Mouse cyclin-dependent kinase (Cdk) 5 is a functional homologue of a yeast Cdk, pho85 kinase

Mouse cyclin-dependent kinase (Cdk) 5 and yeast Pho85 kinase share similarities in structure as well as in the regulation of their activity. We found that mouse Cdk5 kinase produced in pho85Delta mutant cells could suppress some of pho85Delta mutant phenotypes including failure to grow on nonfermentable carbon sources, morphological defects, and growth defect caused by Pho4 or Clb2 overproduction. We also demonstrated that Cdk5 coimmunoprecipitated with Pho85-cyclins including Pcl1, Pcl2, Pcl6, Pcl9, and Pho80, and that the immunocomplex could phosphorylate Pho4, a native substrate of Pho85 kinase. Thus mouse Cdk5 is a functional homologue of yeast Pho85 kinase.     

Cdk5--p39 is a labile complex with the similar substrate specificity to Cdk5--p35

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

Cdk5: a multifaceted kinase in neurodegenerative diseases

Since the identification of cyclin-dependent kinase-5 (Cdk5) as a tau kinase and member of the Cdk family almost 20 years ago, deregulation of Cdk5 activity has been linked to an array of neurodegenerative diseases. As knowledge on the etiopathological mechanisms of these diseases evolved through the years, Cdk5 has also been implicated in additional cellular events that are affected under these pathological conditions. From the role of Cdk5 in the regulation of synaptic functions to its involvement in autophagy deregulation, significant insights have been obtained regarding the role of Cdk5 as a key regulator of neurodegeneration. Here, we summarize recent findings on the involvement of Cdk5 in the pathophysiological mechanisms underlying various neurodegenerative diseases.     

Cdk5 on the brain.

Mammalian brains are highly compartmentalized into groups of functionally specialized neurons. Cell migration and neurite outgrowth must be tightly orchestrated to achieve this level of organization. A small serine/threonine kinase that shows homology to cyclin-dependent kinases (Cdks) has emerged as an important regulator of neuronal migration. Cdk5, unlike other Cdks, is not regulated by cyclins, and its activity is primarily detected in postmitotic neurons in developing and adult nervous systems. This review describes work indicating that Cdk5 links extracellular signaling pathways and cytoskeletal/membrane systems to direct neuronal migration, axon growth, and possibly neurosecretion. Despite its importance, unchecked Cdk5 activity is toxic to neurons, and may underlie some of the pathologies associated with neurodegenerative disorders such as Alzheimer's disease and amyotrophic lateral sclerosis.     

A model of the complex between cyclin-dependent kinase 5 and the activation domain of neuronal Cdk5 activator.

Tau protein kinase II (TPKII) is a heterodimer comprising a catalytic cyclin-dependent kinase subunit (Cdk5) and a regulatory protein called neuronal Cdk5 activator (Nck5a). TPKII is somewhat reminiscent, therefore, of the Cdk2-cyclin complex important in cell cycle regulation. In fact, although the amino acid sequence of Nck5a has little similarity to those of cyclins, recent experimental results obtained by site-directed mutagenesis studies have indicated that its activation domain, Nck5a*, may adopt a conformation of the cyclin-fold structure. Based on this structural inference, a 3-dimensional model of the Cdk5-Nck5a*-ATP complex was derived from the X-ray structure of Cdk2-cyclinA-ATP complex. The computed structure for TPKII is fully compatible with experimental data derived from studies of the Cdk5-Nck5a system, and also predicts which amino acid residues might be involved in formation of the Cdk5-Nck5a* interface and ATP binding pocket in TPKII. The computational structure also shows the interactive region of Nck5a* and the T-loop of Cdk5, a critical region in TPKII which functions as a gate-control-lever of the catalytic cleft. Furthermore, a physical mechanism is put forth to explain why the activation of TPKII is not dependent upon phosphorylation of the Cdk5 subunit, a puzzle long-standing in this area. These findings provide a model with which to consider design of compounds which might serve as inhibitors of TPKII.     

