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Phosphorylation of Gephyrin in Hippocampal Neurons by Cyclin-dependent Kinase CDK5 at Ser-270 Is Dependent on Collybistin

Open AccessPublished:July 09, 2012DOI:https://doi.org/10.1074/jbc.M112.349597
      Gephyrin is a scaffold protein essential for the postsynaptic clustering of inhibitory glycine and different subtypes of GABAA receptors. The cellular and molecular mechanisms involved in gephyrin-mediated receptor clustering are still not well understood. Here we provide evidence that the gephyrin-binding protein collybistin is involved in regulating the phosphorylation of gephyrin. We demonstrate that the widely used monoclonal antibody mAb7a is a phospho-specific antibody that allows the cellular and biochemical analysis of gephyrin phosphorylation at Ser-270. In addition, another neighbored epitope determinant was identified at position Thr-276. Analysis of the double mutant gephyrinT276A,S277A revealed significant reduction in gephyrin cluster formation and altered oligomerization behavior of gephyrin. Moreover, pharmacological inhibition of cyclin-dependent kinases in hippocampal neurons reduced postsynaptic gephyrin mAb7a immunoreactivities. In vitro phosphorylation assays and phosphopeptide competition experiments revealed a phosphorylation at Ser-270 depending on enzyme activities of cyclin-dependent kinases CDK1, -2, or -5. These data indicate that collybistin and cyclin-dependent kinases are involved in regulating the phosphorylation of gephyrin at postsynaptic membrane specializations.

      Introduction

      The formation of clusters of inhibitory glycine receptors (GlyR)
      The abbreviations used are: GlyR
      glycine receptor
      Cb
      Collybistin
      PH
      pleckstrin homology
      CDK
      cyclin-dependent kinase
      VIAAT
      vesicular inhibitory amino acid transporter
      GSK
      glycogen synthase kinase.
      and some subtypes of GABAA receptors at the postsynaptic membrane strictly requires the scaffold protein gephyrin (
      • Feng G.
      • Tintrup H.
      • Kirsch J.
      • Nichol M.C.
      • Kuhse J.
      • Betz H.
      • Sanes J.R
      Dual requirement for gephyrin in glycine receptor clustering and molybdoenzyme activity.
      ). It is believed that gephyrin-dependent cluster formation is achieved by anchoring receptors to the microtubular and actin-based cytoskeleton (
      • Kirsch J.
      • Betz H.
      The postsynaptic localization of the glycine receptor-associated protein gephyrin is regulated by the cytoskeleton.
      ). However, the molecular mechanisms regulating the precise localization and size of gephyrin and GlyR/GABAA receptor clusters within the somatodendritic compartments are not completely understood. Collybistin (Cb), a gephyrin binding guanidine exchange factor for the monomeric GTPase Cdc42 (
      • Kins S.
      • Betz H.
      • Kirsch J.
      Collybistin, a newly identified brain-specific GEF, induces submembrane clustering of gephyrin.
      ,
      • Harvey K.
      • Duguid I.C.
      • Alldred M.J.
      • Beatty S.E.
      • Ward H.
      • Keep N.H.
      • Lingenfelter S.E.
      • Pearce B.R.
      • Lundgren J.
      • Owen M.J.
      • Smart T.G.
      • Lüscher B.
      • Rees M.I.
      • Harvey R.J.
      The GDP-GTP exchange factor collybistin. An essential determinant of neuronal gephyrin clustering.
      ) may regulate local cluster formation by interaction with the cell adhesion protein neuroligin II (
      • Poulopoulos A.
      • Aramuni G.
      • Meyer G.
      • Soykan T.
      • Hoon M.
      • Papadopoulos T.
      • Zhang M.
      • Paarmann I.
      • Fuchs C.
      • Harvey K.
      • Jedlicka P.
      • Schwarzacher S.W.
      • Betz H.
      • Harvey R.J.
      • Brose N.
      • Zhang W.
      • Varoqueaux F.
      Neuroligin 2 drives postsynaptic assembly at perisomatic inhibitory synapses through gephyrin and collybistin.
