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

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. 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. IntroductionPlasticity 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 (1Baranauskas G. Tkatch T. Surmeier D.J. J. Neurosci. 1999; 19: 6394-6404Crossref PubMed Google Scholar, 2Murakoshi H. Trimmer J.S. J. Neurosci. 1999; 19: 1728-1735Crossref PubMed Google Scholar, 3Guan D. Tkatch T. Surmeier D.J. Armstrong W.E. Foehring R.C. J. Physiol. 2007; 581: 941-960Crossref PubMed Scopus (82) Google Scholar), 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 (4Murakoshi H. Shi G. Scannevin R.H. Trimmer J.S. Mol. Pharmacol. 1997; 52: 821-828Crossref PubMed Scopus (127) Google Scholar, 5Misonou H. Mohapatra D.P. Park E.W. Leung V. Zhen D. Misonou K. Anderson A.E. Trimmer J.S. Nat. Neurosci. 2004; 7: 711-718Crossref PubMed Scopus (342) Google Scholar, 6Misonou H. Mohapatra D.P. Menegola M. Trimmer J.S. J. Neurosci. 2005; 25: 11184-11193Crossref PubMed Scopus (140) Google Scholar, 7Misonou H. Menegola M. Mohapatra D.P. Guy L.K. Park K.S. Trimmer J.S. J. Neurosci. 2006; 26: 13505-13514Crossref PubMed Scopus (83) Google Scholar, 8Misonou H. Thompson S.M. Cai X. J. Neurosci. 2008; 28: 8529-8538Crossref PubMed Scopus (52) Google Scholar, 9Mulholland P.J. Carpenter-Hyland E.P. Hearing M.C. Becker H.C. Woodward J.J. Chandler L.J. J. Neurosci. 2008; 28: 8801-8809Crossref PubMed Scopus (55) Google Scholar, 10Aras M.A. Saadi R.A. Aizenman E. Eur. J. Neurosci. 2009; 30: 2250-2257Crossref PubMed Scopus (25) Google Scholar, 11Mulholland P.J. Carpenter-Hyland E.P. Woodward J.J. Chandler L.J. Alcohol. 2009; 43: 45-50Crossref PubMed Scopus (18) Google Scholar, 12Ito T. Nuriya M. Yasui M. Neurobiol. Dis. 2010; 38: 85-91Crossref PubMed Scopus (11) Google Scholar) and leading to homeostatic suppression of neuronal firing (6Misonou H. Mohapatra D.P. Menegola M. Trimmer J.S. J. Neurosci. 2005; 25: 11184-11193Crossref PubMed Scopus (140) Google Scholar, 13Mohapatra D.P. Misonou H. Pan S.J. Held J.E. Surmeier D.J. Trimmer J.S. Channels. 2009; 3: 46-56Crossref PubMed Scopus (72) Google Scholar). Removal of these stimuli leads to recovery of Kv2.1 phosphorylation and clustering (5Misonou H. Mohapatra D.P. Park E.W. Leung V. Zhen D. Misonou K. Anderson A.E. Trimmer J.S. Nat. Neurosci. 2004; 7: 711-718Crossref PubMed Scopus (342) Google Scholar, 7Misonou H. Menegola M. Mohapatra D.P. Guy L.K. Park K.S. Trimmer J.S. J. Neurosci. 2006; 26: 13505-13514Crossref PubMed Scopus (83) Google Scholar, 9Mulholland P.J. Carpenter-Hyland E.P. Hearing M.C. Becker H.C. Woodward J.J. Chandler L.J. J. Neurosci. 2008; 28: 8801-8809Crossref PubMed Scopus (55) Google Scholar, 10Aras M.A. Saadi R.A. Aizenman E. Eur. J. Neurosci. 2009; 30: 2250-2257Crossref PubMed Scopus (25) Google Scholar, 12Ito T. Nuriya M. Yasui M. Neurobiol. Dis. 2010; 38: 85-91Crossref PubMed Scopus (11) Google Scholar). Anesthesia in vivo induces enhanced Kv2.1 phosphorylation (7Misonou H. Menegola M. Mohapatra D.P. Guy L.K. Park K.S. Trimmer J.S. J. Neurosci. 2006; 26: 13505-13514Crossref PubMed Scopus (83) Google Scholar), 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 (14Nataraj K. Le Roux N. Nahmani M. Lefort S. Turrigiano G. Neuron. 