Cdk5 levels oscillate during the neuronal cell cycle: Cdh1 ubiquitination triggers proteosome-dependent degradation during S-phase

When cell cycle re-activation occurs in post-mitotic neurons it places them at increased risk for death. The cell cycle/cell death association has been reported in many neurodegenerative diseases including Alzheimer disease (AD), yet the mechanisms by which a normal neuron suppresses the cycle remain largely unknown. Recently, our laboratory has shown that Cdk5 (cyclin-dependent kinase 5) is a key player in this protective function. When a neuron is under stress, Cdk5 is transported to the cytoplasm; this eliminates its cell cycle suppression activity and the neuron re-enters S-phase. In the current study we show that a similar principle applies during a normal cell cycle. When a neuronal cell enters S phase, Cdk5 is transported to the cytoplasm where it is ubiquitinated by the E3 ligase APC-Cdh1. Ubiquitinated Cdk5 is then rapidly degraded by the proteasome. The ubiquitination site of Cdk5 appears to be in the p35 binding area; in the presence of high levels of p35, the ubiquitination of Cdk5 was blocked, and the degradation in S phase was attenuated. The data suggest an unsuspected role for Cdk5 during the progression of a normal cell cycle and offer new pharmaceutical targets for regulating neuronal cell cycling and cell death.     

p53, Apaf-1, caspase-3, and -9 are dispensable for Cdk5 activation during cell death

Cyclin-dependent kinase 5 (Cdk5) is a member of the cyclin-dependent kinase family that is mostly seen in neurons, does not vary with cell cycle, and is activated in many neurodegenerative disorders and other non-neuronal pathologies, but its relationship to non-neuronal apoptosis is not understood, nor is the control of the activation of Cdk5 by its activators. The most widely studied activator of Cdk5, p35, is cleaved to p25 by calpain, an event that has been linked with activation of Cdk5 and neuronal death. Here we report that calpain-mediated Cdk5/p25 activation accompanies non-neuronal as well as neuronal cell death, suggesting that the p35/calpain/p25/Cdk5 activation sequence is a general feature of cell death. We further demonstrate that Cdk5 can be activated in the absence of p53, Apaf-1, caspase-9, and -3 during cell death, indicating that its activation relates more to cell death than to a specific pathway of apoptosis.     

p25/cyclin-dependent kinase 5 promotes the progression of cell death in nucleus of endoplasmic reticulum-stressed neurons

Dysregulation of cyclin-dependent kinase 5 (Cdk5) by cleavage of its activator p35 to p25 by calpain is involved in the neuronal cell death observed in neurodegenerative disorders, including Alzheimer's disease. However, it is not yet clear how p25/Cdk5 induces cell death, although its cytosolic localization or extended half life are thought to be involved. We show here that endoplasmic reticulum (ER) stress causes the calpain-dependent cleavage of p35 to p25 in primary cultured cortical neurons. Generation of p25 occurred at a cell death execution step in ER-stressed neurons. p25 translocated to the nucleus in ER-stressed neurons, whereas p35/Cdk5 was perinuclear in control neurons. Cdk5 inhibitors or dominant-negative Cdk5 suppressed ER stress-induced neuronal cell death. These findings indicate that p25/Cdk5 is a proapoptotic factor that promotes ER stress-induced neuronal cell death in nuclei.     

Cdk5 and the non-catalytic arrest of the neuronal cell cycle

Cyclin-dependent kinase 5 (Cdk5) is a nontraditional Cdk that is primarily active in postmitotic neurons. An important core function of Cdk5 involves regulating the migration and maturation of embryonic post-mitotic neurons. Initially there is little evidence indicating a role for Cdk5 in normal cell cycle regulation. These development roles are on its kinase activity. Recent data from our lab, however, suggest that Cdk5 plays a crucial role as a cell cycle suppressor in normal post-mitotic neurons and neuronal cell lines. It performs this foundation in a kinase independent manner. Cdk5 normally found in both nucleus and cytoplasm, but it exits the nucleus in neurons risk to death in an AD patient’s brain. The shift in sub-cellular location is accompanied by cell cycle re-entry and neuronal death. This “new” function of Cdk5 raises cautions in the design of Cdk5-directed drugs for the therapy of neurodegenerative diseases.     