      ). Studies of Cb knock-out mice revealed a reduction of gephyrin and γ2-containing GABAA receptor clusters in hippocampus and cerebellum, whereas GlyR clusters in spinal cord neurons were unaltered (
      • Papadopoulos T.
      • Korte M.
      • Eulenburg V.
      • Kubota H.
      • Retiounskaia M.
      • Harvey R.J.
      • Harvey K.
      • O′Sullivan G.A.
      • Laube B.
      • Hülsmann S.
      • Geiger J.R.
      • Betz H.
      Impaired GABAergic transmission and altered hippocampal synaptic plasticity in collybistin-deficient mice.
      ). Thus, the functional role(s) of Cb in different areas of the CNS remains to be established (
      • Papadopoulos T.
      • Soykan T.
      The role of collybistin in gephyrin clustering at inhibitory synapses. Facts and open questions.
      ,
      • Shimojima K.
      • Sugawara M.
      • Shichiji M.
      • Mukaida S.
      • Takayama R.
      • Imai K.
      • Yamamoto T.
      Loss-of-function mutation of collybistin is responsible for X-linked mental retardation associated with epilepsy.
      ).
      In rat, four alternative spliced Cb isoforms have been described (Cb1–4). All variants possess a central tandem dbl-homology (DH)/pleckstrin homology (PH)-domain; apparently a functional PH-, but not DH-domain, is required for proper function (
      • Reddy-Alla S.
      • Schmitt B.
      • Birkenfeld J.
      • Eulenburg V.
      • Dutertre S.
      • Böhringer C.
      • Götz M.
      • Betz H.
      • Papadopoulos T.
      PH-domain-driven targeting of collybistin but not Cdc42 activation is required for synaptic gephyrin clustering.
      ). Moreover, one isoform (Cb2) exists with and without an src-homology-3 (SH3)-domain near the N terminus (
      • Kins S.
      • Betz H.
      • Kirsch J.
      Collybistin, a newly identified brain-specific GEF, induces submembrane clustering of gephyrin.
      ,
      • Harvey K.
      • Duguid I.C.
      • Alldred M.J.
      • Beatty S.E.
      • Ward H.
      • Keep N.H.
      • Lingenfelter S.E.
      • Pearce B.R.
      • Lundgren J.
      • Owen M.J.
      • Smart T.G.
      • Lüscher B.
      • Rees M.I.
      • Harvey R.J.
      The GDP-GTP exchange factor collybistin. An essential determinant of neuronal gephyrin clustering.
      ).
      The domain structure of gephyrin is composed of an N-terminal G-domain and a C-terminal E-domain. Isolated G- and E-domains form trimers and dimers, respectively, and thus were proposed to cause the formation of a hexagonal lattice of gephyrin at the postsynaptic membrane (
      • Sola M.
      • Bavro V.N.
      • Timmins J.
      • Franz T.
      • Ricard-Blum S.
      • Schoehn G.
      • Ruigrok R.W.
      • Paarmann I.
      • Saiyed T.
      • O′Sullivan G.A.
      • Schmitt B.
      • Betz H.
      • Weissenhorn W.
      Structural basis of dynamic glycine receptor clustering by gephyrin.
      ). Both domains are connected by the central C-domain, which harbors binding sites for several gephyrin interacting proteins (
      • Fritschy J.M.
      • Harvey R.J.
      • Schwarz G.
      Gephyrin. Where do we stand, where do we go?.
      ).
      GlyRs and GABAA receptor are pentameric receptor complexes. The homologous subunits contain four transmembrane domains (TM1–4), and the extended cytoplasmic loop between the TM3 and TM4 regions (
      • Betz H.
      • Kuhse J.
      • Schmieden V.
      • Laube B.
      • Kirsch J.
      • Harvey R.J.
      Structure and functions of inhibitory and excitatory glycine receptors.
      ,
      • Luscher B.
      • Fuchs T.
      • Kilpatrick C.L.
      GABAA receptor trafficking-mediated plasticity of inhibitory synapses.
      ) of the GlyR β subunit mediates the binding to gephyrin (
      • Meyer G.
