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Activity-dependent Phosphorylation of Neuronal Kv2.1 Potassium Channels by CDK5*

  • Oscar Cerda
    Affiliations
    Department of Neurobiology, Physiology, and Behavior, University of California, Davis, California 95616
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  • James S. Trimmer
    Correspondence
    To whom correspondence should be addressed: Dept. of Neurobiology, Physiology, and Behavior, 196 Briggs Hall, University of California, One Shields Ave., Davis, CA 95616-8519
    Affiliations
    Department of Neurobiology, Physiology, and Behavior, University of California, Davis, California 95616

    Physiology and Membrane Biology, University of California, Davis, California 95616
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  • Author Footnotes
    * This work was supported, in whole or in part, by National Institutes of Health Grant NS42225 (to J. S. T226).
Open AccessPublished:June 28, 2011DOI:https://doi.org/10.1074/jbc.M111.251942
      Dynamic modulation of ion channel expression, localization, and/or function drives plasticity in intrinsic neuronal excitability. Voltage-gated Kv2.1 potassium channels are constitutively maintained in a highly phosphorylated state in neurons. Increased neuronal activity triggers rapid calcineurin-dependent dephosphorylation, loss of channel clustering, and hyperpolarizing shifts in voltage-dependent activation that homeostatically suppress neuronal excitability. These changes are reversible, such that rephosphorylation occurs after removal of excitatory stimuli. Here, we show that cyclin-dependent kinase 5 (CDK5), a Pro-directed Ser/Thr protein kinase, directly phosphorylates Kv2.1, and determines the constitutive level of Kv2.1 phosphorylation, the rapid increase in Kv2.1 phosphorylation upon acute blockade of neuronal activity, and the recovery of Kv2.1 phosphorylation after stimulus-induced dephosphorylation. We also demonstrate that although the phosphorylation state of Kv2.1 is also shaped by the activity of the PP1 protein phosphatase, the regulation of Kv2.1 phosphorylation by CDK5 is not mediated through the previously described regulation of PP1 activity by CDK5. Together, these studies support a novel role for CDK5 in regulating Kv2.1 channels through direct phosphorylation.

      Introduction

      Plasticity in the intrinsic excitability of neurons comprises experience-dependent changes in how individual neurons integrate and process synaptic input and determine their mode of output, and involves dynamic changes in the expression, localization, and/or functional properties of voltage-gated ion channels. Kv2.1, a delayed rectifier-type voltage-gated potassium or Kv channel expressed in high density clusters in somatodendritic domains of mammalian neurons (
      • Baranauskas G.
      • Tkatch T.
      • Surmeier D.J.
      ,
      • Murakoshi H.
      • Trimmer J.S.
      ,
      • Guan D.
      • Tkatch T.
      • Surmeier D.J.
      • Armstrong W.E.
      • Foehring R.C.
      ), is subjected to rapid activity-dependent, calcineurin-dependent dephosphorylation, resulting in a more hyperpolarized threshold for activation of Kv2.1 currents and loss of clustering (
      • Murakoshi H.
      • Shi G.
      • Scannevin R.H.
      • Trimmer J.S.
      ,
      • Misonou H.
      • Mohapatra D.P.
      • Park E.W.
      • Leung V.
      • Zhen D.
      • Misonou K.
      • Anderson A.E.
      • Trimmer J.S.
      ,
      • Misonou H.
      • Mohapatra D.P.
      • Menegola M.
      • Trimmer J.S.
      ,
      • Misonou H.
      • Menegola M.
      • Mohapatra D.P.
      • Guy L.K.
      • Park K.S.
      • Trimmer J.S.
      ,
      • Misonou H.
      • Thompson S.M.
      • Cai X.
      ,
      • Mulholland P.J.
      • Carpenter-Hyland E.P.
      • Hearing M.C.
      • Becker H.C.
      • Woodward J.J.
      • Chandler L.J.
      ,
      • Aras M.A.
      • Saadi R.A.
      • Aizenman E.
      ,
      • Mulholland P.J.
