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Cyclin-dependent Kinase 5 (Cdk5)-dependent Phosphorylation of p70 Ribosomal S6 Kinase 1 (S6K) Is Required for Dendritic Spine Morphogenesis*

  • Kwok-On Lai
    Correspondence
    To whom correspondence may be addressed: Dept. of Physiology, State Key Laboratory of Brain and Cognitive Sciences, The University of Hong Kong, 21 Sassoon Rd., Hong Kong, China. Tel.: 852-3917-9521;.
    Affiliations
    From the Division of Life Science, Molecular Neuroscience Center and State Key Laboratory of Molecular Neuroscience, The Hong Kong University of Science and Technology, Hong Kong, China
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  • Author Footnotes
    2 Both authors contributed equally to this work.
    Zhuoyi Liang
    Footnotes
    2 Both authors contributed equally to this work.
    Affiliations
    From the Division of Life Science, Molecular Neuroscience Center and State Key Laboratory of Molecular Neuroscience, The Hong Kong University of Science and Technology, Hong Kong, China
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  • Author Footnotes
    2 Both authors contributed equally to this work.
    Erkang Fei
    Footnotes
    2 Both authors contributed equally to this work.
    Affiliations
    From the Division of Life Science, Molecular Neuroscience Center and State Key Laboratory of Molecular Neuroscience, The Hong Kong University of Science and Technology, Hong Kong, China
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  • Huiqian Huang
    Affiliations
    From the Division of Life Science, Molecular Neuroscience Center and State Key Laboratory of Molecular Neuroscience, The Hong Kong University of Science and Technology, Hong Kong, China
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  • Nancy Y. Ip
    Correspondence
    To whom correspondence may be addressed: Division of Life Science, The Hong Kong University of Science and Technology, Clear Water Bay, Hong Kong, China. Tel.: 852-2358-7269; Fax: 852-2358-2765;.
    Affiliations
    From the Division of Life Science, Molecular Neuroscience Center and State Key Laboratory of Molecular Neuroscience, The Hong Kong University of Science and Technology, Hong Kong, China
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  • Author Footnotes
    * This study was supported in part by the Research Grants Council of Hong Kong (Hong Kong University of Science and Technology Grants 660810, 660110, 661010, and 661111), the National Key Basic Research Program of China (Grant 2013CB530900), Theme-based Research Scheme (Grant T13–607/12R), and the S. H. Ho Foundation.
    2 Both authors contributed equally to this work.
Open AccessPublished:April 22, 2015DOI:https://doi.org/10.1074/jbc.M114.627117
      The maturation and maintenance of dendritic spines depends on neuronal activity and protein synthesis. One potential mechanism involves mammalian target of rapamycin, which promotes protein synthesis through phosphorylation of eIF4E-binding protein and p70 ribosomal S6 kinase 1 (S6K). Upon extracellular stimulation, mammalian target of rapamycin phosphorylates S6K at Thr-389. S6K also undergoes phosphorylation at other sites, including four serine residues in the autoinhibitory domain. Despite extensive biochemical studies, the importance of phosphorylation in the autoinhibitory domain in S6K function remains unresolved, and its role has not been explored in the cellular context. Here we demonstrated that S6K in neuron was phosphorylated at Ser-411 within the autoinhibitory domain by cyclin-dependent kinase 5. Ser-411 phosphorylation was regulated by neuronal activity and brain-derived neurotrophic factor (BDNF). Knockdown of S6K in hippocampal neurons by RNAi led to loss of dendritic spines, an effect that mimics neuronal activity blockade by tetrodotoxin. Notably, coexpression of wild type S6K, but not the phospho-deficient S411A mutant, could rescue the spine defects. These findings reveal the importance of cyclin-dependent kinase 5-mediated phosphorylation of S6K at Ser-411 in spine morphogenesis driven by BDNF and neuronal activity.
      Background: The signaling protein S6K undergoes phosphorylation at multiple serine/threonine sites, but the functional significance is unknown.
      Results: Cdk5 phosphorylates S6K at Ser-411 in neuron, and loss of this phosphorylation event reduces the density of dendritic spines.
      Conclusion: Cdk5-mediated phosphorylation of S6K is crucial for spine morphogenesis in neuron.
      Significance: A new signaling pathway in regulating neuronal connectivity is identified.

