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. Author manuscript; available in PMC: 2010 May 11.
Published in final edited form as: Cell Cycle. 2008 Dec 6;7(24):3819–3822. doi: 10.4161/cc.7.24.7215

Cdk1-FOXO1: A mitotic signal takes center stage in post-mitotic neurons

Albert H Kim 1,2, Azad Bonni 1,*
PMCID: PMC2868071  NIHMSID: NIHMS184254  PMID: 19124971

Abstract

An emerging theme in molecular neurobiology is the discovery of post-mitotic functions for proteins classically associated with mitotic transition in cycling cells. Although neurons have departed the cell cycle, they surprisingly express molecules in the cell cycle apparatus throughout development. The major mitotic cyclin-dependent kinase Cdk1 plays a critical role during the period of naturally occurring neuronal death in the nervous system and has been suggested to contribute to the pathogenesis of neurodegenerative diseases. However, the mechanisms by which Cdk1 promotes neuronal apoptosis are incompletely understood. A recent report by Yuan et al., (2008) has identified a direct relationship between this mitotic kinase and forkhead transcription factor FOXO1, a protein previously implicated in cell death, DNA damage repair and tumor suppression. Here we will discuss the key findings of this report and consider the implications of this mechanism to the regulation of other signal transduction pathways in brain development and diseases.

Keywords: Cdk1, FOXO1, neuronal apoptosis, cell cycle, mitosis, 14-3-3, Plk1

Introduction

The developmental selection of neurons appropriately poised to integrate into neural circuits drives the apoptosis of half of all neurons in the mammalian nervous system.1 In the past two decades, significant advances have been made regarding the molecular characterization of the signals involved in this massive wave of naturally occurring cell death.2–4 Comprehensive molecular delineation of this process is meaningful not only to understand the establishment of neuronal connectivity during brain development but also to find insights into neuronal injury in acute and chronic neuropathological contexts as in ischemic stroke, trauma and neurodegenerative diseases.

A growing body of literature has highlighted the key role of cell cycle regulators in developmental and pathological apoptosis in post-mitotic neurons.5–7 Conceptually, it has been proposed that stimuli that activate cell cycle-related molecules in post-mitotic neurons lead to aberrant cell cycle re-entry and subsequent apoptosis in a cell that has permanently exited the cell cycle. In proliferating cells, the tightly regulated, sequential activation of serine/threonine-directed cyclin-dependent kinases drives cells through the cell cycle.8 In a mechanism reminiscent of the G1-S transition in the cell cycle, Greene and colleagues have elegantly defined a pathway through which trophic factor deprivation triggers abnormal activation of Cdk4 and Cdk6, leading to transcriptional activation of the cell death machinery through hyperphosphorylation of Rb family member p130 and consequent E2F derepression.6 Activation of the G1 Cdks contributes to neuronal cell death under other circumstances, including stimuli relevant to neurological diseases.9

The role of Cdk1 in Post-Mitotic Neurons

In cycling cells, Cdk1, along with its regulatory partner, the B-type cyclins, plays a central role in the G2-M transition as well as in mitotic progression. In fact there is evidence to suggest in vivo that Cdk1 alone—in the absence of the other Cdks—can, for the most part, compensate for the activities of all other Cdks to drive the cell cycle, implying that Cdk1 represents the critical Cdk in proliferating cells.10 Earlier reports by Bonni and colleagues found that Cdk1 is expressed in post-mitotic neurons of the mammalian brain during the period of naturally occurring cell death.11,12 They additionally demonstrated that Cdk1, whose expression and catalytic activity are increased by loss of trophic membrane depolarization, is responsible for neuronal death triggered by membrane activity deprivation, suggesting that Cdk1 mediates developmental neuronal death in vivo.11,12 Intriguingly, the aberrant expression of Cdk1 and cyclin B1 has also been reported in human post-mortem brain sections of various neurodegenerative diseases including Alzheimer disease, frontotemporal dementia and progressive supranuclear palsy,13–15 indicating that Cdk1 may be involved in pathological neuronal injury as well as developmental cell death. In a clever Drosophila model of Alzheimer disease, Feany and colleagues demonstrated that coexpression of a dominant negative Cdk1 with either the E2F1 inhibitor Retinoblastoma factor-1 or the Cdk2 inhibitor Dacapo synergistically inhibited mutant tau-induced apoptosis, implicating Cdk1 in this cell death pathway.16

