No abstract

Although the roles of cyclin-dependent kinase 5 (Cdk5) in neurodevelopment and neurodegeneration have been studied extensively, regulation of Cdk5 activity has remained largely unexplored. We report here that glutamate, acting via NMDA or kainate receptors, can induce a transient Ca 2+ / calmodulin-dependent activation of Cdk5 that results in enhanced autophosphorylation and proteasome-dependent degradation of a Cdk5 activator p35, and thus ultimately down-regulation of Cdk5 activity. The relevance of this regulation to synaptic plasticity was examined in hippocampal slices using theta burst stimulation. p35 -/-mice exhibited a lower threshold for induction of long-term potentiation. Thus excitatory glutamatergic neurotransmission regulates Cdk5 activity through p35 degradation, and this pathway may contribute to plasticity.

Cdk5 is a member of the cyclin‐dependent kinases (Cdks), activated by the neuron‐specific activator p39 or p35. The activators also determine the cytoplasmic distribution of active Cdk5, but the mechanism is not yet known. In particular, little is known for p39. p39 and p35 contain localization motifs, such as a second Gly for myristoylation and Lys clusters in the N‐terminal p10 region. Using mutant constructs, we investigated the cellular distribution mechanism. We observed that p39 localizes the active Cdk5 complex in the perinuclear region and at the plasma membrane as does p35. We demonstrated the myristoylation of both p39 and p35, and found that it is a major determinant of their membrane association. Plasma membrane targeting depends on the amino acid sequence containing the Lys‐cluster in the N‐terminal p10 region. In contrast, a non‐myristoylated Ala mutant (p39G2A or p35G2A) showed nuclear localization with stronger accumulation of p39G2A than p35G2A. These results indicate that myristoylation regulates the membrane association of p39 as well as p35 and that the Lys cluster controls their trafficking to the plasma membrane. The differential nuclear accumulation of p39 and p35 suggests their segregated functions, p35–Cdk5 in the cytoplasm and p39–Cdk5 in the nucleus.

Cdk5 is a proline-directed Ser/Thr protein kinase predominantly expressed in postmitotic neurons together with its activator, p35. N-terminal truncation of p35 to p25 by calpain results in deregulation of Cdk5 and contributes to neuronal cell death associated with several neurodegenerative diseases. Previously we reported that p35 occurred as a phosphoprotein, phospho-p35 levels changed with neuronal maturation, and that phosphorylation of p35 affected its vulnerability to calpain cleavage. Here, we identify the p35 residues 138 predominantly defines the susceptibility of p35 to calpain-dependent cleavage and that dephosphorylation of this site is a critical determinant of Cdk5-p25-induced cell death associated with neurodegeneration. Cyclin-dependent kinase 5 (Cdk5)2 is a unique member of the Cdk family. Its activity in postmitotic neurons is completely dependent upon association with one of two neuronal specific activators, p35 or p39. Cdk5/p35 is involved in a panoply of processes critical to central nervous system function both during development and throughout maturity including neuronal migration during corticogenesis, neurite outgrowth, regulation of the synaptic vesicle cycle, neurotransmitter release, and postsynaptic neurotransmitter receptor regulation and signaling (1-3). The mechanisms by which Cdk5 activity is normally regulated remains to be fully delineated. Furthermore, because aberrant Cdk5 activity has been implicated in the etiology of neurodegenerative diseases (4, 5), identifying the biochemical mechanisms contributing to deregulation of Cdk5 is of substantial biomedical relevance.Deregulation of Cdk5 results from removal of the first 98 amino acids of p35 by the Ca 2ϩ -dependent cysteine protease, calpain, leaving Cdk5 associated with the N-terminal truncated form p25. Cleavage of p35 to p25 changes the subcellular distribution of active Cdk5 from membranes to the cytosolic fraction (6, 7), thereby altering substrate specificity. p25 accumulates in neurons undergoing various types of cell death (6 -9). Expression of Cdk5/p25 in cultured cells results in increased phospho-Tau levels in comparison to cells expressing Cdk5/ p35 (6). Furthermore, exogenous overexpression of p25 in transgenic mice results in a neurodegenerative phenotype including the formation of paired helical filaments, Tau aggregation, and neuronal loss similar to that observed in Alzheimer disease (10, 11).Cdk5/p25 has also been implicated in ischemia-induced neuronal loss in the hippocampus via increased phosphorylation of the NR2A subunit of the N-methyl-D-aspartic acid receptor (12). In addition, several recent reports indicate that Cdk5-p25 mediates cell death via translocation to the nucleus (13-15). p25 generation increases nuclear Cdk5 activity in cultured neurons, facilitating phosphorylation and inhibition of the pro-survival transcription factor MEF2 (13,15,16). Aberrant Cdk5 activity may also contribute to neuronal cell death via phosphorylation of other survival factors such as the tumor suppressor protein p53 ...