Cyclin dependent kinase 5 and its interacting proteins in cell death induced in vivo by cyclophosphamide in developing mouse embryos

Activation or inactivation of members of the cyclin-dependent kinase family is important during cell cycle progression. However, Cdk5, a member of this family that was originally identified because of its high structural homology to Cdc2, is activated during cell differentiation and cell death but not during cell cycle progression. We previously demonstrated a correlation between the up-regulation of Cdk5 protein and kinase activity and cell death during development and pathogenesis. We report here that cyclophosphamide (CP) induces massive apoptotic cell death in mouse embryos and that Cdk5 is expressed in apoptotic cells displaying fragmented DNA. During CP-induced cell death, Cdk5 protein expression is substantially increased as detected by immunohistochemistry but not by Western blot, while its mRNA level remains the same as control, and its kinase activity is markedly elevated. The up-regulation of Cdk5 during CP-induced cell death is not due to de novo protein synthesis. We also examined p35, a regulatory protein of Cdk5 in neuronal differentiation. Using a yeast two-hybrid system, we isolated p35, a neuronal differentiation specific protein, as a protein that interacts with Cdk5 in CP-treated embryos. p35 mRNA level does not change, but the protein expression of p25, a truncated form of p35, is elevated during cell death in vivo, as established here, as well as during cell death in vitro. Our results suggest a role for Cdk5 and its regulatory proteins during CP induced cell death. These results further support the view that Cdk5 and its regulation may be key players in the execution of cell death regardless of how the cell dies, whether through biological mechanisms, disease states such as Alzheimer's disease, or induction by CP.     

Identification of nuclear import mechanisms for the neuronal Cdk5 activator

The activation of Cdk5 by p35 plays a pivotal role in a multitude of nervous system activities ranging from neuronal differentiation to degeneration. A fraction of Cdk5 and p35 localizes in the nucleus where Cdk5-p35 exerts its functions via protein phosphorylation, and p35 displays a dynamic localization between the cytoplasm and the nucleus. Here, we examined the nuclear import properties of p35. In nuclear import assays, p35 was actively transported into the nuclei of digitonin-permeabilized HeLa cells and cortical neurons by cytoplasmic carrier-mediated mechanisms. Importin-beta, importin-5, and importin-7 were identified to import p35 into the nuclei via a direct interaction with it. An N-terminal region of p35 was defined to interact with the above importins, serving as a nuclear localization signal. Finally, we show that the nuclear localization of p35 does not require the association of Cdk5. Furthermore, Cdk5 and importin-beta/5/7 are mutually exclusive in binding to p35. These results suggest that p35 employs pathways distinct from that used by Cdk5 for transport to the nucleus.     

Neurodegeneration in an Aβ-induced model of Alzheimer's disease: the role of Cdk5

Cdk5 dysregulation is a major event in the neurodegenerative process of Alzheimer's disease (AD). In vitro studies using differentiated neurons exposed to Aβ exhibit Cdk5-mediated tau hyperphosphorylation, cell cycle re-entry and neuronal loss. In this study we aimed to determine the role of Cdk5 in neuronal injury occurring in an AD mouse model obtained through the intracerebroventricular (icv) injection of the Aβ_1–40 synthetic peptide. In mice icv-injected with Aβ, Cdk5 activator p35 is cleaved by calpains, leading to p25 formation and Cdk5 overactivation. Subsequently, there was an increase in tau hyperphosphorylation, as well as decreased levels of synaptic markers. Cell cycle reactivation and a significant neuronal loss were also observed. These neurotoxic events in Aβ-injected mice were prevented by blocking calpain activation with MDL28170 , which was administered intraperitoneally (ip). As MDL prevents p35 cleavage and subsequent Cdk5 overactivation, it is likely that this kinase is involved in tau hyperphosphorylation, cell cycle re-entry, synaptic loss and neuronal death triggered by Aβ. Altogether, these data demonstrate that Cdk5 plays a pivotal role in tau phosphorylation, cell cycle induction, synaptotoxicity, and apoptotic death in postmitotic neurons exposed to Aβ peptides in vivo , acting as a link between diverse neurotoxic pathways of AD.     

Interplay between the p53 tumor suppressor protein family and Cdk5: novel therapeutic approaches for the treatment of neurodegenerative diseases using selective Cdk inhibitors

