GENENAME : Biological Overview | Regulation | Developmental Biology | Evolutionary Homologs | References
Gene name - Cyclin-dependent kinase 5
Cytological map position - 52A11--B1
Function - signaling
Symbol - Cdk5
FlyBase ID: FBgn0013762
Genetic map position - 2-
Classification - cyclin dependent kinase
Cellular location - cytoplasmic
|Recent literature||Smith-Trunova, S., Prithviraj, R., Spurrier, J., Kuzina, I., Gu, Q. and Giniger, E. (2015). Cdk5 regulates developmental remodeling of mushroom body neurons in Drosophila. Dev Dyn [Epub ahead of print]. PubMed ID: 26394609
During metamorphosis, axons and dendrites of the mushroom body (MB) in the Drosophila central brain are remodeled extensively to support the transition from larval to adult behaviors. This study shows that the neuronal cyclin-dependent kinase, Cdk5, regulates the timing and rate of mushroom body remodeling: reduced Cdk5 activity causes a delay in pruning of MB neurites, while hyperactivation accelerates it. It was shown that Cdk5 cooperates with the ubiquitin-proteasome system in this process. Finally, Cdk5 modulates the first overt step in neurite disassembly, dissolution of the neuronal tubulin cytoskeleton, and it also acts at additional steps of MB pruning. These data show that Cdk5 regulates the onset and extent of remodeling of the Drosophila MB. Given the wide phylogenetic conservation of Cdk5, the study suggests that it is likely to play a role in developmental remodeling in other systems, as well. Moreover, it can be speculated that the well-established role of Cdk5 in neurodegeneration may involve some of the same cellular mechanisms that it employs during developmental remodeling.
|Spurrier, J., Shukla, A. K., McLinden, K., Johnson, K. and Giniger, E. (2018). Altered expression of the Cdk5 activator-like protein, Cdk5alpha, causes neurodegeneration in part by accelerating the rate of aging. Dis Model Mech. PubMed ID: 29469033
Aging is the greatest risk factor for neurodegeneration, but the connection between the two processes remains opaque. This is in part for want of a rigorous way to define physiological age, as opposed to chronological age. This study developed a comprehensive metric for physiological age in Drosophila, based on genome-wide expression profiling. This metric was applied to a model of adult-onset neurodegeneration, increased or decreased expression of the activating subunit of the Cdk5 protein kinase, encoded by the gene Cdk5alpha, the ortholog of mammalian p35. Cdk5alpha-mediated degeneration was associated with a 27-150% acceleration of the intrinsic rate of aging, depending on the tissue and genetic manipulation. Gene ontology analysis and direct experimental tests revealed that affected, age-associated processes included numerous core phenotypes of neurodegeneration, including enhanced oxidative stress and impaired proteostasis. Taken together, these results suggest that Cdk5alpha-mediated neurodegeneration results from accelerated aging, in combination with cell-autonomous neuronal insults. These data fundamentally recast the picture of the relationship between neurodegeneration and its most prominent risk factor, natural aging.
|Shukla, A. K., Spurrier, J., Kuzina, I. and Giniger, E. (2019). Hyperactive innate immunity causes degeneration of dopamine neurons upon altering activity of Cdk5. Cell Rep 26(1): 131-144. PubMed ID: 30605670
Innate immunity is central to the pathophysiology of neurodegenerative disorders, but it remains unclear why immunity is altered in the disease state and whether changes in immunity are a cause or a consequence of neuronal dysfunction. This study identified a molecular pathway that links innate immunity to age-dependent loss of dopaminergic neurons in Drosophila. Altering the expression of the activating subunit of the Cdk5 protein kinase (Cdk5alpha) causes severe disruption of autophagy. Second, this disruption of autophagy is both necessary and sufficient to cause the hyperactivation of innate immunity, particularly expression of anti-microbial peptides. Finally, it is the upregulation of immunity that induces the age-dependent death of dopaminergic neurons. Given the dysregulation of Cdk5 and innate immunity in human neurodegeneration and the conserved role of the kinase in the regulation of autophagy, this sequence is likely to have direct application to the chain of events in human neurodegenerative disease.
Cyclin-dependent kinase 5 (Cdk5) is one of a subfamily of Cdks involved in the control of cell differentiation and morphology rather than cell division. Specifically, Cdk5 and its activating subunit, p35, have been implicated in growth cone motility during axon extension. Both Cdk5 and p35 are expressed in post-mitotic neurons and are localized to growth cones. The Cdk5-p35 complex interacts with the Rac GTPase (see Drosophila Rac1), a protein required for growth cone motility. Studies using cultured neurons have suggested that Cdk5 activity controls the efficiency of neurite extension. Mutant mice lacking p35 exhibit subtle axon-guidance defects, but these mice have severe defects in neuronal migration, making it difficult to define precisely the role of the Cdk5-p35 complex in vivo. Drosophila Cdk5 functions in axon patterning in the Drosophila embryo. Although the data support the idea that Cdk5-p35 is involved in axonogenesis, they do not support the view that Cdk5 simply promotes growth cone motility. Instead, disrupting Cdk5 function causes widespread errors in axon patterning (Connell-Crowley, 2000 and references therein).
The functional consequences of altering Drosophila Cdk5 activity were tested. Cdk5, Cdk5dn and/or Cdk5 activator-like protein (also known as Dp35), the Drosophila counterpart of the vertebrate Cdk5 activating subunit, were ectopically expressed in post-mitotic neurons using the elav-GAL4 driver, and motor nerves innervating lateral and dorsal body wall muscles of stage 16-17 embryos were examined. Altering neuronal Cdk5 activity leads to two important observations. (1) Either increasing or decreasing Cdk5 activity produces errors in axon patterning. (2) There is no simple correlation between Cdk5 activity and axon/dendrite length. When endogenous neuronal Cdk5 is ectopically activated by expressing one copy of Dp35, subtle nerve defects appear in 32%-40% of the embryos. The majority of defects occur in the transverse nerve (TN), with a few defects observed in segmental nerve a (SNa) and the intersegmental nerve (ISN). In contrast to observations in cultured neurons, overextended nerves, stalled nerves, and nerves with errors in pathfinding and target recognition were found. Two copies of Dp35 increases the percentage of embryos with defects, suggesting that the effects of Dp35 are dose dependent. The severity of the phenotype is greatly enhanced by coexpressing Cdk5 with Dp35; 80% of the embryos exhibited nerve defects that were more severe and widespread than when Dp35 alone was expressed. Nevertheless, the spectrum of phenotypes observed (overextended, stalled and misrouted nerves) was similar to that seen when Dp35 alone was expressed (Connell-Crowley, 2000).
Neuronal Cdk5 activity was decreased by expressing dominant negative Cdk5 (Cdk5dn). One copy of Cdk5dn has no effect on axon patterning, but two copies cause 32%-35% of the embryos to exhibit subtle nerve defects. The majority of defects occur in the TN, with some defects in SNa and the ISN. In addition, a similar spectrum of phenotypes as that seen with increased Cdk5 activity is seen. With four copies of Cdk5dn, the percentage of embryos with defects increases to 56%, indicating that the effects of Cdk5dn, like those of Dp35, are dose dependent. Cdk5dn is likely to act, in part, by titrating endogenous Dp35, because coexpression of two copies of Cdk5dn with two copies of Dp35 suppresses the axonal defects produced by two copies of Dp35 alone (31% versus 57%). The phenotypic suppression observed with Cdk5dn/Dp35 coexpression contrasts with the phenotypic enhancement caused by Cdk5/Dp35 coexpression and indicates that the Cdk5/Dp35-induced defects require kinase activity. Additionally, the phenotypes of Cdk5 and Cdk5dn appear to be mutually suppressive because, while the effects of either Cdk5 or Cdk5dn are dose dependent, coexpression of two copies of Cdk5 with two copies of Cdk5dn causes a smaller percentage of embryos to exhibit defects than does four copies of Cdk5dn (26% versus 56) (Connell-Crowley, 2000).
The data presented confirm that Cdk5 is involved in axon development in vivo, consistent with predictions of Cdk5 function from studies using cultured neurons. Analysis of the phenotypes resulting from Cdk5/Dp35 expression in vivo reveal, however, a different picture of Cdk5 function than that observed in cultured neurons. Experiments using cultured neurons suggest a simple correlation between Cdk5 activity and the efficiency of neurite outgrowth. However, in these experiments, it was not possible to examine axon patterning. It is clear that Cdk5 and p35 are not absolutely required for axon growth. Rather, altering Cdk5 activity degrades the accuracy of axon patterning: either increasing or decreasing activity promotes or retards axon growth, and causes errors in pathfinding and target recognition. Perhaps the main function of Cdk5 is to ensure coordination of the many signaling pathways that are active in the growth cone, akin to the 'checkpoint' function of cell-cycle Cdks, and not simply to increase growth cone motility. Such a 'surveillance' mechanism would be consistent with the relatively subtle phenotypes observed when Cdk5 activity is modulated (Connell-Crowley, 2000).
Cdk5, like all Cdks, must associate with a regulatory subunit to become active. Thus, while Cdk5 is expressed ubiquitously during development, the spatial and temporal expression of its regulatory subunit limits where and when Cdk5 can be active. An examination was carried out to see whether a Cdk5 regulatory subunit is present at a time and place consistent with a role in axon outgrowth in the Drosophila embryo (Connell-Crowley, 2000).
Dp35, the activating subunit of Cdk5 is expressed in neurons at a time and place consistent with a role in axon development in vivo. Dp35 is expressed exclusively in the nervous system,with strong expression in the brain,ventral nerve cord and post-mitotic neurons of the peripheral nervous system (PNS). The earliest Dp35 expression was observed at stage 12 in clusters of cells in the central nervous system (CNS) where the neurons that extend the first CNS axons reside. Dp35 expression is not observed in neuroblasts, suggesting that fly Dp35-Cdk5, like mammalian p35-Cdk5, is not involved in cell division. Also examined was the subcellular localization of Dp35-Myc expressed in a small subset of neurons using the 15J2 GAL4 driver. Consistent with the localization of mammalian p35, Dp35-Myc protein expressed in CNS dMP2 neurons is present in the neuron cell body and axon, including the growth cone. Dp35-Myc, expressed in PNS lateral chordotonal neurons, is present in neuron cell bodies, dendrites and axons (Connell-Crowley, 2000).
It is interesting that either increasing or decreasing Cdk5 activity causes qualitatively similar nerve defects. This is reminiscent of the effects produced when activity of the Rac GTPase is increased or decreased. As p35 interacts with Rac, it will be of interest to determine whether the axonal effects of Cdk5/Dp35 in vivo are mediated or regulated by Rac. More generally, the ability to modulate Cdk5 activity in vivo will permit Cdk5 to be placed genetically within the signaling pathways that control growth cone motility and guidance (Connell-Crowley, 2000).
The rest of this essay will consider the role of mammalian Cdk5 in regulating neuronal adhesion and cytoskeletal dynamics (reviewed in Homayouni, 2000). Cdk5 kinase activity is not detected in dividing cells. In fact, the catalytically active form of Cdk5 is present only in differentiated neurons of the brain, where it associates with a neuron-specific 35 kDa regulatory subunit, p35. Cdk5 expression gradually increases during neurogenesis until it reaches maximum levels in the adult brain. Recent evidence, reported by Kwon (2000) suggests that Cdk5 plays a critical role in the regulation of N-cadherin-mediated cell adhesion during cortical development. Insights into the function of Cdk5 and p35 come from studies on mice with targeted disruption of the gene encoding either Cdk5 or p35 (Chae, 1997; Ohshima, 1996). The two mutants were found to exhibit similar abnormalities in the laminar structure of the cerebral cortex. But the cdk5-/- mice have additional defects in other brain structures and died shortly after birth. A small fraction of p35-/- mice die after spontaneous seizures, and greater than 50% died after seizure-inducing treatments that are not fatal in normal mice. The differences between the phenotypes of cdk5-/- and p35-/- mice may be due to the presence of other Cdk5 regulatory subunits in the brain. Thus far, several other Cdk5 regulators have been identified, including p39, Munc18 (p67) and a truncated form of p35, p25. At least in the forebrain, both p35 and Cdk5 play an essential role in the formation of the cortical layers during development (Homayouni, 2000 and references therein).
The mammalian cortex is assembled through a choreographed series of events that ultimately result in the segregation of neurons with similar properties into six layers. In the earliest phase of development, the preplate, composed of Cajal-Retzius and subplate neurons, is formed between the pial surface and the ventricular zone (VZ), where cells are actively dividing. Cells exit the cell cycle in the VZ and migrate radially outward toward the pial surface along glial fibers. The first-born neurons migrate past the subplate, displacing this layer away from the Cajal-Retzius cells in an area known as the marginal zone. Splitting of the preplate requires Reelin, a large extracellular protein secreted by Cajal-Retzius cells. The next wave of post-mitotic neurons migrates along the same fibers, past the subplate and the older neurons in the cortical plate, before inserting beneath the marginal zone. In this manner, the classical inside-out pattern of the neo-cortex arises in which the sequential generation of layers II-VI occurs beneath the marginal zone (Homayouni, 2000).
In the cortices of both p35-/- and cdk5-/- mutant mice,the marginal zone is unaffected and Reelin expression is normal. The preplate splits in p35-/- and cdk5-/- brains, and the first-born population of neurons -- the future layer VI -- appears to migrate correctly past the subplate cells. But the migration of the later-born neurons is impeded, in the mutants, such that the second wave of neurons fails to migrate past the established layer of cortical neurons and, instead, accumulates below the subplate. On the basis of these observations, it has been proposed that p35 and Cdk5 are required for neurons to bypass one another during corticogenesis, although they are not required for splitting of the preplate (Homayouni, 2000).
To form distinct neuronal layers in the developing cortex, one would expect migrating neurons to recognize and preferentially adhere to their cohorts in the respective layers. Indeed, in aggregation assays, early-born cortical neurons selectively associate with one another. A good candidate molecule to mediate this cell-cell adhesion is the Ca 2+-dependent neuronal adhesion molecule N-cadherin (Drosophila homolog: Cadherin-N). N-cadherin is expressed transiently throughout the developing cortical plate, but it persists only in the deepest layers of the postnatal brain. Thus, it is possible that N-cadherin plays a role in maintaining the integrity of the cortical plate at the time newly generated neurons migrate past. This model presents a paradox, however: how can migrating neurons expressing the homophilic protein N-cadherin bypass cortical plate neurons that also express N-cadherin? The recent work of Kwon (2000) on p35-interacting proteins has shed some light on this subject. A novel interaction between p35 and the intracellular regulator of N-cadherin, alpha-catenin was identified. Expression of Cdk5 and p35 in cultured cells decreases the association between alpha-catenin and N-cadherin, resulting in decreased cell adhesion. Conversely, in neuronal aggregation assays, loss of Cdk5 kinase activity by pharmacological inhibition or ablation of p35 resulted in increased cell adhesion. These results provide important insights into the molecular mechanism by which Cdk5 and p35 regulate neuronal migration in the developing cortex (Homayouni, 2000 and references therein).
