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Cdk5 Levels Oscillate during the Neuronal Cell Cycle

Cdh1 UBIQUITINATION TRIGGERS PROTEOSOME-DEPENDENT DEGRADATION DURING S-PHASE*
  • Jie Zhang
    Correspondence
    To whom correspondence may be addressed: Institute of Neuroscience, Xiamen University, SiMing NanLu 422, Xiamen, Fujian, China, 361005. Tel.: 086-592-2180717; Fax: 086-592-2180717;.
    Affiliations
    Institute of Neuroscience, Xiamen University, Xiamen, Fujian, China 361005

    Department of Cell Biology and Neuroscience, Rutgers University, 604 Allison Road, Piscataway, New Jersey 08854
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  • Huifang Li
    Affiliations
    Institute of Neuroscience, Xiamen University, Xiamen, Fujian, China 361005
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  • Tingwen Zhou
    Affiliations
    Institute of Neuroscience, Xiamen University, Xiamen, Fujian, China 361005
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  • Jiechao Zhou
    Affiliations
    Institute of Neuroscience, Xiamen University, Xiamen, Fujian, China 361005
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  • Karl Herrup
    Correspondence
    To whom correspondence may be addressed: Department of Cell Biology and Neuroscience, Rutgers University, 604 Allison Road, Piscataway, New Jersey 08854.
    Affiliations
    Department of Cell Biology and Neuroscience, Rutgers University, 604 Allison Road, Piscataway, New Jersey 08854
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  • Author Footnotes
    * This work was supported, in whole or in part, by National Institutes of Health Grant NS20591 and Rutgers University.
    This article contains supplemental Fig. S1.
Open AccessPublished:May 31, 2012DOI:https://doi.org/10.1074/jbc.M112.343152
      Background: Cdk5 is an atypical Cdk that inhibits the cell cycle in post-mitotic neurons.
      Results: APC-Cdh1 mediates the degradation of Cdk5 during S phase of the cell cycle.
      Conclusion: Maintenance of the proper levels and nuclear location of Cdk5 act to suppress the cell cycle in neuronal cells.
      Significance: Subcellular localization of Cdk5 and p35 may play roles in the pathogenesis of Alzheimer disease.