      • Carpenter-Hyland E.P.
      • Woodward J.J.
      • Chandler L.J.
      ,
      • Ito T.
      • Nuriya M.
      • Yasui M.
      ) and leading to homeostatic suppression of neuronal firing (
      • Misonou H.
      • Mohapatra D.P.
      • Menegola M.
      • Trimmer J.S.
      ,
      • Mohapatra D.P.
      • Misonou H.
      • Pan S.J.
      • Held J.E.
      • Surmeier D.J.
      • Trimmer J.S.
      ). Removal of these stimuli leads to recovery of Kv2.1 phosphorylation and clustering (
      • Misonou H.
      • Mohapatra D.P.
      • Park E.W.
      • Leung V.
      • Zhen D.
      • Misonou K.
      • Anderson A.E.
      • Trimmer J.S.
      ,
      • Misonou H.
      • Menegola M.
      • Mohapatra D.P.
      • Guy L.K.
      • Park K.S.
      • Trimmer J.S.
      ,
      • Mulholland P.J.
      • Carpenter-Hyland E.P.
      • Hearing M.C.
      • Becker H.C.
      • Woodward J.J.
      • Chandler L.J.
      ,
      • Aras M.A.
      • Saadi R.A.
      • Aizenman E.
      ,
      • Ito T.
      • Nuriya M.
      • Yasui M.
      ). Anesthesia in vivo induces enhanced Kv2.1 phosphorylation (
      • Misonou H.
      • Menegola M.
      • Mohapatra D.P.
      • Guy L.K.
      • Park K.S.
      • Trimmer J.S.
      ), showing that bidirectional changes in neuronal activity trigger homeostatic changes in the Kv2.1 phosphorylation state. Modulation of Kv2.1 is the candidate mechanism for plasticity in the intrinsic excitability of visual cortical neurons in response to monocular deprivation and in long term potentiation of intrinsic excitability (
      • Nataraj K.
      • Le Roux N.
      • Nahmani M.
      • Lefort S.
      • Turrigiano G.
      ).
      Liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based analyses have defined a large set of in vivo Ser and Thr Kv2.1 phosphorylation sites (
      • Park K.S.
      • Mohapatra D.P.
      • Misonou H.
      • Trimmer J.S.
      ,
      • Park K.S.
      • Mohapatra D.P.
      • Trimmer J.S.
      ), a subset of which are dephosphorylated upon calcineurin activation and mediate the activity-dependent changes in Kv2.1 localization and function (
      • Misonou H.
      • Menegola M.
      • Mohapatra D.P.
      • Guy L.K.
      • Park K.S.
      • Trimmer J.S.
      ,
      • Park K.S.
      • Mohapatra D.P.
      • Misonou H.
      • Trimmer J.S.
      ). Among these sites, phosphorylation at the Ser-603 residue exhibits extraordinary sensitivity to bidirectional activity-dependent changes in phosphorylation state (
      • Misonou H.
      • Menegola M.
      • Mohapatra D.P.
      • Guy L.K.
      • Park K.S.
      • Trimmer J.S.
      ). The protein phosphatases (PPs)
      The abbreviations used are: PP
      protein phosphatase
      AP
      alkaline phosphatase
      PK
      protein kinase
      RBM
      rat brain membrane
      HBSS
      Hanks' buffered saline solution
      DIV
      days in vitro
      RSB
      reducing SDS sample buffer
      DPBS
      Dulbecco's phosphate-buffered saline
      TTX
      tetrodotoxin.
      PP1 and calcineurin/PP2B have been identified as playing crucial and non-overlapping roles in constitutive and activity-dependent dephosphorylation of Kv2.1, respectively (
      • Misonou H.
      • Mohapatra D.P.
      • Park E.W.
      • Leung V.
      • Zhen D.
      • Misonou K.
      • Anderson A.E.
      • Trimmer J.S.
      ,
      • Misonou H.
      • Menegola M.
      • Mohapatra D.P.
      • Guy L.K.
      • Park K.S.
      • Trimmer J.S.
