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To whom correspondence should be addressed: Dept. of Cell Biology and Neuroscience, Rutgers, The State University of New Jersey, 604 Allison Rd., Piscataway, NJ 08854-8082. Tel.: 732-445-3306; Fax: 732-445-1794
* This work was supported, in whole or in part, by National Institutes of Health Grant R01NS020591. The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1–S6.
Initiation of a cell cycle in an adult neuron leads to cell death, placing great importance on the mechanisms that normally suppress the neuronal cell cycle. We have previously shown that the cyclin-dependent kinase Cdk5 is an important part of this process, but only when it is present in the nucleus. We report here that Cdk5 nuclear localization relies on its binding to the cyclin-dependent kinase inhibitor p27. Cdk5 has no intrinsic nuclear localization signal; in the absence of p27, two weak nuclear export signals that bind CRM1 cause it to shuttle to the cytoplasm. When a neuron is subjected to stress, such as exposure to β-amyloid, the Cdk5-p27 interaction is lost, reducing Cdk5 levels in the nucleus and depriving the neuron of a major cell cycle suppression mechanism. Caspase-3 is activated within hours, but death is not immediate; elevated levels of cytoplasmic Cdk5 appear to retard neuronal death by a mechanism that may involve Bcl2. These data suggest a model in which Cdk5 exerts a double protective function in neurons: chronically suppressing the cell cycle when located in the nucleus and transiently delaying cell death in the cytoplasm.
The Cdks are the catalytic subunits of a family of nine serine/threonine protein kinases: Cdk1–Cdk9. Among all Cdks, Cdk5 is atypical in several ways. First, its activity does not rely on binding to regular cyclins. Instead, Cdk5 is activated by two specific proteins, p35 and p39, that are structurally similar to cyclins yet share no homology at the amino acid level (
). Although Cdk5 does not drive the cell cycle forward, it does hold the cycle in check. As a consequence, the loss of Cdk5 leads to a failure of cell cycle suppression and subsequent neuronal cell death. This is most evident in Cdk5−/− embryonic mouse neocortical neurons, both in vivo and in vitro (
). This distribution changes in neurons that have been shown to re-enter a cell cycle. For example, in the E2f1−/− mouse brain, many neurons in the cerebral cortex have replicated their DNA and continue to express proteins normally found only in cycling cells (
). These data suggest that nuclear/cytoplasmic transport is important to the cell cycle suppressor function of Cdk5 and stimulated our interest in the mechanisms that control Cdk5 localization in the neuron.
We explore here the role of Cdk5 as a nucleocytoplasmic protein. We show that its nuclear localization is dependent on its binding with p27, whereas its cytoplasmic localization is achieved through the NES-CRM-1 nuclear export mechanism. We show that Cdk5 shuttles between the nucleus and the cytoplasm during the cell cycle. In postmitotic neurons in culture, Cdk5 nuclear export is required for cell cycle re-entry, but once in the cytoplasm, Cdk5 may protect against rapid neuronal death. Thus Cdk5 serves a dual protective function in the highly differentiated postmitotic neuron.
There is now a substantial literature documenting the fact that cell cycle re-entrance by postmitotic central nervous system neurons is tightly correlated with neuronal degeneration (
). If developing central nervous system neurons are forced to enter a cell cycle by expression of an oncogene, they will pass into the S phase and synthesize DNA, but rather than divide they will die (
). Despite the frequent correlation of cell cycle and cell death in the same neuronal populations, the mechanistic pathway neurons use to suppress their cell cycle during adult life remains largely undefined. The data presented here begin to address this gap in our knowledge. We have found that cell cycle or cell death stimulation changes the Cdk5 nuclear/cytoplasmic ratio and that this shuttling can actively regulate both the cell cycle and the initiation of the cell death process. This marks the control of Cdk5 transport as a critical part of the maintenance of a normal neuronal homeostasis.
