A decade of CDK5

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Since it was identified a decade ago, cyclin-dependent kinase 5 (CDK5) has emerged as a crucial regulator of neuronal migration in the developing central nervous system. CDK5 phosphorylates a diverse list of substrates, implicating it in the regulation of a range of cellular processes ? from adhesion and motility, to synaptic plasticity and drug addiction. Recent evidence indicates that deregulation of this kinase is involved in the pathology of neurodegenerative diseases.

Key Points

  • Cyclin-dependent kinase 5 (CDK5) is a member of the cyclin-dependent kinase (CDK) family. Monomeric CDK5 displays no enzymatic activity, and requires association with a regulatory partner for activation. Two activators of CDK5 ? called p35 and p39 ? have been identified.

  • Association with its activators, p35 or p39, is necessary and sufficient for maximal activation of CDK5. CDK5 activity is dictated by the temporal and spatial expression and intracellular localization of p35 and p39. Transcriptional and post-translational events also regulate CDK5.

  • The best demonstrated role for CDK5 is in regulating the cytoarchitecture of the central nervous system. To date, about two dozen proteins with diverse functions have been identified as CDK5 substrates, and the kinase has been implicated in the regulation of actin dynamics, microtubule stability and transport, cadherin-mediated adhesion, axon guidance, secretion, membrane transport and dopamine signalling. Several groups have recently demonstrated active C5 in non-neuronal tissues, and proposed a role for CDK5 in myogenesis, haematopoietic cell differentiation, spermatogenesis, insulin secretion, and lens differentiation.

  • Treatment of neurons with neurotoxic insults causes calpain-mediated cleavage of p35 to p25 (a 208-residue carboxy-terminal fragment of p35). Although p25 can bind and activate CDK5, it lacks a myristoylation signal, and is more stable than p35. The generation of p25 therefore causes prolonged activation and mislocalization of CDK5, and hyperphosphorylation of substrates like Tau. Introduction of p25 into neurons produces drastic effects, including neurite retraction, microtubule collapse and apoptosis.

  • In the human brain, elevated levels of p25 correlate with Alzheimer's disease. Increased p25 levels and Cdk5-associated kinase activity are also seen in the spinal cord of transgenic mice expressing a mutant superoxide dismutase that was identified in patients with familial amyotrophic lateral sclerosis (ALS). Production of p25 may therefore be a common neurotoxic factor in the pathology of several neurodegenerative diseases.


Cyclin-dependent kinase 5 (CDK5) was discovered in the early 1990s, and, since then, great progress has been made in identifying its function. The best known role for CDK5 is in regulating the CYTOARCHITECTURE of the central nervous system (CNS), but there is also evidence that links CDK5 activity to regulation of the cytoskeleton, axon guidance, membrane transport, synaptic function, dopamine signalling and drug addiction.

This article discusses the functions of CDK5 and reviews its known protein substrates. It is becoming clear that the kinase activity of CDK5 in the cell is tightly regulated at the levels of protein expression, post-translational modification and proteolytic cleavage of both the kinase and its activators. Several studies have indicated that deregulation of CDK5 might contribute to the pathology of neurodegenerative diseases, such as Alzheimer's disease and amyotrophic lateral sclerosis (ALS). We describe these events here and link them to the physiological and pathogenic functions of CDK5. Finally, we discuss the mechanism for CDK5 deregulation and its role in various neurodegenerative diseases.

CDK5 is a unique member of the CDK family

CDK5, also known as neuronal CDC2-like kinase (NCLK), is a member of the small serine/threonine cyclin-dependent kinase (CDK) family (Box 1). CDK5 was initially identified by biochemical purification from bovine brain and by virtue of its close sequence homology to human CDC2 (Refs 1,2). Despite having 60% sequence identity with CDC2 and CDK2, a role for CDK5 in cell-cycle regulation has not yet been identified. CDK5 is expressed in all tissues (Box 2), although its highest expression and associated kinase activity are detected in the nervous system3,4,5.

Like other CDKs, monomeric CDK5 shows no enzymatic activity and requires association with a regulatory partner for activation. Two activators of CDK5, p35 and p39, have been identified. p35 (NCK5a, neuronal CDK5 activator), was identified as a CDK5 binding partner in brain extract, and association of p35 with CDK5 is sufficient to activate the kinase6,7,8. p39 (NCK5ai, neuronal CDK5 activator isoform) was identified by its sequence homology to p35, with which it shares 57% amino-acid identity9. Like p35, p39 interacts with and activates CDK5 (Refs 9,10). Evolutionarily, CDK5 and p35/p39 seem to be conserved across eukaryotic species, and ORTHOLOGUES of CDK5 and its activators have been identified in other vertebrates, Xenopus laevis11,12,13, Drosophila melanogaster14,15,16, Caenorhabditis elegans and Saccharomyces cerevisiae17,18,19 (Box 3).

CDK5 is a proline-directed kinase that phosphorylates serines and threonines immediately upstream of a proline residue. In addition to an absolute requirement for proline in the +1 position, CDK5 shows a marked preference for a basic residue in the +3 position and phosphorylates the consensus sequence (S/T)PX(K/H/R), where S or T are the phosphorylatable serine or threonine, X is any amino acid, and P is the proline residue in the +1 position20,21. CDK1 and CDK2 have an identical substrate specificity to that described above for CDK5 (Ref. 22).

