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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. These developmental roles are dependent on its kinase activity. Initially, there was little evidence indicating a role for Cdk5 in normal cell cycle regulation. 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 (Cdk5) is a proline directed serine/threonine kinase. Structurally, it is similar to cdc2,1 but functions differently from traditional cyclin dependent kinases.2 As a non-traditional Cdk, The activity of Cdk5 is not dependent on traditional cyclins but relies on two specific activator proteins—p35 and p39—that are structurally similar to cyclins yet share no homology at the amino acid level. The p35 and p39 double knockout mice exhibit phenotypes identical to those of the Cdk5 null mutant mice, which suggests that p35 and p39 are normally the only activators of Cdk5.3 Cdk5 protein is found in many cell types,4,5 however, its activity is primarily detected in the nervous system where the levels of p35 and p39 are highest.6-8 Cdk5 can phosphorylate serine or threonine in the motif S/TPXK/R where S and T are serine or threonine residents that can be phosphorylated (X is any amino acid and P is the obligatory proline present at position +1).9,10 The serine at 159 position site determines its specific activators;11 the comparable site on Cdk2 is threonine 160. The specific partner of cdk5 endow it total new function compared with other Cdks. Cdk5 has also been shown to bind other traditional cyclins (D1, D2, D3 and E), but it is not activated by them.12-15 The known functions of Cdk5 are diverse; they include neuronal migration,16 axonal growth17 and synaptic function18 (reviewed in ref. 19). Since its discovery in the 1990s, Cdk5 has been viewed a having no role in cell cycle regulation. In support of this view, ectopic expression of Cdk5 in either mammalian or yeast cells does not promote cell cycle progression.20,21 This lack of cell cycle activity identified Cdk5 as unique among the CDK family, and has promoted its identification as an atypical Cdk.
In the CNS, once the developing neurons leave the ventricular zone (VZ) and the subventricular zone (SVZ), they will be permanently postmitotic, and never complete a full cell cycle again. But what would happen if a neuron lost its control of the cell cycle and attempted to divide? Feddersen et al. were probably first describe what will happen if a neuron is forced to leave G0. After forcing expression of an the SV40 T antigen oncogene in maturing cerebellar Purkinje cell, the result was the induction of a cell cycle related neuronal death (CRND) rather than cell division.22-24 More and more laboratories have confirmed this basic finding: when mature and fully differentiated neurons attempt to reenter a cell cycle, their destiny is death rather than cell division.22,25-27 The CRND phenomenon suggests that, contrary to expectations, a mature neuron is not permanently postmitotic after all. This suggestion emphasizes how little is known about the molecular basis of neuronal cell cycle arrest.
As mentioned above, Cdk5 was not considered to play a role in cell cycle regulation. We were surprised, therefore, when we discovered a crucial role for Cdk5 in neuronal cell cycle control. The homozygous Cdk5 null mutation is embryonic lethal; the embryos die at E18 with defective neuronal migration, differentiation and survival.28,29 Analysis of Cdk5−/− (E16.5) mice, however, revealed that loss of Cdk5 also leads to loss of cell cycle control and death of normally postmitotic embryonic mouse neocortical neurons both in vivo and in vitro. Cell cycle activity was documented by the abnormal expression of cell cycle proteins such as cyclin D, cyclin A, and PCNA as well as by BrdU incorporation.30 This implies that Cdk5 must be somehow necessary to suppress the cell cycle in a normal CNS neuron.
Cdk5 protein was first observed in the axons of postmitotic neurons suggesting a cytoplasmic localization.31 Latter studies showed that Cdk5 is also detectable in the neuronal nucleus.30,32,33 O’Hare et al. found that Cdk5 has different functions in these different localizations.34 Compared to the cytoplasm, however, the amount of Cdk5 in the nucleus is relatively small. But this small nuclear fraction endows Cdk5 with a new and different function. By using serum withdrawal to manipulate the cell cycle of NIH 3T3 cells, we find that the nuclear/cytoplasmic ratio changes with the phase of the cell cycle.35 In log phase, most Cdk5 is in cytoplasm. When the cell cycle is arrested by serum starvation, the total amount of Cdk5 protein barely changes, but the subcellular distribution is significantly altered. The fraction of Cdk5 in the nucleus increases whereas the cytoplasmic fraction decreases. This distribution is restored after serum is added back to induce cell cycle reentry. This translocation of Cdk5 suggests its localization is dynamic. The cellular distribution of Cdk5 was also investigated in vivo. In wild type mouse brain, Cdk5 immunoactivity is detected in both nucleus and cytoplasm. But when we examined the location of Cdk5 in neurons in either, (1) the R1.40 mouse model of Alzheimer’s disease, (2) the brain of AD patients or (3) the neocortex and cerebellum of the E2f1 knockout mouse in which there is ectopic neuronal cell cycle reentry,36,37 a consistent pattern emerged. In brain regions where cell cycle events had begun we found that the nuclei of “cycling” neurons were largely devoid Cdk5. The evaluation of Cdk5 levels in the nucleus during G0 phase shows that the nuclear localization of Cdk5 is a pivotal determinant in neuronal cell cycle arrest.