      Introduction

      Dendritic spines are specialized structures that serve as the postsynaptic sites of excitatory synapses. Spine morphogenesis depends on neuronal activity and protein synthesis. Blockade of spontaneous neuronal activity by TTX
      The abbreviations used are: TTX
      tetrodotoxin
      mTOR
      mammalian target of rapamycin
      S6K
      p70 ribosomal S6 kinase 1
      Cdk5
      cyclin-dependent kinase 5
      p-
      phospho-
      DIV
      days in vitro
      mEPSC
      miniature excitatory postsynaptic current
      ANOVA
      analysis of variance.
      or inhibition of dendritic protein synthesis through depletion of elongation factor kinase eEF2K leads to the appearance of immature spines (
      • Papa M.
      • Segal M.
      Morphological plasticity in dendritic spines of cultured hippocampal neurons.
      ,
      • Verpelli C.
      • Piccoli G.
      • Zibetti C.
      • Zanchi A.
      • Gardoni F.
      • Huang K.
      • Brambilla D.
      • Di Luca M.
      • Battaglioli E.
      • Sala C.
      Synaptic activity controls dendritic spine morphology by modulating eEF2-dependent BDNF synthesis.
      ). Activity-dependent protein synthesis involves activation of the kinase mTOR, which is required for long lasting forms of synaptic plasticity, such as long term potentiation and metabotropic glutamate receptor-dependent long term depression (
      • Richter J.D.
      • Klann E.
      Making synaptic plasticity and memory last: mechanisms of translational regulation.
      ), as well as dendritic spine maturation (
      • Tavazoie S.F.
      • Alvarez V.A.
      • Ridenour D.A.
      • Kwiatkowski D.J.
      • Sabatini B.L.
      Regulation of neuronal morphology and function by the tumor suppressors Tsc1 and Tsc2.
      ). One of the mTOR substrates is S6K. Upon activation, S6K phosphorylates multiple substrates that are involved in protein synthesis, including ribosomal protein S6, eEF2K, and the translation initiation factor eIF4B (
      • Magnuson B.
      • Ekim B.
      • Fingar D.C.
      Regulation and function of ribosomal protein S6 kinase (S6K) within mTOR signalling networks.
      ). Compared with mTOR, the function of S6K in synapse function and plasticity is less clear. S6K-deficient mice surprisingly display normal long term potentiation and metabotropic glutamate receptor-dependent long term depression (
      • Antion M.D.
      • Hou L.
      • Wong H.
      • Hoeffer C.A.
      • Klann E.
      mGluR-dependent long-term depression is associated with increased phosphorylation of S6 and synthesis of elongation factor 1A but remains expressed in S6K-deficient mice.
      ,
      • Antion M.D.
      • Merhav M.
      • Hoeffer C.A.
      • Reis G.
      • Kozma S.C.
      • Thomas G.
      • Schuman E.M.
      • Rosenblum K.
      • Klann E.
      Removal of S6K1 and S6K2 leads to divergent alterations in learning, memory, and synaptic plasticity.
      ) but show impaired memory formation and hypoactive exploratory behavior (
      • Antion M.D.
      • Merhav M.
      • Hoeffer C.A.
      • Reis G.
      • Kozma S.C.
      • Thomas G.
      • Schuman E.M.
      • Rosenblum K.
      • Klann E.
      Removal of S6K1 and S6K2 leads to divergent alterations in learning, memory, and synaptic plasticity.
      ). S6K is hyperactivated in Fragile X mental retardation protein knock-out mice, the mouse model of fragile X syndrome, and genetic removal of S6K is able to correct the phenotypes of these mice (
      • Bhattacharya A.
      • Kaphzan H.
      • Alvarez-Dieppa A.C.
      • Murphy J.P.
      • Pierre P.
      • Klann E.
      Genetic removal of p70 S6 kinase 1 corrects molecular, synaptic, and behavioral phenotypes in fragile X syndrome mice.
      ). Given the abnormal spine morphogenesis in Fragile X mental retardation protein deficient neurons and fragile X syndrome patients, it is conceivable that the precise control of S6K is critical to the development of dendritic spines.
      The regulation of S6K appears to be highly complicated. Upon extracellular stimulation, S6K is phosphorylated by mTOR at Thr-389. S6K can also undergo phosphorylation at as many as seven other Ser/Thr sites, including four serine residues in the C-terminal autoinhibitory domain. Phosphorylation of the autoinhibitory domain was originally proposed to open up the S6K conformation, allowing phosphorylation by mTOR and subsequent S6K activation (
      • Dennis P.B.
      • Pullen N.
      • Pearson R.B.
      • Kozma S.C.
      • Thomas G.