To date, about two dozen proteins with diverse functions have been identified as substrates of CDK5 (Table 1). Some of these proteins are also substrates of CDK1 or CDK2. Despite the overlapping protein substrates that are phosphorylated by these kinases, ectopic expression of p35?CDK5 in mammalian cells or yeast does not promote cell-cycle progression2,23, indicating that CDK5 has a distinct function from the mitotic CDKs.

Table 1 Substrates of CDK5

Regulation of CDK5

The main mode of regulation for all CDK family members is by association with the cyclin activator. Transcriptional and post-translational events also regulate the mitotically active CDKs. Recent evidence indicates that similar regulatory strategies also extend to CDK5 (Fig. 1), but with novel modifications that have probably evolved to regulate the functions of CDK5 in post-mitotic cells.

Figure 1: Regulation of CDK5.

CDK5 is recruited to the membrane through its interaction with the activator p35, which is myristoylated. p35 is a short-lived protein, and its phosphorylation by CDK5 targets it for ubiquitin-mediated proteolysis. Post-translational modifications, such as phosphorylation by c-Abl (through an Abl-binding adaptor protein called Cables), can regulate CDK5 kinase activity. Engagement of the integrin receptors by laminin can stimulate transcription of p35 and p39. Treatment of neurons with nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) induces the extracellular signal-regulated kinase (ERK)-mediated expression of p35, which requires the transcription factor EGR1. The transcription factor δFOSB promotes CDK5 expression in response to chronic cocaine and electroconvulsive seizure (ECS) treatments, implicating the dopaminergic signalling pathway. (Green and red arrows represent stimulatory and inhibitory regulatory mechanisms, respectively.)

Association with the regulatory subunit. CDK5 binds to the two non-cyclin proteins p35 and p39, for activation of its kinase activity. Despite the fact that p35 and p39 have little sequence similarity to the cyclins ? the classical activators of CDKs ? computer modelling and mutagenesis studies have predicted that p35 might adopt a cyclin-like tertiary structure24,25. Recent crystallization of a complex between CDK5 and p25 (a 208-residue carboxy-terminal proteolytic product of p35) has confirmed these predictions, showing that p25 folds into a conformation very similar to that of a cyclin-box fold26 (Box 1).

In addition to interaction with cyclins, maximal activation of CDKs requires phosphorylation of its activation loop (T-loop) at residue Thr160, with the unphosphorylated complex being 200-fold less active27,28. The crystal structure of p25?CDK5 showed that the interaction with the regulatory subunit is sufficient to stretch the activation loop of unphosphorylated CDK5 into a fully extended, active conformation, indistinguishable from phosphorylated CDK2?cyclin-A complexes26. This confirmed earlier biochemical studies that showed that phosphorylation of the equivalent Ser159 in the activation loop of CDK5 is not required for its maximal activation29.

Spatial and temporal expression of p35 and p39. High levels of CDK5 kinase activity are detected primarily in the nervous system, despite widespread expression of the protein4. This observation is explained by the expression pattern of p35 and p39, which is highest in post-mitotic neurons of the nervous system30. The spatial and temporal expression of p35 and p39 in the brain seems to be complementary30,31,32,33,34, and cell-fractionation studies indicate that they localize to overlapping but distinct subcellular compartments in growth cones, synapses, and detergent-insoluble cytoskeleton and membrane fractions10,35,36,37. It is not clear, however, whether p35 and p39 can confer discrete substrate specificity to CDK5. Interestingly, p39 can compensate for some (but not all) functions of p35 (Ref. 38); however, the lack of outward phenotype of the p39-mutant mice argues that p35 can mask the absence of p39.

Membrane targeting by a myristoylation signal. Immunocytochemistry shows that the p35 signal is intense at the cell periphery, including the LAMELLIPODIA and FILOPODIA structures39,40, and that p35 and p39 are enriched in membrane fractions10,36,37. There is an amino-terminal myristoylation signal motif in p35 and p39 that targets these proteins to cell membranes, and a p35 mutant that cannot be myristoylated is absent from the cell periphery41. CDK5, when overexpressed, does not show a specific distribution pattern, but becomes localized to the cell periphery when co-expressed with p35. The subcellular distribution of CDK5 is therefore dictated by p35 and p39. Furthermore, the physiological substrates of CDK5 are likely to be transmembrane or membrane-associated proteins.

Degradation of p35. PULSE?CHASE EXPERIMENTS in primary neuronal cultures and in transfected cells show that p35 is an unstable protein with a half-life of 20?30 minutes42. p35 is multi-ubiquitylated and degraded through the UBIQUITIN?PROTEASOME PATHWAY. Inhibition of CDK5 kinase activity increases the stability of p35 several-fold. This is likely to be due to the decrease in phosphorylation of p35 by CDK5, as p35 mutants that lack CDK5 phosphorylation sites have a longer half-life42. These observations hint at the possibility of a negative-feedback regulation of CDK5, and show that the kinase activity of CDK5 is stringently regulated in neurons.