Until recently, however, little was known about the targets of Cdk1 in post-mitotic neurons. Konishi et al., (2002) identified proapoptotic Bcl-2 family member BAD as a direct target of Cdk1-mediated phosphorylation and activation, defining a non-transcriptional Cdk1-dependent cell death pathway in neurons.11 However, given the number and diversity of Cdk1 substrates in cycling cells, it is plausible that multiple substrates for Cdk1 also exist in post-mitotic neurons.

FOXO Transcription Factors

The FOXO proteins (FOXO1, 3, 4 and 6) comprise a subfamily of forkhead transcription factors, which represent the mammalian orthologs of C. elegans protein DAF-16.17,18 As with other fork-head family members, FOXOs carry the forkhead DNA-binding domain, which binds as a monomer to consensus sequence 5′-TTGTTTAC-3′.19,20 In mammals, FOXO1, 3 and 6 are known to be expressed in post-mitotic neurons in the brain.21 Earlier studies implicated FOXOs in neuronal apoptosis, as forced expression of an activated mutant of FOXO3 caused apoptosis in primary neurons,22 and acute knockdown of FOXO3 by short hairpin RNA (shRNA) protected primary neurons against hydrogen peroxide-induced apoptosis.23 Indeed, consistent with a role in apoptosis, knockout of FOXO1, 3 and 4 promotes thymic and endothelial tumorigenesis, indicating these proteins can act as tumor suppressors.24 In other, nonneuronal cell types, however, FOXOs can initiate the transcription of genes involved in DNA damage repair and protection against reactive oxygen species, two aspects of FOXO function that may contribute to an evolutionarily conserved role in organismal longevity.23,25,26 A specific function for endogenous FOXO1 or its regulation in post-mitotic neurons remained unknown.

Linking Cdk1 to FOXO1 in Postmitotic Neurons

Based upon the delayed time course of apoptosis triggered by membrane activity deprivation and the predominantly cytoplasmic localization of neuronal Cdk1, Yuan et al., (2008) reasoned that a transcription factor that shuttles between the cytosol and nucleus might be regulated by Cdk1 to execute a transcriptional cell death program.27 Given that FOXO transcription factors have been implicated in cell death, Yuan and colleagues noted that a conserved Cdk1 phosphorylation site lies in the Forkhead domain of FOXO1 (PEGGKSGKSPRRRAASMD, human, residues 241–258). Cdk1 efficiently phosphorylated FOXO1 at the predicted serine residue, Ser249, in vitro (Fig. 1A). Using a specific antibody raised against the Ser249 phospho-epitope, the authors discovered that endogenous FOXO1 is phosphorylated at Ser249 in primary neurons subjected to neuronal activity withdrawal, a stimulus known to activate Cdk1.11 Consistent with a requirement for Cdk1 in FOXO1 phosphorylation, RNAi directed against Cdk1 or pharmacological inhibition of Cdk1 abrogated FOXO1 Ser249 phosphorylation.

Figure 1.

Figure 1

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FOXO1 Ser249 phosphorylation is essential for activity withdrawal-induced apoptosis in post-mitotic neurons. (A) Recombinant GST or GST-FOXO1 was incubated with cyclin B and Cdk1 (B/Cdk1) in an in vitro kinase assay and was immunoblotted with the phosphoS249-FOXO1 or GST antibody. *GST-FOXO1 degradation products. (B) COS cells were transfected with the U6/foxo RNAi targeting FOXO1 or control U6 plasmid along with wild type FOXO1 or an RNAi-resistant FOXO1 plasmid (FOXO1-Res), and lysates were immunoblotted with the FOXO1 or 14-3-3 antibody. (C) Granule neurons transfected with the U6/foxo RNAi plasmid along with FOXO1-Res or the FOXO1-ResS249A mutant were subjected to membrane-depolarizing medium (30 mM KCl) or deprived of membrane depolarization (5 mM KCl) for 30 hours and then assessed for apoptosis (mean + SEM). FOXO1-Res, but not FOXO1-ResS249A, triggered apoptosis in activity-deprived neurons in the context of FOXO RNAi (ANOVA; p < 0.05). Reprinted with permission from Science.