      ). However, the specific protein kinases (PKs) responsible for constitutive and activity-dependent phosphorylation of Kv2.1 have not been identified.
      Among the identified Kv2.1 phosphorylation sites, almost half (including Ser-603) are adjacent to a C-terminal Pro residue, suggesting phosphorylation by Pro-directed Ser/Thr PKs. Among these, cyclin-dependent kinase 5 (CDK5) is a neuronal PK whose activity depends on association with myristoyl-anchored p35 and p39 cofactors and whose activity underlies diverse aspects of neuronal biology, including neurogenesis, neuronal migration and survival, synaptic plasticity, and neurodegeneration (
      • Tsai L.H.
      • Delalle I.
      • Caviness Jr., V.S.
      • Chae T.
      • Harlow E.
      ,
      • Cai X.H.
      • Tomizawa K.
      • Tang D.
      • Lu Y.F.
      • Moriwaki A.
      • Tokuda M.
      • Nagahata S.
      • Hatase O.
      • Matsui H.
      ,
      • Lai K.O.
      • Ip N.Y.
      ). Here, we investigate the role of CDK5 in the constitutive and activity-dependent phosphorylation of Kv2.1 and define a new role for CDK5 in regulating neuronal function through direct phosphorylation of a voltage-gated ion channel crucial to activity-dependent plasticity in intrinsic neuronal excitability.

      DISCUSSION

      Plasticity in the intrinsic excitability of neurons is based on dynamic changes in the expression, localization, and/or functional properties of voltage-gated ion channels. Kv channels are the most diverse family of voltage-gated channels and as such are primary determinants of diversity of overall neuronal excitability and of the input-output relationships in mammalian neurons (
      • Johnston J.
      • Forsythe I.D.
      • Kopp-Scheinpflug C.
      ). A number of recent studies have provided valuable insights into the role of specific Kv channel subtypes in the processing and integration of synaptic input within the somatodendritic domain (
      • Johnston D.
      • Christie B.R.
      • Frick A.
      • Gray R.
      • Hoffman D.A.
      • Schexnayder L.K.
      • Watanabe S.
      • Yuan L.L.
      ), initiation and propagation of axonal action potentials (
      • Kress G.J.
      • Mennerick S.
      ), and regulation of neurotransmitter release (
      • Dodson P.D.
      • Forsythe I.D.
      ). Modulation of the abundance, subcellular distribution, and gating of Kv channels through reversible multisite phosphorylation has emerged as a common theme for dynamic regulation of neuronal function (
      • Johnston J.
      • Forsythe I.D.
      • Kopp-Scheinpflug C.
      ,
      • Cerda O.
      • Trimmer J.S.
      ,
      • Shah M.M.
      • Hammond R.S.
      • Hoffman D.A.
      ) by allowing for integration between cell signaling pathways impacting the activity of specific neuronal PKs and PPs and the ion channels crucial for regulating neuronal excitability. Prominent examples include enhanced excitatory synaptic activity causing PKA-dependent phosphorylation and internalization of Kv4.2 in dendritic spines that results in enhancement of mEPSCs in hippocampal neurons (
      • Kim J.
      • Jung S.C.
      • Clemens A.M.
      • Petralia R.S.
      • Hoffman D.A.
      ,
      • Hammond R.S.
      • Lin L.
      • Sidorov M.S.
      • Wikenheiser A.M.
      • Hoffman D.A.
      ) and high frequency auditory stimulation causing rapid dephosphorylation of Kv3.1, leading to the enhancement of Kv3.1 activity needed to support high frequency spiking in auditory neurons (
      • Song P.
      • Yang Y.
      • Barnes-Davies M.
      • Bhattacharjee A.
      • Hamann M.
      • Forsythe I.D.
      • Oliver D.L.
      • Kaczmarek L.K.
      ). As detailed above, Kv2.1 is subjected to extensive bidirectional activity-dependent changes in phosphorylation state, changing its localization and function to homeostatically regulate neuronal excitability.