Transcriptional regulation of p35 and CDK5. The unstable nature of the p35 protein underscores the importance of regulating p35 expression levels. Treatment of cultured cerebellar macroneurons and the SH-SY5Y neuroblastoma cell line with the extracellular-matrix glycoprotein, laminin, results in an increase in p35 protein and messenger RNA levels, as well as CDK5-associated kinase activity43,44,45. The engagement of laminin with integrin receptors can therefore induce p35 transcription. The signalling pathway downstream from integrins that is responsible for p35 induction remains a mystery.

Recently, it was shown that nerve growth factor (NGF) induces p35 expression in PC12 cells46. The extracellular-signal-regulated kinase (ERK) cascade seems to be required for NGF-induced p35 expression, as pharmacological inhibition of the ERK pathway blocks this effect. Furthermore, constitutive activation of the ERK pathway is sufficient for induction of p35. Early growth response 1 (EGR1), a member of a family of zinc-finger transcription factors induced by NGF, can bind to the promoter region of p35. So, NGF-induced upregulation of p35 might be mediated by EGR1. In addition to NGF, brain-derived neurotrophic factor (BDNF) can also induce CDK5 kinase activity in cultured primary neurons47.

Drugs of abuse, such as cocaine, achieve some of their addictive actions by modifying signalling downstream of the dopamine receptors48. Chronic cocaine administration to rats increases expression of p35 and CDK5 (Ref. 49). A sustained increase in levels of the transcription factor δFOSB is seen after chronic cocaine treatment, and CDK5 mRNA and protein are elevated in the striatum and hippocampus of transgenic mice that express δFosB49,50. Administration of repeated electroconvulsive seizures (ECS), a treatment that induces δFOSB expression, also increases CDK5 expression50. In addition, overexpression of δFOSB in cells induces expression from the CDK5 promoter50, indicating that CDK5 might be one of the targets by which δFOSB mediates some of its long-lasting physiological effects in the brain.

Phosphorylation of CDK5. The mitotic CDKs are regulated by three distinct phosphorylation events (Fig. 2). Phosphorylation of Thr14 and Tyr15 by the dual-specificity kinases Wee1 and Myt1 inhibits CDK activity, whereas phosphorylation of Thr160 of CDK2 (or Thr161 of CDK1) by the CDK-activated kinase (CAK) is necessary for maximal activation51. Although Thr14 and Tyr15 are conserved in CDK5, CDK5 is not phosphorylated by Wee1 in vitro29. Tyr15 on CDK5 can be phosphorylated by c-Abelson (c-Abl), and this phosphorylation is enhanced by association of CDK5 with an Abl-binding adaptor protein called Cables52. Unexpectedly, phosphorylation of CDK5 on Tyr15 is stimulatory, and increases CDK5 kinase activity52. An inhibitory protein kinase was purified from bovine thymus cytosol and shown to phosphorylate and inactivate CDK5, as well as CDC2 and CDK2 on Thr14 (Ref. 53). The existing structure of p25?CDK5 is, unfortunately, poorly resolved at Thr14 and Tyr15 (Ref. 26), and further structural analysis will be necessary to determine how phosphorylation on two adjacent residues of CDK5 can result in an opposite effect on its activity.

Figure 2: Regulation of CDK5 and CDK2 by phosphorylation.

Phosphorylation of Thr160 on CDK2 by CDK-activating kinase (CAK) results in a 200-fold activation of the kinase. CAK does not phosphorylate CDK5 on the equivalent Ser159. Structural analysis of the p25?CDK5 complex indicates that phosphorylation on Ser159 would be inhibitory. The dual-specificity kinases Wee1 and Myt1 phosphorylate CDK2 on Thr14 and Tyr15, resulting in an inhibition of kinase activity. Neither of these kinases phosphorylates CDK5. However, Tyr15 of CDK5 is phosphorylated by c-Abl, which stimulates its kinase activity. A kinase activity purified from bovine thymus can phosphorylate Thr14 on both CDK5 and CDK2, resulting in inhibition of activity. Green and red arrows represent stimulatory and inhibitory phosphorylation events, respectively.

While phosphorylation of Thr160 in the T-loop of CDKs is required for maximal activation, phosphorylation of the equivalent Ser159 on CDK5 is dispensable29,54. The retention of serine in all CDK5 orthologues ? from yeast to human ? indicates that the presence of a phosphate acceptor at this position might be relevant for CDK5 regulation. The structure of p25?CDK5 shows that Ser159 in the T-loop of CDK5 is in very close proximity to p25, and the addition of a phosphate group on Ser159 would generate steric clashes and adversely affect association with the activator. Indeed, mutating Ser159 to glutamic acid, or even threonine, abolishes binding to CDK5, whereas substitution to a smaller residue, such as alanine, has no effect26. This indicates that phosphorylation of Ser159 of CDK5, if occurring, would probably negatively regulate CDK5 kinase activity.