On the basis of these findings a new molecular model can be put forward to describe cortical development. As neurons exit the cell cycle, they express p35, which activates Cdk5, causing down-regulation of N-cadherin-mediated cell adhesion. This allows the neurons to migrate past their predecessors in the cortical plate, which express large amounts of N-cadherin. In cdk5-/- and p35-/- brains, the migrating neurons are unable to down-regulate N-cadherin, so they cannot bypass the earlier-born cortical plate neurons. This may explain why later-born neurons accumulate beneath the subplate in these mutant mice (Homayouni, 2000 and references therein).
One question remaining is how does a migrating neuron that has reached the marginal zone down-regulate the p35/Cdk5 kinase pathway and reactivate N-cadherin mediated adhesion? Reelin has been proposed to function as a stop signal for migrating neurons. It is possible that Reelin acts as a stop cue by inhibiting the p35/Cdk5 kinase pathway. Thus, Reelin may activate N-cadherin-mediated cell adhesion to terminate migration and initiate homotypic adhesion between the new arrivals and neurons resident in the cortical plate. Recent results by Hiesberger (1999) support this hypothesis. Tau, a substrate of Cdk5, is hyperphosphorylated in reeler mutant brains. One explanation for this observation is that Reelin down-regulates p35/Cdk5 kinase activity under normal conditions. Therefore, it is possible that, in reeler mice, p35/Cdk5 kinase remains active in migrating neurons, resulting in a failure of post-mitotic neurons to adhere to one another in the presumptive marginal zone. This may in part explain the disruption of cortical layers observed in reeler brains (Homayouni, 2000 and references therein).
Increasing evidence points to the fact that Cdk5 is a key regulator of neuronal function. It modulates cell adhesion and cytoskeletal dynamics, processes that are essential during development and in the adult nervous system. Not surprisingly, deregulation of Cdk5 has severe consequences for brain development, causing disruption of cell positioning and perinatal lethality. In the adult nervous system, aberrant activation of Cdk5 by p25 is associated with neurodegeneration. Cdk5 family members play critical roles in the control of cell division and they have attracted attention as a target for anti-cancer therapies. Perhaps Cdk5 plays a similarly critical role in postmitotic, developing and adult neurons. It may also attract attention as a therapeutic target for neurodegenerative diseases (Homayouni, 2000).
A fly Cdk5 regulatory subunit (Dp35; GenBank accession number AF231134) was identified from the Drosophila expressed sequence tag (EST) database using DNA sequence of clones obtained by degenerate PCR followed by cDNA and genomic library screens. Dp35 encodes a ~52 kDa protein with 31%-40% overall identity to other p35 family members, and 55%-66% identity in its carboxy-terminal half, where the Cdk5-binding and activation domains reside. Dp35 also has the amino-terminal myristoylation motif, which is conserved in p35 family members, indicating that the cDNA is full length (Connell-Crowley, 2000).
Dp35 and Cdk5 associate and cooperate functionally in vitro and in vivo. Dp35 has been shown to bind Cdk5. In an in vitro glutathione-S-transferase (GST) pull-down assay, fly Cdk5 associates with GST-Dp35 but not with GST alone, as does a mutant Cdk5 (Cdk5K33A) that is predicted to be kinase inactive. Moreover, Myc-epitope-tagged Dp35 (Dp35-Myc) expressed in human 293T cells associates with endogenous human Cdk5 in vivo, but not with Cdks 1, 2, 4 or 6. Dp35 activates Cdk5. Anti-Myc antibody immunoprecipitates from human 293T cells containing Dp35-Myc and human Cdk5 phosphorylated histone H1. Furthermore, anti-FLAG antibody immunoprecipitates from human 293T cells containing fly FLAG-epitope-tagged Cdk5 (Cdk5-FLAG) exhibit H1 kinase activity in the presence, but not in the absence, of Dp35. In contrast, Cdk5K33A-FLAG is catalytically inactive, although it does associate with Dp35. Dp35 interacts genetically with Cdk5 in the Drosophila eye (Connell-Crowley, 2000).
Expression of one copy of Cdk5-FLAG in all cells of the eye using the GMR-GAL4 driver has no effect on eye surface morphology, whereas one copy of Dp35-Myc causes a slight rough eye phenotype, presumably by activation of endogenous, low-level Cdk5. In contrast, coexpression of Cdk5-FLAG with Dp35-Myc severely disrupts eye morphology. This synthetic phenotype requires Cdk5 kinase activity because coexpression with two copies of Cdk5K33A-FLAG suppresses rather than enhances the mild rough eye phenotype observed with two copies of Dp35-Myc. Thus, like their mammalian orthologs, fly Cdk5 is activated by Dp35, whereas Cdk5K33A appears to act as a dominant-negative mutant by titrating Dp35 and will be referred to below as Cdk5dn (Connell-Crowley, 2000).
Cyclin-dependent kinase 5 activator (Cdk5alpha), is an activator of Cdk5 kinase activity and its expression is restricted to neurons. The complex of Ckd5/Cdk5alpha is essential for neurite outgrowth during neuronal differentiation and possibly also for neuronal degeneration. Drosophila Cdk5alpha-like, dCdk5alpha, has been isolated and characterized. The gene encoding this molecule is localized in the Drosophila chromosome region of 31D1-31D2. The expression of this gene is differentially regulated with a very low level at earlier developmental stages and reaches the highest level in the adult. The C-terminal of this protein shares high homology with the mammalian Cdk5alpha molecule. Constitutive over-expression of dCdk5alpha in transgenic flies significantly prolongs their recovery time from a 5 minute O2 deprivation or anoxia in older flies (15 days). Recovery times are not prolonged when younger flies (4 days old) are deprived of O2. In addition, anoxia up-regulates the expression of this gene. Taken together, the results in this report and others provide a framework for genetically dissecting the functions of Cdk5alpha/Cdk5 complex in the CNS (Ma, 1999).
Multisite hyperphosphorylation of tau has been implicated in the pathogenesis of neurodegenerative diseases including Alzheimer's disease (AD). However, the phosphorylation events critical for tau toxicity and mechanisms regulating these events are largely unknown. Drosophila PAR-1 kinase is shown to initiate tau toxicity by triggering a temporally ordered phosphorylation process. PAR-1 directly phosphorylates tau at S262 and S356. This phosphorylation event is a prerequisite for the action of downstream kinases, including glycogen synthase kinase 3 (GSK-3) and cyclin-dependent kinase-5 (Cdk5), to phosphorylate several other sites and generate disease-associated phospho-epitopes. The initiator role of PAR-1 is further underscored by the fact that mutating PAR-1 phosphorylation sites causes a much greater reduction of overall tau phosphorylation and toxicity than mutating S202, one of the downstream sites whose phosphorylation depends on prior PAR-1 action. These findings begin to differentiate the effects of various phosphorylation events on tau toxicity and provide potential therapeutic targets (Nishimura, 2004).
Drosophila has established itself as a model system for studying human neurodegenerative disorders. Fly models of tauopathy have been created by expressing wild-type or FTDP-linked mutant forms of h-tau. Using such models and based largely on overexpression experiments, it has been shown that Shaggy (GSK-3) can promote neurofibrillary tangle (NFT) pathology in photoreceptor neurons (Jackson, 2002). Whether GSK-3 and NFT are necessary for tau-mediated neurodegeneration, however, remains uncertain. Other studies have shown that tau-mediated neurodegeneration could occur without NFT and that GSK-3ß-induced tau hyperphosphorylation in mice could correlate inversely with neuropathology (Nishimura, 2004 and references therein).
Critical testing for a functional role of phosphorylation in tau-mediated neuropathology will require identifying the physiological tau kinase and assessing the consequence of removing this kinase activity on the disease process. Through loss-of-function and overexpression genetic studies and biochemical analysis, it has been shown that PAR-1 is a physiological tau kinase that plays a central role in regulating tau phosphorylation and toxicity in Drosophila. PAR-1 is a Ser/Thr kinase originally identified in C. elegans for its role in regulating cell polarity and asymmetric cell division. PAR-1 homologs have been found in eukaryotes ranging from yeast to mammals and exert essential cellular and developmental functions. MARK kinase, the mammalian homolog of PAR-1, regulates MT dynamics, epithelial cell polarity, and neuronal differentiation. Drosophila PAR-1 plays important roles in MT organization, oocyte differentiation, anterior-posterior axis formation, and Wingless signaling. While analyzing the neuronal function of PAR-1, it was found that Drosophila PAR-1 is a physiological kinase for fly Tau and h-tau. Overexpression of PAR-1 leads to elevated tau phosphorylation and enhanced toxicity, whereas removing PAR-1 function or mutating PAR-1 phosphorylation sites in tau abolishes tau toxicity. Furthermore, an initiator role for PAR-1 has been uncovered in a multisite phosphorylation process that generates pathogenic forms of tau. In this process, phosphorylation by PAR-1 precedes and is obligatory for downstream phosphorylation events, including those carried out by GSK-3 and Cdk5, to generate toxic tau. Consistent with PAR-1 playing an initiator role in the process, mutating PAR-1 phosphorylation sites causes a much more dramatic reduction of overall tau phosphorylation and toxicity than mutating one of the downstream Cdk5/GSK-3 phosphorylation sites. These findings have important implications for understanding the biogenesis of pathogenic tau in neurons and for developing mechanism-based therapeutic strategies (Nishimura, 2004).
Recent transgenic animal studies have implicated two kinases, GSK-3 and Cdk5, in the phosphorylation of tau in vivo. Analyses of tau phosphorylation status in transgenic mice overexpressing GSK-3 or Cdk5 have detected increased phosphorylation at certain sites previously identified as their in vitro phosphorylation sites. For example, S202 and PHF-1 sites (S396 and S404) have been shown to be prominent Cdk5 and GSK-3 phosphorylation sites, respectively, and the two kinases may have overlapping specificity at these sites. Tests were performed to see whether these sites in h-tauM were also phosphorylated by the corresponding fly kinases. The activity of Cdk5 is regulated by its binding with neuron-specific activators. Overexpression of Drosophila P35 activator has been shown to elevate endogenous Cdk5 activity. In P35 and h-tauM coexpression flies, the level of phosphorylation at S202 recognized by CP13 antibody is elevated. In addition, phosphorylation at AT270 sites was also significantly increased. Phosphorylation at AT100, AT180, and PHF-1 sites was relatively unchanged. Thus, phosphorylation at S202 and T181 responds to changes in Cdk5 levels. The eye morphology of P35 and h-tauM coexpressing flies appearssimilar to that of flies expressing h-tauM alone, suggesting that elevated Cdk5 activity does not significantly enhance tau toxicity. Shaggy and h-tauM coexpression flies were analyzed next. Coexpression of Shaggy and h-tau results in enhanced eye degeneration phenotypes. In the coexpression flies, significantly increased tau phosphorylation was observed at PHF-1, CP13, AT180, and AT100 sites. It is concluded that these phospho-epitopes contain GSK-3 phosphorylation sites and that elevated phosphorylation at these sites enhances tau toxicity (Nishimura, 2004).
The fact that many of the above-tested phosphorylation sites for GSK-3 and Cdk5 kinases are affected in S2A suggests that phosphorylation by the two kinases is regulated by prior PAR-1 action. To test this idea further, the phosphorylation status of GSK-3 and Cdk5 phosphorylation sites was analyzed in PAR-1 and h-tauM coexpression flies. In addition to 12E8 sites, significant increase of phosphorylation was observed at CP13 and PHF-1 sites in these flies. In contrast, phosphorylation at other sites such as AT100 sites was little changed, suggesting that PAR-1 is not a rate-limiting factor for these phosphorylation events. Since in vitro kinase assays showed that PAR-1 is incapable of directly phosphorylating the CP13 and PHF-1 sites, the elevated phosphorylation at these sites in PAR-1 coexpressing flies are likely mediated by downstream kinases such as Cdk5 and GSK-3 (Nishimura, 2004).
Whether coexpression of PAR-1, GSK-3, or Cdk5 has any modulating effect on S2A toxicity was further tested in vivo. PAR-1 and S2A coexpression flies showed a mild rough eye phenotype similar to PAR-1 overexpression alone, indicating that PAR-1 overexpression does not confer additional toxicity to S2A. Co-overexpression of GSK-3 or Cdk-5 also did not change S2A toxicity. These results further support the notion that phosphorylation by PAR-1 at S262 and S356 is a prerequisite for the subsequent phosphorylation by downstream kinases such as GSK-3 and Cdk5 to generate toxic tau species (Nishimura, 2004).
Since the S2A mutation disrupts tau phosphorylation at multiple downstream sites, it does not allow distinguishing the contribution of individual phosphorylation sites to tau toxicity. This issue was addressed by making point mutations in the downstream phosphorylation sites. Focus was placed on the S202 site because it is phosphorylated by Cdk5 and GSK-3 in vivo and because AT8 antibody, which is sensitive to phosphorylation at this site, was considered an Alzheimer-diagnostic antibody. Transgenic flies were generated that express h-tauM containing an Ala substitution at S202 (S202A). Western blot analysis demonstrated that, as predicted, S202A protein was no longer recognized by CP13 or AT8 antibodies. Significantly, phosphorylation at 12E8, AT100, PHF-1, AT180, and AT270 sites was unaffected by S202A mutation. This suggests that unlike S262 and S356 sites, the phosphorylation state of S202 does not influence that of other sites. Examination of external eye morphology by SEM and photoreceptor staining of eye sections has shown that, unlike S2A, S202A is as toxic as h-tauM. This suggests that phosphorylation by GSK-3 and Cdk5 at S202 site plays a rather limited role in conferring tau toxicity. This result supports the notion that PAR-1 plays an initiator role in the pathogenic phosphorylation process and further suggests that phosphorylation at downstream sites other than S202 or a combination of those downstream phosphorylation events makes a major contribution to tau toxicity (Nishimura, 2004).
Thus PAR-1, the fly homolog of mammalian MARK kinase, plays a central role in conferring tau toxicity in vivo. This study reveals PAR-1 function in triggering a temporally ordered phosphorylation process that is responsible for generating toxic forms of tau. This multisite phosphorylation process involves downstream kinases such as Cdk5 and GSK-3, whose action depends on prior phosphorylation of h-tau by PAR-1. A nonphosphorylatable mutation at S202, one of the downstream GSK-3/Cdk5 target sites whose phosphorylation depends on prior PAR-1 action, has a much smaller impact on overall tau phosphorylation and toxicity than mutations at PAR-1 phosphorylating sites. This strongly supports the initiator role of PAR-1 in generating toxic species of tau and further implies that the toxic form of tau may be phosphorylated at a subset or all of the other downstream sites (Nishimura, 2004).