      Introduction

      The cell division cycle of eukaryotic cells is conventionally divided into four phases: G1, S, G2, and M. Cyclins and cyclin-dependent kinases (Cdks)
      The abbreviations used are: Cdk
      cyclin-dependent kinase
      NES
      nuclear export signal.
      are two key protein families that help determine the pace of progression through these phases. Cyclins function as the regulatory proteins of the proline-directed serine/threonine kinases. They are up- or down-regulated, depending on the phase of the cell cycle, by both transcriptional and post-transcriptional mechanisms. In a well-coordinated cell cycle, the timely degradation of cyclin proteins is as important as their synthesis. This degradation is usually mediated by a highly specific ubiquitin-dependent proteolysis (
      • Peters J.M.
      The anaphase promoting complex/cyclosome: a machine designed to destroy.
      ).
      The mammalian Cdk family consists of 10 members: Cdk1 to Cdk9 and Cdk11 (
      • Malumbres M.
      • Barbacid M.
      Mammalian cyclin-dependent kinases.
      ). Among these Cdks, Cdk5 is regarded as non-traditional. Though its substrate preferences are typical for a Cdk kinase, its activity relies on two specific activator proteins, p35 and p39, not traditional cyclins. These two proteins are structurally similar to cyclins, but share no homology at the amino acid level. Cdk5 is found in many cell types (
      • Philpott A.
      • Porro E.B.
      • Kirschner M.W.
      • Tsai L.H.
      The role of cyclin-dependent kinase 5 and a novel regulatory subunit in regulating muscle differentiation and patterning.
      ,
      • Gao C.Y.
      • Zakeri Z.
      • Zhu Y.
      • He H.
      • Zelenka P.S.
      Expression of Cdk5, p35, and Cdk5-associated kinase activity in the developing rat lens.
      ); however its activity is primarily detected in the nervous system (
      • Lew J.
      • Huang Q.Q.
      • Qi Z.
      • Winkfein R.J.
      • Aebersold R.
      • Hunt T.
      • Wang J.H.
      A brain-specific activator of cyclin-dependent kinase 5.
      ,
      • Tang D.
      • Yeung J.
      • Lee K.Y.
      • Matsushita M.
      • Matsui H.
      • Tomizawa K.
      • Hatase O.
      • Wang J.H.
      An isoform of the neuronal cyclin-dependent kinase 5 (Cdk5) activator.
      ,
      • Tsai L.H.
      • Delalle I.
      • Caviness Jr., V.S.
      • Chae T.
      • Harlow E.
      p35 is a neural-specific regulatory subunit of cyclin-dependent kinase 5.
      ) where the levels of p35 and p39 are highest. In addition to the brain, low levels of Cdk5 kinase activity are also present in the adult mouse prostate and embryonic limb buds (
      • Zhang Q.
      • Ahuja H.S.
      • Zakeri Z.F.
      • Wolgemuth D.J.
      Cyclin-dependent kinase 5 is associated with apoptotic cell death during development and tissue remodeling.
      ). The function of Cdk5 has been widely investigated during development where it has a recognized role in the phosphorylation of a variety of cytoskeletal proteins (
      • Hosoi T.
      • Uchiyama M.
      • Okumura E.
      • Saito T.
      • Ishiguro K.
      • Uchida T.
      • Okuyama A.
      • Kishimoto T.
      • Hisanaga S.
      Evidence for cdk5 as a major activity phosphorylating tau protein in porcine brain extract.
      ,
      • Veeranna
      • Shetty K.T.
      • Link W.T.
      • Jaffe H.
      • Wang J.
      • Pant H.C.
      Neuronal cyclin-dependent kinase-5 phosphorylation sites in neurofilament protein (NF-H) are dephosphorylated by protein phosphatase 2A.
      ). In recent years, additional roles for Cdk5 have been discovered in the post-mitotic neuron. Most of these are related to the ability of Cdk5 to phosphorylate numerous synaptic proteins (
      • Cheng K.
      • Ip N.Y.
      Cdk5: a new player at synapses.
      ,
      • Fischer A.
      • Sananbenesi F.
      • Spiess J.
      • Radulovic J.
      Cdk5: a novel role in learning and memory.
      ,
      • Smith D.S.
      • Tsai L.H.
      Cdk5 behind the wheel: a role in trafficking and transport?.
      ); there is no reported linkage of Cdk5 activity with the promotion of a normal cell cycle. Despite its apparent outlier status, however, our laboratory has demonstrated that Cdk5 does play a role in cell cycle regulation. It has an unexpected non-catalytic function of inhibiting rather than advancing the cell cycle in post-mitotic neurons (