The ability of the Cdk5 kinase to phosphorylate a wide variety of substrates underlies its function in neuronal migration,28 axonal growth,17 synaptic function18 and stress response.38 To our surprise, however, Cdk5 activity does not appear to be involved in the neuronal cell cycle arrest process. Since, roscovitine (the Cdk5 activity inhibitor) does not affect its cell cycle blocking ability, there were early hints that the role of Cdk5 in cell cycle inhibition involves the protein but not its kinase activity.35 The nuclear localization-dependent but kinase-independent characteristic suggests that Cdk5 interacts with other proteins in the nucleus to function as a cell cycle supressor.
The transitions between cell cycle phases are driven by the activities of cyclin/cyclin dependent kinase (CDK) complexes.39 Cyclin-CDK inhibitors (CKIs) are key regulators of CDKs and modulate their activity.40 As mentioned above, if postmitotic neuronal cells leave the quiescent, G0 phase of the cell cycle and enter into G1, they stop at the G1/S checkpoint and then undergo either re-differentiation or apoptosis.41 One important CKI, p27Kip1 (p27) tightly regulates the transition from G0, through G1, into S phase.42-44 In neurons, as in cell lines, high levels of p27 in the nucleus are essential for G0 arrest, and any re-entrance into a neuronal cell cycle would likely require that these levels be reduced. Indeed, during the G0–G1/S transition, p27 translocates from the nucleus to the cytoplasm where it is rapidly degraded.47,48 Evidence suggests that there is an interaction between p27 and Cdk5 in the nucleus,45,46 and we have found that Cdk5 is also transported out of the nucleus during the G0 to G1 transition. Thus, Cdk5 and p27 move into and out of the same compartments during the cell cycle. These data raise the possibility the Cdk5 stops the cell cycle by binding and stabilizing nuclear p27.
The transition out of G0 also requires the complexing of G1 cyclins (D and E) with their Cdk partner proteins (Cdk4 or Cdk6). D-type cyclins are facilitated by40,49 and possibly require50 p27 to form an effective partnership with their Cdk. We know that the translocation of p27 from the nucleus to the cytoplasm is necessary for the G0–G1/S transition,47,48 but the precise mechanism by which this translocation occurs is not known. One recent report found that the translocation of p27 is orchestrated by phosphorylated cyclin D2 at residue Thr280.48 This is intriguing as the interaction between Cdk5 and D-type cyclins has been previously reported.12,46 The binding to cyclin D may have a negative effect on cyclin D phosphorylation thus blocking the p27 translocation and its degradation. Another possible mechanism is that the binding of Cdk5 to other cyclins (including D-type cyclins) may buffer their access to traditional cell cycle kinases (such as Cdk4/6). A recent finding about the ability of the KLF6 tumor suppressor to block the cell cycle fits this model. These authors suggest that KLF6 blocks the cell cycle by binding with D-type cyclins, and disrupting the Cdk4/cyclin D complex.51
The Rb-E2F pathway is also tightly involved in the G0/G1 to S phase transition.52 The E2F family of transcription factors is well known for its linkage with cell cycle control and apoptosis through the regulation of a variety of different target genes.53-55 Our lab recently found that E2F1 functions as a cell cycle suppressor in CNS neurons.36 Cell cycle reentry is common in cultured cortical neurons from E2f1−/− embryos; FISH results directly demonstrate neuronal DNA replication in the E2f1−/− brain. Ectopic expression of the cell cycle proteins PCNA and cyclin A were found in both Purkinje cells and cortical neurons. With these observations as background we investigated whether Cdk5 was involved in the dysregulation of the cell cycle E2f1−/− neurons. We found that the “cycling neurons” (PCNA/cyclin A positive) had weak nuclear and elevated cytoplasmic Cdk5 immunostaining in E2f1−/− cortex. This correlation is suggestive, but it is unclear whether the loss of nuclear Cdk5 is the cause or the consequence of the E2f1−/− neurons’ re-entry into the cell cycle. Our finding that a small fraction of the E2f1−/− neurons have cytoplasmic Cdk5 but no cell cycle activity is consistent with the idea that the shift in Cdk5 location precedes and is necessary for cell cycle initiation.36