Cyclin-dependent kinases (Cdks) play a key role in orchestrating the coordination of cell cycle progression in proliferating cells. The escape from the proper control of the cell cycle by the upregulation of cyclins or aberrant activation of Cdks leads to malignant transformation. In quiescent cells and/or terminally differentiated cells, the expression pattern and activity of Cdks is altered. In postmitotic neurons, expression of mitotic kinases is downregulated, whereas Cdk5 expression becomes upregulated. Similarly to other Cdks, free Cdk5 displays no enzymatic activity and requires complex formation with a specific regulatory subunit. Two activators of Cdk5 have been identified. p35 and its isoform p39 bind to, and thereby activate, Cdk5. Unlike mitotic kinases, Cdk5 does not require activating phosphorylation within the T-loop. Because p35 is a short-lived protein, the p35/Cdk5 complexes are unstable. The stability of the p35 protein is regulated by its Cdk5-mediated phosphorylation of p35. Activated p35/Cdk5 kinase phosphorylates numerous physiological targets. The proper phosphorylation of the most important substrates, such as tau protein and neurofilament H, is essential for the correct regulation of the cytoskeletal organization, thereby regulating cell adhesion, motility, and synaptic plasticity. Moreover, Cdk5 regulates the activity of the p53 tumor suppressor via phosphorylation. p53 is upregulated in multiple neuronal death paradigms, including hypoxia, ischemia, and excitotoxicity, and plays a key role in the induction of apoptosis. On the other hand, an abnormally high expression and elevated activity of Cdk5 was observed in neurodegenerative diseases, suggesting the application of Cdk inhibitors for their therapy. Considering the action of some Cdk inhibitors on the expression and activity of the p53 protein, their therapeutic efficacy must be carefully evaluated.     

Interplay between the p53 tumor suppressor protein family and Cdk5

Cyclin-dependent kinases (Cdks) play a key role in orchestrating the coordination of cell cycle progression in proliferating cells. The escape from the proper, control of the cell cycle by the upregulation of cyclins or aberrant activation of Cdks leads to malignant transformation. In quiescent cells and/or terminally differentiated cells, the expression pattern and activity of Cdks is altered. In postmitotic neurons, expression of mitotic kinases is downregulated, whereas Cdk5 expression becomes upregulated. Similarly to other Cdks, free Cdk5 displays no enzymatic activity and requires complex formation with a specific regulatory subunit. Two activators of Cdk5 have been identified. p35 and its isoform p39 bind to, and thereby activate, Cdk5. Unlike mitotic kinases, Cdk5 does not require activating phosphorylation within the T-loop. Because p35 is a short-lived protein, the p35/Cdk5 complexes are unstable. The stability of the p35 protein is regulated by its Cdk5-mediated phosphorylation of p35. Activated p35/Cdk5 kinase phosphorylates numerous physiological targets. The proper phosphorylation of the most important substrates, such as τ protein and neurofilament H, is essential for the correct regulation of the cytoskeletal organization, thereby regulating cell adhesion, motility, and synaptic plasticity. Moreover, Cdk5 regulates the activity of the p53 tumor suppressor via phosphorylation. p53 is upregulated in multiple neuronal death paradigms, including hypoxia, ischemia, and excitotoxicity, and plays a key role in the induction of apoptosis. On the other hand, an abnormally high expression and elevated activity of Cdk5 was observed in neurodegenerative diseases, suggesting the application of Cdk inhibitors for their therapy. Considering the action of some Cdk inhibitors on the expression and activity of the p53 protein, their therapeutic efficacy must be carefully evaluated.     

Interplay Between the p53 Tumor Suppressor Protein Family and Cdk5: Novel Therapeutic Approaches for the Treatment of Neurodegenerative Diseases Using Selective Cdk Inhibitors.

Cyclin-dependent kinases (Cdks) play a key role in orchestrating the coordination of cell cycle progression in proliferating cells. The escape from the proper control of the cell cycle by the upregulation of cyclins or aberrant activation of Cdks leads to malignant transformation. In quiescent cells and/or terminally differentiated cells, the expression pattern and activity of Cdks is altered. In postmitotic neurons, expression of mitotic kinases is downregulated, whereas Cdk5 expression becomes upregulated. Similarly to other Cdks, free Cdk5 displays no enzymatic activity and requires complex formation with a specific regulatory subunit. Two activators of Cdk5 have been identified. p35 and its isoform p39 bind to, and thereby activate, Cdk5. Unlike mitotic kinases, Cdk5 does not require activating phosphorylation within the T-loop. Because p35 is a short-lived protein, the p35/Cdk5 complexes are unstable. The stability of the p35 protein is regulated by its Cdk5-mediated phosphorylation of p35. Activated p35/Cdk5 kinase phosphorylates numerous physiological targets. The proper phosphorylation of the most important substrates, such as tau protein and neurofilament H, is essential for the correct regulation of the cytoskeletal organization, thereby regulating cell adhesion, motility, and synaptic plasticity. Moreover, Cdk5 regulates the activity of the p53 tumor suppressor via phosphorylation. p53 is upregulated in multiple neuronal death paradigms, including hypoxia, ischemia, and excitotoxicity, and plays a key role in the induction of apoptosis. On the other hand, an abnormally high expression and elevated activity of Cdk5 was observed in neurodegenerative diseases, suggesting the application of Cdk inhibitors for their therapy. Considering the action of some Cdk inhibitors on the expression and activity of the p53 protein, their therapeutic efficacy must be carefully evaluated.     