2010; 68: 750-762Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar).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 (15Park K.S. Mohapatra D.P. Misonou H. Trimmer J.S. Science. 2006; 313: 976-979Crossref PubMed Scopus (225) Google Scholar, 16Park K.S. Mohapatra D.P. Trimmer J.S. Channels. 2007; 1: 59-61Crossref PubMed Scopus (19) Google Scholar), a subset of which are dephosphorylated upon calcineurin activation and mediate the activity-dependent changes in Kv2.1 localization and function (7Misonou H. Menegola M. Mohapatra D.P. Guy L.K. Park K.S. Trimmer J.S. J. Neurosci. 2006; 26: 13505-13514Crossref PubMed Scopus (83) Google Scholar, 15Park K.S. Mohapatra D.P. Misonou H. Trimmer J.S. Science. 2006; 313: 976-979Crossref PubMed Scopus (225) Google Scholar). Among these sites, phosphorylation at the Ser-603 residue exhibits extraordinary sensitivity to bidirectional activity-dependent changes in phosphorylation state (7Misonou H. Menegola M. Mohapatra D.P. Guy L.K. Park K.S. Trimmer J.S. J. Neurosci. 2006; 26: 13505-13514Crossref PubMed Scopus (83) Google Scholar). The protein phosphatases (PPs) 2The abbreviations used are: PPprotein phosphataseAPalkaline phosphatasePKprotein kinaseRBMrat brain membraneHBSSHanks' buffered saline solutionDIVdays in vitroRSBreducing SDS sample bufferDPBSDulbecco's phosphate-buffered salineTTXtetrodotoxin. PP1 and calcineurin/PP2B have been identified as playing crucial and non-overlapping roles in constitutive and activity-dependent dephosphorylation of Kv2.1, respectively (5Misonou H. Mohapatra D.P. Park E.W. Leung V. Zhen D. Misonou K. Anderson A.E. Trimmer J.S. Nat. Neurosci. 2004; 7: 711-718Crossref PubMed Scopus (342) Google Scholar, 7Misonou H. Menegola M. Mohapatra D.P. Guy L.K. Park K.S. Trimmer J.S. J. Neurosci. 2006; 26: 13505-13514Crossref PubMed Scopus (83) Google Scholar). 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 (17Tsai L.H. Delalle I. Caviness Jr., V.S. Chae T. Harlow E. Nature. 1994; 371: 419-423Crossref PubMed Scopus (805) Google Scholar, 18Cai X.H. Tomizawa K. Tang D. Lu Y.F. Moriwaki A. Tokuda M. Nagahata S. Hatase O. Matsui H. Neurosci. Res. 1997; 28: 355-360Crossref PubMed Scopus (47) Google Scholar, 19Lai K.O. Ip N.Y. Biochim. Biophys. Acta. 2009; 1792: 741-745Crossref PubMed Scopus (72) Google Scholar). 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.DISCUSSIONPlasticity 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 (34Johnston J. Forsythe I.D. Kopp-Scheinpflug C. J. Physiol. 2010; 588: 3187-3200Crossref PubMed Scopus (196) Google Scholar). 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 (35Johnston D. Christie B.R. Frick A. Gray R. Hoffman D.A. Schexnayder L.K. Watanabe S. Yuan L.L. Philos. Trans. R. Soc. Lond. B Biol. Sci. 2003; 358: 667-674Crossref PubMed Scopus (211) Google Scholar), initiation and propagation of axonal action potentials (36Kress G.J. Mennerick S. Neuroscience. 2009; 158: 211-222Crossref PubMed Scopus (69) Google Scholar), and regulation of neurotransmitter release (37Dodson P.D. Forsythe I.D. Trends Neurosci. 2004; 27: 210-217Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar). 