To determine the biological function of the Cdk1-induced FOXO1 Ser249 phosphorylation in post-mitotic neurons, the authors first specifically knocked down the FOXO1 protein in primary neurons. Both activity withdrawal- and cyclin B/Cdk1 overexpression-induced apoptosis were attenuated by FOXO RNAi. To evaluate the role of FOXO1 Ser249 phosphorylation, a rescue experiment was performed utilizing a FOXO1 expression plasmid resistant to RNAi (FOXO1-rescue) (Fig. 1B). In the background of FOXO1 RNAi, activity deprivation-induced cell death was restored by expression of FOXO1-rescue (Fig. 1C, column 2). On the other hand, expression of FOXO1-rescue with a Ser-to-Ala mutation at Ser249 in the background of FOXO1 RNAi did not restore activity deprivation-induced cell death (Fig. 1C, column 4), indicating that phosphorylation of FOXO1 Ser249 is critical for the execution of Cdk1-mediated apoptosis.

The proximity between the Cdk1 phosphorylation site and the Akt phosphorylation site on FOXO1 (seven residues apart) immediately suggested a mechanism by which Cdk1 might activate FOXO1. A major regulator of FOXO subcellular localization and subsequent transcriptional activity is the evolutionarily conserved phosphati-dylinositol-3 kinase (PI3K)-Akt pathway. The Akt family of kinases directly phosphorylates FOXO1 at Thr24, Ser256 and Ser319, yielding phospho-epitopes that serve as protein-protein interaction motifs for the 14-3-3 family of proteins.22,28,29 Interaction of FOXO proteins with 14-3-3 proteins leads to nuclear exclusion and therefore inhibition of transcriptional activity.22,29 Yuan et al., thus tested the hypothesis that Cdk1 phosphorylation at FOXO1 Ser249 might disrupt 14-3-3 binding to the phospho-epitope surrounding the Akt site, Ser256. Indeed, overexpression of cyclin B/Cdk1 with FOXO1 disrupted the interaction between FOXO1 and endogenous 14-3-3 in heterologous cells. In primary neurons, activity deprivation increased FOXO1 Ser249 phosphorylation while decreasing the endogenous interaction between FOXO1 and 14-3-3. Consistent with these biochemical data, activity deprivation in neurons increased the nuclear localization of epitope-tagged FOXO1, an event which could be reversed by Cdk1 RNAi. Together, these experiments indicated that Cdk1-mediated FOXO1 phosphorylation impairs FOXO1 interaction with 14-3-3 and thereby stimulates nuclear accumulation of FOXO1.

The Cdk1-triggered nuclear accumulation of FOXO1 would predict increased FOXO1 transcriptional activity. Consistent with this conclusion, the activity of two different FOXO-responsive promoters was found to be inhibited by Cdk1 knockdown. Additionally, expression of the FOXO1 transcriptional target gene BIM, which is induced by activity withdrawal, was inhibited by overexpression of the dominant-negative FOXO1 Ser249Ala mutant but not wildtype FOXO1, implying a critical role for FOXO1 Ser249 in endogenous Bim induction. Thus in neurons, FOXO1 Ser249 phosphorylation by Cdk1 leads to transcriptional activation of FOXO1.

Identification of the Cdk1-FOXO1 signaling connection provides a molecular basis for how Cdk1 triggers a transcription-dependent mechanism of cell death in post-mitotic neurons. Cdk1 phosphorylates FOXO1 at a site that is nearby the Akt site of phosphorylation and thereby disrupts the FOXO1-14-3-3 interaction. Among the FOXO family of transcription factors, Serine 249 is found specifically in FOXO1. Interestingly, Cdk1 also activates the BH3-only protein BAD by phosphorylating BAD at Ser128, a site that dissociates Akt-induced Ser136-phosphorylated BAD from 14-3-3 proteins.30 Strikingly, the arrangement of the Cdk1 and Akt sites of phosphorylation in FOXO1 and BAD is very similar, suggesting the conservation of a peptide motif that subjects proteins to dual regulation by Cdk1 and Akt (Table 1). Thus, Cdk1 and Akt may act on a common set of target proteins that operate as integrators of apoptotic and survival signals in neurons, and the net effect of Cdk1- and Akt-induced phosphorylation may determine the balance between cell death and survival (Fig. 2).