A Jekyll and Hyde kinase: roles for Cdk5 in brain development and disease

Cyclin-dependent kinase 5 (Cdk5) is a multi-faced kinase implicated in both development and disease of the mammalian central nervous system. These different faces of Cdk5 are preferentially regulated by the activation of Cdk5 by its different binding partners. The precise molecular and cellular mechanisms governing the role of Cdk5 in brain development and disease are unclear. Emerging evidence is now unraveling how Cdk5 normally orchestrates new signaling pathways that dictate the proper maturation and maintenance of the central nervous system. Under pathological conditions, however, Cdk5 activity goes awry and the malevolent face of Cdk5 surfaces. Recently developed animal models that display this deregulated Cdk5 activity reveal the intimate involvement of Cdk5 in tau pathology and neuronal cell death, and underscore the importance of phosphorylation in the progression of neurodegenerative diseases.     

Cdk5/p25(nck5a) interaction with synaptic proteins in bovine brain

Cyclin-dependent kinase 5 (Cdk5) exists in large multimeric complexes, but its function and binding partners in these complexes are unclear. We explored these issues by chromatographic and immunochemical analyses of Cdk5 and p25(nck5a) (a neuronal Cdk5 activator) and their associated proteins from bovine brain. Mono-S column enzyme eluates were divided into three fractions and analyzed by gel filtration. The majority of p25(nck5a) from Mono-S fractions I, II, and III eluted from the gel filtration column at approximately 60, 200, and 400 kDa, respectively, and Cdk5 was abundant in fractions >400 kDa. We characterized these macromolecular structures by immunoprecipitating p25(nck5a), followed by a second immunoprecipitation of remaining unbound proteins using a Cdk5 antibody. The p25(nck5a) immunoprecipitates showed association with Cdk5. Amphiphysin was detected in the 400-kDa complex and synapsin I in the >400 kDa structure. The Cdk5 immunoprecipitates, however, revealed abundant retained Cdk5 but no remaining p25(nck5a), indicating that Cdk5 in macromolecular structures is mostly unassociated with p25(nck5a). Thus, we demonstrate: an amphiphysin-associated 400-kDa Cdk5/p25(nck5a) complex, a synapsin I-associated >400-kDa Cdk5/p25(nck5a) complex, and nck5a-free Cdk5 complexes (200 to >400 kDa). Amphiphysin acts as a Cdk5/p25(nck5a) substrate in the 400-kDa complex and we speculate that Cdk5/p25(nck5a) participates in amphiphysin-mediated endocytosis.     

Protein kinase CK2 is an inhibitor of the neuronal Cdk5 kinase

The complex of Cdk5 and its neuronal activator p35 is a proline-directed Ser/Thr kinase that plays an important role in various neuronal functions. Deregulation of the Cdk5 enzymatic activity was found to associate with a number of neurodegenerative diseases. To search for regulatory factors of Cdk5-p35 in the brain, we developed biochemical affinity isolation using a recombinant protein comprising the N-terminal 149 amino acids of p35. The catalytic alpha-subunit of protein kinase CK2 (formerly known as casein kinase 2) was identified by mass spectrometry from the isolation. The association of CK2 with p35 and Cdk5 was demonstrated, and the CK2-binding sites were delineated in p35. Furthermore, CK2 displayed strong inhibition toward the Cdk5 activation by p35. The Cdk5 inhibition is dissociated from the kinase function of CK2 because the kinase-dead mutant of CK2 displayed the similar Cdk5 inhibitory activity as the wild-type enzyme. Further characterization showed that CK2 blocks the complex formation of Cdk5 and p35. Together, these findings suggest that CK2 acts as an inhibitor of Cdk5 in the brain.     

Glutathione-S-transferase P1 is a critical regulator of Cdk5 kinase activity

Cyclin dependent kinase-5 (Cdk5) activity is deregulated in Alzheimer's disease (AD) and contributes to all three hallmarks: neurotoxic β-amyloid formation, neurofibrillary tangles, and neuronal death. However, the mechanism leading to Cdk5 deregulation remains controversial. Cdk5 deregulation in AD is usually linked to the formation of p25, a proteolysis product of Cdk5 activator p35, which leads to Cdk5 mislocalization and hyperactivation. A few studies have indeed shown increased p25 levels in AD brains; however, others have refuted this observation. These contradictory findings suggest that additional factors contribute to Cdk5 deregulation. This study identified glutathione-S-transferase pi 1 (GSTP1) as a novel Cdk5 regulatory protein. We demonstrate that it is a critical determinant of Cdk5 activity in human AD brains and various cancer and neuronal cells. Increased GSTP1 levels were consistently associated with reduced Cdk5 activity. GSTP1 directly inhibits Cdk5 by dislodging p25/p35, and indirectly by eliminating oxidative stress. Cdk5 promotes and is activated by oxidative stress, thereby engaging a feedback loop which ultimately leads to cell death. Not surprisingly, GSTP1 transduction conferred a high degree of neuroprotection under neurotoxic conditions. Given the critical role of oxidative stress in AD pathogenesis, an increase in GSTP1 level may be an alternative way to modulate Cdk5 signaling, eliminate oxidative stress, and prevent neurodegeneration.     