      • Cicero S.
      • Herrup K.
      Cyclin-dependent kinase 5 is essential for neuronal cell cycle arrest and differentiation.
      ). Further, this newly discovered activity of Cdk5 appears to be neuroprotective since the unscheduled reactivation of neuronal cell cycle activity is closely linked to neurodegenerative disease (
      • Nagy Z.
      • Esiri M.M.
      • Cato A.M.
      • Smith A.D.
      Cell cycle markers in the hippocampus in Alzheimer's disease.
      ,
      • Vincent I.
      • Jicha G.
      • Rosado M.
      • Dickson D.W.
      Aberrant expression of mitotic cdc2/cyclin B1 kinase in degenerating neurons of Alzheimer disease brain.
      ,
      • Busser J.
      • Geldmacher D.S.
      • Herrup K.
      Ectopic cell cycle proteins predict the sites of neuronal cell death in Alzheimer disease brain.
      ). This linkage and the failure of cell cycle suppression in Cdk5 deficiency has led our laboratory to focus on the biochemistry of neuronal cell cycle regulation in post-mitotic neurons.
      We find that Cdk5 normally prevents neuronal cell cycle re-entry by disrupting the E2F1-DP1 complex, inhibiting its access to the promoters of cell cycle genes (
      • Zhang J.
      • Herrup K.
      Cdk5 and the non-catalytic arrest of the neuronal cell cycle.
      ,
      • Zhang J.
      • Cicero S.A.
      • Wang L.
      • Romito-Digiacomo R.R.
      • Yang Y.
      • Herrup K.
      Nuclear localization of Cdk5 is a key determinant in the postmitotic state of neurons.
      ,
      • Zhang J.
      • Li H.
      • Yabut O.
      • Fitzpatrick H.
      • D'Arcangelo G.
      • Herrup K.
      Cdk5 suppresses the neuronal cell cycle by disrupting the E2F1-DP1 complex.
      ,
      • Zhang J.
      • Li H.
      • Herrup K.
      Cdk5 nuclear localization is p27-dependent in nerve cells: implications for cell cycle suppression and caspase-3 activation.
      ). We also find that Cdk5 is a nucleocytoplasmic protein. Its nuclear import depends on its binding the Cdk inhibitor, p27; its nuclear export depends on an endogenous nuclear export signal (NES) and the activity of CRM1. This nucleocytoplasmic shuttling is tightly correlated with the cell cycle. Cdk5 moves to the cytoplasm either during S-phase of a normal cell cycle, or during the DNA replication that occurs when a post-mitotic neuron re-enters the cell cycle (
      • Zhang J.
      • Herrup K.
      Nucleocytoplasmic Cdk5 is involved in neuronal cell cycle and death in post-mitotic neurons.
      ). This neuroprotective function, however, contrasts with the finding that Cdk5 hyperactivity, triggered in part by elevated levels of p25 (a breakdown product of p35), is neurotoxic over a timeframe of weeks. Indeed, this toxicity has been proposed as a pathogenic force in neurodegenerative diseases such as Alzheimer disease (
      • Patrick G.N.
      • Zukerberg L.
      • Nikolic M.
      • de la Monte S.
      • Dikkes P.
      • Tsai L.H.
      Conversion of p35 to p25 deregulates Cdk5 activity and promotes neurodegeneration.
      ,
      • Lee M.S.
      • Kwon Y.T.
      • Li M.
      • Peng J.
      • Friedlander R.M.
      • Tsai L.H.
      Neurotoxicity induces cleavage of p35 to p25 by calpain.
      ). Paradoxically, our own short-term studies offered hints that over a timeframe of hours, cytoplasmic Cdk5 is neuroprotective (
      • Zhang J.
      • Li H.
      • Herrup K.
      Cdk5 nuclear localization is p27-dependent in nerve cells: implications for cell cycle suppression and caspase-3 activation.
      ,
      • O'Hare M.J.
      • Kushwaha N.
      • Zhang Y.
      • Aleyasin H.
      • Callaghan S.M.
      • Slack R.S.
      • Albert P.R.
      • Vincent I.
      • Park D.S.
      Differential roles of nuclear and cytoplasmic cyclin-dependent kinase 5 in apoptotic and excitotoxic neuronal death.
      ). This discrepancy led us to an interest in the fate of Cdk5 once it moved to the neuronal cytoplasm. In the current study we show that cytoplasmic Cdk5 is an unstable protein and that its stability is regulated by ubiquitin-dependent proteasomal degradation. We suggest that the failure of this normal clearance pathway would worsen the consequence of any elevation of Cdk5 kinase activity and, coupled with the loss of neuronal cell cycle suppression, suggests that Cdk5 plays multiple roles in the pathway leading to neurodegenerative disease.