Although CDK5 shares many features with the regulatory network that impinges on its family members, important differences, emphasized above, are beginning to emerge. A principal mechanism for CDK regulation involves a diverse family of CDK-inhibitory subunits (CKIs) that bind and inactivate CDK?cyclin complexes55. The CKIs p21 and p27 effectively inhibit CDK2, but have minimal effect on p35?CDK5 (Ref. 56), supporting the idea that interaction with a non-cyclin activator might allow CDK5 to escape inhibition from the CKIs. Protein fractionation and gel-filtration chromatography of bovine brain yields an inactive p35?CDK5 complex that is part of a macromolecular structure with a molecular mass of approximately 670 kDa (Ref. 57). Active p35?CDK5 can be obtained by subjecting the complex to gel-filtration chromatography in the presence of 10% ethylene glycol. This indicates that the macromolecular complex could contain a CDK5 inhibitory factor. Identification of CDK5-specific inhibitory proteins remains an intriguing possibility.

CDK5 function in CNS development

Gene-targeting experiments demonstrate an essential role of CDK5 in the cytoarchitecture of the CNS. Mice that are deficient in Cdk5 die just before or after birth, and show widespread disruptions in neuronal layering of many brain structures ? including the cerebral cortex, hippocampus, cerebellum and olfactory bulb? indicating a possible impairment in neuronal migration58,59,60. In the cerebral cortex, the laminar organization of neurons is inverted with a superficial ectopic subplate (Fig. 3). The lethality of the Cdk5-deficient mice is likely to stem from defects in the nervous system, as it can be completely rescued by expressing the Cdk5 transgene under the p35 promoter61. Mice that lack p35 show an inverted layering of cortical neurons similar to that seen in the Cdk5−/− mice, but have only mild disruptions in the hippocampus and have a fairly normal cerebellum62,63. In contrast to the Cdk5?null mutants, the p35−/− mice are viable and fertile, although they have increased susceptibility to seizures.

Figure 3: Corticogenesis in wild-type and Cdk5−/− mice.

The cerebral cortex is characterized by six distinct layers of neurons. This laminated structure is produced by the birth of post-mitotic neurons in successive waves throughout the course of corticogenesis following a highly orchestrated migration programme. a | During early corticogenesis, dividing cells occupy the ventricular zone (VZ), and a thin mantle layer of cortical primordium forms the preplate. b | The first cohort of neurons exit the cell-division cycle, move out of the ventricular zone and take up residence in the middle of the preplate. This splits the preplate into three distinct compartments: the cell-sparse marginal zone (MZ) on the surface; the cortical plate (CP) that accommodates the newly arrived neurons; and the subplate (SP) underneath. In the Cdk5−/− mice, the preplate seems to split normally. c?e | Wild type. Successive waves of neurons migrate along radial glial fibres, transit the subplate and navigate through the cortical plate to take up more superficial positions. So, the cortex is essentially assembled in an 'inside?out' gradient, such that early-born neurons reside in deeper layers and later-born neurons in more superficial layers. c?e | Cdk5−/−. Later-born neurons cannot migrate past their predecessors to occupy superficial positions, and stack up underneath the pre-existing neurons. This results in an inversion of the cortex. The intermediate zone (IZ), which is cell sparse in the wild-type mice, has a higher density of neurons in the Cdk5-deficient mice.

p35-mutant mice also have defects in the FASCICULATION of several prominent axon tracts64. CALLOSAL AXONS defasciculate prematurely from the CORPUS CALLOSUM after crossing the midline, and the thalamocortical AFFERENTS also appear defasciculated. In Drosophila, an increase or decrease of Cdk5 activity results in errors in axon pathfinding and target recognition of abdominal motor nerves16. These observations support a role for Cdk5 in axon guidance and targeting.

Finally, CHROMATOLYTIC CHANGES, such as a ballooned cell soma with eccentric nucleus, are observed in motor neurons of Cdk5-deficient mice. This is accompanied by accumulation of neurofilament immunoreactivity58. These observations hint at transport defects in motor neurons that lack CDK5, and also indicate that CDK5 function is crucial for the survival of motor neurons.

The apparent discrepancy in the phenotypes of the p35−/− and Cdk5−/− mice can be explained by the compensatory role of p39. While p39-deficient mice do not show any noticeable defects, the phenotype of the p35/p39 double-mutant mice is indistinguishable from that of Cdk5?null mice38. These models show that, together, p35 and p39 are necessary and sufficient for Cdk5 function.

CDK5 and pro- and anti-migratory processes

The crucial role of CDK5 in corticogenesis, described above, leads to the hypothesis that CDK5 promotes migration by acting positively on pro-migratory signals, and possibly by antagonizing anti-migratory signals. Reduction of Cdk5 kinase activity by expression of dominant-negative Cdk5 mutants, or antisense oligonucleotides of Cdk5, p35 and p39 in cultured primary neurons, inhibits neurite outgrowth39,65. Suppression of CDK5 in cerebellar macroneurons results in reduced axonal elongation, further indicating that CDK5 has a stimulatory role in axonal extension43,44. Studies of migration in neurons and other cell types have shown that regulation of actin, microtubule and intermediate-filament cytoskeletal components, and modulation of cell adhesion, transport and intracellular signalling, all impinge on this process. Interestingly, CDK5 has been implicated in all of the above (Fig. 4).

Figure 4: Cellular processes regulated by Cdk5.