      Here we show that CDK5 is the key PK for determining the Kv2.1 phosphorylation state in neurons, including at the Ser-603 site that is key to phosphorylation-dependent regulation of Kv2.1 gating (
      • Park K.S.
      • Mohapatra D.P.
      • Misonou H.
      • Trimmer J.S.
      ) and at other sites that regulate Kv2.1 clustering. CDK5 can directly phosphorylate the recombinant Kv2.1 C terminus as well as Kv2.1 purified from mammalian brain. Moreover, we show here that CDK5 is responsible for Kv2.1 phosphorylation under diverse conditions of neuronal activity, including determining the constitutive level of Kv2.1 phosphorylation, the enhanced Kv2.1 phosphorylation that occurs after acute activity blockade, and the recovery of Kv2.1 phosphorylation after activity-dependent dephosphorylation. As such, CDK5 is poised to be a key determinant of the activity-dependent changes in Kv2.1 expression, localization, and function that have been found to underlie certain forms of plasticity in intrinsic excitability. Previous studies have established a clear role for CDK5 activity in nervous system development, such that inhibition, ablation, or knockdown of CDK5 leads to defects in neuronal migration, maturation, and survival (
      • Jessberger S.
      • Gage F.H.
      • Eisch A.J.
      • Lagace D.C.
      ). CDK5 has also been implicated as a key player in synaptic plasticity, with actions on both postsynaptic neurotransmitter receptors and presynaptic neurotransmitter release (
      • Lai K.O.
      • Ip N.Y.
      ). Although CDK5 has been recently implicated in regulating constitutive biosynthetic trafficking of neuronal Kv1 channels to the axon initial segment (
      • Vacher H.
      • Yang J.W.
      • Cerda O.
      • Autillo-Touati A.
      • Dargent B.
      • Trimmer J.S.
      ), a role for CDK5 in dynamic, reversible modulation of Kv channels or of other neuronal ion channels has not been described previously.
      We show here that CDK5 activity is required for the recovery of the phosphorylation and clustering of Kv2.1 protein after an episode of activity-induced, calcineurin-dependent dephosphorylation. Excitatory stimulation (e.g. glutamatergic stimulation or depolarization) has been found to reduce CDK5 activity in neurons, due to degradation of p35 and p39 regulatory subunits (
      • Schuman E.M.
      • Murase S.
      ,
      • Wei F.Y.
      • Tomizawa K.
      • Ohshima T.
      • Asada A.
      • Saito T.
      • Nguyen C.
      • Bibb J.A.
      • Ishiguro K.
      • Kulkarni A.B.
      • Pant H.C.
      • Mikoshiba K.
      • Matsui H.
      • Hisanaga S.
      ). Subsequent recovery of the expression level of these subunits and therefore of CDK5 activity begins at 90 min after washout of the stimulus (
      • Hosokawa T.
      • Saito T.
      • Asada A.
      • Ohshima T.
      • Itakura M.
      • Takahashi M.
      • Fukunaga K.
      • Hisanaga S.
      ), similar to the kinetics shown here and elsewhere (
      • Misonou H.
      • Mohapatra D.P.
      • Park E.W.
      • Leung V.
      • Zhen D.
      • Misonou K.
      • Anderson A.E.
      • Trimmer J.S.
      ,
      • Mulholland P.J.
      • Carpenter-Hyland E.P.
      • Hearing M.C.
      • Becker H.C.
      • Woodward J.J.
      • Chandler L.J.
      ) for recovery of Kv2.1 phosphorylation. As such, our findings are consistent with a mechanism whereby de novo synthesis of obligatory p35/p39 subunits and their association in active CDK5 complexes is the rate-limiting step in the recovery of Kv2.1 phosphorylation after calcineurin-dependent dephosphorylation in response to excitatory stimulation. Whether changes in the activity of neuronal PPs (PP1 and/or calcineurin) or PKs other than CDK5 are also involved in determining other aspects of the recovery of Kv2.1 phosphorylation is at yet unknown. It is intriguing, given their opposing roles in regulating Kv2.1, that CDK5 and calcineurin also have counteracting activity-dependent effects on synaptic vesicle endocytosis, via phosphorylation of components of the release machinery (
      • Tan T.C.