Table 1.

Proteins subject to dual phosphorylation by Cdk1 and Akt kinases

graphic file with name nihms184254f3.jpg
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BAD and FOXO1 have been experimentally confirmed to be Cdk1 and Akt substrates.11,27 Tau and TSC2 (tuberous sclerosis 2 or tuberin) harbor predicted Cdk1 and Akt consensus phosphorylation sites in close proximity to each other.

Figure 2.

Figure 2

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Differential phosphorylation of cellular substrates by Cdk1 and Akt kinases may determine cell death vs. cell survival.

Cdk1-FOXO1 Signaling in the Cell Cycle

The identification of a neuronal signaling link between Cdk1 and FOXO1 raised the interesting possibility that Cdk1-FOXO1 signaling might also occur during the cell cycle. Yuan et al., (2008) therefore moved to cycling cells to ascertain whether FOXO1 Ser249 phosphorylation might be relevant during the period of high Cdk1 activity, G2/M. In normal diploid cells as well as a prostate cancer cell line, high levels of FOXO1 Ser249 phosphorylation were found enriched in nuclei during G2/M, suggesting that Cdk1-mediated phosphorylation promotes FOXO1 nuclear accumulation at G2/M. Additionally, a novel G2/M target of FOXO1, Polo-like kinase 1 (Plk1), was identified by quantitative PCR and chromatin immunoprecipitation. In 3T3 cells, Plk1 expression was confirmed to be dependent upon both FOXO1 by RNAi and FOXO1 Ser249 phosphorylation through rescue experiments using the FOXO1 Ser249Ala rescue mutant in the background of FOXO1 RNAi. Taken together, these results indicate that the Cdk1-FOXO1 signaling cassette is conserved between post-mitotic neurons and cycling cells and that Plk1 is a newly discovered transcriptional target of FOXO1 during G2/M.

Since Plk plays a critical role in promoting the normal progression of mitosis,31 endogenous Cdk1-FOXO1 signaling in proliferating cells may ensure the proper transition of cells during mitosis. Intriguingly, FOXO1 Ser249 phosphorylation by overexpressed Cdk2 reportedly contributes to cell cycle arrest following DNA damage.32 However, the FOXO1 Ser249 phosphorylation is intact in Cdk2-deficient cycling cells, suggesting that Cdk2 may not be required for the FOXO1 Ser249 phosphorylation.27

Beyond the control of neuronal apoptosis and the cell cycle, the characterization of a signaling link between Cdk1 and FOXO1 suggests novel functions for the Cdk1 as well as related kinases and FOXO1. Since the Cdk1-related kinase Cdk5 is highly expressed in the nervous system beyond the peak period of neuronal apoptosis,33 the potential for Cdk5-induced phosphorylation of FOXO1 raises the intriguing possibility that FOXO1 might function in neural development beyond the control of cell death, including neuronal migration, axon outgrowth and synapse differentiation. Moreover, the transcription factor FOXO1 regulates cell differentiation, metabolism and angiogenesis.34–36 Thus, by phosphorylating and activating FOXO1, Cdk1 and related kinases may serve a direct role in diverse biological processes. More speculatively, the identification of a Cdk1-FOXO1 cassette suggests the tantalizing idea that additional cell cycle proteins operative during G2/M in proliferating cells may play as-of-yet undiscovered roles in the biology of post-mitotic neurons.

Acknowledgments

This work was supported by an NIH grant to A.B. NS047188 as well as a Ruth L. Kirschstein NRSA Research Training grant (NCI) and Brain Science Foundation grant to A.H.K. We thank members of the Bonni laboratory for helpful discussions during the preparation of this manuscript.

Abbreviations

Cdk

cyclin-dependent kinase

shRNA

short hairpin RNA

PI3K

phosphatidylinositol-3 kinase

Plk1

polo-like kinase

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