      Phosphorylation sites in the autoinhibitory domain participate in p70s6k activation loop phosphorylation.
      ). Other studies, however, have challenged this hypothesis, and the importance of autoinhibitory domain phosphorylation remains obscure (
      • Magnuson B.
      • Ekim B.
      • Fingar D.C.
      Regulation and function of ribosomal protein S6 kinase (S6K) within mTOR signalling networks.
      ). It is noteworthy that virtually all previous studies on the regulatory mechanisms of S6K were limited to an in vitro kinase assay, and the significance of the autoinhibitory domain phosphorylation has not been explored in the cellular context.
      In the present study, we examined the regulation and functional role of Ser-411 phosphorylation within the autoinhibitory domain of S6K in neuron. Multiple proline-directed serine/threonine kinases can phosphorylate S6K at Ser-411 in vitro (
      • Mukhopadhyay N.K.
      • Price D.J.
      • Kyriakis J.M.
      • Pelech S.
      • Sanghera J.
      • Avruch J.
      An array of insulin-activated, proline-directed serine/threonine protein kinases phosphorylate the p70 S6 kinase.
      ,
      • Shah O.J.
      • Ghosh S.
      • Hunter T.
      Mitotic regulation of ribosomal S6 kinase 1 involves Ser/Thr, Pro phosphorylation of consensus and non-consensus sites by Cdc2.
      • Hou Z.
      • He L.
      • Qi R.Z.
      Regulation of S6 kinase 1 activation by phosphorylation at Ser-411.
      ). One of them is Cdk5, which is an emerging key player in regulating spine morphogenesis through phosphorylation of multiple substrates (
      • Kim Y.
      • Sung J.Y.
      • Ceglia I.
      • Lee K.W.
      • Ahn J.H.
      • Halford J.M.
      • Kim A.M.
      • Kwak S.P.
      • Park J.B.
      • Ho Ryu S.
      • Schenck A.
      • Bardoni B.
      • Scott J.D.
      • Nairn A.C.
      • Greengard P.
      Phosphorylation of WAVE1 regulates actin polymerization and dendritic spine morphology.
      • Fu W.Y.
      • Chen Y.
      • Sahin M.
      • Zhao X.S.
      • Shi L.
      • Bikoff J.B.
      • Lai K.O.
      • Yung W.H.
      • Fu A.K.
      • Greenberg M.E.
      • Ip N.Y.
      Cdk5 regulates EphA4-mediated dendritic spine retraction through an ephexin1-dependent mechanism.
      ,
      • Yamashita N.
      • Morita A.
      • Uchida Y.
      • Nakamura F.
      • Usui H.
      • Ohshima T.
      • Taniguchi M.
      • Honnorat J.
      • Thomasset N.
      • Takei K.
      • Takahashi T.
      • Kolattukudy P.
      • Goshima Y.
      Regulation of spine development by semaphorin3A through cyclin-dependent kinase 5 phosphorylation of collapsin response mediator protein 1.
      ,
      • Lai K.O.
      • Ip N.Y.
      Recent advances in understanding the roles of Cdk5 in synaptic plasticity.
      • Lai K.O.
      • Wong A.S.
      • Cheung M.C.
      • Xu P.
      • Liang Z.
      • Lok K.C.
      • Xie H.
      • Palko M.E.
      • Yung W.H.
      • Tessarollo L.
      • Cheung Z.H.
      • Ip N.Y.
      TrkB phosphorylation by Cdk5 is required for activity-dependent structural plasticity and spatial memory.
      ). Here we report that the phosphorylation of S6K by Cdk5 at Ser-411 is regulated by neuronal activity and the neurotrophin BDNF and that this phosphorylation event is crucial for spine morphogenesis.

      Discussion

      Dysregulation of mTOR and S6K is implicated in neurodevelopmental disorders, such as autism (
      • Bhattacharya A.
      • Kaphzan H.
      • Alvarez-Dieppa A.C.
      • Murphy J.P.
      • Pierre P.
      • Klann E.
      Genetic removal of p70 S6 kinase 1 corrects molecular, synaptic, and behavioral phenotypes in fragile X syndrome mice.
      ,
      • Kelleher 3rd, R.J.
      • Bear M.F.
      The autistic neuron: troubled translation?.
      ), and absence of S6K in rodents is associated with abnormal behavior (
      • Antion M.D.
      • Merhav M.
      • Hoeffer C.A.
      • Reis G.
      • Kozma S.C.
      • Thomas G.
      • Schuman E.M.
      • Rosenblum K.