Regulation and role of cyclin-dependent kinase activity in neuronal survival and death

Cyclin-dependent kinase (Cdk)5 is a proline-directed Ser/Thr protein kinase that functions mainly in neurons and is activated by binding to a regulatory subunit, p35 or p39. Kinase activity is mainly determined by the amount of p35 available, which is controlled by a balance between synthesis and degradation. Kinase activity is also regulated by Cdk5 phosphorylation, but the activity of phosphorylated Cdk5 is in contrast to that of cycling Cdks. Cdk5 is a versatile protein kinase that regulates multiple neuronal activities including neuronal migration and synaptic signaling. Further, Cdk5 plays a role in both survival and death of neurons. Long-term inactivation of Cdk5 triggers cell death, and the survival activity of Cdk5 is apparent when neurons suffer from stress. In contrast, hyper-activation of Cdk5 by p25 promotes cell death, probably by reactivating cell-cycle machinery in the nucleus. The pro-death activity is suppressed by membrane association of Cdk5 via myristoylation of p35. Appropriate activity, localization, and regulation of Cdk5 may be critical for long-term survival of neurons, which is more than 80 years in the case of humans.     

Phosphorylation of Cyclin-dependent Kinase 5 (Cdk5) at Tyr-15 Is Inhibited by Cdk5 Activators and Does Not Contribute to the Activation of Cdk5

Cdk5 is a member of the cyclin-dependent kinase (Cdk) family. In contrast to other Cdks that promote cell proliferation, Cdk5 plays a role in regulating various neuronal functions, including neuronal migration, synaptic activity, and neuron death. Cdks responsible for cell proliferation need phosphorylation in the activation loop for activation in addition to binding a regulatory subunit cyclin. Cdk5, however, is activated only by binding to its activator, p35 or p39. Furthermore, in contrast to Cdk1 and Cdk2, which are inhibited by phosphorylation at Tyr-15, the kinase activity of Cdk5 is reported to be stimulated when phosphorylated at Tyr-15 by Src family kinases or receptor-type tyrosine kinases. We investigated the activation mechanism of Cdk5 by phosphorylation at Tyr-15. Unexpectedly, however, it was found that Tyr-15 phosphorylation occurred only on monomeric Cdk5, and the coexpression of activators, p35/p25, p39, or Cyclin I, inhibited the phosphorylation. In neuron cultures, too, the activation of Fyn tyrosine kinase did not increase Tyr-15 phosphorylation of Cdk5. Further, phospho-Cdk5 at Tyr-15 was not detected in the p35-bound Cdk5. In contrast, expression of active Fyn increased p35 in neurons. These results indicate that phosphorylation at Tyr-15 is not an activation mechanism of Cdk5 but, rather, indicate that tyrosine kinases could activate Cdk5 by increasing the protein amount of p35. These results call for reinvestigation of how Cdk5 is regulated downstream of Src family kinases or receptor tyrosine kinases in neurons, which is an important signaling cascade in a variety of neuronal activities.     

The regulation of cyclin-dependent kinase 5 activity through the metabolism of p35 or p39 Cdk5 activator

Cyclin-dependent kinase 5 (Cdk5) displays kinase activity predominantly in post-mitotic neurons and its physiological roles are unrelated to cell cycle progression. Cdk5 is activated by its binding to a neuron-specific activator, p35 or p39. The protein amount of p35 or p39 is a primary determinant of the Cdk5 activity in neurons, with the amount of p35 or p39 being determined by its synthesis and degradation. The expression of p35 is induced in differentiated neurons and is enhanced by extracellular stimuli such as neurotrophic factors or extracellular matrix molecules, specifically those acting on the ERK/Erg pathway. p35 is a short-lived protein and its degradation determines the life span. Degradation is mediated by the ubiquitin/proteasome system, similar to that for cyclins in proliferating cells. Autophosphorylation of p35 by Cdk5 is a signal for ubiquitination/degradation, and the degradation of p35 is triggered by glutamate treatment in cultured neurons. p35 is cleaved to p25 by calpain at the time of neuronal cell death, and this limited cleavage is suggested to be the cause of neurodegenerative diseases such as Alzheimer's disease. Active Cdk5 changes the cellular localization by cleavage of p35 to p25; p35/Cdk5 is associated with membrane or cytoskeletons, but p25/Cdk5 is a soluble protein. Cleavage also increases the life span of p25 and changes the activity or substrate specificity of Cdk5. p25/Cdk5 shows higher phosphorylating activity to tau than p35/Cdk5 in a phosphorylation site-specific manner. Phosphorylation of p35 suppresses cleavage by calpain. Thus, phosphorylation of p35 modulates its proteolytic pattern, stimulates proteasomal degradation and suppresses calpain cleavage. Phosphorylation is age dependent, as p35 is phosphorylated in foetal brains, but unphosphorylated in adult brains. Therefore, foetal phosphorylated p35 is turned over rapidly, whereas adult unphosphorylated p35 has a long life and is easily cleaved to p25 when calpain is activated. p39 is also a short-lived protein and cleaved to the N-terminal truncation form of p29 by calpain. How the metabolism of p39 is regulated, however, is a future problem to be investigated.     