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 (34Johnston J. Forsythe I.D. Kopp-Scheinpflug C. J. Physiol. 2010; 588: 3187-3200Crossref PubMed Scopus (196) Google Scholar, 38Cerda O. Trimmer J.S. Neurosci. Lett. 2010; 486: 60-67Crossref PubMed Scopus (36) Google Scholar, 39Shah M.M. Hammond R.S. Hoffman D.A. Trends Neurosci. 2010; 33: 307-316Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar) 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 (40Kim J. Jung S.C. Clemens A.M. Petralia R.S. Hoffman D.A. Neuron. 2007; 54: 933-947Abstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar, 41Hammond R.S. Lin L. Sidorov M.S. Wikenheiser A.M. Hoffman D.A. J. Neurosci. 2008; 28: 7513-7519Crossref PubMed Scopus (71) Google Scholar) 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 (42Song P. Yang Y. Barnes-Davies M. Bhattacharjee A. Hamann M. Forsythe I.D. Oliver D.L. Kaczmarek L.K. Nat. Neurosci. 2005; 8: 1335-1342Crossref PubMed Scopus (105) Google Scholar). 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 (15Park K.S. Mohapatra D.P. Misonou H. Trimmer J.S. Science. 2006; 313: 976-979Crossref PubMed Scopus (225) Google Scholar) 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 (43Jessberger S. Gage F.H. Eisch A.J. Lagace D.C. Trends Neurosci. 2009; 32: 575-582Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). CDK5 has also been implicated as a key player in synaptic plasticity, with actions on both postsynaptic neurotransmitter receptors and presynaptic neurotransmitter release (19Lai K.O. Ip N.Y. Biochim. Biophys. Acta. 2009; 1792: 741-745Crossref PubMed Scopus (72) Google Scholar). Although CDK5 has been recently implicated in regulating constitutive biosynthetic trafficking of neuronal Kv1 channels to the axon initial segment (44Vacher H. Yang J.W. Cerda O. Autillo-Touati A. Dargent B. Trimmer J.S. J. Cell Biol. 2011; 192: 813-824Crossref PubMed Scopus (53) Google Scholar), 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 (28Schuman E.M. Murase S. Philos. Trans. R. Soc. Lond. B Biol. Sci. 2003; 358: 749-756Crossref PubMed Scopus (33) Google Scholar, 30Wei F.Y. Tomizawa K. Ohshima T. Asada A. Saito T. Nguyen C. Bibb K. H.C. K. Matsui H. S. J. 2005; PubMed Scopus Google Scholar). recovery of the level of these subunits and of CDK5 activity at after of the T. Saito T. Asada A. Ohshima T. M. M. K. S. J. Neurosci. Res. 2006; PubMed Scopus Google Scholar), to the here and (5Misonou H. Mohapatra D.P. Park E.W. Leung V. Zhen D. Misonou K. Anderson A.E. Trimmer J.S. Nat. Neurosci. 2004; 7: 711-718Crossref PubMed Scopus (342) Google Scholar, 9Mulholland P.J. Carpenter-Hyland E.P. Hearing M.C. Becker H.C. Woodward J.J. Chandler L.J. J. Neurosci. 