      • Valova V.A.
      • Malladi C.S.
      • Graham M.E.
      • Berven L.A.
      • Jupp O.J.
      • Hansra G.
      • McClure S.J.
      • Sarcevic B.
      • Boadle R.A.
      • Larsen M.R.
      • Cousin M.A.
      • Robinson P.J.
      ,
      • Tomizawa K.
      • Sunada S.
      • Lu Y.F.
      • Oda Y.
      • Kinuta M.
      • Ohshima T.
      • Saito T.
      • Wei F.Y.
      • Matsushita M.
      • Li S.T.
      • Tsutsui K.
      • Hisanaga S.
      • Mikoshiba K.
      • Takei K.
      • Matsui H.
      ).
      We found that the CDK inhibitor roscovitine blocks the increased phosphorylation of Kv2.1 induced by acute neuronal activity blockade. That Kv2.1 is a direct substrate for CDK5 in vitro suggests that the enhanced Kv2.1 phosphorylation upon activity blockade is due to increased CDK5 activity and direct CDK5 phosphorylation of Kv2.1. Whereas the activity of most other PKs is under the control of either second messengers (e.g. cAMP, Ca2+, DAG, etc.) or phosphorylation cascades involving other PKs or PPs, either of which can be rapidly modulated by neuronal signaling pathways, all available evidence suggests that CDK5 activity is exclusively regulated by the levels of its obligatory p35 and p39 subunits, as determined by their de novo synthesis and degradation (
      • Hisanaga S.
      • Saito T.
      ). Rapid activity-dependent down-regulation of CDK5 activity is achieved by triggered degradation of p35/p39 in response to neuronal depolarization (
      • Schuman E.M.
      • Murase S.
      ,
      • Tan T.C.
      • Valova V.A.
      • Malladi C.S.
      • Graham M.E.
      • Berven L.A.
      • Jupp O.J.
      • Hansra G.
      • McClure S.J.
      • Sarcevic B.
      • Boadle R.A.
      • Larsen M.R.
      • Cousin M.A.
      • Robinson P.J.
      ) and NMDA stimulation (
      • Wei F.Y.
      • Tomizawa K.
      • Ohshima T.
      • Asada A.
      • Saito T.
      • Nguyen C.
      • Bibb J.A.
      • Ishiguro K.
      • Kulkarni A.B.
      • Pant H.C.
      • Mikoshiba K.
      • Matsui H.
      • Hisanaga S.
      ). Acute up-regulation of CDK5 activity would therefore require rapid de novo synthesis of p35 and/or p39 and their association with plasma membrane-localized CDK5, events unlikely to occur within the short time frame of acute activity blockade (15 min) that we found induces enhanced Kv2.1 phosphorylation. Extensive relocalization of CDK5 in response to excitotoxic stimulation occurs through calpain-mediated cleavage of the myristoylated region from p35, leading to loss of the plasma membrane association of the CDK5 complex and its translocation into the cytoplasm and resulting in phosphorylation of non-physiological CDK5 substrates and neurotoxicity nucleus (
      • Lee M.S.
      • Kwon Y.T.
      • Li M.
      • Peng J.
      • Friedlander R.M.
      • Tsai L.H.
      ). Although changes in the subcellular localization of the obligatory subunits or of preexisting active CDK5 complexes to sites of high density Kv2.1 clustering could underlie the rapid increase in Kv2.1 phosphorylation upon acute activity blockade, relocalization of CDK5 and its regulatory subunits to access different plasma membrane substrates has not been described. Interestingly, a previous study revealed a slight increase in DARPP-32 phosphorylation at Thr-75, a known CDK5 phosphorylation site, in response to short (10-min) TTX treatment (
      • Nishi A.
      • Bibb J.A.
      • Matsuyama S.
      • Hamada M.
      • Higashi H.
      • Nairn A.C.