      DISCUSSION

      The regulation of all Cdk activities is critical for a cell to properly navigate a cell cycle. During a normal cell cycle, the concentration of the Cdks themselves remains relatively constant. The needed regulation is achieved through dynamic changes in the concentrations of the cyclins and Cdk inhibitors (such as p27 or p16) (
      • Hochegger H.
      • Takeda S.
      • Hunt T.
      Cyclin-dependent kinases and cell-cycle transitions: does one fit all?.
      ). This picture applies to most of the Cdks we examined during the N2a cell cycle. The levels of Cdk1, Cdk2, and Cdk4 protein all remained constant while the levels of their cyclin partners oscillated substantially (Fig. 1).
      The pattern of Cdk constancy during the cell cycle is violated, however, by the Cdk5 kinase whose levels decrease substantially during S-phase of the N2a cell cycle. In an almost cyclin-like pattern its levels follow a near perfect inverse relationship with Cyclin A, an S-phase partner of Cdk2. Fig. 2 shows that this relationship applies not only to cycling neuroblastoma cells, but also to non-cycling primary neurons in culture. In these cells, endogenous Cdk5 and BrdU (an S-phase marker) also show a strong inverse correlation with one another. This is significant as there is considerable data documenting that if such post-mitotic neurons are forced to re-enter a cell cycle they will die rather than divide, as they do in several different neurodegenerative diseases and their mouse models (
      • Vincent I.
      • Jicha G.
      • Rosado M.
      • Dickson D.W.
      Aberrant expression of mitotic cdc2/cyclin B1 kinase in degenerating neurons of Alzheimer disease brain.
      ,
      • Busser J.
      • Geldmacher D.S.
      • Herrup K.
      Ectopic cell cycle proteins predict the sites of neuronal cell death in Alzheimer disease brain.
      ). Combined with our previous data documenting a cell cycle suppressor function for Cdk5 when it is present in the nucleus (
      • Zhang J.
      • Li H.
      • Herrup K.
      Cdk5 nuclear localization is p27-dependent in nerve cells: implications for cell cycle suppression and caspase-3 activation.
      ), we conclude that from the time they are mitotic precursors until their maturation into adult post-mitotic cells and beyond, neurons rely on Cdk5 to meet a life-long need to suppress re-entrance into S-phase.
      Unlike most Cdks, but similar to most cyclins, the levels of Cdk5 are mediated primarily by its degradation at a specific moment in the cell cycle. Cyclin degradation during the cell cycle proceeds by ubiquitin-mediated proteolysis (
      • Peters J.M.
      The anaphase promoting complex/cyclosome: a machine designed to destroy.
      ), and our data show that the same is true for Cdk5. It is particularly intriguing that Cdh1 is the E3 ligase responsible for adding ubiquitin to Cdk5 to target its degradation. The E3 ubiquitin-ligases are responsible for substrate recognition (
      • Kerscher O.
      • Felberbaum R.
      • Hochstrasser M.
      Modification of proteins by ubiquitin and ubiquitin-like proteins.
      ), and the two principal E3-ligases involved in cell cycle control are the SCF and APC/C (anaphase-promoting complex/cyclosome) complexes (
      • Ang X.L.
      • Wade Harper J.
      SCF-mediated protein degradation and cell cycle control.
      ). The SCF complex is used throughout the cell cycle whereas APC/C is active from mitosis only through early G1 (
      • Harper J.W.
      • Burton J.L.
      • Solomon M.J.
      The anaphase-promoting complex: it's not just for mitosis any more.
      ). APC/C is a multi-protein complex that can switch between two major activator proteins, Cdc20 and Cdh1, depending on the cell cycle phase (
      • Fang G.
      • Yu H.
      • Kirschner M.W.
      Direct binding of CDC20 protein family members activates the anaphase-promoting complex in mitosis and G1.
      ,
      • Visintin R.
      • Prinz S.
      • Amon A.
      CDC20 and CDH1: a family of substrate-specific activators of APC-dependent proteolysis.
      ,
      • Huang X.
      • Summers M.K.
      • Pham V.
      • Lill J.R.
      • Liu J.
      • Lee G.
      • Kirkpatrick D.S.
      • Jackson P.K.
      • Fang G.
      • Dixit V.M.
      Deubiquitinase USP37 is activated by CDK2 to antagonize APC(CDH1) and promote S phase entry.
      ); Cdh1 is used primarily in early M-phase while Cdc20 functions during late M- and early G1-phase.
      Superficially, this offers a rationale for APCCdh1 being the enzyme to regulate Cdk5 levels during the N2a cell cycle. But the timing of Cdk5 ubiquitination is out of phase with the reported changes in APCCdh1 activity. APCCdh1 is usually inactivated prior to initiation of S phase (
      • S⊘rensen C.S.
      • Lukas C.
      • Kramer E.R.
      • Peters J.M.
      • Bartek J.
      • Lukas J.
      A conserved cyclin-binding domain determines functional interplay between anaphase-promoting complex-Cdh1 and cyclin A-Cdk2 during cell cycle progression.
      ), resulting in the stabilization of cyclin A (
      • Hsu J.Y.
      • Reimann J.D.
      • S⊘rensen C.S.
      • Lukas J.
      • Jackson P.K.
      E2F-dependent accumulation of hEmi1 regulates S phase entry by inhibiting APC(Cdh1).