      • Kirsch J.
      • Betz H.
      • Langosch D.
      Identification of a gephyrin binding motif on the glycine receptor beta subunit.
      ,
      • Kirsch J.
      • Kuhse J.
      • Betz H.
      Targeting of glycine receptor subunits to gephyrin-rich domains in transfected human embryonic kidney cells.
      ), whereas the homologous region of the GlyR α2 subunit may bind different kinases and calcineurin (
      • Bluem R.
      • Schmidt E.
      • Corvey C.
      • Karas M.
      • Schlicksupp A.
      • Kirsch J.
      • Kuhse J.
      Components of the translational machinery are associated with juvenile glycine receptors and are redistributed to the cytoskeleton upon aging and synaptic activity.
      ). Whereas the binding of gephyrin to the GlyR was evident from GlyR purification studies and was analyzed in great detail (
      • Sola M.
      • Bavro V.N.
      • Timmins J.
      • Franz T.
      • Ricard-Blum S.
      • Schoehn G.
      • Ruigrok R.W.
      • Paarmann I.
      • Saiyed T.
      • O′Sullivan G.A.
      • Schmitt B.
      • Betz H.
      • Weissenhorn W.
      Structural basis of dynamic glycine receptor clustering by gephyrin.
      ,
      • Schrader N.
      • Kim E.Y.
      • Winking J.
      • Paulukat J.
      • Schindelin H.
      • Schwarz G.
      Biochemical characterization of the high affinity binding between the glycine receptor and gephyrin.
      ,
      • Kim E.Y.
      • Schrader N.
      • Smolinsky B.
      • Bedet C.
      • Vannier C.
      • Schwarz G.
      • Schindelin H.
      Deciphering the structural framework of glycine receptor anchoring by gephyrin.
      ), it has only recently been shown that a similar site on gephyrin also binds directly to GABAA receptor subunits α1, α2, and α3 (
      • Tretter V.
      • Jacob T.C.
      • Mukherjee J.
      • Fritschy J.M.
      • Pangalos M.N.
      • Moss S.J.
      The clustering of GABA(A) receptor subtypes at inhibitory synapses is facilitated via the direct binding of receptor α2 subunits to gephyrin.
      ,
      • Saiepour L.
      • Fuchs C.
      • Patrizi A.
      • Sassoè-Pognetto M.
      • Harvey R.J.
      • Harvey K.
      Complex role of collybistin and gephyrin in GABAA receptor clustering.
      ,
      • Maric H.M.
      • Mukherjee J.
      • Tretter V.
      • Moss S.J.
      • Schindelin H.
      Gephyrin-mediated GABA(A) and glycine receptor clustering relies on a common binding site.
      ).
      Phosphorylation of gephyrin was first reported in 1992 (
      • Langosch D.
      • Hoch W.
      • Betz H.
      The 93 kDa protein gephyrin and tubulin associated with the inhibitory glycine receptor are phosphorylated by an endogenous protein kinase.
      ). More recently, the binding of gephyrin to the peptidyl-prolyl cis/trans-isomerase Pin1 was analyzed and allowed the identification of amino acid residues (Ser-188, Ser-194, and Ser-200) in the C-domain as major determinants of phosphorylation dependent Pin1 binding and also of gephyrin clustering (
      • Zita M.M.
      • Marchionni I.
      • Bottos E.
      • Righi M.
      • Del Sal G.
      • Cherubini E.
      • Zacchi P.
      Post-phosphorylation prolyl isomerization of gephyrin represents a mechanism to modulate glycine receptors function.
      ). In addition, Ser-270 was identified as another phosphorylation site that was reported to be phosphorylated by GSK3β (
      • Tyagarajan S.K.
      • Ghosh H.
      • Yévenes G.E.
      • Nikonenko I.
      • Ebeling C.
      • Schwerdel C.
      • Sidler C.
      • Zeilhofer H.U.
      • Gerrits B.
      • Muller D.
      • Fritschy J.M.
      Regulation of GABAergic synapse formation and plasticity by GSK3beta-dependent phosphorylation of gephyrin.