It was previously shown that PAR-1 regulates the Wingless/Wnt pathway in Drosophila and Xenopus by phosphorylating the core component Dishevelled. It is thus interesting that GSK-3, another core component of Wingless pathway, acts downstream of PAR-1 to phosphorylate h-tau. These results are consistent with the notion that the Wingless pathway may be involved in regulating tau phosphorylation. It has been proposed that the pathway components are utilized differently in tau phosphorylation than in canonical Wnt signaling. The data indicate that PAR-1 and GSK-3 directly phosphorylate tau in an ordered fashion, with PAR-1 action preceding that of GSK-3. One parsimonious explanation for the requirement of prior phosphorylation by PAR-1 is that PAR-1 phosphorylation reduces the affinity of tau for MT and releases it from the MT network, therefore allowing easy access by other kinases. If that is the case, the mechanism may operate in a region-specific manner since certain phosphorylation sites do not depend on prior PAR-1 action. The data are also consistent with the idea that PAR-1 phosphorylation at 12E8 sites provides docking sites for intermediary kinase(s) and/or adaptor molecule(s), which facilitate subsequent phosphorylation by GSK-3 and Cdk5. It appears that the phosphorylation at certain downstream sites is achieved through a complex process. For example, phosphorylation at AT100 sites depends on prior PAR-1 action, but PAR-1 co-overexpression does not increase phosphorylation at these sites. Instead, co-overexpression of GSK-3 can lead to increased phosphorylation at AT100 sites. Previous in vitro studies have shown that the generation of AT100 epitope requires a PHF-like conformation of tau and the sequential phosphorylation by GSK-3 and PKA. It remains to be determined whether GSK-3 and PKA act downstream of PAR-1 to phosphorylate AT100 sites in flies (Nishimura, 2004).
Although Abl functions in mature neurons, work to date has not addressed Abl's role on Cdk5 in neurodegeneration. β-amyloid (Aβ42) initiates Abl kinase activity and blockade of Abl kinase rescues both Drosophila and mammalian neuronal cells from cell death. Activated Abl kinase is necessary for the binding, activation, and translocalization of Cdk5 in Drosophila neuronal cells. Conversion of p35 into p25 is not observed in Aβ42-triggered Drosophila neurodegeneration, suggesting that Cdk5 activation and protein translocalization can be p25-independent. These genetic studies also showed that abl mutations repress Aβ42-induced Cdk5 activity and neurodegeneration in Drosophila eyes. Although Aβ42 induces conversion of p35 to p25 in mammalian cells, it does not sufficiently induce Cdk5 activation when c-Abl kinase activity is suppressed. Therefore, it is proposed that Abl and p35/p25 cooperate in promoting Cdk5-pY15, which deregulates Cdk5 activity and subcellular localization in Aβ42-triggered neurodegeneration (Lin, 2007).
Like Cdk5, cellular Abl functions in neural development and its kinase activity and subcellular localization are tightly regulated. This study shows that Abl appears to be essential for Aβ42-triggered Drosophila neurodegeneration both in vivo and in vitro. It is of interest in this regard that Abl may serve as a putative molecular target to stop the progress of neurodegeneration. Interestingly, the anti-leukemic agent Abl kinase inhibitor, STI571, has been shown to rescue the Aβ42-induced neurodegeneration in both Drosophila and mammalian cells. However, STI571 is probably not an ideal reagent for testing this idea in vivo because of its low penetration capability through the blood-brain barrier. Another previous link between Aβ42 and Abl inhibition by STI571 has been reported. Aβ42 production is reduced by STI571 in neuronal cultures and in guinea-pig brain. Therefore, it is reasonable to speculate that Abl kinases might affect amyloid signaling at various points including Aβ42 production (Lin, 2007).
The cyclin-dependent kinase Cdk5 has attracted a great deal of attention both because of its roles in cell migration and axon patterning, and the extensive data implicating it in adult-onset neurodegeneration in mammals. Both the kinase activity and the biological effects of Cdk5 are absolutely dependent on association with an activating subunit, called p35. Drosophila lacking the Cdk5 activator, D-p35, display a wide range of defects in embryonic axon patterning. While viable and fertile, p35 mutant adults display progressive, age-dependent loss of motor function and have a significantly shortened lifespan (Connell-Crowley, 2007).
Previous studies employing overexpression of dominant transgenic constructs suggested that reduction of Cdk5 activity leads to errors in axon guidance in vivo. The p35 null phenotype reported here supports those findings, confirming the necessary role of Cdk5 and p35 in embryonic axon guidance. Moreover, absence of p35 causes age-dependent deficits in motor function, as demonstrated by progressive degradation of motor function, culminating in periods of rigidity, and associated with premature death (Connell-Crowley, 2007).
The axon patterning defects observed in the p35 null mutant mimic closely, both in kind and in severity, the defects produced by neuronal expression of a kinase-inactive Cdk5. This suggests that the dominant negative Cdk5 was both faithful and effective in revealing the function of Cdk5/p35. Moreover, while it is clear from the Drosophila genome sequence and from molecular studies that there is only a single p35 family member in Drosophila, the observation that expression of the dominant-negative Cdk5 in the p35 null background does not yield a stronger phenotype than either the mutant or the transgene alone suggests that there is not likely to be another major Cdk5 activator protein of some other molecular nature in growing Drosophila axons (Connell-Crowley, 2007).
Careful observation of the p35 null stock suggested the presence of behavioral deficits and reduced adult vigor. Quantitative behavioral assays supported this impression, revealing that while motor function in the mutants was nearly wild-type at eclosion, it declined swiftly with advancing adult age, to be followed by periods of unmoving rigidity and then by premature death. The cellular basis of these phenotypes remains unclear. The neuron-specific pattern of p35 expression, however, and the ability to mimic the lifespan phenotype of p35 null mutants by specific expression of dominant negative Cdk5 in postmitotic neurons demonstrate that it is the function of p35 in mature, postmitotic neurons that is essential for normal lifespan. Experiments are currently in progress to test whether the lifespan and behavioral phenotypes of p35 are a delayed, secondary consequence of developmental miswiring, or whether they reflect a required adult function of this protein (Connell-Crowley, 2007).
It is noteworthy that one of the obvious behavioral defects in p35 mutant adults is a tendency of the adult flies to lie on their backs, unable to right themselves. This phenomenon has been observed previously in the Mediteranean fruit fly, Ceratitis capitata, and has been termed the 'supine' phenotype. In that system, as well, the behavior was associated with reduced lifespan and it was further shown that assumption of the supine phenotype by a particular individual was an early marker of approaching death (Connell-Crowley, 2007).
The pattern of behavioral phenotypes observed in p35 null adults bear intriguing parallels with observable features of mammalian neurodegenerative diseases, some of which have been linked to altered activity of mammalian Cdk5. Does neurodegeneration play a role in the progressive phenotype of p35 null mutants? Can the wild-type functions and mutant phenotypes of p35 and Cdk5 in Drosophila shed any light on mammalian neurodegenerative diseases? If detailed histological and ultrastructural analysis of p35 mutants reveals anatomical degeneration with morphological similarity to mammalian neurodegeneration, it holds the promise of applying the uniquely precise tools of Drosophila genetics to investigate fundamental cellular and molecular mysteries of neurodegeneration, including the earliest events in neuropathology, cell autonomy, and the molecular genetic basis of the disease process (Connell-Crowley, 2007).
In addition to the previously identified Drosophila cdc2 and cdc2c genes, four additional cdc2-related genes have been identified with low stringency and polymerase chain reaction approaches. Sequence comparisons suggest that the four putative kinases represent the Drosophila homologs of vertebrate cdk4/6, cdk5, PCTAIRE, and PITSLRE kinases. Although the similarity between human and Drosophila homologs is extensive in the case of cdk5, PCTAIRE, and PITSLRE kinases (78%, 58%, and 65% identity in the kinase domain), only limited conservation is observed for Drosophila cdk4/6 (47% identity). Northern blot analysis indicated that the four Drosophila kinases are expressed throughout embryogenesis. Expression in early embryogenesis appears to be ubiquitous according to in situ hybridization. Abundant expression already at the start of embryogenesis and long before neuron differentiation is also observed in the case of cdk5 protein, which has been described as predominantly neuron specific in mice. Sequence conservation and expression pattern, therefore, suggest that all of these kinases perform important cellular functions (Sauer, 1996).
Neuronal communication requires the coordinated assembly of polarized structures including axons, dendrites, and synapses. This study reports the identification of a ubiquitin ligase mind bomb 1 (Mib1) in the postsynaptic density and the characterization of its role in neuronal morphogenesis. Expression of Rat Mib1 inhibits neurite outgrowth in cell culture and its gene deletion enhances synaptic growth at the neuromuscular junction in Drosophila. The analysis of Rat Mib1 interactome by mass spectrometry revealed that Mib1 primarily interacts with membrane trafficking proteins [e.g., EEA1 (early endosomal antigen 1), Rab11-interacting proteins, and SNAP25 (synaptosomal-associated protein of 25 kDa)-like protein] and cell adhesion components (e.g., catenin, coronin, dystrobrevin, and syndecan), consistent with its previously reported function in protein sorting. More interestingly, Mib1 is associates with deubiquitinating enzymes, BRCC36 and the mammalian ortholog of fat facets, and a number of kinases, such as casein kinase II, MARK (microtubule affinity regulating kinase)/PAR1, and cyclin-dependent kinase 5 (CDK5). Further characterization of the Mib1-CDK5 interaction indicated that the N-terminal domain of Mib1 directly binds to the regulatory subunit p35 of the CDK5 complex. In cell culture, Mib1 induces the relocalization of p35/CDK5 without affecting its degradation. Surprisingly, p35/CDK5 downregulates the protein level of Mib1 by its kinase activity, and completely rescues the Mib1-induced inhibitory effect on neurite morphology. p35/CDK5 also genetically interacts with Mib1 in the fly according to the rough-eye phenotype. The data strongly support that the negative interplay between Mib1 and p35/CDK5 may integrate the activities of multiple pathways during neuronal development (Choe, 2007; full text of article).
A Drosophila model with Mib1 loss-of-function was used to examine its physiological role and interaction with p35/CDK5. A P-element insertion was identified in the fly gene of CG5841, the Drosophila ortholog of Mib1. The homozygote was affirmed to be a mib1-null allele, because it showed pupal lethality that was rescued by precise removal of the transposon in the 5' untranslated region of the gene, or by the expression of transgenic mib1. The lack of full-length mib1 expression in the allele was verified by Western blotting. In Drosophila, the synaptic structure at the larval neuromuscular junction (NMJ) is a well defined system with which to study synaptic structure and neurotransmission, and the number of synaptic boutons is an established index for synaptic growth. By counting synaptic boutons in the wild-type and the mutant larvae, it was found that the loss of mib1 causes the synaptic overgrowth, and the number of synaptic boutons increased 85%. This finding suggests that Mib1 plays a conserved negative role in the formation of synaptic structure (Choe, 2007).
The genetic interaction between p35/CDK5 and mib1 was tested by monitoring adult eye phenotype. Overexpression of p35/CDK5 causes a rough eye phenotype, whereas the heterozygotic line of the mib1 mutant had a smooth eye phenotype. Crossing the p35/CDK5 transgenic line with the mib1 mutant line showed that the partial loss of mib1 in the heterozygote enhances the rough eye phenotype induced by p35/CDK5. This result also supports the negative regulation between p35/CDK5 and Mib1 in neuronal development (Choe, 2007).
Dictyostelium Crp is a member of the cyclin-dependent kinase (Cdk) family of proteins. It is most related in sequence to mammalian Cdk5, which unlike other members of the family, has functions that are unrelated to the cell cycle. In order to better understand the function of Crp in Dictyostelium, a dominant negative form, Crp-D144N, was overexpressed under the control of the actin 15 promoter. Cells overexpressing Crp-D144N exhibit a reduced growth rate in suspension culture and reduced rates of fluid-phase endocytosis and phagocytosis. There is no reduction in Cdc2 kinase activity in extracts from cells overexpressing Crp-D144N, suggesting that the growth defect is not due to inhibition of Cdc2. In addition to the growth defect, the act15::crp-D144N transformants aggregate at a slower rate than wild-type cells and form large aggregation streams. These eventually break up to form small aggregates and most of these do not produce mature fruiting bodies. The aggregation defect is fully reversed in the presence of wild-type cells but terminal differentiation is only partially rescued. In act15::crp-D144N transformants, the countin component of the counting factor, a secreted protein complex that regulates the breakup of streams, mostly appears outside the cell as degradation products and the reduced level of the intact protein may at least partially account for the initial formation of the large aggregation streams. These observations indicate that Crp is important for both endocytosis and efflux and that defects in these functions lead to reduced growth and aberrant development (Sharma, 2002b).
Polarized trafficking of synaptic proteins to axons and dendrites is crucial to neuronal function. Through forward genetic analysis in C. elegans, a cyclin (CYY-1) and a cyclin-dependent Pctaire kinase (PCT-1) necessary for targeting presynaptic components to the axon was identified. Another cyclin-dependent kinase, CDK-5, and its activator p35, act in parallel to and partially redundantly with the CYY-1/PCT-1 pathway. Synaptic vesicles and active zone proteins mostly mislocalize to dendrites in animals defective for both PCT-1 and CDK-5 pathways. Unlike the kinesin-3 motor, unc-104/Kif1a mutant, cyy-1 cdk-5 double mutants have no reduction in anterogradely moving synaptic vesicle precursors (SVPs) as observed by dynamic imaging. Instead, the number of retrogradely moving SVPs is dramatically increased. Furthermore, this mislocalization defect is suppressed by disrupting the retrograde motor, the cytoplasmic dynein complex. Thus, PCT-1 and CDK-5 pathways direct polarized trafficking of presynaptic components by inhibiting dynein-mediated retrograde transport and setting the balance between anterograde and retrograde motors (Ou, 2010).
Phosphorylation of the neurofilament proteins of high and medium relative molecular mass, as well as of the Alzheimer's tau protein, is thought to be catalysed by a protein kinase with Cdc2-like substrate specificity. A novel Cdc2-like kinase has been purified from bovine brain; it is capable of phosphorylating both the neurofilament proteins and tau. The purified enzyme is a heterodimer of cyclin-dependent kinase 5 (Cdk5) and a novel regulatory subunit, p25. When overexpressed and purified from Escherichia coli, p25 can activate Cdk5 in vitro. Unlike Cdk5, which is ubiquitously expressed in human tissue, the p25 transcript is expressed only in brain. A full-length complementary DNA clone showed that p25 is a truncated form of a larger protein precursor, p35, which seems to be the predominant form of the protein in crude brain extract. Cdk5/p35 is the first example of a Cdc2-like kinase with neuronal function (Lew, 1994).
Cyclin-dependent kinase 5 (Cdk5) was originally isolated through its structural homology to human Cdc2, a key regulator of cell-cycle progression. In tissue samples from adult mice, Cdk5 protein is found at the highest level in brain, at an intermediate level in testis, and at low or undetectable levels in all other tissues, but brain is the only tissue that shows Cdk5 histone H1 kinase activity. No equivalent kinase activity has been found in tissue culture cell lines despite high levels of Cdk5. This raised the possibility that a Cdk5 regulatory subunit was responsible for the activation of Cdk5 in brain. The cloning and characterization of a regulatory subunit for Cdk5, known as p35, is described. p35 displays a neuronal cell-specific pattern of expression; it associates physically with Cdk5 in vivo and activates the Cdk5 kinase. p35 differs from the mammalian cyclins and thus represents a new type of regulatory subunit for cyclin-dependent kinase activity (Tsai, 1994).