The association between cell cycle events with neuronal cell death has been reported many times. In recent years, in a variety of disorders, cell cycle proteins reappear in neurons at risk for degeneration. These disorders include stroke,56 amyotrophic lateral sclerosis,57 ataxia-telangiectasia,58 spinal cord injury (SCI),59 Parkinson’s disease60 and especially Alzheimer’s disease (AD). The literature on the re-expression of cell cycle proteins in neurons from patients with AD is extensive. Such proteins include cyclins,61-64 Cdk kinases,65 PCNA,61,62 Ki67,66 and Cdk inhibitors.67,68
An inappropriate reactivation of the cell cycle is an early and important event in the development of AD. The ectopic cell cycle events appear early in human AD64 and precede the formation of amyloid plaques in most mouse models.37 In fact, it may well be among the earliest pathological changes found in AD mouse model. Although the presence of ectopic cell cycle events in AD is certain, the mechanism that drives this CRND during the disease is still unclear. The biggest question is what triggers a neuron’s “final cycle”. The mitogenic signals that we presume to exist in the AD patient or mouse model have not yet been identified. One possible candidate for a signal to cycle is DNA damage. It was also reported that cell cycle activation in postmitotic neurons is essential for DNA repair.69 Several different agents that induce DNA damage can directly activate a cortical neuron cell cycle.70 Significantly, this neuronal cell cycle reentry can be prevented by administration of inhibitors of the DNA damage checkpoint kinase, ATM.
Cdk5 has been implicated in Alzheimer’s disease pathogenesis through a possible contribution to the aberrant phosphorylation of the micortubule associate protein, tau and the resultant formation of neurofibrillary tangles.19,71 While this may prove correct, we have recently found that the localization of Cdk5 may play as big a role in the disease as does its kinase activity. The Cdk5 nuclear/cytoplasmic ratio is dramatically changed in both human AD and its mouse model (R1.40).25 Double staining of neurons with both Cdk5 and PCNA reveals that cycling neurons (PCNA-positive) have a predominantly cytoplasmic distribution of Cdk5. Consistent with these observations, treatment of either N2a cells or primary cultured neurons with the amyoloidogenic Aβ peptide can also drive Cdk5 translocation to the cytoplasm. At present we know little about the factors that regulate the translocation of Cdk5. There is tantalizing preliminary evidence, however, that Cdk5 translocation may be regulated by ATM. Pharmacological inactivation of ATM activity appears to block Cdk5 translocation into the nucleus. We have no proof that Cdk5 is a direct ATM substrate; indeed there is no S/TQ ATM target site on Cdk5. Nonetheless, p35, the activating partner of Cdk5 does contain a ST/Q site on it, and we have recently shown using bimolecular fluorescent complementation that Cdk5 and p35 form a complex in the nucleus. The model that emerges from these data is one in which the translocation of Cdk5 and p35 occurs as a complex after these partners separate from a larger complex of proteins with a potential mass as large as 680 kD. This has potentially important clinical implications. On the positive side, blocking the translocation of Cdk5 from nucleus to cytoplasm could provide new pharmacological strategies for blocking or delaying ectopic cell cycles and hence CRND in AD or other neurodegenerative diseases.
Cdk5 has been studied for more than 20 years. Almost all Cdk5 research has been focused on its kinase activity while less has been done to study its characteristics as a protein, its localization or its transport. Our recent data support the concept that Cdk5 actively shuttles between nucleus and cytoplasm. The different locations may determine the identity of the Cdk5 binding partners and thus endow Cdk5 with different functions. We suggest that Cdk5 plays a crucial role as a cell cycle suppressor in normal post-mitotic neurons and neuronal cell lines and that it does so in a kinase-independent manner. This is diagrammed in Figure 1. In G0/G1 phase, Cdk5 is located in the nucleus where it blocks the export of p27 and maintains E2F1 transcriptional activity. When mitotic signals appear, as in AD, this triggers Cdk5 to move from nucleus to cytoplasm, eliminating its interaction with p27 or E2F1. This change releases the cell cycle suppression. As in the neurons of the AD patient brain, this 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.