      ). Exchange of Ser-270 by alanine in that study increased gephyrin cluster numbers, suggesting an inhibitory role of phosphorylation at this site. Moreover, another study demonstrated that the inhibition of phosphatase 1 decreased gephyrin cluster size in cultured hippocampal neurons (
      • Bausen M.
      • Weltzien F.
      • Betz H.
      • O′Sullivan G.A.
      Regulation of postsynaptic gephyrin cluster size by protein phosphatase 1.
      ).
      The precise role of Cb and Cdc42 in receptor clustering is still not well understood. One current model of Cb function proposes that the N-terminal SH3 domain present in the Cb splice variants Cb1, Cb2+SH3, and Cb3 inactivates Cb. The binding to SH3-domain-interacting proteins, like neuroligin II or the cytoplasmic loop of the GABAA receptor α2 subunit, may activate Cb, which consequently would mediate local specific gephyrin clustering by an unknown molecular mechanism (
      • Poulopoulos A.
      • Aramuni G.
      • Meyer G.
      • Soykan T.
      • Hoon M.
      • Papadopoulos T.
      • Zhang M.
      • Paarmann I.
      • Fuchs C.
      • Harvey K.
      • Jedlicka P.
      • Schwarzacher S.W.
      • Betz H.
      • Harvey R.J.
      • Brose N.
      • Zhang W.
      • Varoqueaux F.
      Neuroligin 2 drives postsynaptic assembly at perisomatic inhibitory synapses through gephyrin and collybistin.
      ,
      • Tretter V.
      • Jacob T.C.
      • Mukherjee J.
      • Fritschy J.M.
      • Pangalos M.N.
      • Moss S.J.
      The clustering of GABA(A) receptor subtypes at inhibitory synapses is facilitated via the direct binding of receptor α2 subunits to gephyrin.
      ). A conditional Cdc42 knock-out mouse revealed no alteration of gephyrin cluster formation (
      • Reddy-Alla S.
      • Schmitt B.
      • Birkenfeld J.
      • Eulenburg V.
      • Dutertre S.
      • Böhringer C.
      • Götz M.
      • Betz H.
      • Papadopoulos T.
      PH-domain-driven targeting of collybistin but not Cdc42 activation is required for synaptic gephyrin clustering.
      ). More recently, however, cotransfection experiments with Cb mutants and constitutively active Cdc42 suggested an important contribution of Cdc42 to gephyrin clustering, possibly by forming a ternary complex with gephyrin and Cb2-SH3 (
      • Tyagarajan S.K.
      • Ghosh H.
      • Harvey K.
      • Fritschy J.M.
      Collybistin splice variants differentially interact with gephyrin and Cdc42 to regulate gephyrin clustering at GABAergic synapses.
      ).
      To study the functional role of Cb, we performed Cb-shRNA knockdown experiments in cultured hippocampal neurons. Interestingly, a decrease of Cb expression resulted in a reduction of gephyrin phosphorylation. Pharmacological inhibition of specific kinase pathways showed that cyclin-dependent kinases (CDKs) are involved in phosphorylation of gephyrin at Ser-270. Thus, our results are consistent with a model of cluster formation at inhibitory postsynaptic membrane specializations in which Cb increases gephyrin phosphorylation at Ser-270, a process that can be monitored by the phosphospecific antibody mAb7a (
      • Pfeiffer F.
      • Simler R.
      • Grenningloh G.
      • Betz H.
      Monoclonal antibodies and peptide mapping reveal structural similarities between the subunits of the glycine receptor of rat spinal cord.
      ).

      DISCUSSION

      The data presented in this paper provide novel information for understanding the gephyrin-dependent formation of GlyR and GABAA receptor clusters at postsynaptic membrane specializations. First, we demonstrate for the first time that Cb affects the phosphorylation of gephyrin. Second, we show that phosphorylation of gephyrin at position Ser-270 can be detected using the monoclonal antibody mAb7a. Third, we identify CDK5 as novel binding protein of gephyrin and provide evidence that CDK5 and other related CDKs may be involved in the phosphorylation of gephyrin at position Ser-270.