Apoptosis-associated tyrosine kinase is a Cdk5 activator p35 binding protein

A 3(')-terminal fragment of a splice variant of KIAA0641, a human homologue of apoptosis-associated tyrosine kinase (AATYK), was screened from human brain cDNA libraries by a yeast two-hybrid system using a Cdk5 activator p35 as a bait. The cloned cDNA encoded 477 amino acids, composed of internal 458 amino acids of KIAA0641 and 19 amino acids unique to this variant after splicing, then referred to this clone as hAATYKs-p35BP (human AATYK short isoform-p35 binding polypeptide). Using GST-fusion protein, hAATYKs-p35BP was shown to bind to Cdk5/p35 in a rat brain extract. hAATYKs made by fusing the kinase domain of KIAA0641 to the N-terminus of hAATYKs-p35BP was used for binding to Cdk5/p35 in HEK293 cells. Both hAATYKs and KIAA0641 bound to and were phosphorylated by Cdk5/p35. These results suggest that both isoforms of hAATYK are novel Cdk5/p35-binding and substrate proteins.     

Aurora B kinase exists in a complex with survivin and INCENP and its kinase activity is stimulated by survivin binding and phosphorylation

Aurora B regulates chromosome segregation and cytokinesis and is the first protein to be implicated as a regulator of bipolar attachment of spindle microtubules to kinetochores. Evidence from several systems suggests that Aurora B is physically associated with inner centromere protein (INCENP) in mitosis and has genetic interactions with Survivin. It is unclear whether the Aurora B and INCENP interaction is cell cycle regulated and if Survivin physically interacts in this complex. In this study, we cloned the Xenopus Survivin gene, examined its association with Aurora B and INCENP, and determined the effect of its binding on Aurora B kinase activity. We demonstrate that in the Xenopus early embryo, all of the detectable Survivin is in a complex with both Aurora B and INCENP throughout the cell cycle. Survivin and Aurora B bind different domains on INCENP. Aurora B activity is stimulated >10-fold in mitotic extracts; this activation is phosphatase sensitive, and the binding of Survivin is required for full Aurora B activity. We also find the hydrodynamic properties of the Aurora B/Survivin/INCENP complex are cell cycle regulated. Our data indicate that Aurora B kinase activity is regulated by both Survivin binding and cell cycle-dependent phosphorylation.     

Identification of a neuronal Cdk5 activator-binding protein as Cdk5 inhibitor

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

Tyrosine hydroxylase expression and Cdk5 kinase activity in ataxic cerebellum

Ataxia has been associated with abnormalities in neuronal differentiation and migration, which are regulated by Cyclin-dependent kinase 5 (Cdk5). The cerebellum of mice lacking Cdk5 or its activator, p35, resembles those of ataxic reeler and scrambler mice, suggesting that Cdk5 may contribute to ataxic pathology. As with other ataxic mice, the pogo/pogo mouse shows aberrant cerebellar tyrosine hydroxylase (TH) expression. Since Cdk5 phosphorylates and upregulates TH expression, we sought to analyze (i) Cdk5 activity in the pogo cerebellum, which exhibits abnormal TH expression, and (ii) TH expression in the cerebellum of p35-/- and p39-/- mice, which display reduced Cdk5 activity. Interestingly, we found that increased TH expression in the pogo cerebellum coincided with reduced Cdk5 activity. However, reduced Cdk5 activity in both p35-/- and p39-/- cerebellum did not correspond to defects in TH expression. Together, these suggest that abnormal TH expression in the cerebellum might be regulated by mechanisms other than Cdk5 activity.     

The role of the Cdk5--p35 kinase in neuronal development.

Cyclin-dependent kinase 5 (Cdk5) plays a key role in proper development of the nervous system. To be activated, Cdk5 associates with regulatory subunits not related to cyclins, such as p35 (the regulatory subunit of Cdk5). In this article, we review some of the experimental evidence supporting a central role for the Cdk5/p35 kinase in neuronal migration and process formation.     