Neuronal Cdc2-like kinase is a heterodimer of Cdk5 and a 25-kDa subunit that is derived from a 35-kDa brain- and neuron-specific protein called the neuronal Cdk5 activator (p35/p25nck5a). Upon screening of a human hippocampus library with a bovine Nck5a cDNA, a distinct clone encoding a 39-kDa isoform of Nck5a was uncovered. The isoform, designated the neuronal Cdk5 activator isoform (p39nck5ai), shows a high degree of sequence similarity to p35nck5a, with 57% amino acid identity. Northern blot analysis detected its mRNA transcript in bovine and rat cerebrum and cerebellum, but not in any other rat tissues examined. In situ hybridization has shown that Nck5ai is enriched in CA1 to CA3 of the hippocampus, but absent in the fimbria of hippocampal formation. Among seven cell lines in proliferating cultures, only PC12 and N2A, two cell lines capable of differentiating into neuron-like cells, were found to contain Nck5ai mRNA. A 30-kDa truncated form of Nck5ai (expressed as a glutathione S-transferase fusion protein in Escherichia coli) was found to associate with Cdk5 to form an active Cdk5 kinase. Thus, the isoform shares many common characteristics with p35nck5a, including Ckd5 activating activity and brain- and neuron-specific expression. Both proteins show limited sequence homology to cyclins, suggesting that they define a new family of cyclin-dependent kinase-activating proteins (Tang, 1995).
Cyclin-dependent kinase 5 (Cdk5) is activated by the neuronal-specific activator protein, p35. The proteolytic active fragment of p35, p25 (residues 91-307) as well as the slightly smaller fragment containing residues 109-291, is sufficient to bind and activate Cdk5. Distinct regions in p35 required for binding to Cdk5 or activation of Cdk5 have been identified. Residues approximately 150-200 of p35 are sufficient for binding to Cdk5, but residues approximately 279-291 are needed in addition for activation of Cdk5 in vitro (Poon, 1997).
Cyclin-dependent kinase 5 (Cdk5) was originally isolated by its close homology to the human CDC2 gene, which is a key regulator of cell cycle progression. However, unlike other Cdks, the activity of Cdk5 is required in post-mitotic neurons. The neuronal-specific p35 protein, which shares no homology to cyclins, was identified by virtue of its association with and activation of Cdk5. Gene targeting studies in mice have shown that the p35/Cdk5 kinase is required for the proper neuronal migration and development of the mammalian cortex. The regulation of the p35/Cdk5 kinase has been investigated. p35, the activator of Cdk5, is a short-lived protein with a half-life (t1/2) of 20 to 30 min. Specific proteasome inhibitors such as lactacystin greatly stabilize p35 in vivo. Ubiquitination of p35 can be readily demonstrated in vitro and in vivo. Inhibition of Cdk5 activity by a specific Cdk inhibitor, roscovitine, or by overexpression of a dominant negative mutant of Cdk5 increases the stability of p35 by 2- to 3-fold. Furthermore, phosphorylation mutants of p35 also stabilize p35 2- to 3-fold. Together, these observations demonstrate that the p35/Cdk5 kinase can be subject to rapid turnover in vivo and suggest that phosphorylation of p35 upon Cdk5 kinase activation plays an autoregulatory role in p35 degradation mediated by ubiquitin-mediated proteolysis (Patrick, 1998).
The ubiquitously expressed cyclin-dependent kinase 5 (cdk5) is essential for brain development. Bioactivation of cdk5 in the brain requires the presence of one of two related regulatory subunits, p35 and p39. Since either protein alone can activate cdk5, the significance of their coexistence as cdk5 kinase activators is unclear. To determine whether the two activators are expressed in different cells throughout the nervous system and during development, the tissue distributions of cdk5, p35, and p39 mRNAs in the rat were compared using in situ hybridization. In the adult rat, expression levels of p35 mRNA are generally higher in the brain than in the spinal cord, while the converse is observed for p39 mRNA. During neurogenesis, both p35 and p39 transcripts can be detected as early as embryonic day 12 (E12) in the marginal zone, but are absent from the ventricular zone, which may restrict cdk5 activation to the postmitotic neural cells in the developing brain. The expression levels of p35 and p39 mRNAs in the marginal zone increase by E15 and E17, paralleling the neurogenetic timetable. One exception is in the rostral forebrain, where p35 mRNA expression levels are high, suggesting that p35 may be the major activator for cdk5 during telencephalic morphogenesis. A significant level of p35 mRNA is present in the myotome at E12 and p35 expression persists in the premuscle mass and mature musculature at later stages, suggesting that p35 may also activate cdk5 during myogenesis (Zheng, 1998).
Cyclin-dependent kinase 5 (CDK5) is a unique CDK, the activity of which can be detected in postmitotic neurons. To date, CDK5 purified from mammalian brains has always been associated with a truncated form of the 35-kDa major brain specific activator (p35, also known as nck5a) of CDK5, known as p25. In this study, it is reported that p35 can be cleaved to p25 both in vitro and in vivo by calpain. In a rat brain extract, p35 is cleaved to p25 by incubation with Ca(2+). This cleavage is inhibited by a calpain inhibitor peptide derived from calpastatin and is ablated by separating the p35.CDK5 from calpain by centrifugation. The p35 recovered in the pellet after centrifugation can then be cleaved to p25 by purified calpain. Cleavage of p35 is also induced in primary cultured neurons by treatment with a Ca(2+) ionophore and Ca(2+) and inhibited by calpain inhibitor I. The cleavage changes the solubility of the CDK5 active complex from the particulate fraction to the soluble fraction but does not affect the histone H1 kinase activity. Increased cleavage is detected in cultured neurons undergoing cell death, suggesting a role of the cleavage in neuronal cell death (Kusakawa, 2000).
CDK5 plays an indispensable role in the central nervous system, and its deregulation is involved in neurodegeneration. The crystal structure of a complex between CDK5 and p25, a fragment of the p35 activator, is reported in this study. Despite its partial structural similarity with the cyclins, p25 displays an unprecedented mechanism for the regulation of a cyclin-dependent kinase. p25 tethers the unphosphorylated T loop of CDK5 in the active conformation. Residue Ser159, equivalent to Thr160 on CDK2, contributes to the specificity of the CDK5-p35 interaction. Its substitution with threonine prevents p35 binding, while the presence of alanine affects neither binding nor kinase activity. Evidence is provided that the CDK5-p25 complex employs a distinct mechanism from the phospho-CDK2-cyclin A complex to establish substrate specificity (Tarricone, 2001).
The interaction of p25 with CDK5 stabilizes an active conformation of the T loop, which is indistinguishable from those observed in phosphorylated CDK2 and ERK2. Evidence that Ile153 and Ser159 in the T loop of CDK5 are critical for p25 and p35 recognition and might contribute to the selectivity of the CDK5-p35 interaction. Retention of serine in all CDK5 homologs from yeast to human suggests that the presence of a phosphate acceptor at position 159 might be relevant for CDK5 regulation. It is predicted that phosphorylation of Ser159CDK5, if occurring, would negatively regulate kinase activity. Phosphorylation of Tyr15 on CDK5 by Abl is stimulatory, while phosphorylation of Tyr15 and Thr14 by Wee1 family kinases is inhibitory for CDK1 and 2. When taken together, these observations indicate that phosphorylation and dephosphorylation events similar to those impinging on mitotic CDKs regulate CDK5 in a completely distinct fashion. Structural and biochemical evidence is provided that the CDK5-p25 complex has devised a novel and distinct mechanism for substrate recognition and specificity entailing the participation of the activator subunit. An important difference between the activation mechanism of CDK5 and that of other proline-directed kinases, such as ERK2 and CDK2, is that the active T loop conformation of CDK5 is not stabilized by phosphorylation but by extensive interactions with the regulatory moiety (Tarricone, 2001).
Cyclin-dependent kinase 5 (Cdk5) plays a pivotal role in brain development and neuronal migration. Cdk5 is abundant in postmitotic, terminally differentiated neurons. The ability of Cdk5 to phosphorylate substrates is dependent on activation by its neuronal-specific activators p35 and p39. There exist striking differences in the phenotypic severity of Cdk5-deficient mice and p35-deficient mice. Cdk5-null mutants show a more severe disruption of lamination in the cerebral cortex, hippocampus, and cerebellum. In addition, Cdk5-null mice display perinatal lethality, whereas p35-null mice are viable. These discrepancies have been attributed to the function of other Cdk5 activators, such as p39. To understand the roles of p39 and p35, p39-null mice and p35/p39 compound-mutant mice were created. Interestingly, p39-null mice show no obvious detectable abnormalities, whereas p35-/-p39-/- double-null mutants are perinatal lethal. The p35-/-p39-/- mutants exhibit phenotypes identical to those of the Cdk5-null mutant mice. Other compound-mutant mice with intermediate phenotypes allow for the determination of the distinct and redundant functions between p35 and p39. The data strongly suggest that p35 and p39 are essential for Cdk5 activity during the development of the nervous system. Thus, p35 and p39 are likely to be the principal, if not the only, activators of Cdk5 (Ko, 2001).
Cyclin-dependent kinase 5 (Cdk5) null mice exhibit a unique phenotype characterized by perinatal mortality, disrupted cerebral cortical layering attributable to abnormal neuronal migration, lack of cerebellar foliation, and chromatolytic changes of neurons in the brainstem and the spinal cord. Because Cdk5 is expressed in both neurons and astrocytes, it has been unclear whether this phenotype is primarily attributable to defects in neurons or in astrocytes. Cdk5 expression has been reconstructed in neurons in Cdk5 null mice, and its effect on the null phenotype was examined. Unlike the Cdk5 null mice, the reconstituted Cdk5 null mice that express the Cdk5 transgene under the p35 promoter (TgKO mice) are viable and fertile. Because Cdk5 expression is mainly limited to neurons in these mice and rescues the defects in the nervous system of the Cdk5 null phenotype, it clearly demonstrates that Cdk5 activity is necessary for normal development and survival of p35-expressing neurons (Tanaka, 2001).
Cultures of cerebellar macroneurons were used to study the expression, activity, subcellular localization, and function of cdk5 during neuronal morphogenesis. The results obtained indicate that in non-polarized neurons, cdk5 is restricted to the cell body but as soon as polarity is established it becomes highly concentrated at the distal tip of growing axons where it associates with microtubules and the subcortical cytoskeleton. In addition, laminin, an extracellular matrix molecule capable of stimulating axonal extension and promoting MAP1b phosphorylation, accelerates the redistribution of cdk5 to the axonal tip and dramatically increases its activity. Finally, these results indicate that cdk5 suppression by antisense oligonucleotide treatment selectively reduces axonal elongation and decreases the phosphorylation status of MAP1b, as well as its binding to microtubules. Taken collectively, these observations suggest that cdk5 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 formation (Pigiono, 1997).
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. In addition, 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 linking agent between environmental signals (e.g., laminin) and constituents of the intracellular machinery (e.g., MAP1B) involved in axonal elongation (Paglini, 1998).
Cyclin-dependent kinase 5 (Cdk5) is a multifunctional neuronal protein kinase that is required for neurite outgrowth and cortical lamination and that plays an important role in dopaminergic signaling in the neostriatum through phosphorylation of Thr-75 of DARPP-32 (dopamine and cAMP-regulated phosphoprotein, molecular mass 32 kDa). Casein kinase 1 (CK1) has been implicated in a variety of cellular functions such as DNA repair, circadian rhythm, and intracellular trafficking. In the neostriatum, CK1 has been found to phosphorylate Ser-137 of DARPP-32. However, first messengers for the regulation of Cdk5 or CK1 have remained unknown. Both Cdk5 and CK1 are regulated by metabotropic glutamate receptors (mGluRs) in neostriatal neurons. (S)-3,5-dihydroxyphenylglycine (DHPG), an agonist for group I mGluRs, increases Cdk5 and CK1 activities in neostriatal slices, leading to the enhanced phosphorylation of Thr-75 and Ser-137 of DARPP-32, respectively. The effect of DHPG on Thr-75, but not on Ser-137, is blocked by a Cdk5-specific inhibitor, butyrolactone. In contrast, the effects of DHPG on both Thr-75 and Ser-137 are blocked by CK1-7 and IC261, specific inhibitors of CK1, suggesting that activation of Cdk5 by mGluRs requires CK1 activity. In support of this possibility, the DHPG-induced increase in Cdk5 activity, measured in extracts of neostriatal slices, is abolished by CK1-7 and IC261. Treatment of acutely dissociated neurons with DHPG enhances voltage-dependent Ca2+ currents. This enhancement is eliminated by either butyrolactone or CK1-7 and is absent in DARPP-32 knockout mice. Together these results indicate that a CK1-Cdk5-DARPP-32 cascade may be involved in the regulation by mGluR agonists of Ca2+ channels (Liu, 2001).
Numerous motile cell functions depend on signaling from the cytoskeleton to the nucleus. Myocardin-related transcription factors (MRTFs) translocate to the nucleus in response to actin polymerization and cooperate with serum response factor (Srf) to regulate the expression of genes encoding actin and other components of the cytoskeleton. This study shows that MRTF-A (Mkl1) and MRTF-B (Mkl2) redundantly control neuronal migration and neurite outgrowth during mouse brain development. Conditional deletion of the genes encoding these Srf coactivators disrupts the formation of multiple brain structures, reflecting a failure in neuronal actin polymerization and cytoskeletal assembly. These abnormalities were accompanied by dysregulation of the actin-severing protein gelsolin and Pctaire1 (Cdk16) kinase, which cooperates with Cdk5 to initiate a kinase cascade that governs cytoskeletal rearrangements essential for neuron migration and neurite outgrowth. Thus, the MRTF/Srf partnership interlinks two key signaling pathways that control actin treadmilling and neuronal maturation, thereby fulfilling a regulatory loop that couples cytoskeletal dynamics to nuclear gene transcription during brain development (Mokalled, 2010).
Cyclin-dependent kinase 5 (Cdk5) and its neuron-specific regulator p35 are essential for neuronal migration and for the laminar configuration of the cerebral cortex. In addition, p35/Cdk5 kinase concentrates at the leading edges of axonal growth cones and regulates neurite outgrowth in cortical neurons in culture. The Rho family of small GTPases is implicated in a range of cellular functions, including cell migration and neurite outgrowth. The p35/Cdk5 kinase is shown to co-localize with Rac (see Drosophila Rac1) in neuronal growth cones. Furthermore, p35 associates directly with Rac in a GTP-dependent manner. Another Rac effector, Pak1 kinase, is also present in the Rac-p35/Cdk5 complexes and co-localizes with p35/Cdk5 and Rac at neuronal peripheries. The active p35/Cdk5 kinase causes Pak1 hyperphosphorylation in a Rac-dependent manner, which results in down-regulation of Pak1 kinase activity. Because the Rho family of GTPases and the Pak kinases are implicated in actin polymerization, the modification of Pak1, imposed by the p35/Cdk5 kinase, is likely to have an impact on the dynamics of the reorganization of the actin cytoskeleton in neurons, thus promoting neuronal migration and neurite outgrowth (Nikolic, 1998).