The pleiotropic nature of Cdk5 is evident in its involvement in several cellular processes. a | Neuronal migration. Defects in neuronal positioning in the Cdk5- and p35-deficient mice show that Cdk5 is crucial for neuronal migration. b | Actin dynamics. Phosphorylation of the Rac effector, Pak1, by Cdk5 can regulate actin dynamics, and Cdk5 activity promotes neurite outgrowth. c | Microtubule stability and transport. Several microtubule-associated proteins (MAPs) are substrates of Cdk5, and regulate microtubule polymerization. Inhibition of Cdk5 decreases anterograde transport, and Nudel and Neurofilament proteins are substrates of Cdk5. d | Cell adhesion. Cdk5 phosphorylates β-catenin, disrupts the N-cadherin?β-catenin complex and inhibits adhesion. e | Axon guidance. p35-mutant mice have defects in fasciculation of several axon tracts, and, in Drosophila, increase or decrease of Cdk5 activity results in errors in axon pathfinding. f | Synaptic structure and plasticity. Cdk5 phosphorylates several synaptic proteins, modulates dopaminergic signalling and development of the neuromuscular junction. g | Myogenesis. Cdk5 kinase acitivity induces expression of MyoD and Mrf4, master regulators of myogenesis. h | Membrane transport. Cdk5 phosphorylates the Munc18?syntaxin1 complex and amphyphysin, indicating a possible role in membrane fusion, neurosecretion and endocytosis.

Regulation of cytoskeletal dynamics. When overexpressed in fibroblasts, p35 and p39 induce pronounced actin reorganization and the appearance of lamellipodia and filopodia10,40. The Rho family of small GTPases is important in regulating actin dynamics, and members of this family are directly implicated in axon guidance, neurite outgrowth and cell motility66. In cultured neurons, p35 and CDK5 are conspicuous in the periphery of growth cones, colocalizing with F-actin, the small GTPase RAC and its effector PAK1 (Refs 39,40). CDK5 induces PAK1 hyperphosphorylation in a RAC-dependent manner, which results in downregulation of PAK1 kinase activity40. Because the Rho family of GTPases and the PAK kinases are implicated in actin polymerization, this modification of PAK1 is likely to regulate actin cytoskeleton dynamics in neurons, promoting neuronal migration and neurite outgrowth.

CDK5 is also associated with the microtubule cytoskeleton, and can be purified from bovine brain microtubules67,68. Several microtubule-associated proteins (MAPs), including MAP1B and Tau, are substrates of CDK5 (Refs 8,43,44,69,70). Phosphorylation of MAP1B by CDK5 in cerebellar macroneurons is implicated in neurite extension43,44. Phosphorylation of Tau ? a neuron-specific MAP ? by CDK5 reduces the binding of Tau to microtubules41, inhibits the ability of Tau to promote microtubule assembly71 and decreases MICROTUBULE-NUCLEATION activity72. It is plausible that modulation of microtubule stability by phosphorylation of several MAPs is part of the programme by which CDK5 exerts its effect on neuronal migration.

CDK5, along with other proline-directed kinases, such as CDC2, also phosphorylates the intermediate and heavy chain of neurofilament polymers (NFM and NFH, respectively) in vitro6,73,74,75. Neurofilaments have a well-established function in the control of axon calibre, and there is growing evidence that they can affect the dynamics of microtubules and actin filaments. It has been suggested that phosphorylation of the carboxy-terminal KSP-rich domain of neurofilament subunits can influence their integration into the cytoskeleton76, and phosphorylation of NFH by CDK5 reduces its association with microtubules77.

Cadherin-mediated adhesion. A feature of the Cdk5- and p35-deficient mice is the failure of later-born neurons to migrate past pre-existing neurons in the cortex. One possible mechanism that could account for this phenotype is an upregulation of cellular adhesion. In fact, loss of cadherin-mediated adhesion has been implicated in promoting migration in several cell types78,79,80. N-cadherin belongs to a family of transmembrane molecules that promote cell adhesion by their calcium-dependent homophilic interactions. The cytoplasmic tail of the classical cadherins associates with α-, β-, γ-catenin, α-actinin and the actin cytoskeleton to form the ADHERENS JUNCTION complexes81.

A yeast two-hybrid screen isolated β-catenin as a p35-binding protein82,83. The p35?Cdk5 complex associates with β-catenin and N-cadherin, and inhibition of CDK5 activity causes an increase in N-cadherin-mediated aggregation of neurons. Conversely, active CDK5 dissociates β-catenin from N-cadherin, which is accompanied by a loss of adhesion82. β-catenin is a substrate of CDK5 (Ref. 83), and phosphorylation by CDK5 might be the event that regulates its association with the cadherins. Therefore, the role of CDK5 in neuronal migration might, in part, be mediated by its regulation of the cadherin?catenin complexes.

Regulation of transport. CDK5 was recently linked to axonal transport by the identification of Nudel as a CDK5 substrate in vitro and in vivo36,84. Nudel is expressed at high levels in the brain, where it associates with cytoplasmic dynein ? the main ATP-dependent RETROGRADE MOTOR. Nudel was identified in a yeast two-hybrid screen as a LIS1-interacting protein36,84. The LIS1 protein binds to microtubules, and modulation of LIS1 levels has a marked influence on microtubule organization and dynein distribution85. Expression of a non-phosphorylatable mutant of Nudel in neurons results in the appearance of swellings along neuritic processes36, reminiscent of those observed when dynein function is disrupted in Drosophila neurons86. This raises an intriguing hypothesis that inhibition of Nudel phosphorylation by CDK5 could lead to suppression of dynein-based axonal transport along microtubules. Although definitive evidence for such a model has yet to be obtained, these results indicate that Nudel might represent the point of convergence for the CDK5-mediated and the LIS1-mediated neuronal migration pathways. A role for CDK5 in axonal transport is further supported by the observation that its inhibition causes a decrease in ANTEROGRADE TRANSPORT in giant axons of squid87.