2008; 28: 8801-8809Crossref PubMed Scopus (55) Google Scholar) for recovery of Kv2.1 phosphorylation. As such, are with a mechanism of subunits and their association in CDK5 is the in the recovery of Kv2.1 phosphorylation after calcineurin-dependent dephosphorylation in response to excitatory changes in the activity of neuronal PPs and/or or PKs other CDK5 are also in determining other aspects of the recovery of Kv2.1 phosphorylation is at is their roles in regulating Kv2.1, that CDK5 and calcineurin also have activity-dependent on synaptic phosphorylation of of the release G. S.J. B. R.A. M.A. P.J. Nat. Cell Biol. 2003; PubMed Scopus Google Scholar, K. S. Lu Y.F. Y. M. Ohshima T. Saito T. F.Y. M. K. S. K. K. Matsui H. J. Cell Biol. 2003; 813-824Crossref PubMed Scopus Google found that the the phosphorylation of Kv2.1 by acute neuronal activity Kv2.1 is a direct for CDK5 in that the enhanced Kv2.1 phosphorylation upon activity blockade is due to CDK5 activity and direct CDK5 phosphorylation of the activity of most other PKs is under the of (e.g. or phosphorylation other PKs or of which can be by neuronal signaling that CDK5 activity is by the of its p35 and p39 as by their and degradation S. Saito T. 2003; PubMed Scopus Google Scholar). activity-dependent of CDK5 activity is by degradation of in response to neuronal (28Schuman E.M. Murase S. Philos. Trans. R. Soc. Lond. B Biol. Sci. 2003; 358: 749-756Crossref PubMed Scopus (33) Google Scholar, G. S.J. B. R.A. M.A. P.J. Nat. Cell Biol. 2003; PubMed Scopus Google Scholar) and stimulation F.Y. Tomizawa K. Ohshima T. Asada A. Saito T. Nguyen C. Bibb K. H.C. K. Matsui H. S. J. 2005; PubMed Scopus Google Scholar). of CDK5 activity rapid of p35 and/or p39 and their association with to within the of acute activity blockade that we found induces enhanced Kv2.1 phosphorylation. of CDK5 in response to stimulation occurs through of the from leading to loss of the association of the CDK5 and its into the and resulting in phosphorylation of CDK5 and M.S. M. J. L.H. Nature. PubMed Scopus Google Scholar). Although changes in the subcellular localization of the subunits or of CDK5 to sites of high density Kv2.1 clustering underlie the rapid increase in Kv2.1 phosphorylation upon acute activity blockade, of CDK5 and its regulatory subunits to has not been a a increase in phosphorylation at a CDK5 phosphorylation in response to A. Bibb S. M. H. P. J. PubMed Scopus Google Scholar), for rapid activation of CDK5 in response to acute activity studies determine the CDK5 activity can be by acute activity blockade and how phosphorylation of Kv2.1 and other to mediate to neuronal found that of PP1 in neurons phosphorylation of Kv2.1 at the Ser-603 phosphorylation is in to calcineurin which have on constitutive phosphorylation of Kv2.1 the rapid dephosphorylation of Kv2.1 in response to excitatory stimuli (5Misonou H. Mohapatra D.P. Park E.W. Leung V. Zhen D. Misonou K. Anderson A.E. Trimmer J.S. Nat. Neurosci. 2004; 7: 711-718Crossref PubMed Scopus (342) Google Scholar, 7Misonou H. Menegola M. Mohapatra D.P. Guy L.K. Park K.S. Trimmer J.S. J. Neurosci. 2006; 26: 13505-13514Crossref PubMed Scopus (83) Google Scholar). We found that PP1 leads to Kv2.1 phosphorylation in and that calcineurin (7Misonou H. Menegola M. Mohapatra D.P. Guy L.K. Park K.S. Trimmer J.S. J. Neurosci. 2006; 26: 13505-13514Crossref PubMed Scopus (83) Google Scholar), can directly Kv2.1 in PP1 is in diverse aspects of neuronal plasticity S. Lett. 2004; PubMed Scopus (72) Google Scholar), and ion channels are for dephosphorylation S. J.W. Physiol. 