The p35-Cdk5 kinase has been implicated in a variety of functions in the central nervous system (CNS), including axon outgrowth, axon guidance, fasciculation, and neuronal migration during cortical development. In p35(-/-) mice, embryonic cortical neurons are unable to migrate past their predecessors, leading to an inversion of cortical layers in the adult cortex. In order to identify molecules important for p35-Cdk5-dependent function in the cortex, a screen was undertaken for p35-interacting proteins using the two-hybrid system. In this study, the identification of a novel interaction between p35 and the versatile cell adhesion signaling molecule beta-catenin is reported. The p35 and beta-catenin proteins interact in vitro and colocalize in transfected COS cells. In addition, the p35-Cdk5 kinase is associated with a beta-catenin-N-cadherin complex in the cortex. In N-cadherin-mediated aggregation assays, inhibition of Cdk5 kinase activity using the Cdk5 inhibitor roscovitine leads to the formation of larger aggregates of embryonic cortical neurons. This finding was recapitulated in p35(-/-) cortical neurons, which aggregate to a greater degree than wild-type neurons. In addition, introduction of active p35-Cdk5 kinase into COS cells leads to a decreased beta-catenin-N-cadherin interaction and loss of cell adhesion. The association between p35-Cdk5 and an N-cadherin adhesion complex in cortical neurons and the modulation of N-cadherin-mediated aggregation by p35-Cdk5 suggests that the p35-Cdk5 kinase is involved in the regulation of N-cadherin-mediated adhesion in cortical neurons (Kwon, 2000).
Several models can be proposed to account for the regulation of cadherin-mediated adhesion by the p35-Cdk5 kinase. As p35-Cdk5 is a protein serine/threonine kinase, it may phosphorylate one or more components of the cadherin-adhesion complex, ultimately leading to decreased cell adhesion. Indeed, beta-catenin contains three minimal consensus sites for phosphorylation by Cdks. However, there is little evidence to support a role for serine/threonine phosphorylation as a modulator of cadherin-mediated adhesion in cortical neurons. On the other hand, evidence of regulation of tyrosine phosphorylation of beta-catenin and the cadherins by the EGF receptor, the kinase Src, and the phosphatases PTP1B and LAR suggests that tyrosine phosphorylation may serve as an important mechanism to regulate cadherin-mediated adhesion. The role of tyrosine phosphorylation in cadherin-mediated adhesion is interesting in light of the recent identification of a Cdk5-interacting protein, Cables, which bridges Cdk5 and the non-receptor tyrosine kinase Abl (L. Zukerberg, G. Patrick, M. Nicolic, S. Humbert, L. Lanier, F. Gertler, et al., unpublished observations cited in Kwon, 2000). Additionally, the p35-Cdk5 kinase may function as a scaffold to assemble molecules that act to destabilize N-cadherin-mediated adhesion. For instance, p35-Cdk5 interacts with the active form of the small GTPase Rac, and modulates Pak1 kinase activity. As Rac activity is necessary for cadherin-mediated adhesion, it is possible that the p35-Cdk5 kinase may regulate cadherin-mediated adhesion by regulating a Rac-dependent signaling pathway (Kwon, 2000).
Only about 1% of endogenous beta-catenin binds to p35, whereas more than 10% of total p35 binds to beta-catenin in the embryonic brain lysates. This observation suggests that whereas the beta-catenin-N-cadherin complex may be one of the major targets for the p35-Cdk5 kinase, only a small fraction of the beta-catenin-N-cadherin complex in the developing cortex is actually regulated by p35-Cdk5. Indeed, no difference was detected in the overall levels and association of beta-catenin and N-cadherin in membrane extracts derived from the embryonic and adult cortices of p35-/- mice. Thus, it is possible that p35-Cdk5 regulation of N-cadherin mediated adhesion is only crucial in a very specific population of migrating cortical neurons. In fact, it may be that p35-Cdk5 regulation of N-cadherin-mediated adhesion is relevant to neurons only when they traverse the intermediate zone of the developing cortex (Kwon, 2000).
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 serine/threonine and tyrosine kinases. Cyclin dependent kinase-5 (Cdk5) associates with and phosphorylates NR2A subunits at Ser-1232 in vitro and in intact cells. Moreover, roscovitine, a selective Cdk5 inhibitor, blocks both long-term potentiation induction and NMDA-evoked 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 (Li, 2001).
The Munc-18/syntaxin 1A complex has been postulated to act as a negative control on the regulated exocytotic process because its formation blocks the interaction of syntaxin with vesicle SNARE proteins. However, the formation of this complex is simultaneously essential for the final stages of secretion as evidenced by the necessity of Munc-18's homologs in Saccharomyces cerevisiae (Sec1p), Drosophila (ROP), and Caenorhabditis elegans (Unc-18) for proper secretion in these organisms. As such, any event that regulates the interaction of these two proteins is important for the control of secretion. One candidate for such regulation is cyclin-dependent kinase 5 (Cdk5), a member of the Cdc2 family of cell division cycle kinases that has recently been copurified with Munc-18 from rat brain. The present study shows that Cdk5 bound to its neural specific activator p35 not only binds to Munc-18 but utilizes it as a substrate for phosphorylation. Furthermore, it is demonstrated that Munc-18, when it has been phosphorylated by Cdk5, has a significantly reduced affinity for syntaxin 1A. Cdk5 can also bind to syntaxin 1A and a complex of Cdk5, p35, Munc-18, and syntaxin 1A can be fashioned in the absence of ATP and promptly disassembled upon the addition of ATP. These results suggest a model in which p35-activated Cdk5 becomes localized to the Munc-18/syntaxin 1A complex by its affinity for both proteins so that it may phosphorylate Munc-18 and thus permit the positive interaction of syntaxin 1A with upstream protein effectors of the secretory mechanism (Shuang, 1998).
Cyclin-dependent kinase, Cdk5, has been identified in neural tissue in connection with neurofilament and tau protein phosphorylation. This report describes the characterization of a 62-kDa protein that copurifies with Cdk5 from rat spinal cord homogenates. Dissociation of the protein from neural Cdk5 is concomitant with a reversible loss in kinase activity. Amino acid sequence information from tryptic peptide fragments was used to clone the complementary DNA from rat brain. A single full-length cDNA was characterized coding for a 67.5-kDa protein (p67). Exogenously expressed p67 stimulates Cdk5 kinase activity in vitro in a dose-dependent manner and when presented as an affinity matrix, selectively adsorbes Cdk5 from a cleared rat brain homogenate. In situ hybridization analysis of E18 rat embryos and adult rat brain demonstrates that p67 transcript expression is restricted to neural tissue. Immunohistochemical staining with an amino-terminal peptide-specific antibody further indicates that p67 is exclusively expressed in neurons. Localization in vivo and in cultured rat hippocampal neurons shows that p67 is highly enriched in axons. It is proposed that p67, by virtue of its regulation of Cdk5, participates in the dynamics of axonal architecture through the modulation of phosphorylation of cytoskeletal components (Shetty, 1999).
Disruption of one allele of the LIS1 gene (see Drosophila Lissencephaly-1)causes a severe developmental brain abnormality, type I lissencephaly. In Aspergillus nidulans, the LIS1 homolog, NUDF, and cytoplasmic dynein are genetically linked and regulate nuclear movements during hyphal growth. Recently, it has been demonstrated that mammalian LIS1 regulates dynein functions. NUDEL is a novel LIS1-interacting protein with sequence homology to gene products also implicated in nuclear distribution in fungi. Like LIS1, NUDEL is robustly expressed in brain, enriched at centrosomes and neuronal growth cones, and interacts with cytoplasmic dynein. Furthermore, NUDEL is a substrate of Cdk5, a kinase known to be critical during neuronal migration. Inhibition of Cdk5 modifies NUDEL distribution in neurons and affects neuritic morphology. These findings point to cross-talk between two prominent pathways that regulate neuronal migration (Niethammer, 2000).
Cyclin-dependent kinase 5 (Cdk5) is a small serine/threonine kinase that plays a pivotal role during development of the CNS. Cables (Cdk5 and Abl enzyme substrate), a novel protein, interacts with Cdk5 in brain lysates. Cables also binds to and is a substrate of the c-Abl tyrosine kinase. Cables displays little sequence homology to other known proteins in the databases. It does, however, show weak homology to cyclin A and weaker homology to cyclin C over an ~200 amino acid stretch in the C-terminal third of the protein, which may be the Cdk-interacting region. Cables also contains six PXXP motifs, defined as the minimal consensus for SH3 domain binding, and two tyrosine-based sorting motifs (YXXLE), which have been implicated in axonal growth cone sorting. It contains three serine proline/threonine proline minimal Cdk phosphorylation sites and at least one potential c-Abl phosphorylation site (YXXP). Active c-Abl kinase leads to Cdk5 tyrosine phosphorylation, and this phosphorylation is enhanced by Cables. Phosphorylation of Cdk5 by c-Abl occurs on tyrosine 15 (Y15), which is stimulatory for p35/Cdk5 kinase activity. Expression of antisense Cables in primary cortical neurons inhibits neurite outgrowth. Furthermore, expression of active Abl results in lengthening of neurites. The data provide evidence for a Cables-mediated interplay between the Cdk5 and c-Abl signaling pathways in the developing nervous system (Zukerberg, 2000).
These data suggest that Cables serves as an adaptor molecule, facilitating Cdk5 tyrosine phosphorylation and regulation by c-Abl. Phosphorylation of key substrates involved in actin and microtubule dynamics by active Cdk5 is likely to contribute to its role in neuronal migration and neurite outgrowth. Furthermore, Cdk5 has been shown to downregulate N-cadherin-mediated cell adhesion. Data presented in this communication suggest that Cables mediates an interaction between c-Abl and Cdk5, and may positively affect brain development and neurite outgrowth by enhancing Cdk5 tyrosine phosphorylation and upregulation of kinase activity. Cables may also mediate an interaction between Cdk5 and mDab1 by binding to both Cdk5 and c-Abl (Zukerberg, 2000).
The physiological state of the cell is controlled by signal transduction mechanisms which regulate the balance between protein kinase and protein phosphatase activities. A single protein can, depending on which particular amino-acid residue is phosphorylated, function either as a kinase or phosphatase inhibitor. DARPP-32 (dopamine and cyclic AMP-regulated phospho-protein, relative molecular mass 32,000) is converted into an inhibitor of protein phosphatase 1 when it is phosphorylated by protein kinase A (PKA) at threonine 34. DARPP-32 is converted into an inhibitor of PKA when phosphorylated at threonine 75 by cyclin-dependent kinase 5 (Cdk5). Cdk5 phosphorylates DARPP-32 in vitro and in intact brain cells. Phospho-Thr 75 DARPP-32 inhibits PKA in vitro by a competitive mechanism. Decreasing phospho-Thr 75 DARPP-32 in striatal slices, either by a Cdk5-specific inhibitor or by using genetically altered mice, results in increased dopamine-induced phosphorylation of PKA substrates and augmented peak voltage-gated calcium currents. Thus DARPP-32 is a bifunctional signal transduction molecule which, by distinct mechanisms, controls a serine/threonine kinase and a serine/threonine phosphatase (Bibb, 1999).
The Pak kinases are targets of the Rho GTPases Rac and Cdc42, which regulate cell shape and motility. It is increasingly apparent that part of this function is due to the effect Pak kinases have on microtubule organization and dynamics. Overexpression of Xenopus Pak5 enhances microtubule stabilization, and Pak1 may inhibit a microtubule-destabilizing protein, Op18/Stathmin. A specific phosphorylation site has been identified on mammalian Pak1, T212, which is targeted by the neuronal p35/Cdk5 kinase. Pak1 phosphorylated on T212, Pak1T212(PO4), is enriched in axonal growth cones and colocalizes with small peripheral bundles of microtubules. Cortical neurons overexpressing a Pak1A212 mutant display a tangled neurite morphology, which suggests that the microtubule cytoskeleton is affected. Cyclin B1/Cdc2 phosphorylates Pak1 in cells undergoing mitosis. In the developing cortex and in cultured fibroblasts, Pak1T212(PO4) is enriched in microtubule-organizing centers and along parts of the spindles. In living cells, a peptide mimicking phosphorylated T212 accumulates at the centrosomes and spindles and causes an increased length of astral microtubules during metaphase or following nocodazole washout. It is proposed that the region surrounding phosphorylated T212 contains a protein binding site, since the phosphorylated peptide is enriched in spindles and MTOCs and competes with endogenous Pak1 for this location.Together these results suggest that similar signaling pathways regulate microtubule dynamics in a remodeling axonal growth cone and during cell division (Banerjee, 2002).
Neurotoxic insults deregulate Cdk5 activity, which leads to neuronal apoptosis and may contribute to neurodegeneration. The biological activity of Cdk5 has been ascribed to its phosphorylation of cytoplasmic substrates. However, its roles in the nucleus remain unknown. The mechanism by which Cdk5 promotes neuronal apoptosis has been investigated. The prosurvival transcription factor MEF2 has been identified as a direct nuclear target of Cdk5. Cdk5 phosphorylates MEF2 at a distinct serine in its transactivation domain to inhibit MEF2 activity. Neurotoxicity enhances nuclear Cdk5 activity, leading to Cdk5-dependent phosphorylation and inhibition of MEF2 function in neurons. MEF2 mutants resistant to Cdk5 phosphorylation restore MEF2 activity and protect primary neurons from Cdk5 and neurotoxin-induced apoptosis. These studies reveal a nuclear pathway by which neurotoxin/Cdk5 induces neuronal apoptosis through inhibiting prosurvival nuclear machinery (Gong, 2003).
Mutations in the doublecortin (DCX) gene in human or targeted disruption of the cdk5 gene in mouse lead to similar cortical lamination defects in the developing brain. Dcx is phosphorylated by Cdk5. Dcx phosphorylation is developmentally regulated and corresponds to the timing of expression of p35, the major activating subunit for Cdk5. Mass spectrometry and Western blot analysis indicate phosphorylation at Dcx residue Ser297. Phosphorylation of Dcx lowers its affinity to microtubules in vitro, reduces its effect on polymerization, and displaces it from microtubules in cultured neurons. Mutation of Ser297 blocks the effect of Dcx on migration in a fashion similar to pharmacological inhibition of Cdk5 activity. These results suggest that Dcx phosphorylation by Cdk5 regulates its actions on migration through an effect on microtubules (Tanaka, 2004).
The relationship between cdk5 activity and regulation of the mitogen-activated protein (MAP) kinase pathway has been studied. 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 results 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, an increase is observed in the phosphorylation of NF-M subunit of neurofilament proteins that correlate with an up-regulation of MEK1 and ERK1/2 activity. The activity of a constitutively active MEK1 with threonine 286 mutated to alanine (within a TPXK cdk5 phosphorylation motif in the proline-rich domain) is not affected by cdk5 phosphorylation, suggesting that Thr286 might be the cdk5/p35 phosphorylation-dependent regulatory site. These findings support the hypothesis that cdk5 and the MAP kinase pathway cross-talk in the regulation of neuronal functions. Moreover, these data have prompted the proposal of a model for feedback down-regulation of the MAP kinase signal cascade by cdk5 inactivation of MEK1 (Sharma, 2002a).