Neurofibrillary tangles are one of the major pathological hallmarks of Alzheimer disease (AD).1 Neurofibrillary tangles are bundles of paired helical filament composed of the microtubule (MT)-associated protein tau in a hyperphosphorylated state (1, 2). Intracellular inclusions made of tau are also found in several other neurodegenerative diseases, including Pick disease, progressive supranuclear palsy, corticobasal degeneration, and frontotemporal dementia and Parkinsonism linked to chromosome 17 (FTDP-17), collectively called tauopathies (3, 4). Exonic and intronic mutations in the tau gene have recently been identified in familial FTDP-17 (5-7), indicating that dysfunction of the tau protein can cause the above mentioned neurodegenerative diseases. Many of the exonic mutations reduce the ability of tau to promote MT assembly (8 -10), but why these mutations cause the formation of tau inclusions is unknown.The tau protein in inclusions is hyperphosphorylated. Around 25 phosphorylation sites have been identified in paired helical filament tau from AD brains (11-13). Characteristic phosphorylation sites are Ser or Thr residues followed by Pro that are phosphorylated by the proline-directed protein kinase activities of extracellular signal-regulated kinase, glycogen synthase kinase 3␤ (GSK3␤), and cyclin-dependent kinase 5 (Cdk5). Phosphorylation reduces the ability of tau to bind to and polymerize MTs, resulting in an increase in the soluble form of tau dissociated from MTs. However, it is unclear how phosphorylated soluble tau assembles into filamentous aggregates of neurofibrillary tangles and then induces neurodegeneration. Elucidating the relationship between tau phosphorylation and aggregate formation is critical to our understanding of the pathogenesis. Phosphorylation of FTDP-17 mutant tau has been studied mainly with GSK3␤ or in non-neuronal cultured cells (14 -17). Mutant tau proteins were not phosphorylated more than wild-type (WT) tau either in transfected cultured cells or in vitro. Among them, R406W mutant tau showed significantly reduced phosphorylation in those experiments. However, both mutant tau and wildtype tau deposited in FTDP-17 brains are also hyperphosphorylated (18,19). It is important to resolve the discrepancy between the reduced phosphorylation of mutant tau in vitro and in cultured neurons and the high phosphorylation of mutant tau in pathological brains.Cdk5 is a proline-directed Ser/Thr kinase activated by a p35 or p39 Cdk5 activator (20 -22). Cdk5 activity is primarily detected in differentiated neurons because p35 and p39 show limited expression in neurons. As described above, Cdk5 is one of the tau protein kinases that phosphorylate tau in living neurons and is also able to generate several paired helical filament epitopes of tau in AD (23,24). However, the phosphorylation of FTDP-17 mutant tau by Cdk5 has not yet been examined. This question should be addressed because the involvement of Cdk5 in the pathogenic phosphorylation of tau has recently become more evident (25,26). In ...