Cdk5/p35 expression in the mouse ovary

Cyclin-dependent kinase 5 (Cdk5) is primarily associated with brain development but it is also implicated in lens and muscle differentiation. We found that Cdk5 is also expressed in mouse ovary, and explored the possibility that it plays a role in that tissue. We show by Western blotting and immunohistochemistry that the known Cdk5 activator, p35, is also present in the mouse ovary. Cdk5 and p35 were detected in oocytes at all stages of the follicle. While Cdk5 was present in the cytoplasm and nucleus of the oocyte, p35 was observed only in the cytoplasm. Both proteins were detected in the cytoplasm of luteinized cells in the corpus luteum. Immunoprecipitation and histone H1 kinase assays revealed that they form an ovarian complex with considerable kinase activity. Phosphorylation assays showed that several ovarian proteins are substrates for Cdk5/p35 in vitro. Together our findings suggest that p35-associated Cdk5 activity plays an important role in the ovary, where it may regulate cell differentiation and apoptosis as it does in the brain.     

Mutant superoxide dismutase 1 causes motor neuron degeneration independent of cyclin-dependent kinase 5 activation by p35 or p25

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by selective loss of motor neurons in the brain and spinal cord. Neurotoxicity mediated by glutamate is thought to play a role in the neuronal death through intracellular calcium-dependent signaling cascades. Cyclin-dependent kinase 5 (Cdk5) has been proposed as one of the calcium-dependent mediators that may cause neuronal death observed in this disease. Cdk5 is activated in neurons by the association with its activators, p35 or p39. The calcium-activated protease calpain cleaves p35 to its truncated product, p25, which eventually causes the cellular mislocalization and prolonged activation of Cdk5. This deregulated Cdk5 induces cytoskeletal disruption and apoptosis. To examine whether inhibition of the calpain-mediated conversion of p35 to p25 can delay the disease progression of ALS, we generated double transgenic mice in which ALS-linked mutant copper/zinc superoxide dismutase 1 (SOD1G93A) was expressed in a p35-null background. The absence of p35 neither affected the onset and progression of motor neuron disease in the mutant SOD1 mice nor ameliorated the pathological lesions in these mice. Our results provide direct evidence that the pathogenesis of motor neuron disease in the mutant SOD1 mice is independent of the Cdk5 activation by p35 or p25.     

Cdk5 is required for the positioning and survival of GABAergic neurons in developing mouse striatum

Cyclin‐dependent kinase 5 (Cdk5) is a serine/threonine kinase, and its activity is dependent upon an association with a neuron‐specific activating subunit. It was previously reported that Cdk5^−/− mice exhibit perinatal lethality and defective neuronal positioning. In this study, they focused on the analysis of neuronal positioning of GABAergic neurons in the forebrain. Defective formation of the ventral striatum, nucleus accumbens, and olfactory tubercles was found in Cdk5^−/− embryos. To further study this abnormal development, we generated and analyzed Dlx5/6‐Cre p35 conditional KO (cKO); p39^−/− mice in which forebrain GABAergic neurons have lost their Cdk5 kinase activity. Defective formation of the nucleus accumbens and olfactory tubercles as well as neuronal loss in the striatum of Dlx5/6‐Cre p35cKO; p39^−/− mice was found. Elevated levels of phosphorylated JNK were observed in neonatal striatal samples from Dlx5/6‐Cre p35cKO; p39^−/− mice, suggestive of neuronal death. These results indicate that Cdk5 is required for the formation of the ventral striatum in a cell‐autonomous manner, and loss of the kinase activity of Cdk5 causes GABAergic neuronal death in the developing mouse forebrain. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 77: 419–437, 2017