Nucleocytoplasmic proteins usually have both an NLS and an NES signal. During typical nuclear import, an importin family member binds to the NLS of its cargo and moves the importin-cargo complex into the nucleus (
). Although there is a potential NLS located in amino acids 33–36 of Cdk5 (KRVR), our results show that the N31 truncation mutation containing the KRVR residues nonetheless localizes to the cytoplasm (supplemental Fig. S1). In support of this, Fu et al. (
). Indeed we located several potential phosphorylation sites on Cdk5 with the use of the NetPhos web-based program. Mutation of these sites, however, did not change Cdk5 localization (supplemental Fig. S6). Instead, our data show that it is its interaction with p27 that localizes Cdk5 to the nucleus.
As with most nucleocytoplasmic proteins, its subcellular location is also regulated by its ability to bind CRM1, a key mediator of nuclear export (
). Truncation mutation studies indicate that the export of Cdk5 relies on two atypical NES motifs, located between amino acids 64 and 83 and amino acids 128 and 147. Their atypical form may help to explain why the 1–83 construct is only partially localized to the cytoplasm (Fig. 3, B and C). Thus unlike its nuclear import, which relies on a second protein (p27), nuclear export of Cdk5 is directed by its endogenous NES signals.
Our cell cycle data illustrate that there are important functional consequences to the loss of the Cdk5-p27 interaction. First, in the absence of p27, the localization of Cdk5 becomes cytoplasmic. Second, this translocation relieves the Cdk5-dependent suppression of the cell cycle. We find it intriguing that if Cdk5 cannot bind p27, even if it is forced into the nucleus with an independent NLS, it cannot suppress the cell cycle. Exactly how the interaction of p27 and Cdk5 serves to arrest the cell cycle is unclear. Further work will be needed to answer this question. The consequences of Cdk5 nuclear export extend beyond the loss of cell cycle suppression. Our caspase-3 data illustrate that cytoplasmic but not nuclear Cdk5 slows the time course of β-amyloid toxicity. Taken together our results suggest that Cdk5 plays a protective function in a nerve cell during the process of cell cycle-related neuronal death. Cdk5 in the nucleus suppresses the cell cycle, whereas Cdk5 in the cytoplasm delays caspase-3 activation and Bcl2 degradation.
Ours is not the only evidence that Cdk5 may have a protective function in neurons. Cdk5 has been shown to protect against excitotoxic death in cerebellar granule neurons (
). The present work broadens this evidence by showing that (i) in our conditions the nuclear and cytoplasmic portions of Cdk5 both contribute to its protective effect and (ii) the cytoplasmic action is independent of its effects on the cell cycle. Questions remain unanswered as to how cytoplasmic Cdk5 serves to protect the neuron. One potential mechanism, suggested by the data in Fig. 5, is that Cdk5 contributes to the stabilization of Bcl-2, which is localized mainly in the cytoplasm. This suggestion is consistent with previous models (
) report that nuclear Cdk5 phosphorylates and destabilizes the anti-apoptotic factor, MEF2, thus contributing to the excitotoxic cell death of cultured neurons. In addition, several labs have argued that the hyperactivation of Cdk5 through the calpain cleavage of p35 to p25 is also responsible for neuronal cell death (
). The integration of these disparate findings is crucial to our full understanding of the action of Cdk5 in neurons.
We find that the movement of Cdk5 precedes or is contemporaneous with cell cycle initiation and precedes caspase cleavage. If this is correct, then part of the linkage between cycle and death might occur through Cdk5 itself. Our current model is diagrammed in Fig. 9. When a postmitotic neuron is subjected to cell cycle or cell death stimulation (such as in Alzheimer disease), the physical interaction between Cdk5 and p27 is lost. When this happens, Cdk5 and p27 are both transported into the cytoplasm by CRM1 (Fig. 7, B and C). The reduction in nuclear Cdk5 and p27 deprives the neuron of its cell cycle suppression activity. The stressed neuron re-enters the cell cycle, but with the enhanced levels of cytoplasmic Cdk5, the neurons are temporarily protected from cell death.
An important remaining challenge is to link this model to the situation in the Alzheimer disease brain. Cdk5 protein levels are much higher in adult brain than in developing brain (
). We propose that cytoplasmic Cdk5 contributes to this delayed cell death in Alzheimer disease and perhaps other neurodegenerative diseases.
We acknowledge the generosity of the many laboratories that shared reagents with us. We also thank Gabriella D'Arcangelo, Jianmin Chen, and Jiali Li for thoughtful comments and suggestions during the preparation of this manuscript.