2009; PubMed Scopus Google Scholar). PP1 activity is by CDK5 phosphorylation, phosphorylation at the site on PP1 M. D.A. P. N. Sci. 1994; PubMed Scopus Google Scholar, T. J. Biol. 2007; Full Text Full Text PDF PubMed Scopus Google Scholar). As such, of CDK5 to enhanced PP1 activity, the dephosphorylation of Kv2.1 an mechanism that is not on Kv2.1 phosphorylation. We here that the of a PP1 that is to from of a role for direct phosphorylation of Kv2.1 as the primary determinant of the rapid increase in Kv2.1 phosphorylation upon acute activity blockade and the recovery Kv2.1 dephosphorylation. However, PP1 activity to be in the level of Kv2.1 phosphorylation, its to regulating Kv2.1 phosphorylation these other be studies the between CDK5 and neuronal such as PP1 and in determining the phosphorylation state of Kv2.1 and of other ion channels whose modulation intrinsic excitability and that as the for intrinsic neuronal IntroductionPlasticity 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 (1Baranauskas G. Tkatch T. Surmeier D.J. J. Neurosci. 1999; 19: 6394-6404Crossref PubMed Google Scholar, 2Murakoshi H. Trimmer J.S. J. Neurosci. 1999; 19: 1728-1735Crossref PubMed Google Scholar, 3Guan D. Tkatch T. Surmeier D.J. Armstrong W.E. Foehring R.C. J. Physiol. 2007; 581: 941-960Crossref PubMed Scopus (82) Google Scholar), 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 (4Murakoshi H. Shi G. Scannevin R.H. Trimmer J.S. Mol. Pharmacol. 1997; 52: 821-828Crossref PubMed Scopus (127) Google Scholar, 5Misonou H. Mohapatra D.P. Park E.W. Leung V. Zhen D. Misonou K. Anderson A.E. Trimmer J.S. Nat. Neurosci. 2004; 7: 711-718Crossref PubMed Scopus (342) Google Scholar, 6Misonou H. Mohapatra D.P. Menegola M. Trimmer J.S. J. Neurosci. 2005; 25: 11184-11193Crossref PubMed Scopus (140) Google Scholar, 7Misonou H. Menegola M. Mohapatra D.P. Guy L.K. Park K.S. Trimmer J.S. J. Neurosci. 2006; 26: 13505-13514Crossref PubMed Scopus (83) Google Scholar, 8Misonou H. Thompson S.M. Cai X. J. Neurosci. 2008; 28: 8529-8538Crossref PubMed Scopus (52) Google Scholar, 9Mulholland P.J. Carpenter-Hyland E.P. Hearing M.C. Becker H.C. Woodward J.J. Chandler L.J. J. Neurosci. 2008; 28: 8801-8809Crossref PubMed Scopus (55) Google Scholar, 10Aras M.A. Saadi R.A. Aizenman E. Eur. J. Neurosci. 2009; 30: 2250-2257Crossref PubMed Scopus (25) Google Scholar, 11Mulholland P.J. Carpenter-Hyland E.P. Woodward J.J. Chandler L.J. Alcohol. 2009; 43: 45-50Crossref PubMed Scopus (18) Google Scholar, 12Ito T. Nuriya M. Yasui M. Neurobiol. Dis. 2010; 38: 85-91Crossref PubMed Scopus (11) Google Scholar) and leading to homeostatic suppression of neuronal firing (6Misonou H. Mohapatra D.P. Menegola M. Trimmer J.S. J. Neurosci. 2005; 25: 11184-11193Crossref PubMed Scopus (140) Google Scholar, 13Mohapatra D.P. Misonou H. Pan S.J. Held J.E. Surmeier D.J. Trimmer J.S. Channels. 2009; 3: 46-56Crossref PubMed Scopus (72) Google Scholar). Removal of these stimuli leads to recovery of Kv2.1 phosphorylation and clustering (5Misonou H. Mohapatra D.P. Park E.W. Leung V. Zhen D. Misonou K. Anderson A.E. Trimmer J.S. Nat. Neurosci. 2004; 7: 711-718Crossref PubMed Scopus (342) Google Scholar, 7Misonou H. Menegola M. Mohapatra D.P. Guy L.K. Park K.S. Trimmer J.S. J. Neurosci. 