Increasing evidence implicates cyclin-dependent kinase 5 (Cdk5) in neuronal synaptic function. We searched for Cdk5 substrates in synaptosomal fractions prepared from mouse brains. Mass spectrometric analysis after two-dimensional SDS-PAGE identified several synaptic proteins phosphorylated by Cdk5-p35; one protein identified was Sept5 (CDCrel-1). Although septins were isolated originally as cell division-related proteins in yeast, Sept5 is expressed predominantly in neurons and is implicated in exocytosis. We confirmed that Sept5 is phosphorylated by Cdk5-p35 in vitro and identified Ser 17 of adult type Sept5 (Sept5_v1) as a major phosphorylation site. We found that Ser 17 of Sept5_v1 is phosphorylated in mouse brains. Coimmunoprecipitation from synaptosomal fractions and glutathione S-transferase-syntaxin-1A pulldown assays of Sept5_v1 expressed in COS-7 cells showed that phosphorylation of Sept5_v1 by Cdk5-p35 decreases the binding to syntaxin-1. These results indicate that the interaction of Sept5 with syntaxin-1 is regulated by the phosphorylation of Sept5_v1 at Ser 17 by Cdk5-p35.Cyclin-dependent kinase 5 (Cdk5) 3 is a proline-directed Ser/ Thr kinase that is activated by binding to a neuron-specific activator, p35 or p39 (1-3). In contrast to other members of the Cdk family, which are known as cell cycle promoting factors, Cdk5 plays a role in neuronal activities unrelated to cell cycle progression. Cdk5 is involved in neuronal migration during brain development and neurodegeneration in aged brains (4 -8). Recent evidence indicates that Cdk5 also participates in synaptic transmission (9). Cdk5-p35 inhibits neurotransmitter release by phosphorylating the P/Q type calcium channels (10) and inhibits their endocytotic recycling by phosphorylation of dynamin 1 and amphiphysin 1 in the presynaptic region (11-13). Cdk5 is also thought to regulate exocytosis by inhibiting the interaction of Munc18 with syntaxin-1A by phosphorylation (14 -16). Cdk5 also phosphorylates several postsynaptic proteins including NR2A subunit of N-methyl-D-aspartate (NMDA) receptor (17, 18), postsynaptic density-95 (19), protein phosphatase 1 inhibitor-1 (20), and DARPP-32 (dopamineand cAMP-regulated phosphoprotein of 32 kDa) (21). However, the precise function of Cdk5-p35 in synaptic activity has not been elucidated fully.Sept5 (also known as CDCrel-1) is a member of the septin family of cytoplasmic 40 -60-kDa proteins comprising a highly conserved GTPase motif at the N terminus and a coiled-coil domain at the C terminus. Septins were first discovered in mutants of budding yeast (Saccharomyces cerevisiae) that cannot complete cell division (22). Yeast septins form hetero-oligomeric filaments that contribute to bud site selection and neck stability (23-25). Septins play an essential role in cytokinesis in Drosophila (26) and mammals (27). Mammalian septins are involved in many other cellular activities including membrane dynamics, apoptosis, and cytoskeletal remodeling (28 -30). Several septins are also expressed in postmitotic neurons....

Glycogen synthase kinase 3β (GSK3β) is a multifunctional protein kinase involved in many cellular activities including development, differentiation and diseases. GSK3β is thought to be constitutively activated by autophosphorylation at Tyr216 and inactivated by phosphorylation at Ser9. The GSK3β activity has previously been evaluated by inhibitory Ser9 phosphorylation, but it does not necessarily indicate the kinase activity itself. Here, we applied the Phos-tag SDS-PAGE technique to the analysis of GSK3β phosphoisotypes in cells and brains. There were three phosphoisotypes of GSK3β; double phosphorylation at Ser9 and Tyr216, single phosphorylation at Tyr216 and the nonphosphorylated isotype. Active GSK3β with phosphorylation at Tyr216 represented half or more of the total GSK3β in cultured cells. Although levels of phospho-Ser9 were increased by insulin treatment, Ser9 phosphorylation occurred only in a minor fraction of GSK3β. In mouse brains, GSK3β was principally in the active form with little Ser9 phosphorylation, and the phosphoisotypes of GSK3β changed depending on the regions of the brain, age, sex and disease conditions. These results indicate that the Phos-tag SDS-PAGE method provides a simple and appropriate measurement of active GSK3β in vivo, and the activity is regulated by the mechanism other than phosphorylation on Ser9.

Cyclin-dependent kinase 5 (Cdk5) is a proline-directed Ser/ Thr kinase that plays important roles in various neuronal activities, including neuronal migration, synaptic activity, and neuronal cell death. Cdk5 is activated by association with a neuron-specific activator, p35 or its isoform p39, but little is known about the kinase activity of Cdk5-p39. In fact, kinaseactive Cdk5-p39 was not prepared from rat brain extracts nor from HEK293 cells expressing Cdk5 and p39 by immunoprecipitation in the presence of non-ionic detergent, under conditions with which active Cdk5-p35 could be isolated. p39 dissociated from Cdk5 in the presence of detergent, indicating that p39 has a lower binding affinity for Cdk5 than p35. We developed a method for purifying kinase-active Cdk5-p39 from Sf9 cells infected with baculovirus encoding Cdk5 and p39. The purified Cdk5-p39 complex showed similar substrate specificity to that of Cdk5-p35, but with opposite sensitivity to detergent. Cdk5-p39 was inactivated by Triton X-100, whereas Cdk5-p35 was activated. The N-terminal deletion from p35 and p39, the amino acid sequences of which are different, did not change the stability or substrate specificity of either Cdk5 complex. The different stability between Cdk5-p35 and Cdk5-p39 suggests their distinct roles under different regulation mechanisms in neurons.