      ). Unexpectedly, our data demonstrates that APCCdh1 mediates the degradation of Cdk5 in early S-phase. This represents a previously unrecognized G1/S-phase activity for the ligase and raises the question of how it is regulated. One possibility is that the protein components of the APCCdh1 complex change leading to an altered substrate specificity that favors Cdk5 over more traditional targets. It is also possible that there is a change in the subcellular location of APC/C. Cdk5 mediates cell cycle suppression only if located in the nucleus. In adult neurons, APC/C has a cytoplasmic location. Indeed, cytoplasmic APC/CCdh1 and APC/CCdc20 have been shown to engage in reciprocal activities. APCCdh1 functions as an inhibitor of axonal growth and patterning (
      • Konishi Y.
      • Stegmüller J.
      • Matsuda T.
      • Bonni S.
      • Bonni A.
      Cdh1-APC controls axonal growth and patterning in the mammalian brain.
      ) and synaptic control (
      • van Roessel P.
      • Elliott D.A.
      • Robinson I.M.
      • Prokop A.
      • Brand A.H.
      Independent regulation of synaptic size and activity by the anaphase-promoting complex.
      ), while APCCdc20 plays a role in the suppression of dendrite growth (
      • Konishi Y.
      • Stegmüller J.
      • Matsuda T.
      • Bonni S.
      • Bonni A.
      Cdh1-APC controls axonal growth and patterning in the mammalian brain.
      ,
      • Huynh M.A.
      • Stegmüller J.
      • Litterman N.
      • Bonni A.
      Regulation of Cdh1-APC function in axon growth by Cdh1 phosphorylation.
      ). Additional complexity is added by the demonstration that Cdk5 can phosphorylate Cdh1. This reduces APCCdh1 activity and leads to the accumulation of Cyclin B1 in neurons (
      • Maestre C.
      • Delgado-Esteban M.
      • Gomez-Sanchez J.C.
      • Bolaños J.P.
      • Almeida A.
      Cdk5 phosphorylates Cdh1 and modulates cyclin B1 stability in excitotoxicity.
      ), followed by their re-entrance into a lethal cell cycle (
      • Almeida A.
      • Bolaños J.P.
      • Moreno S.
      Cdh1/Hct1-APC is essential for the survival of postmitotic neurons.
      ).
      This complexity underscores the key role of protein-protein interactions in regulating the levels of Cdk5 protein. Cdk5 binding to either p35 or its p25 breakdown product occludes the Cdh1 ubiquitination site and thus protects Cdk5 from ubiquitin-dependent proteolysis, maintaining its presence in the cell. The importance of p35 binding is demonstrated by the finding that the ΔP35 mutant, which is unable to bind with Cdk5 (
      • Fu X.
      • Choi Y.K.
      • Qu D.
      • Yu Y.
      • Cheung N.S.
      • Qi R.Z.
      Identification of nuclear import mechanisms for the neuronal Cdk5 activator.
      ), is unable to block the interaction between Cdk5 and Cdh1 and thus unable to block the ubiquitination of Cdk5. In support of this model, the truncation constructs (Fig. 4) that bind p35 are all protected from ubiquitination and degradation; those that cannot bind p35 are not. As the 4-protein E2F1-p35-Cdk5-p27 complex reported by our laboratory requires p35 to form (
      • Zhang J.
      • Cicero S.A.
      • Wang L.
      • Romito-Digiacomo R.R.
      • Yang Y.
      • Herrup K.
      Nuclear localization of Cdk5 is a key determinant in the postmitotic state of neurons.
      ,
      • Zhang J.
      • Li H.
      • Yabut O.
      • Fitzpatrick H.
      • D'Arcangelo G.
      • Herrup K.
      Cdk5 suppresses the neuronal cell cycle by disrupting the E2F1-DP1 complex.
      ,
      • Zhang J.
      • Li H.
      • Herrup K.
      Cdk5 nuclear localization is p27-dependent in nerve cells: implications for cell cycle suppression and caspase-3 activation.
      ), we assume that this higher order association also provides protection, but our finding that p25 also protects Cdk5 from ubiquitination and degradation suggests that the other members of the 4-protein complex are not required.
      Different cyclins are only capable of activating the kinase activities of specific Cdks. In this context it is noteworthy that, even though p35 can bind to Cdk1–6, it only attenuates the ubiquitination of Cdk5 and Cdk1. This suggests that despite the high degree of structural similarity between p35 and the traditional cyclins, there are Cdk-specific changes that are realized by the p35 protein. The outcome of these interactions is that, in the presence of p35 or p25, the degradation of Cdk5 in S phase is significantly reduced. Fig. 8 summarizes our findings. In either dividing neuronal cell lines, or in mature non-mitotic neurons, the entrance into S-phase is preceded by the translocation of Cdk5 from nucleus to cytoplasm. This is accompanied by the dissociation of Cdk5 from the 4-protein complex and from p35/25, followed by its association with, and ubiquitination by, APCCdh1 leading to its ultimate degradation by the ubiquitin-proteasome pathway. As the Cdk5 kinase has been proposed to play a critical role in the pathogenesis of Alzheimer disease, our data offer new insight into the different ways in which Cdk5 levels can be regulated.
      Figure thumbnail gr8
      FIGURE 8A diagram of our model of the degradation mechanism of Cdk5. When a neuronal cell enters S-phase, Cdk5 is transported into cytoplasm where it is ubiquitinated by the E3 ligase, APC-Cdh1. The ubiquitinated Cdk5 is then targeted for degradation by the proteasome. Based on the protection data of full-length and truncated Cdk5 constructs, we propose that the ubiquitination site of Cdk5 is in the p35 binding area. Thus, in the presence of p35, the ubiquitination of Cdk5 is blocked, and the degradation of Cdk5 during S phase is attenuated.

      Acknowledgments

      We thank the many laboratories that shared reagents with us. Too numerous to list here, they are described in the methods.

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