Synaptic functions and membrane cycling

CDK5, p35 and p39 are all abundantly expressed in the adult brain. Moreover, they are present in subcellular fractions that are enriched for synaptic membranes, and they localize to pre- and postsynaptic compartments10,36. The increased sensitivity of p35-null mice to seizure also indicates that CDK5 might be involved in synaptic functions62. Indeed, several synaptic proteins have been shown to be CDK5 substrates (Table 1).

Presynaptic function. Three proteins at the presynaptic compartment that are involved in aspects of neurosecretion and endocytosis ? synapsin 1, MUNC18 and amphiphysin ? are substrates of CDK5 (Refs 88?91). Synapsin 1 is an abundant phosphoprotein of synaptic vesicles that interacts with many cytoskeletal proteins, including actin filaments, spectrin, tubulin and neurofilaments92. Despite much work, the function of synapsin 1 in exocytosis, and the consequences of its phosphorylation, remain unclear.

MUNC18 interacts with the target SNARE protein, syntaxin, and this inhibits the vesicle?SNARE interactions of the vesicle with syntaxin that are required for secretory vesicles to achieve competence for membrane fusion and secretion93. Phosphorylation of Munc18 by CDK5 on Thr574 results in disassembly of the Munc18?syntaxin 1 complex, indicating a role for CDK5 in modulating neurosecretion89,90. Amphiphysin 1 is a phosphoprotein in neurons that participates in synaptic vesicle endocytosis; it interacts with p35 and is phosphorylated by CDK5 (Refs 91,94). These observations indicate that CDK5 might have an evolutionarily conserved role in membrane transport and secretion.

Postsynaptic function. Recently, a requirement for CDK5 in the development of the neuromuscular junction was shown37. CDK5 and p35 are highly concentrated at the neuromuscular junction, where they co-localize with the acetylcholine receptor on the postsynaptic muscle membrane. Neuregulin upregulates CDK5 kinase in C2 cells, indicating that CDK5 is a component of neuregulin signalling. Interestingly, p35?CDK5 binds and phosphorylates ERBB, the neuregulin receptor. Inhibition of CDK5 kinase activity attenuates activation of ERBB and the downstream mitogen-activated protein kinase (MAPK) cascade, and blocks neuregulin-induced expression of the acetylcholine receptor37. These observations indicate that CDK5 is indispensable in neuregulin-mediated development of the neuromuscular junction. CDK5 could have an analogous role in the CNS, and conditional knockout experiments will be necessary to discern such a function.

Regulation of dopaminergic signalling. CDK5 phosphorylates DARPP32, a NEOSTRIATUM-specific protein that modulates dopamine signalling in dopaminoceptive neurons48. Phosphorylation of DARPP32 by protein kinase A (PKA) on Thr34 makes it a potent inhibitor of protein phosphatase-1 (PP-1), and potentiates dopamine-induced phosphorylation of PKA substrates. In contrast, phosphorylation of Thr75 by CDK5 renders it an inhibitor of PKA95. Inhibition of CDK5 activity results in an augmented dopamine response in striatal slices. Protein phosphatase inhibitor-1 (PP inhibitor-1) is also phosphorylated by both PKA and CDK5 with antagonistic consequences96. Phosphorylation by PKA converts it into an inhibitor of PP-1, whereas phosphorylation by CDK5 makes it a worse substrate for PKA. So, CDK5 and PKA seem to exert opposing effects on dopamine signalling through phosphorylation of DARPP32, PP inhibitor-1 and possibly other proteins in the pathway.

As mentioned earlier, drugs like cocaine achieve some of their addictive actions by modifying dopaminergic transmission and, surprisingly, Cdk5 and p35 expression is induced in rats that are administered cocaine chronically49. The increase in Cdk5 and p35 expression is accompanied by upregulated phosphorylation of Darpp32 at Thr75 and decreased phosphorylation of PKA-dependent target proteins. Furthermore, inhibition of Cdk5 in the rat model potentiates the locomotor effects of chronic cocaine administration in mice and rats49. These observations raise the intriguing possibility that CDK5 has a role in drug addiction by modulating dopamine signalling through phosphorylation of DARPP32.

Deregulation of CDK5 and neurodegeneration

Tau protein kinase II (TPKII) was purified on the basis of its Tau-phosphorylating activity from bovine brain microtubules67. Subsequently, TPKII was shown to be identical to the p25?CDK5 complex8,97. Abnormal phosphorylation of Tau has been implicated in the pathology of several neurodegenerative diseases, such as Alzheimer's disease, Down's syndrome, progressive supranuclear palsy and Parkinson's disease. A pathological hallmark of these degenerative disorders is the presence of neurofibrillary tangles, which are primarily composed of paired helical filaments of hyperphosphorylated Tau (PHF-Tau). TPKII?CDK5 was shown to be associated with neurofibrillary tangles in vivo98, and phosphorylates Tau on sites found in PHF-Tau69. Several groups have therefore postulated that CDK5 kinase activity might be elevated during neurodegeneration, and there is growing evidence that deregulation of CDK5 kinase activity is detrimental to cell survival.

