the p35 and p25 protein levels coincided with decreases in cdk5 activity and tau phosphorylation after treatment with HNE and etoposide. However, the relationship between the p35 and p25 levels and cdk5 activity was complex. We conclude that neuronal apoptosis is accompanied with a decrease in the levels of p35, p25, and cdk5, and tau phosphorylation. These changes may reinforce the neuronal damage.

Processing of cdk5 Activator p35 to Its Truncated Form (p25) by Calpain in Acutely Injured Neuronal Cells

Recently, it was shown that conversion of cdk5 activator protein p35 to a C-terminal fragment p25 promotes a deregulation of cdk5 activity, which may contribute to neurodegeneration in Alzheimer's disease. In this study, we present evidence that calpain is a protease involved in the conversion of p35 to p25. To activate calpain, rat cerebellar granule neurons were treated with maitotoxin (MTX). A C-terminus-directed anti-p35 antibody detected that p35 conversion to p25 paralleled the formation of calpain-generated alpha-spectrin (alpha-fodrin) breakdown products (SBDP's) in a maitotoxin-dose-dependent manner. Two calpain inhibitors (MDl28170 and SJA6017) reduced p35 processing but were unchanged when exposed to the caspase inhibitor carbobenzoxy-Asp-CH2OC(O)-2,6-dichlorobenzene or the proteasome inhibitors (lactacystin and Z-Ile-Glu(OtBu)Ala-Leu-CHO). p35 protein was also degraded to p25 when rat brain lysate was subjected to in vitro digestion with purified μ- and m-calpains. Additionally, in a rat temporary middle cerebral artery occlusion model, p35 processing to p25 again paralleled SBDP formation in the ischemic core. Lastly, in malonate-injured rat brains, the ipsilateral side showed a striking correlation of SBDP formation with p35 to p25 conversion and tau phosphorylation (at Ser202 and Thr205) increase. These data suggest that calpain is a major neuronal protease capable of converting p35 to p25 and might play a pathological role of activating cdk5 and its phosphorylation of tau in Alzheimer's disease.

Evidence for the Participation of the Neuron-Specific CDK5 Activator P35 during Laminin-Enhanced Axonal Growth

Cultures of cerebellar macroneurons were used to study the pattern of expression, subcellular localization, and function of the neuronal cdk5 activator p35 during laminin-enhanced axonal growth. The results obtained indicate that laminin, an extracellular matrix molecule capable of selectively stimulating axonal extension and promoting MAP1B phosphorylation at a proline-directed protein kinase epitope, selectively stimulates p35 expression, increases its association with the subcortical cytoskeleton, and accelerates its redistribution to the axonal growth cones. Besides, suppression of p35, but not of a highly related isoform designated as p39, by antisense oligonucleotide treatment selectively reduces cdk5 activity, laminin-enhanced axonal elongation, and MAP1b phosphorylation. Taken collectively, the present results suggest that cdk5/p35 may serve as an important regulatory linker between environmental signals (e.g., laminin) and constituents of the intracellular machinery (e.g., MAP1B) involved in axonal elongation.

Association of Neurofilament Proteins with Neuronal Cdk5 Activator

Cdk5 exists in brain extracts in multiple forms, one of which is a macromolecular protein complex comprising Cdk5, neuron-specific Cdk5 activator p35nck5a and other protein components (Lee, K.-Y., Rosales, J. L., Tang, D., and Wang, J.H. (1996) J. Biol. Chem. 271, 1538-1543). The yeast two-hybrid system was employed to identify p35nck5a-interacting proteins from a human brain cDNA library. One of the isolated clones encodes a fragment of glial fibrillary acidic protein, which is a glial-specific protein. Sequence alignment revealed significant homology between the p35nck5a-binding fragment of glial fibrillary acidic protein and corresponding regions in neurofilaments. The association between p35nck5a and neurofilament medium molecular weight subunit (NF-M) was confirmed by both the yeast two-hybrid assay and direct binding of the bacteria-expressed proteins. The p35nck5a binding site on NF-M was mapped to a carboxyl-terminal region of the rod domain, in close proximity to the putative Cdk5 phosphorylation sites in NF-M. A region immediately amino-terminal to the kinase-activating domain in p35nck5a is required for its binding with NF-M. In in vitro binding assays, NF-M binds both monomeric p35nck5a and the Cdk5/p35nck5a complex. The binding of NF-M has no effect on the kinase activity of Cdk5/p35nck5a.