      • Greengard P.
      ), providing additional suggestive evidence for rapid activation of CDK5 in response to acute activity blockade. Future studies will determine the mechanisms whereby CDK5 activity can be rapidly stimulated by acute activity blockade and how it then acts via phosphorylation of Kv2.1 and other substrates to mediate responses to decreased neuronal activity.
      We found that pharmacological inhibition of PP1 in unstimulated neurons dramatically increases phosphorylation of Kv2.1 at the CDK5-dependent Ser-603 phosphorylation site. This is in sharp contrast to calcineurin inhibitors, which have little effect on constitutive phosphorylation of Kv2.1 but abolish the rapid dephosphorylation of Kv2.1 in response to excitatory stimuli (
      • Misonou H.
      • Mohapatra D.P.
      • Park E.W.
      • Leung V.
      • Zhen D.
      • Misonou K.
      • Anderson A.E.
      • Trimmer J.S.
      ,
      • Misonou H.
      • Menegola M.
      • Mohapatra D.P.
      • Guy L.K.
      • Park K.S.
      • Trimmer J.S.
      ). We found that PP1 overexpression leads to decreased Kv2.1 phosphorylation in heterologous cells and that PP1, like calcineurin (
      • Misonou H.
      • Menegola M.
      • Mohapatra D.P.
      • Guy L.K.
      • Park K.S.
      • Trimmer J.S.
      ), can directly dephosphorylate immunopurified Kv2.1 in vitro. PP1 is involved in diverse aspects of neuronal plasticity (
      • Munton R.P.
      • Vizi S.
      • Mansuy I.M.
      ), and ion channels are prominent targets for PP1-mediated dephosphorylation (
      • Dai S.
      • Hall D.D.
      • Hell J.W.
      ). Interestingly, PP1 activity is negatively regulated by CDK5 phosphorylation, via inhibitory phosphorylation at the Thr-320 site on PP1 (
      • Dohadwala M.
      • da Cruz e Silva E.F.
      • Hall F.L.
      • Williams R.T.
      • Carbonaro-Hall D.A.
      • Nairn A.C.
      • Greengard P.
      • Berndt N.
      ,
      • Li T.
      • Chalifour L.E.
      • Paudel H.K.
      ). As such, inhibition of CDK5 could lead to enhanced PP1 activity, driving the dephosphorylation of Kv2.1 via an indirect mechanism that is not dependent on reduced CDK5-mediated Kv2.1 phosphorylation. We showed here that the effects of expressing a mutant PP1 (PP1-T320A) that is resistant to CDK5-mediated inhibition were indistinguishable from those of WT PP1, supporting a role for direct CDK5-mediated phosphorylation of Kv2.1 as the primary determinant of the rapid increase in Kv2.1 phosphorylation seen upon acute activity blockade and during the recovery period following excitation-induced, calcineurin-mediated, Kv2.1 dephosphorylation. However, because PP1 activity does seem to be important in establishing the steady-state level of Kv2.1 phosphorylation, its possible contributions to regulating Kv2.1 phosphorylation during these other processes cannot be completely discounted. Future studies will reveal the precise interplay between CDK5 and neuronal PPs, such as PP1 and calcineurin, in determining the phosphorylation state of Kv2.1 and of other ion channels whose modulation alters intrinsic excitability and that act as the molecular substrates for intrinsic neuronal plasticity.

      Acknowledgments

      We thank Dr. Li-Huei Tsai (MIT) for providing the pcDNA-GFP-CDK5-D144N, pcDNA3-GFP-CDK5, and pCMV-myc-p35 plasmids (via Addgene, plasmids 1344, 1346, and 1347, respectively) and Dr. Hemant Paudel (McGill University) for providing the pcDNA-myc-PP1 and pcDNA-myc-PP1 (T320A). Mass spectrometry was performed at the University of California Davis Proteomics Facility. We are grateful to Ashleigh Evans for critical comments on the manuscript and to Jesus Aguado, Ada Kan, Linda Sah, and Rachel Silverman for technical assistance.

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