The kinase activity of CDK5 is dictated by the temporal and spatial expression and intracellular localization of p35. The best example of CDK5 deregulation is through the proteolytic cleavage of p35 to generate p25. Accumulation of p25 has been linked to neurodegenerative diseases, including Alzheimer's disease and ALS (Fig. 5).

Figure 5: Cleavage of p35 to p25 is neurotoxic.

p35 targets CDK5 to the membrane, where it phosphorylates several substrates. p35 is short lived and turned over by ubiquitin-mediated proteolysis. Exposure of neurons to oxidative stress, amyloid-β (Aβ) peptides or excitotoxicity activates the calcium-dependent protease calpain, which converts p35 into p25. p25 lacks the myristoylation signal and is mislocalized to the cytoplasm, allowing CDK5 access to substrates away from the membrane. In addition, p25 has a longer half-life (T1/2) than p35, causing sustained activation of CDK5. So, conversion of p35 to p25 translates into elevated and mislocalized CDK5 activity, hyperphosphorylation of the Neurofilament and Tau proteins, cytoskeletal disruption and neuronal death.

p25 is induced by neurotoxicity. p25 is produced by neurotoxic insults, and is generated by H2O2, glutamate, maitotoxin and ionomycin treatments of cultured primary neurons99,100,101. In rats, neuronal injury that is caused by ischaemia after occlusion of cerebral arteries also generates p25. All treatments described above have been shown to disrupt calcium homeostasis in the cell, and adding calcium to fresh brain lysate is enough to promote the cleavage of p35 to p25. Generation of p25 following neurotoxic treatments can be blocked by specific inhibitors to the calcium-dependent cysteine protease, calpain. Furthermore, immunodepletion of calpain markedly reduced the conversion of p35 to p25 in calcium-treated brain lysates. These results indicate that calpain might be responsible for the proteolytic processing of p35 to p25 after disruption of calcium homeostasis.

p25 is neurotoxic. p25 contains all the elements necessary for CDK5 binding, and it efficiently activates CDK5 in vitro and in vivo29,54. However, several in vivo properties of p25 are distinct from those of p35 (Ref. 41). First, p25 has a substantially longer half-life than p35, which is very unstable. In addition, p25 lacks the amino-terminal myristoylation site and is concentrated in the cell body and nucleus, whereas p35 is present throughout the entire cell, including the tips of processes in neurons.

The generation of p25 is, therefore, likely to disrupt the normal regulation of CDK5, causing prolonged activation and mislocalization of CDK5. Indeed, introduction of p25 into cultured primary neurons produces drastic effects, including neurite retraction, microtubule collapse and apoptosis41. The mechanisms that underlie the neurotoxic effect of p25 remain to be determined. One hypothesis is that p25 could redirect CDK5 to alternative substrates to initiate an apoptotic programme.

In transfected cells and cultured neurons, p35?CDK5 poorly phosphorylates Tau, whereas Tau is potently phosphorylated by p25?CDK5 (Ref. 41). CDK5-phosphorylated Tau shows decreased binding to microtubules41,71. Transgenic expression of p25 in mice results in hyper-phosphorylation of Tau and NFH, as well as cytoskeletal disruptions102. Interestingly, there is no evidence for hyperphosphorylation of Tau in triple transgenic mice that express p35, Cdk5 and tau, despite elevated Cdk5 kinase activity103. Finally, Tau phosphorylation is not altered in the forebrain of p35?p39 double-mutant embryos38. Together, these observations argue that Tau is not a physiological substrate of p35?CDK5; under pathological conditions, p25 is produced, allowing CDK5 to phosphorylate Tau.

This scenario might also apply to NFH, which is phosphorylated by p25?CDK5 and p35?CDK5. Like Tau, no decrease in phosphorylation of NFH was detectable in either the p35?p39 compound mutant or Cdk5-null mice38,58, although NFH is hyperphosphorylated in p25 transgenic mice102. p25?CDK5 might also phosphorylate other proteins that are not substrates of p35?CDK5, and this could then contribute to cytoskeletal disruption and cell death.

p25 and Alzheimer's disease. In the human brain, elevated levels of p25 strongly correlate with Alzheimer's disease41. Furthermore, both CDK5 and p25 are present in neurons that contain NFTs. Particularly strong CDK5 immunoreactivity is associated with pre-tangle neurons and neurons that bear early-stage NFTs98, supporting the hypothesis that p25?CDK5 might contribute to NFTs by hyperphosphorylation of Tau.