      • Klann E.
      Removal of S6K1 and S6K2 leads to divergent alterations in learning, memory, and synaptic plasticity.
      ). Nonetheless, the regulation and cellular function of S6K in neuron remain unclear. Our study showed that S6K is crucial for activity-dependent spine development. We further found that Ser-411 of S6K is phosphorylated by Cdk5 in response to neuronal activity or BDNF, and this phosphorylation event is required for dendritic spine morphogenesis. Our study therefore suggests that, in addition to the well known mTOR pathway that phosphorylates S6K at Thr-389, a parallel pathway that involves Ser-411 phosphorylation by Cdk5 is also required for S6K function in neuron.
      Although phosphorylation of S6K in the autoinhibitory loop, including Ser-411, has been reported for a long time, its functional significance remains unresolved. One study showed that S6K that is completely deficient in phosphorylation of the autoinhibitory domain retains comparable kinase activity after insulin stimulation (
      • Mahalingam M.
      • Templeton D.J.
      Constitutive activation of S6 kinase by deletion of amino-terminal autoinhibitory and rapamycin sensitivity domains.
      ). Others have reported reduced S6K activity while lacking phosphorylation of the autoinhibitory domain, but the kinase activity of the mutant can still be induced by serum, suggesting that the autoinhibitory domain phosphorylation is not a prerequisite for kinase activation (
      • Han J.W.
      • Pearson R.B.
      • Dennis P.B.
      • Thomas G.
      Rapamycin, wortmannin, and the methylxanthine SQ20006 inactivate p70s6k by inducing dephosphorylation of the same subset of sites.
      ,
      • Weng Q.P.
      • Kozlowski M.
      • Belham C.
      • Zhang A.
      • Comb M.J.
      • Avruch J.
      Regulation of the p70 S6 kinase by phosphorylation in vivo. Analysis using site-specific anti-phosphopeptide antibodies.
      ). The importance of the Ser-411 phosphorylation therefore has not been resolved. More importantly, those studies only examined the phospho-deficient SA mutant function using in vitro kinase assay, and it is essential to address the function of the S6K SA mutant in a cellular context. Our observations in the RNAi rescue experiments represent the first demonstration that Ser-411 phosphorylation is indeed crucial for S6K function in a cellular context. Inhibition of Cdk5 specifically blocked the BDNF-induced Ser-411 phosphorylation without affecting the induction of Thr-389 phosphorylation, suggesting that Ser-411 does not serve as the priming phosphorylation event for subsequent Thr-389 phosphorylation by mTOR. One possible role of Ser-411 phosphorylation might be the regulation of substrate specificity of S6K. In this context, it is noteworthy that although both S6 ribosomal protein and eEF2K are downstream substrates of S6K only phosphorylation of S6, but not eEF2K, is reduced in the brains of Cdk5-null mice. With the identification of more S6K substrates (
      • Magnuson B.
      • Ekim B.
      • Fingar D.C.
      Regulation and function of ribosomal protein S6 kinase (S6K) within mTOR signalling networks.
      ), further studies on the interaction between different substrates and the wild type versus various phospho-deficient mutants of S6K should provide important insights into how S6K function is precisely regulated by multiple phosphorylation events.
      S6K promotes dendritic arborization in young neurons (
      • Jaworski J.
      • Spangler S.
      • Seeburg D.P.
      • Hoogenraad C.C.
      • Sheng M.
      Control of dendritic arborization by the phosphoinositide-3′-kinase-Akt-mammalian target of rapamycin pathway.
      ). Using shRNA to knock down S6K expression in mature neurons, we observed a significant reduction of dendritic spine density. The spine defects could be rescued upon coexpression of RNAi-resistant S6K, indicating that the impaired spine morphogenesis is specifically attributed to the depletion of S6K. Interestingly, dendritic arborization and spine morphogenesis appear to be normal in S6K knock-out mice (
      • Bhattacharya A.
      • Kaphzan H.
      • Alvarez-Dieppa A.C.
      • Murphy J.P.
      • Pierre P.
      • Klann E.
      Genetic removal of p70 S6 kinase 1 corrects molecular, synaptic, and behavioral phenotypes in fragile X syndrome mice.
      ). It is likely that other proteins, such as the closely related S6K2 and p90RSK, can compensate for the absence of S6K in the knock-out mice, and the role of S6K in dendritic arborization and spine morphogenesis can only be revealed upon acute knockdown of the protein.