Cables links Cdk5 and c-Abl and facilitates Cdk5 tyrosine phosphorylation, kinase upregulation, and neurite outgrowth

Cyclin-dependent kinase 5 (Cdk5) is a small serine/threonine kinase that plays a pivotal role during development of the CNS. Cables, a novel protein, interacts with Cdk5 in brain lysates. Cables also binds to and is a substrate of the c-Abl tyrosine kinase. 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 inhibited neurite outgrowth. Furthermore, expression of active Abl resulted 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.     

The Cdk5-p35 kinase associates with the Golgi apparatus and regulates membrane traffic

We show here that an active Cdk5-p35 kinase is present in Golgi membranes, where it associates with a detergent-insoluble fraction containing actin. In addition, Cdk5-p35-dependent phosphorylation of α-PAK immunoreactive protein species was detected in Golgi membranes, as well as an interaction with the small GTPase, Cdc42. Moreover, antisense oligonucleotide suppression of Cdk5 or p35 in young cultured neurons, as well as inhibition of Cdk5 activity with olomoucine, blocks the formation of membrane vesicles from the Golgi apparatus. Taken together, these results show a novel subcellular localization of this kinase and suggest a role for Cdk5-p35 in membrane traffic during neuronal process outgrowth.     

Structural Insights into Cdk5 activation by a neuronal Cdk5 activator.

Although Cdk5 shows high sequence identity to Cdk1 and Cdk2, it can be fully activated by its neuronal activators p35/p25(nck5a) and p39(nck5ai) in a phosphorylation-independent manner. To understand structural basis of the Cdk5/p25(nck5a) activation, the complex is modelled to assume either an obstructed or an opened conformation based on X-ray structures of the unphosphorylated or the phosphorylated Cdk2/cyclin A complex, respectively. Comparison and analysis of the two models, along with mutagenesis studies of p25(nck5a), suggest that the opened form represents more closely the structure of active Cdk5/p25(nck5a). The results provide a rationale basis for understanding the phosphorylation-independent activation of Cdk5/p25(nck5a).     

The complex p25/Cdk5 kinase in neurofibrillary degeneration and neuronal death: the missing link to cell cycle

Emergence of the cell cycle hypothesis in neurodegenerative disease comes from the numerous lines of evidence showing a tight link between "cell cycle-like reactivation" and neuronal death. Terminally differentiated neurons remain in G0 phase and display, compared to proliferating cells, an opposite regulation pattern of cell cycle markers in that most of the key activators and inhibitors are respectively down- and up-regulated. It has been clearly established that any experimental attempt to force terminally differentiated neurons to divide ultimately leads to their death. Conversely, cell cycle blockade in experimental models of neuronal death is able to rescue neurons. Hence, cell cycle deregulation is certainly among mechanisms governing neuronal death. However, many questions remain unresolved, especially those related to which molecular mechanisms trigger cell cycle deregulation and how this deregulation leads to cell death. In the present review, we focus on neurodegeneration in Alzheimer's disease and discuss the cell cycle deregulation related to this neurodegenerative pathology. Finally, we emphasize the role of p25/Cdk5 kinase complex in this pathological process through retinoblastoma protein phosphorylation and derepression of E2F-responsive genes and other actors such as cdc2, cyclins, and MCM proteins.     

Cyclin-dependent protein kinase 5 (Cdk5) and the regulation of neurofilament metabolism.

Cyclin-dependent kinase 5 (Cdk5), a complex of Cdk5 and its activator p35 (Cdk5/p35), phosphorylates diverse substrates which have multifunctional roles in the nervous system. During development, it participates in neuronal differentiation, migration, axon outgrowth and synaptogenesis. Cdk5, acting together with other kinases, phosphorylates numerous KSPXK consensus motifs in diverse cytoskeletal protein target molecules, including neurofilaments, and microtubule associated proteins, tau and MAPs. Phosphorylation regulates the dynamic interactions of cytoskeletal proteins with one another during all aspects of neurogenesis and axon radial growth. In this review we shall focus on Cdk5 and its regulation as it modulates neurofilament metabolism in axon outgrowth, cytoskeletal stabilization and radial growth. We suggest that Cdk5/p35 forms compartmentalized macromolecular complexes of cytoskeletal substrates, other neuronal kinases, phosphatases and activators ('phosphorylation machines') which facilitate the dynamic molecular interactions that underlie these processes.