Cyclin-dependent kinase 5 (Cdk5) plays a key role in the development of the mammalian nervous system; it phosphorylates a number of targeted proteins involved in neuronal migration during development to synaptic activity in the mature nervous system. Its role in the initial stages of neuronal commitment and differentiation of neural stem cells (NSCs), however, is poorly understood. This study shows that Cdk5 phosphorylation of p27(Kip1) at Thr187 is crucial to neural differentiation because (A) neurogenesis is specifically suppressed by transfection of p27(Kip1) siRNA into Cdk5(+/+) NSCs; (B) reduced neuronal differentiation in Cdk5(-/-) compared with Cdk5(+/+) NSCs; (C) Cdk5(+/+) NSCs, whose differentiation is inhibited by a nonphosphorylatable mutant, p27/Thr187A, are rescued by cotransfection of a phosphorylation-mimicking mutant, p27/Thr187D; (D) transfection of mutant p27(Kip1) (p27/187A) into Cdk5(+/+) NSCs inhibits differentiation. These data suggest that Cdk5 regulates the neural differentiation of NSCs by phosphorylation of p27(Kip1) at the Thr187 site. Additional experiments exploring the role of Ser10 phosphorylation by Cdk5 suggest that together with Thr187 phosphorylation, Ser10 phosphorylation by Cdk5 promotes neurite outgrowth as neurons differentiate. Cdk5 phosphorylation of p27(Kip1), a modular molecule, may regulate the progress of neuronal differentiation from cell cycle arrest through differentiation, neurite outgrowth and migration (Zheng, 2010).
Hyperphosphorylation of microtubule-associated proteins such as tau and neurofilament may underlie the cytoskeletal abnormalities and neuronal death seen in several neurodegenerative diseases, including Alzheimer's disease. One potential mechanism of microtubule-associated protein hyperphosphorylation is augmented activity of protein kinases known to associate with microtubules, such as cdk5 or GSK3beta. Tau and neurofilament are hyperphosphorylated in transgenic mice that overexpress human p25, an activator of cdk5. The p25 transgenic mice display silver-positive neurons using the Bielschowsky stain. Disturbances in neuronal cytoskeletal organization are apparent at the ultrastructural level. These changes are localized predominantly to the amygdala, thalamus/hypothalamus, and cortex. The p25 transgenic mice display increased spontaneous locomotor activity and differences from control mice in the elevated plus-maze test. The overexpression of an activator of cdk5 in transgenic mice results in increased cdk5 activity that is sufficient to produce hyperphosphorylation of tau and neurofilament as well as cytoskeletal disruptions reminiscent of Alzheimer's disease and other neurodegenerative diseases (Ahlijanian, 2000).
Phosphorylation of tau (a heat-stable neuron-specific microtubule-associated protein) by cdk5 is stimulated in the presence of microtubules (MTs). This stimulation is due to an increased phosphorylation rate but there is no increase in the total amount of phosphorylation. Two-dimensional phosphopeptide map analysis shows that MTs stimulate phosphorylation of a specific peptide. Using Western blotting with antibodies that recognize phosphorylation-dependent epitopes within tau, the phosphorylation sites stimulated by the presence of MTs were found to be Ser202 and Thr205 (numbered according to the human tau isoform containing 441 residues). MT-dependent phosphorylation at Thr205 is observed in situ in rat cerebrum primary cultured neurons. Stimulated phosphorylation at Ser202 and Thr205 decreases the MT-nucleation activity of tau, which is in contrast to MT-independent phosphorylation at Ser235 and Ser404 (Wada, 1998).
Recent work has shown that high molecular weight neurofilament (NF) proteins are phosphorylated in their carboxy-terminal tail portion by the enzyme cyclin-dependent kinase 5 (CDK-5). The tail domain of neurofilaments contains 52 tripeptide repeats, namely, Lys-Ser-Pro, which mainly exist as KSPXK and KSPXXX motifs (X = amino acid). CDK-5 specifically phosphorylates the serine residues within the KSPXK sites. The structural basis for this type of substrate selectivity was probed by studying the conformation of synthetic peptides containing either KSPXK or KSPXXX repeats designed from native neurofilament sequences. Synthetic peptides with KSPXK repeats are phosphorylated on serine with a recombinant CDK-5/p25 complex, whereas those with KSPXXX repeats are unreactive in this system. Circular dichroism (CD) studies in 50% TFE/H2O reveal a predominantly helical conformation for the KSPXXX-containing peptides, whereas the CD spectra for KSPXK-containing peptides indicates the presence of a high population of extended structures in water and 50% TFE solutions. However, detailed NMR analysis of one such peptide, which includes two such KSPXK repeats, suggests a turn-like conformation encompassing the first KSPXK repeat. Restrained molecular dynamics calculations yield an unusually stable, folded structure with a double S-like bend incorporating the central residues of the peptide. The data suggest that a transient reverse turn or loop-type structure may be a requirement for CDK-5-promoted phosphate transfer to neurofilament-specific peptide segments (Sharma, 1998).
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. 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 reveals 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 (Qi, 1998).
Neurofilament proteins, the major cytoskeletal components of large myelinated axons, are highly phosphorylated by second messenger-dependent and -independent kinases. These kinases, together with tubulins and other cytoskeletal proteins, have been shown to bind to neurofilament preparations. Cdk5 and Erk2, proline-directed kinases in neuronal tissues, phosphorylate the Lys-Ser-Pro (KSP) repeats in tail domains of NF-H, NF-M, and other axonal proteins such as tau and synapsin. In neurofilament and microtubule preparations from rat brain, it has been demonstrated by Western blot analysis that cdk5, a neuronal cyclin dependent kinase and Erk1/2 are associated with complexes of NF proteins, tubulins and tau. Using P13(suc1) affinity chromatography, a procedure known to bind cdc2-like kinases in proliferating cells with high affinity, a P13 complex was obtained from a rat brain extract exhibiting the same profiles of cdk5 and Erk2 bound to cytoskeletal proteins. The phosphorylation activities of these preparations and the effect of the cdk5 inhibitor, butyrolactone, are consistent with the presence of active kinases. Finally, during a column fractionation and purification of Erk kinases from rat brain extracts, fractions enriched in Erk kinase activity also exhibit co-elution of phosphorylated NF-H, tubulin, tau and cdk5. It is suggested that in mammalian brain, different kinases, their regulators and phosphatases form multimeric complexes with cytoskeletal proteins and regulate multisite phosphorylation from synthesis in the cell body to transport and assembly in the axon (Veeranna, 1999).
During axonal growth, repulsive guidance cues cause growth cone collapse and retraction. In the chick embryo, membranes from the posterior part of the optic tectum containing ephrins are original collapsing factors for axons growing from the temporal retina. Signal transduction pathways were investigated in retinal axons underlying this membrane-evoked collapse. Perturbation experiments using pertussis toxin (PTX) show that membrane-induced collapse is mediated via G(o/i) proteins, as is the case for semaphorin/collapsin-1-induced collapse. Studies with Indo-1 reveal that growth cone collapse by direct activation of G(o/i) proteins with mastoparan does not cause elevation of the intracellular Ca(2+) level, and thus this signal transduction pathway is Ca(2+) independent. Application of the protein phosphatase inhibitor okadaic acid alone induces growth cone collapse in retinal culture, suggesting signals involving protein dephosphorylation. In addition, pretreatment of retinal axons with olomoucine, a specific inhibitor of cdk5 (tau kinase II), prevents mastoparan-evoked collapse. Olomoucine also blocks caudal tectal membrane-mediated collapse. These results suggest that rearrangement of the cytoskeleton is mediated by tau phosphorylation. Immunostaining visualized complementary distributions of tau phospho- and dephosphoisoforms within the growth cone, which also supports the involvement of tau. Taking these findings together, it is concluded that cdk5 and tau phosphorylation probably lie downstream of growth cone collapse signaling mediated by PTX-sensitive G proteins (Nakayama, 1999).
Phosphorylation of the neurofilament-H subunit (NF-H) was investigated in rat embryonic brain neurons in culture. A portion of the NF-H is phosphorylated in vivo by embryonic day 17 when brain neurons were prepared. When the neurons were isolated and cultured, the NF proteins disappear and then reappear over the next several days in the following order: (1) NF-L/NF-M; (2) dephosphorylated NF-H and (3) phosphorylated NF-H. Phosphorylation of NF-H began around 4 days after cell plating, at about the time of synapse formation. Treatments that appear to modulate the timing of synapse formation also affect the timing of NF-H phosphorylation: (1) earlier phosphorylation is observed at higher neuronal cell density; (2) earlier phosphorylation is observed in neurons cultured on a coating substrate that promotes rapid neurite extension, and (3) phosphorylation is suppressed when neurite extension is inhibited by brefeldin A. Three possible synapse formation-induced events, excitation, cell-cell contact through adhesion proteins and elevated concentrations of neurotrophic factors, were examined for their possible involvement in generating the signal for NF-H phosphorylation. Neither excitation nor cell contact enhances NF-H phosphorylation. Neurotrophic factors, brain-derived neurotrophic factor (BDNF) and neurotrophin 3 (NT3) stimulate phosphorylation of NF-H. The BDNF-stimulated phosphorylation is inhibited by an anti-BDNF antibody and K252a, an inhibitor of BDNF receptor TrkB tyrosine kinase. Among known NF-H kinases of cyclin-dependent kinase 5 (CDK5), external signal-regulated protein kinase (ERK) and stress-activated protein kinase (SAPK), CDK5 and SAPK show an increase in kinase activity or an active form with a time course similar to NF-H phosphorylation in control culture. BDNF stimulates the kinase activity of CDK5 and induces appearance of an active form of ERK transiently. These results suggest a possibility that synapse formation induces NF-H phosphorylation, at least in part, through activation of CDK5 by BDNF (Tokuoka, 2000).
Cyclin-dependent kinase 5(cdk5) is highly homologous to other members of the cdk family that are known to function in proliferating cells. Despite the structural similarity, cdk5-associated histone H1 kinase activity is only detectable in postmitotic neurons of the central nervous system (CNS). p35 is a neuronal-specific cdk5 regulator that activates cdk5 kinase activity upon association. The cdk5/p35 kinase activity increases during the progression of CNS neurogenesis, suggesting a function of cdk5 in neuronal differentiation. Both cdk5 and p35 proteins are present in the growth cones of developing neurons. The staining pattern of cdk5 in the growth cones is similar to that of actin filaments but not microtubules. To address the functional significance of the cdk5/p35 kinase in neurogenesis, wild-type or mutant kinases were ectopically expressed in cortical cultures. Expression of dominant-negative mutants of cdk5 (cdk5N144 and cdk5T33) inhibits neurite outgrowth, which is rescued by coexpression of the wild-type proteins. A similar extent of neurite outgrowth inhibition is obtained by transfection of an antisense p35 construct, which in turn is only rescued by p35 but not cdk5 coexpression. In contrast, longer neurites were elaborated in neurons that coexpressed exogenous cdk5 and p35. These observations suggest that the cdk5/p35 kinase plays a critical role in neurite outgrowth during neuronal differentiation (Nikolic, 1996).
The expression, activity and localization of cyclin dependent kinase 5 (cdk5) during myogenesis was examined. Cdk5 protein is expressed in adult mouse muscle. In murine C2 cells, both the protein level and kinase activity of cdk5 shows a marked increase during early myogenesis with a peak between 36 and 48 hours of differentiation, decreasing as myotubes fuse after 60 to 72 hours. This increase in cdk5 protein level is specific for differentiation and not simply related to cell cycle arrest since it is not observed in fibroblasts grown for 48 hours in low serum medium. Anti-cdk5 antibodies showe a low level cytoplasmic staining in proliferative myoblasts, a rapid increase in nuclear staining during the initial 12 hours of differentiation and a predominant nuclear staining in myotubes. Microinjection of plasmids encoding wild-type cdk5 into C2 myoblasts enhances differentiation as assessed by both myogenin and troponin T expression after 48 hours of differentiation. In contrast, microinjection of plasmids encoding a dominant negative mutant of cdk5 inhibits the onset of differentiation. These data imply a previously unsuspected role for cdk5 protein kinase as a positive modulator of early myogenesis (Lazaro, 1997).
The adult mammalian cortex is characterized by a distinct laminar structure generated through a well-defined pattern of neuronal migration. Successively generated neurons are layered in an 'inside-out' manner to produce six cortical laminae. p35, the neuronal-specific activator of cyclin-dependent kinase 5, plays a key role in proper neuronal migration. Mice lacking p35, and thus p35/cdk5 kinase activity, display severe cortical lamination defects and suffer from sporadic adult lethality and seizures. Histological examination reveals that the mutant mice lack the characteristic laminated structure of the cortex. Neuronal birth-dating experiments indicate a reversed packing order of cortical neurons such that earlier born neurons reside in superficial layers and later generated neurons occupy deep layers. The phenotype of p35 mutant mice thus demonstrates that the formation of cortical laminar structure depends on the action of the p35/cdk5 kinase (Chae, 1997).
Although cyclin-dependent kinase 5 (Cdk5) is closely related to other cyclin-dependent kinases, its kinase activity is detected only in postmitotic neurons. Cdk5 expression and kinase activity are correlated with the extent of differentiation of neuronal cells in the developing brain. Cdk5 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. Cdk5(-/-) mice were generated through gene targeting. Cdk5(-/-) mice exhibit unique lesions in the central nervous system associated with perinatal mortality. The brains of Cdk5(-/-) 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 (Ohshima, 1996).
The cerebral cortex of mice with a targeted disruption in the gene for cyclin-dependent kinase 5 (cdk5) is abnormal in its structure. Bromodeoxyuridine labeling reveals that the normal inside-out neurogenic gradient is inverted in the mutants; earlier born neurons are most often found superficial to those born later. Despite this, the early preplate layer separates correctly and neurons with a normal, pyramidal morphology can be found between true marginal zone and subplate. Consistent with their identity as layer VI corticothalamic neurons, they can be labeled by DiI injections into thalamus. The DiI injections also reveal that the trajectories of the cdk5(-/-) thalamocortical axons are oblique and cut across the entire cortical plate, instead of being oriented tangentially in the subcortical white matter. A model is proposed in which the cdk5(-/-) defect blocks cortical development at a heretofore undescribed intermediate stage, after the splitting of the preplate, but before the migration of the full complement of cortical neurons (Gilmore, 1998).
The p35/cdk5 neuronal-specific kinase complex has been shown to play an important role in the laminar configuration of cortical neurons. Mice lacking either p35 or cdk5 exhibit a disrupted cortical lamination pattern. It has previously been shown that nstead of the normal 'inside-out' layering pattern of cortical neurons, cortical neurons are layered from 'outside-in' in p35 mutant mice. To gain insight into the mechanisms that underlie these defects, the organization of landmark structures formed during cortical development and the migratory behavior of p35(-/-) cortical neurons were examined by using bromodeoxyuridine labeling. Reelin localization in the marginal zone is normal in p35 mutant mice. Furthermore, the preplate properly splits into the marginal zone and subplate, a developmental event that fails to occur in reeler mice. Finally, the migration of the earliest born cortical plate neurons is normal in p35 mutant mice; cortical neurons subsequently generated remain underneath these neurons. These data suggest that the p35/cdk5 kinase is required for cortical plate neurons to migrate past preexisting neurons and take up superficial positions to constitute the inside-outside layering order of cortical lamination (Kwon, 1998).