      Knockdown of S6K mimicked and occluded the effect of TTX on spine loss and abolished the effect of BDNF on spine formation. Our findings therefore suggest that S6K is required for spine formation and maintenance in response to neuronal activity. BDNF signaling has been implicated in activity-dependent spine maintenance, and we also observed reduced BDNF expression in S6K knockdown neurons (data not shown). However, in contrast to the dramatic (∼50%) loss of dendritic spines after S6K knockdown or TTX treatment, neurons incubated with TrkB-IgG for 24 h only displayed modest reduction of spine density (less than 10%; data not shown). Therefore, although S6K is crucial to mediate the effect of BDNF on spine formation, it is likely that the observed spine loss after S6K knockdown or TTX treatment is caused by mechanisms other than reduced BDNF signaling.
      Most excitatory synapses are located on dendritic spines. Whereas knockdown of S6K led to a roughly 50% reduction of spine density, surprisingly excitatory synaptic transmission remained unaffected as indicated by normal mEPSC frequency and amplitude. This suggests that excitatory synaptic transmission can be dissociated from dendritic spines. One possibility is that the mEPSCs are being generated at sites in the dendritic arbor spatially distinct from the regions chosen for quantifying spines. Alternatively the excitatory synapses might be shifted from the spines to dendritic shaft. This is consistent with a recent study showing a drastic reduction of spine density and abnormal distribution of excitatory synapses in neuron upon overexpression of the nuclear receptor Nr4a1 but no change in excitatory synaptic transmission (
      • Chen Y.
      • Wang Y.
      • Ertürk A.
      • Kallop D.
      • Jiang Z.
      • Weimer R.M.
      • Kaminker J.
      • Sheng M.
      Activity-induced Nr4a1 regulates spine density and distribution pattern of excitatory synapses in pyramidal neurons.
      ). Besides simply acting as a site for excitatory synapse, dendritic spines offer additional properties for excitatory synaptic transmission, such as forming isolated compartments for Ca2+ signaling and allowing modulation of synaptic strength as a result of rapid changes in spine size (
      • Yuste R.
      Dendritic spines and distributed circuits.
      ). It therefore remains possible that the properties of excitatory synapses on the dendritic shaft differ from those on the spines, and the spine location of excitatory synapses might be important for specific features, such as synaptic plasticity.
      How S6K promotes spine morphogenesis remains to be determined. One possibility is that S6K is crucial for global de novo protein synthesis in neuron. We think this is unlikely because in situ metabolic labeling using the azide-bearing non-canonical amino acid followed by click reaction revealed that the general protein synthesis is not affected in neurons upon knockdown of S6K by shRNA (data not shown). Conversely, it is plausible that S6K might be required for the induced synthesis of specific proteins by BDNF. For example, the mTOR-dependent synthesis of LIMK1 has been implicated in BDNF-induced spine maturation (
      • Schratt G.M.
      • Tuebing F.
      • Nigh E.A.
      • Kane C.G.
      • Sabatini M.E.
      • Kiebler M.
      • Greenberg M.E.
      A brain-specific microRNA regulates dendritic spine development.
      ). BDNF also induces the expression of Arc, which can modulate phosphorylation of the actin-binding protein cofilin (
      • Bramham C.R.
      Local protein synthesis, actin dynamics, and LTP consolidation.
      ) and represents another potential candidate that promotes spine formation and maintenance. Alternatively, S6K might regulate spine morphogenesis in a protein synthesis-independent manner. S6K has been shown to interact with neurabin and the Rac guanine nucleotide exchange factor Tiam1 that are known to directly regulate spine growth (
      • Burnett P.E.
      • Blackshaw S.
      • Lai M.M.
      • Qureshi I.A.
      • Burnett A.F.
      • Sabatini D.M.
      • Snyder S.H.
      Neurabin is a synaptic protein linking p70 S6 kinase and the neuronal cytoskeleton.
      ,
      • Buchsbaum R.J.
      • Connolly B.A.
      • Feig L.A.
      Regulation of p70 S6 kinase by complex formation between the Rac guanine nucleotide exchange factor (Rac-GEF) Tiam1 and the scaffold spinophilin.
      ). It will be of significant interest to further elucidate how S6K affects spine morphogenesis through phosphorylation of different substrates.

      ACKNOWLEDGMENTS

      We are grateful to Ashok Kulkarni and Tom Curran for the Cdk5-null mice and Robert Qi for the S6K construct. We thank Jenny Wang, Ye Wang, Elaine Cheng, Cara Kwong, Bowie Lai, and Busma Butt for excellent technical assistance.

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