Another pathological hallmark of Alzheimer's disease is the amyloid plaque, which represents the extracellular deposition of the fibrillogenic amyloid-β (Aβ) peptides104. Aβ peptides have been shown to cause death in cell-culture models. Remarkably, treating dissociated primary cortical neurons with Aβ peptides results in the activation of calpain105, generation of p25 (Ref. 100) and hyperphosphorylation of Tau106. Furthermore, Aβ-associated neurotoxicity can be diminished by inhibition of CDK5 (Refs 100,107). These observations indicate that activation of calpain and deregulation of CDK5 might mediate, at least in part, the observed neurotoxicity of Aβ peptides. This is consistent with the current hypothesis that the action of Aβ peptides is upstream of Tau hyperphosphorylation and NFTs in the pathogenesis of Alzheimer's disease104, and raises the exciting possibility that production of p25 involves a molecular link between the plaque and tangle pathologies.

p25 and ALS. Elevation of p25 levels and Cdk5-associated kinase activity was also demonstrated in the spinal cord of transgenic mice expressing mutant superoxide dismutase 1 (Sod1G37R) (Ref. 108). This mutation was identified in patients with familial ALS, which is characterized by severe motor-neuron degeneration109,110. The mechanism by which Sod1G37R generates p25 is unclear. However, degeneration of neurons in ALS has been linked to a disruption in calcium homeostasis, oxidative damage and excitotoxicity from impaired clearing of glutamate. An increase in calcium levels could induce calpain-mediated production of p25.

Cdk5 and p25 colocalize with PERIKARYAL neurofilament inclusions in the motor neurons of Sod1G37R mice. Although accumulation of hyperphosphorylated neurofilaments in the perikarya of motor neurons is the hallmark of ALS, previous work has indicated a surprising correlation of accumulation of neurofilament inclusions and extension of lifespan in Sod1-mutant mice110. Accumulation of hyperphosphorylated neurofilaments has an inverse relationship with Tau hyperphosphorylation in the Sod1-mutant mice. These observations raise the possibility that aberrant activation of Cdk5 by virtue of p25 accumulation might be involved in the pathogenesis of ALS, and that perikaryal neurofilament inclusions serve as a 'phosphorylation sink' that diverts p25?Cdk5 from phosphorylating Tau ? and perhaps other substrates contributing to apoptosis ? which, in turn, prolongs the lifespan of the mutant Sod1 mice. Although p25-mediated elevation of CDK5 activity might contribute to motor-neuron degeneration in ALS, there is evidence that loss of p35?CDK5 function is also detrimental to motor-neuron survival38,58. Rigorous control of CDK5 kinase activity is therefore crucial for motor-neuron function and survival.


CDK5 was identified as a member of the family of cyclin-dependent kinases that are primarily implicated in regulating the cell cycle. A decade later, it is clear that CDK5 is neither cyclin dependent, nor is it involved in regulating the cell cycle. Instead, it has a pivotal role in modulating the complex migration programme of post-mitotic neurons. Several substrates of CDK5 have been identified, but given the heterogeneity of CDK5 function, the list is probably incomplete. Identification of CDK5 substrates will clarify the prevailing view of its role in the nervous system and uncover new areas of regulation for this versatile kinase. Until recently, very little was known of regulatory mechanisms upstream of CDK5, and today we have only a rudimentary understanding of how this kinase is modulated in neurons. But one thing is clear ? deregulation of CDK5 has dire consequences and might contribute to neurodegeneration. Further study of CDK5 function and its regulation will provide exciting insights into the molecular basis of neurodevelopment and the progression of disease.


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Correspondence to Li-Huei Tsai.

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Cellular organization of a tissue.


Genes in different species that are homologous because they are derived from a common ancestral gene.


Differentiation and development of muscle.


Thin, sheet-like extensions of the cytoplasm, temporarily put forward by some eukaryotic cells (such as fibroblasts) when moving.


Fine, thread-like extensions of the cytoplasm of eukaryotic cells.


A radioactive small molecule is added to a cell for a brief period (the pulse), during which it is incorporated into macromolecules. The fate of the newly synthesized radioactive macromolecule is examined when the radioactive small molecule is removed and replaced by an excess of the same molecule, but unlabelled (the chase).


A small protein, ubiquitin, becomes covalently linked to a cellular protein, which is then targeted for degradation by a multiprotein complex of proteolytic enzymes (called the proteasome).


Bundling of nerve fibres.


An axon of the corpus callosum.


A wide tract of fibres that connects the two cerebral hemispheres, and is involved in the transfer of information from one hemisphere to the other.


A sensory nerve that brings impulses towards the central nervous system.


Following injury of axons, several changes occur in the cell body of a neuron. It swells and could even double in size. The nucleus swells and moves to an eccentric position, usually opposite the axon hillock. The rough endoplasmic reticulum breaks apart and moves to the periphery of the swollen cell body.


Microtubules are assembled by polymerization of α- and β- tubulin dimers. The addition of nuclei in the form of microtubule fragments to a solution of α- and β-tubulin dimers greatly accelerates the polymerization rate and is called microtubule nucleation.


Cell?cell adhesive junctions that are linked to cytoskeletal filaments of the microfilament type.


A motor protein that moves cargo in axons of neurons towards the cell body of neurons.


Transport of cargo in axons of neurons away from the cell body.


Soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) proteins are a family of membrane-tethered coiled-coil proteins that regulate fusion reactions and target specificity in the vacuolar system.


The input nuclei for the basal ganglia, which participates in the control of movement and receives input mainly from the cerebral cortex.


The cell body containing the nucleus in nerve cells.

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