Mice lacking p35, an activator of cdk5 in the central nervous system (CNS), exhibit defects in a variety of CNS structures, most prominently characterized by a disruption in the laminar structure of the neocortex. In addition, alterations of certain axonal fiber tracts are found in the cortex of p35 mutant mice. Notably, the corpus callosum appears bundled at the midline, but dispersed lateral to the midline. Tracer injection experiments in adult p35 mutant mice reveal that projecting cortical axons fail to assimilate into the corpus callosum, and take oblique paths to the midline. After crossing the midline, cortical axons defasciculate prematurely from the corpus callosum and take similarly oblique paths through the cortex. This callosal phenotype is not detected in reeler mice, which also exhibit defects in cortical lamination, suggesting that the lack of fasciculation of callosal axons is not an inherent manifestation of a disruption of cortical lamination. The embryonic callosal axon tract is defasciculated before crossing the midline, suggesting that axon guidance may be affected during embryonic development of the corpus callosum. In addition, embryonic thalamocortical afferents also exhibit a defasciculated phenotype. These results suggest that defective axonal fasciculation and guidance may be primary responses to the loss of p35 in the cortex. Furthermore, this study postulates a role for the p35/cdk5 kinase in molecular signaling pathways necessary for proper guidance of selective axons during embryonic development (Kwon, 1999).
In spite of the clarification in the temporal and spatial expression pattern of cyclin-dependent kinase 5 (Cdk5) and its neuron-specific activator, p35, in the CNS, these expression patterns remain to be elucidated in the PNS. In addition, it is not known whether Cdk5 activity exists in the PNS. Therefore, Cdk5 and p35 expression and activity in the PNS were examined by immunoblot analysis, immunohistochemistry, and in vitro kinase assay. Immunoblot analysis indicates the expression of Cdk5 and p35 proteins in both the dorsal root ganglion (DRG) and sciatic nerve in the CNS. By immunohistochemistry, both proteins are present in the cell body and axon (sciatic nerve) of both DRG neurons and anterior horn cells. A co-immunoprecipitation study indicates the in vivo association between Cdk5 and p35 in both DRG and sciatic nerve. However, Cdk5 kinase activity is found only in DRG, and not in sciatic nerve. These results suggest that Cdk5 kinase activity exists and functions physiologically in the PNS and may be regulated by unknown mechanisms other than the availability of p35 as reported in developing brains (Terada, 1998).
The expansion of the mammalian cerebral cortex is safeguarded by a concerted balance between amplification and neuronal differentiation of intermediate progenitors (IPs). Nonetheless, the molecular controls governing these processes remain unclear. This study found that the scaffold protein Axin is a critical regulator that determines the IP population size and ultimately the number of neurons during neurogenesis in the developing cerebral cortex. The increase of the IP pool is mediated by the interaction between Axin and GSK-3 in the cytoplasmic compartments of the progenitors. Importantly, as development proceeds, Axin becomes enriched in the nucleus to trigger neuronal differentiation via beta-catenin activation. The nuclear localization of Axin and hence the switch of IPs from proliferative to differentiative status are strictly controlled by the Cdk5-dependent phosphorylation of Axin at Thr485. The results demonstrate an important Axin-dependent regulatory mechanism in neurogenesis, providing potential insights into the evolutionary expansion of the cerebral cortex (Fang, 2013).
The fate decision of NPCs between amplification and differentiation controls the number of neurons produced during brain development and ultimately determines brain size. However, it is unclear how the NPCs make this fundamental choice. This study shows that the subcellular localization of a signaling scaffold protein, Axin, defines the activation of specific signaling networks in NPCs, thereby determining the amplification or neuronal differentiation of NPCs during embryonic development. Cytoplasmic Axin in NPCs enhances IP generation, which ultimately leads to increased neuron production, whereas nuclear Axin in IPs promotes neuronal differentiation. Intriguingly, the Cdk5-dependent phosphorylation of Axin facilitates the nuclear accumulation of the protein, thereby functioning as a 'brake' to prevent the overproduction of IPs and induce neuronal differentiation (Fang, 2013).
The expansion of cortical surface may result from increased numbers of neuroepithelial (NE) cells and radial glial cells (RGs) or from an amplified IP pool. NE/RG augmentation evidently controls the global enlargement of cortical surface. The amplification of a subset of RGs expressing the transcription factor Cux2 was recently suggested to facilitate upper-layer neuron expansion (Franco, 2012). However, there is a lack of experimental evidence indicating whether IP amplification also substantially contributes to the expansion of upper-layer cortical neurons and the cerebral cortex. Nonetheless, upper-layer neurons are generated during mid- and late neurogenesis, at which time IPs play the primary role in neuron production. Moreover, the enlargement of IP-residing SVZ is temporally correlated with the increased number of upper-layer neurons and expanded cortical surface. Therefore, it is tempting to speculate that the amplification of IPs during mid- and late corticogenesis has facilitated the evolutionary expansion of the cerebral cortex. The present findings demonstrate that increasing Axin levels during midcorticogenesis, which leads to the transient amplification of IPs without affecting the RG pool, is sufficient to expand the surface of the neocortex. Previous studies show that Axin expression is tightly regulated by different posttranslational modifications including deubiquitination, SUMOylation, methylation, and phosphorylation, which increase the stability of Axin; meanwhile, polyubiquitination and poly-ADP-ribosylation lead to its degradation. Thus, the adaptive evolution of the Axin gene that regulates its posttranslational modifications and hence its expression level might be involved in the evolutionary expansion of the cerebral cortex (Fang, 2013).
To ensure the development of a cerebral cortex of the proper size, the amplification and neuronal differentiation of IPs need to be precisely controlled. A reduced number of IPs due to precocious depletion of NEs/RGs or inhibition of IP generation/proliferation ultimately lead to the generation of fewer cortical neurons, resulting in a smaller cortex - a characteristic feature of human microcephalic syndromes (Fang, 2013).
In contrast, the overexpansion of IPs generates an excessive number of neurons, which is associated with macrocephaly and autism. The current findings demonstrate that Axin strictly controls the process of indirect neurogenesis to ensure the production of a proper number of neurons. Although cytoplasmic Axin simultaneously maintains the RG pool and promotes IP amplification to sustain rapid and long-lasting neuron production, subsequent enrichment of Axin in the nuclei of IP daughter cells triggers neuronal differentiation and prevents the overexpansion of IPs. In addition, the results demonstrate that Cdk5-mediated phosphorylation regulates the nucleocytoplasmic shuttling of Axin, thereby controlling the switching of NPCs from proliferative to differentiation status (Fang, 2013).
The findings show that Axin phosphorylation in IPs triggers neuronal differentiation in a rostrolateral-high to caudo-medial-low gradient correlated with the spatial gradient of neurogenesis. Thus, the gradient of Axin phosphorylation may provide a quantitative tool for evaluating the temporal and spatial gradient of IP differentiation into neurons. Importantly, nuclear Axin phosphorylation is rapidly induced in IP daughter cells in the G1 phase, which is the stage when progenitor cells actively respond to neurogenic signals; this suggests that the timing of Axin phosphorylation-dependent IP differentiation is regulated by diffusible extracellular signals. Therefore, understanding how Axin phosphorylation is regulated in IPs by extracellular cues and niches should shed new light on the molecular basis underlying the gradient-specific differentiation of IPs (Fang, 2013).
The findings also highlight the importance of Cdk5 in embryonic neurogenesis. Although Cdk5 plays critical roles in neuronal development and is implicated in the neurogenesis of cultured neural stem cells, it remains unclear whether Cdk5 regulates embryonic neurogenesis. The current findings provide in vivo evidence that Cdk5 is required for the neuronal differentiation of IPs, at least in part through phosphorylating Axin. Intriguingly, although cdk5/ cortices exhibited an accumulation of IPs and reduced neuron production during early-mid neurogenesis, the brain size of these mutant mice remained unchanged by the end of neurogenesis. This may be due to the compensatory increase of neuron production from the expanded pool of IPs during the mid-to-late neurogenesis stages. Therefore, elucidating how Cdk5 is involved in different stages of neurogenesis may provide insights into the molecular control of neuronal number and subtypes (Fang, 2013).
Several factors that regulate the generation and amplification of IPs have been identified. Nonetheless, key questions remain open: how RGs determine to differentiate into IPs instead of neurons, how RG-to-IP transition and IP differentiation are coordinated, and how IP amplification and differentiation are balanced. The present results show that the interaction between cytoplasmic Axin and GSK-3β maintains the RG pool and promotes IP production. The signaling mechanisms underlying the action of Axin-GSK-3β interaction require further investigation. It is hypothesized that Axin regulates IP differentiation from RGs via various molecular mechanisms. First, the Axin-GSK-3β complex may reduce the level of Notch receptor or β-catenin, leading to the suppression of Notch- and Wnt-mediated signaling, respectively. Given that Axin and GSK-3β can associate with the centrosome and mitotic spindle, Axin-GSK-3β interaction may also modulate cleavage plane orientation. Furthermore, Axin-GSK-3β can interact with and affect the microtubule-binding activity of adenomatous polyposis coli (APC), which is required for establishing the apical-basal polarity and asymmetric division of RGs. Finally, interaction with Axin can cause GSK-3β inhibition, which may enhance IP amplification through the activation of Shh signaling (Fang, 2013).
The timing of IPs to undergo cell-cycle exit balances the proliferative and neurogenic divisions of IPs and switches the RG-to- IP transition to the neuronal differentiation of IPs. This study shows that the interaction between Axin and β-catenin in the nucleus switches the division of IPs from proliferative to neurogenic by enhancing the neurogenic transcriptional activity of β-catenin. Indeed, Axin and β-catenin are required for the signal transduction of Wnt, RA, and TGF-β, which triggers and promotes neuronal differentiation. Thus, Axin in the nucleus may serve to transduce and converge multiple neurogenic signaling pathways to β-catenin during neurogenesis. However, the mechanism by which nuclear Axin enhances the transcriptional activity of β-catenin requires further investigation. Given that β-catenin exerts its transcriptional regulation of target genes through association with T cell factor/lymphoid enhancer factor (Tcf/Lef), it is hypothesized that nuclear Axin facilitates β-catenin/Tcf/Lef complex formation to enhance transcription (Fang, 2013).
Although Axin was previously recognized as a negative regulator of canonical Wnt signaling, suppressing cell division by recruiting GSK-3β and β-catenin into the β-catenin destruction complex for β-catenin degradation, the present results show that cytoplasmic Axin and nuclear Axin act distinctly from canonical Wnt signaling through specific binding to GSK-3β and β-catenin, respectively. Therefore, the current findings corroborate the notion that Wnt signaling components play multifaceted roles in NPCs during neurogenesis, independent of canonical Wnt signaling as demonstrated in previous studies (Fang, 2013).
In conclusion, the present study identified distinct roles of Axin in IP amplification and neuron production. The results demonstrate that the modulation of Axin levels, subcellular localization, phosphorylation, and its interaction with key signaling regulators (e.g., GSK-3β and β-catenin) in NPCs ultimately control neuron production and expansion of the cerebral cortex. Given that Axin is a key regulator of the switch from IP amplification to differentiation, the characterization of the signals that control this switch will not only advance current understanding of how the cerebral cortex expands during evolution but also provide important insights into neurodevelopmental disorders such as microcephaly (Fang, 2013).
Cyclin-dependent kinase-5 (cdk-5) is a serine/threonine kinase that displays neuron-specific activity. Experimental manipulation of cdk-5 expression in neurons has shown that cdk-5 is essential for proper development of the nervous system and, in particular, for outgrowth of neurites. Such observations suggest that cdk-5 activity must be tightly controlled during development of the nervous system. To identify possible regulators of cdk-5, the yeast two-hybrid system was used to search for proteins that interact with cdk-5. In two independent yeast transformation events, cyclin D2 interacts with cdk-5. Immunoprecipitation experiments confirm that cyclin D2 and cdk-5 interact in mammalian cells. Cyclin D2 did not activate cdk-5 as assayed using three different substrates; this is in contrast to a known cdk-5 activator, p35. However, cyclin D2 expression leads to a decrease in cdk-5/p35 activity in transfected cells. Since cyclin D2 and cdk-5 are known to share overlapping patterns of expression during development of the CNS, the results presented here suggest a role for cyclin D2 in modulating cdk-5 activity in postmitotic developing neurons (Guidato, 1998).
The mammalian cerebral cortex consists of six layers that are generated via coordinated neuronal migration during the embryonic period. Recent studies identified specific phases of radial migration of cortical neurons. After the final division, neurons transform from a multipolar to a bipolar shape within the subventricular zone-intermediate zone (SVZ-IZ) and then migrate along radial glial fibres. Mice lacking Cdk5 exhibit abnormal corticogenesis owing to neuronal migration defects. When GFP was introduced into migrating neurons at E14.5 by in utero electroporation, migrating neurons were observed in wild-type but not in Cdk5-/- embryos after 3-4 days. Introduction of the dominant-negative form of Cdk5 into the wild-type migrating neurons confirmed specific impairment of the multipolar-to-bipolar transition within the SVZ-IZ in a cell-autonomous manner. Cortex-specific Cdk5 conditional knockout mice showed inverted layering of the cerebral cortex and the layer V and callosal neurons, but not layer VI neurons, had severely impaired dendritic morphology. The amount of the dendritic protein Map2 was decreased in the cerebral cortex of Cdk5-deficient mice, and the axonal trajectory of cortical neurons within the cortex was also abnormal. These results indicate that Cdk5 is required for proper multipolar-to-bipolar transition, and a deficiency of Cdk5 results in abnormal morphology of pyramidal neurons. In addition, proper radial neuronal migration generates an inside-out pattern of cerebral cortex formation and normal axonal trajectories of cortical pyramidal neurons (Ohshima, 2007).
The role of cyclin-dependent kinases in cell death has been investigated and the expression of cyclin-dependent kinase 5 (Cdk5) has been found to be associated with apoptotic cell death in both adult and embryonic tissues. By double labeling immunohistochemistry and confocal microscopy, the expression of Cdk5 was specifically associated with dying cells. The association of Cdks with cell death is unique to Cdk5, since this association is not found with the other Cdks (Cdk 1-8) and cell death. The differential increase in Cdk5 expression is at the level of protein only, and no differences can be detected at the level of mRNA. Using the limbs of mutant mice detective in the pattern of interdigital cell death and limbs with increased interdigital cell death as a result of retinoic acid treatment, the specificity of Cdk5 protein expression in dying cells has been confirmed. To investigate the regulation of Cdk5 during cell death, the expression of a regulatory protein of Cdk5, p35, was examined. p35 was found to be expressed in the dying cells as well. Similar to Cdk5, there is also no specific differential expression of the p35 mRNA in dying cells. These results suggest a role for Cdk5 and p35 proteins in cell death. This protein complex may function in the rearrangement of the cytoskeleton during apoptosis (Ahuja, 1998).
Cyclin-dependent kinase 5 (Cdk5) is required for proper development of the mammalian central nervous system. To be activated, Cdk5 has to associate with its regulatory subunit, p35. p25, a truncated form of p35, accumulates in neurons in the brains of patients with Alzheimer's disease. This accumulation correlates with an increase in Cdk5 kinase activity. Unlike p35, p25 is not readily degraded, and binding of p25 to Cdk5 constitutively activates Cdk5, changes its cellular location and alters its substrate specificity. In vivo the p25/Cdk5 complex hyperphosphorylates tau, which reduces tau's ability to associate with microtubules. Moreover, expression of the p25/Cdk5 complex in cultured primary neurons induces cytoskeletal disruption, morphological degeneration and apoptosis. These findings indicate that cleavage of p35, followed by accumulation of p25, may be involved in the pathogenesis of cytoskeletal abnormalities and neuronal death in neurodegenerative diseases (Patrick, 1999).