2006; 26: 13505-13514Crossref PubMed Scopus (83) Google Scholar, 9Mulholland P.J. Carpenter-Hyland E.P. Hearing M.C. Becker H.C. Woodward J.J. Chandler L.J. J. Neurosci. 2008; 28: 8801-8809Crossref PubMed Scopus (55) Google Scholar, 10Aras M.A. Saadi R.A. Aizenman E. Eur. J. Neurosci. 2009; 30: 2250-2257Crossref PubMed Scopus (25) Google Scholar, 12Ito T. Nuriya M. Yasui M. Neurobiol. Dis. 2010; 38: 85-91Crossref PubMed Scopus (11) Google Scholar). Anesthesia in vivo induces enhanced Kv2.1 phosphorylation (7Misonou H. Menegola M. Mohapatra D.P. Guy L.K. Park K.S. Trimmer J.S. J. Neurosci. 2006; 26: 13505-13514Crossref PubMed Scopus (83) Google Scholar), 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 (14Nataraj K. Le Roux N. Nahmani M. Lefort S. Turrigiano G. Neuron. 2010; 68: 750-762Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar).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 (15Park K.S. Mohapatra D.P. Misonou H. Trimmer J.S. Science. 2006; 313: 976-979Crossref PubMed Scopus (225) Google Scholar, 16Park K.S. Mohapatra D.P. Trimmer J.S. Channels. 2007; 1: 59-61Crossref PubMed Scopus (19) Google Scholar), a subset of which are dephosphorylated upon calcineurin activation and mediate the activity-dependent changes in Kv2.1 localization and function (7Misonou H. Menegola M. Mohapatra D.P. Guy L.K. Park K.S. Trimmer J.S. J. Neurosci. 2006; 26: 13505-13514Crossref PubMed Scopus (83) Google Scholar, 15Park K.S. Mohapatra D.P. Misonou H. Trimmer J.S. Science. 2006; 313: 976-979Crossref PubMed Scopus (225) Google Scholar). Among these sites, phosphorylation at the Ser-603 residue exhibits extraordinary sensitivity to bidirectional activity-dependent changes in phosphorylation state (7Misonou H. Menegola M. Mohapatra D.P. Guy L.K. Park K.S. Trimmer J.S. J. Neurosci. 2006; 26: 13505-13514Crossref PubMed Scopus (83) Google Scholar). The protein phosphatases (PPs) 2The abbreviations used are: PPprotein phosphataseAPalkaline phosphatasePKprotein kinaseRBMrat brain membraneHBSSHanks' buffered saline solutionDIVdays in vitroRSBreducing SDS sample bufferDPBSDulbecco's phosphate-buffered salineTTXtetrodotoxin. PP1 and calcineurin/PP2B have been identified as playing crucial and non-overlapping roles in constitutive and activity-dependent dephosphorylation of Kv2.1, respectively (5Misonou H. Mohapatra D.P. Park E.W. Leung V. Zhen D. Misonou K. Anderson A.E. Trimmer J.S. Nat. Neurosci. 2004; 7: 711-718Crossref PubMed Scopus (342) Google Scholar, 7Misonou H. Menegola M. Mohapatra D.P. Guy L.K. Park K.S. Trimmer J.S. J. Neurosci. 2006; 26: 13505-13514Crossref PubMed Scopus (83) Google Scholar). 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 (17Tsai L.H. Delalle I. Caviness Jr., V.S. Chae T. Harlow E. Nature. 1994; 371: 419-423Crossref PubMed Scopus (805) Google Scholar, 18Cai X.H. Tomizawa K. Tang D. Lu Y.F. Moriwaki A. Tokuda M. Nagahata S. Hatase O. Matsui H. Neurosci. Res. 1997; 28: 355-360Crossref PubMed Scopus (47) Google Scholar, 19Lai K.O. Ip N.Y. Biochim. Biophys. Acta. 2009; 1792: 741-745Crossref PubMed Scopus (72) Google Scholar). 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.

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