Cyclin-dependent kinase 5 (cdk5) is a serine/threonine kinase activated by associating with its neuron-specific activators p35 and p39. Analysis of cdk5-/- and p35-/- mice has demonstrated that both cdk5 and p35 are essential for neuronal migration, axon pathfinding and the laminar configuration of the cerebral cortex, suggesting that the cdk5-p35 complex may play a role in neuron survival. However, the targets of cdk5 that regulate neuron survival have been unknown. This study shows that cdk5 directly phosphorylates c-Jun N-terminal kinase 3 (JNK3) on Thr131 and inhibits its kinase activity, leading to reduced c-Jun phosphorylation. Expression of cdk5 and p35 in HEK293T cells inhibits c-Jun phosphorylation induced by UV irradiation. These effects can be restored by expression of a catalytically inactive mutant form of cdk5. Moreover, cdk5-deficient cultured cortical neurons exhibit increased sensitivity to apoptotic stimuli, as well as elevated JNK3 activity and c-Jun phosphorylation. Taken together, these findings show that cdk5 may exert its role as a key element by negatively regulating the c-Jun N-terminal kinase/stress-activated protein kinase signaling pathway during neuronal apoptosis (Li, 2002).
Cyclin-dependent kinase 5 (Cdk5) and its regulatory subunit p35 are integral players in the proper development of the mammalian central nervous system. Proteolytic cleavage of p35 generates p25, leading to aberrant Cdk5 activation. The accumulation of p25 is implicated in several neurodegenerative diseases. In primary neurons, p25 causes apoptosis and tau hyperphosphorylation. Current mouse models expressing p25, however, fail to rigorously recapitulate these phenotypes in vivo. In this study, inducible transgenic mouse lines were generated overexpressing p25 in the postnatal forebrain. Induction of p25 preferentially directs Cdk5 to pathological substrates. These animals exhibit neuronal loss in the cortex and hippocampus, accompanied by forebrain atrophy, astrogliosis, and caspase-3 activation. Endogenous tau is hyperphosphorylated at many epitopes, aggregated tau accumulates, and neurofibrillary pathology develops progressively in these animals. These cumulative findings provide compelling evidence that in vivo deregulation of Cdk5 by p25 plays a causative role in neurodegeneration and the development of neurofibrillary pathology (Cruz, 2003).
Synaptogenesis is a highly regulated process that underlies formation of neural circuitry. Considerable work has demonstrated the capability of some adhesion molecules, such as SynCAM and Neurexins/Neuroligins, to induce synapse formation in vitro. Furthermore, Cdk5 gain of function results in an increased number of synapses in vivo. To gain a better understanding of how Cdk5 might promote synaptogenesis, potential crosstalk between Cdk5 and the cascade of events mediated by synapse-inducing proteins was investigated in a mammalian system. One protein recruited to developing terminals by SynCAM and Neurexins/Neuroligins is the MAGUK family member CASK. It was found that Cdk5 phosphorylates and regulates CASK distribution to membranes. In the absence of Cdk5-dependent phosphorylation, CASK is not recruited to developing synapses and thus fails to interact with essential presynaptic components. Functional consequences include alterations in calcium influx. Mechanistically, Cdk5 regulates the interaction between CASK and liprin-α. These results provide a molecular explanation of how Cdk5 can promote synaptogenesis (Samuels, 2007).
Homologs of liprin-α proteins are essential for presynaptic terminal formation in C. elegans and Drosophila . Mutations in C. elegans syd-2 result in a diffuse localization of several presynaptic proteins and abnormally sized active zones, and loss- and gain-of-function experiments demonstrate that presynaptic organization is dependent on syd-2. Likewise, Dliprin-α is required for normal synaptic morphology including the size and shape of the presynaptic active zone in Drosophila . Cdk5-dependent phosphorylation of CASK occurs in both the CaMK and L27 domains, and only mutation of both sites yields a localization phenotype. Since liprin-α proteins require the presence of both domains to interact with CASK, the phosphorylation sites are in a prime spot to mediate the interaction. According to the model described in this study, liprin-α is required for initial CASK localization to presynaptic terminals. Since, liprin-α binds directly to the kinesin motor KIF1A and in Drosophila liprin-α mutant axons there is decreased anterograde processivity resulting in reduced levels of presynaptic markers at terminals, it is feasible that liprin-α acts as a cargo receptor that delivers CASK, as well as other components, to and within the developing synapse. Cdk5-dependent phosphorylation could then act to coordinate distinct pools of CASK that are bound to liprin-α or are bound to other components of the presynaptic machinery. Importantly, it is not believed that Cdk5 loss of function generally affects liprin-α-mediated transport since synaptophysin, a marker of synaptic vesicles, is still properly localized within synaptosomes. In this model, there would be advantages of having locally enhanced Cdk5 activity within the presynaptic terminal relative to some other cellular compartments. Supporting this idea, phospho-CASK is particularly enriched at synaptic membranes, and Cdk5 has been shown to phosphorylate and regulate several proteins, including Munc-18, Dynamin-1, Amphiphysin-1, and Synaptojanin-1, that function to control multiple rounds of the synaptic vesicle cycle. Synapsin-1 is also a Cdk5 substrate. With regard to the role of liprin-α, it will ultimately be essential to assay synapse formation and CASK localization in mammalian liprin-α loss-of-function models (Samuels, 2007).
Learning is accompanied by modulation of postsynaptic signal transduction pathways in neurons. Although the neuronal protein kinase cyclin-dependent kinase 5 (Cdk5) has been implicated in cognitive disorders, its role in learning has been obscured by the perinatal lethality of constitutive knockout mice. Conditional knockout of Cdk5 in the adult mouse brain improved performance in spatial learning tasks and enhanced hippocampal long-term potentiation and NMDA receptor (NMDAR)-mediated excitatory postsynaptic currents. Enhanced synaptic plasticity in Cdk5 knockout mice is attributed to reduced NR2B degradation, which causes elevations in total, surface and synaptic NR2B subunit levels and current through NR2B-containing NMDARs. Cdk5 facilitates the degradation of NR2B by directly interacting with both it and its protease, calpain. These findings reveal a previously unknown mechanism by which Cdk5 facilitates calpain-mediated proteolysis of NR2B and may control synaptic plasticity and learning (Hawasli, 2007).
Homeostatic plasticity keeps neuronal spiking output within an optimal range in the face of chronically altered levels of network activity. Little is known about the underlying molecular mechanisms, particularly in response to elevated activity. In hippocampal neurons experiencing heightened activity, the activity-inducible protein kinase Polo-like kinase 2 (Plk2, also known as SNK) is required for synaptic scaling-a principal mechanism underlying homeostatic plasticity. Synaptic scaling also requires CDK5, which acts as a 'priming' kinase for the phospho-dependent binding of Plk2 to its substrate SPAR, a postsynaptic RapGAP and scaffolding molecule that is degraded following phosphorylation by Plk2. RNAi knockdown of SPAR weakens synapses, and overexpression of a SPAR mutant resistant to Plk2-dependent degradation prevents synaptic scaling. Thus, priming phosphorylation of the Plk2 binding site in SPAR by CDK5, followed by Plk2 recruitment and SPAR phosphorylation-degradation, constitutes a molecular pathway for neuronal homeostatic plasticity during chronically elevated activity (Seeburg, 2008).
Cyclin-dependent kinase 5 regulates numerous neuronal functions with its activator, p35. Under neurotoxic conditions, p35 undergoes proteolytic cleavage to liberate p25, which has been implicated in various neurodegenerative diseases. This study shows that p25 is generated following neuronal activity under physiological conditions in a GluN2B- and CaMKIIalpha-dependent manner. Moreover, a knockin mouse model was developed in which endogenous p35 is replaced with a calpain-resistant mutant p35 (Deltap35KI) to prevent p25 generation. The Deltap35KI mice exhibit impaired long-term depression and defective memory extinction, likely mediated through persistent GluA1 phosphorylation at Ser845. Finally, crossing the Deltap35KI mice with the 5XFAD mouse model of Alzheimer's disease (AD) resulted in an amelioration of beta-amyloid (Aβ)-induced synaptic depression and cognitive impairment. Together, these results reveal a physiological role of p25 production in synaptic plasticity and memory and provide new insights into the function of p25 in Aβ-associated neurotoxicity and AD-like pathology (Seo, 2014).
Passage of normal cells in culture leads to senescence, an irreversible cell cycle exit characterized by biochemical changes and a distinctive morphology. Cellular stresses, including oncogene activation, can also lead to senescence. Consistent with an antioncogenic role for this process, the tumor suppressor pRb plays a critical role in senescence. Reexpression of pRb in human tumor cells results in senescence-like changes, including cell cycle exit and shape changes. Senescence is accompanied by increased expression and altered localization of ezrin, an actin binding protein involved in membrane-cytoskeletal signaling. pRb expression results in the stimulation of CDK5-mediated phosphorylation of ezrin with subsequent membrane association and induction of cell shape changes, linking pRb activity to cytoskeletal regulation in senescent cells (Yang, 2003).
Normal human somatic cells do not divide indefinitely, but rather, have a limited capacity to replicate in culture. The finite replicative lifespan of cells leads to an arrest of cell division by a process termed senescence, clearly distinct from differentiation, in which cells remain metabolically active indefinitely. The irreversible arrest of cell division that accompanies cellular senescence may be tumor suppressive, and escape of cells from senescence accompanies immortalization and oncogenesis. Indeed, a premature senescence is observed following oncogene introduction into primary human cells, and this antiproliferative response must be overcome if cells are to become transformed. Further, senescence may play a significant role in response to cancer therapy (Yang, 2003 and references therein).
Despite this potentially critical role for senescence in tumor formation, knowledge of the biochemical pathways responsible for the acquisition of cellular senescence is rudimentary. The retinoblastoma tumor suppressor protein, pRb, plays a fundamental role in cellular senescence, consistent with a critical role for pRb in the cellular machinery that controls passage from G1 into S phase of the cell cycle. Senescent cells accumulate active pRb, fail to inactivate pRb upon mitogenic stimulation, and consequently cannot enter S phase. Indeed, reintroduction of pRb into Rb-/- tumor cell lines induces senescence, even in cells that do not contain wild-type p53. Similarly, overexpression of p16INK4a can induce senescence in pRb-positive tumor cells. Loss of p16INK4a or pRb function appears to be required for immortalization of at least some human cell types, apparently as an obligate step in preventing senescence (Yang, 2003 and references therein).
RB-transfected SAOS-2 osteosarcoma cells serve as a model system of senescence. Reintroduction of pRb into SAOS-2 cells results in an immediate G1 arrest and subsequent expression of characteristic markers of senescence. The first indication of pRb-induced senescence to be recognized in this system was 'flat cell' formation, typified by an increased cell area and a flattened appearance. This phenotype appears identical to that observed during classical senescence, where a morphological alteration from spindle shape to an enlarged, flattened, and irregular shape is taken as an indicator of the senescent state (Yang, 2003 and references therein).
Despite the universality of morphological changes observed in a wide variety of senescent cells, little is known about the induction of this phenotype nor about its potential contribution to the establishment or maintenance of the irreversible growth arrest that accompanies senescence. Nevertheless, considerable work has clearly indicated a significant role for cell shape in cellular proliferation, largely as a consequence of communication between the cytoskeleton and its associated proteins. One such set of cytoskeletal-associated proteins that has recently emerged as important in proliferation control is the ezrin-radixin-moesin (ERM) family of cytoskeleton-membrane crosslinking proteins, including the related protein NF-2/merlin, an established tumor suppressor. These proteins play a role in the formation of microvilli, cell-cell junctions, and membrane ruffles, and also regulate substrate adhesion and motility. It has most recently become clear that the ERM proteins regulate and respond to proliferative signals, both in a positive and negative manner (Yang, 2003 and references therein).
ERM proteins possess two conserved domains that have been termed N- and C- ERM association domains, or ERMADs. The NH2-terminal domain associates with several transmembrane adhesion molecules, whereas the COOH-terminal domain contains an F-actin binding site. These binding sites are masked in cytoplasmic, inactive ERMs due to an intramolecular N/C-ERMAD interaction. Regulation of ERMs is thought to occur through conformational changes consequent to posttranslational modifications that inhibit association of the N-ERMAD with the C-ERMAD. This scheme has been supported by solution of the crystal structure of the relevant domains of moesin. This work reveals a globular conformation for the N-ERMAD domain and an extended conformation for the C-ERMAD, which mutually mask binding sites for other cellular proteins (Yang, 2003 and references therein).
Phosphorylation has been proposed to regulate ERM activation, since phosphorylation of ERM proteins correlates with their cytoskeletal association. Several observations have suggested that phosphorylation of serine/threonine residues is important for the activity of ERM proteins. Phosphorylation of T567 in ezrin has been found to be critical for conversion of ezrin to the active, open form competent for membrane localization and actin binding. Indeed, structural studies suggest that phosphorylation of T567 would sterically interfere with N-ERMAD/C-ERMAD interactions. Furthermore, induction of apoptosis induces a serine/threonine dephosphorylation of ezrin. This dephosphorylation is essential for the translocation of ezrin from the plasma membrane to the cytoplasm. Thus, regulation of ERM proteins through phosphorylation is likely critical to membrane-cytosekeleton signaling, and this in turn will have a pleiotropic impact on cell shape, motility, and proliferation (Yang, 2003 and references therein).
Ezrin regulation has been linked to pRb function in the senescent phenotype. Ezrin expression increases upon pRb-induced senescence, and more significantly, ezrin becomes membrane associated concomitant with acquisition of the senescent phenotype. This membrane association appears to be the consequence of direct phosphorylation of T235 of ezrin by CDK5, which is activated in response to pRb expression. Phosphorylation of T235 prevents the intermolecular N/C ERMAD association in a manner analogous to and cooperative with phosphorylation of T567, likely allowing ezrin to participate in cytoskeleton-related signaling events germane to senescence (Yang, 2003).
Calpain-mediated Cdk5 activation is critical for mitochondrial toxin-induced dopaminergic death. This study reports a target that mediates this loss. Prx2, an antioxidant enzyme, binds Cdk5/p35. Prx2 is phosphorylated at T89 in neurons treated with MPP+ and/or MPTP in animals in a calpain/Cdk5/p35-dependent manner. This phosphorylation reduces Prx2 peroxidase activity. Consistent with this, p35-/- neurons show reduced oxidative stress upon MPP+ treatment. Expression of Prx2 and Prx2T89A, but not the phosphorylation mimic Prx2T89E, protects cultured and adult neurons following mitochondrial insult. Finally, downregulation of Prx2 increases oxidative stress and sensitivity to MPP+. A mechanistic model is proposed by which mitochondrial toxin leads to calpain-mediated Cdk5 activation, reduced Prx2 activity, and decreased capacity to eliminate ROS. Importantly, increased Prx2 phosphorylation also occurs in nigral neurons from postmortem tissue from Parkinson's disease patients when compared to control, suggesting the relevance of this pathway in the human condition (Qu, 2007).
Search PubMed for articles about Drosophila cdk5
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date revised: 10 June 2014
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