      We performed shRNA-mediated Cb knockdown experiments that resulted in a robust reduction of mAb7a immunoreactivity at postsynaptic sites, suggesting an almost complete loss of gephyrin clusters in most infected neurons. Immunoblot analysis revealed that the loss of mAb7a immunoreactivity was not due to a reduction of total gephyrin protein expression, as the use of antibody Ab-175 revealed comparable gephyrin levels in control and Cb knockdown cell cultures. Dephosphorylation experiments showed that mAb7a immunoreactivity was dependent on the phosphorylation of gephyrin. This finding is supported by the fact that mutation of one putative phosphorylation site (Ser-270) abolished mAb7a immunoreactivity. In addition, the inhibition of different kinase pathways in HEK293T cells expressing gephyrin and Cb resulted in a strong reduction of mAb7a binding to gephyrin, demonstrating that mAb7a detects kinase-dependent phosphorylation of gephyrin. Moreover, the coexpression of gephyrin with Cb2-SH3 strongly increased the mAb7a-specific signals in immunoblots, supporting our hypothesis that the phosphorylation of gephyrin is dependent on the presence of Cb. Interestingly, in 2000, Kins et al. (
      • Kins S.
      • Betz H.
      • Kirsch J.
      Collybistin, a newly identified brain-specific GEF, induces submembrane clustering of gephyrin.
      ) showed that coexpression of Cb2-SH3 with gephyrin in HEK293 cells induced the formation of submembranous gephyrin microclusters instead of gephyrin “blobs” which were observed upon expression of gephyrin alone. Thus, future experiments have to disclose whether these observations are functionally related.
      Besides the strong increase in mAb7a immunoreactivity upon coexpression of gephyrin with Cb2-SH3, the migration behavior of gephyrin in SDS-PAGE was also altered. One single band was detected with increasing intensity, whereas a second, slower migrating band appeared in addition when comparing the single expression of gephyrin with coexpression of gephyrin and Cb2-SH3 (see Fig. 4). As the reduction of mAb7a immunoreactivity of both bands was observed in neurons upon Cb knockdown, these results support the view that one functional role of collybistin is to mediate the phosphorylation of gephyrin by one or more different kinases and that gephyrin may exist in at least two different forms resulting from post-translational modifications.
      The use of different kinase inhibitors in HEK293T expression experiments showed that kinases like GSK3β and CDKs might be involved in the phosphorylation of gephyrin. Pharmacological inhibition of CDKs in cultured hippocampal neurons resulted in the reduction of mAb7a puncta upon prolonged inhibition at div9, whereas the general staining pattern of presynaptic markers from inhibitory synapses (VIAAT) or postsynaptic markers from excitatory synapses (PSD95, Homer) were not altered. However, the results using another antibody (Ab-175) specific for gephyrin were different. Although the mean values of the number of gephyrin puncta were reduced, this reduction did not reach significance levels. Therefore, it might be possible that the formation or stability of gephyrin clusters is independent of CDK-mediated phosphorylation. On the other hand, one might speculate that the remaining phosphorylation of gephyrin under our experimental conditions was sufficient to allow gephyrin cluster formation.
      In addition, we demonstrated that shorter incubation (1–6 h) with higher concentrations of inhibitors reduced mAb7a-specific puncta at div14 stages. Again this reduction of mAb7a immunoreactivity is not correlated with a similar decrease of Ab-175 immunoreactivity, suggesting that dephosphorylation of existing gephyrin clusters may reduce mAb7a immunoreactivity. Alternatively, it may be possible that mAb7a clusters have a short half-life and the turn-over of gephyrin at existing clusters is independent of CDK-mediated phosphorylation. From our inhibition experiments, we conclude that the reduced phosphorylation of gephyrin at position Ser-270 in Cb knockdown hippocampal neurons may not play a major role for the reduction of gephyrin clusters (see also Ref.
      • Körber C.
      • Richter A.
      • Kaiser M.
      • Schlicksupp A.
      • Mükusch S.
      • Kuner T.
      • Kirsch J.
      • Kuhse J.
      Effects of distinct collybistin isoforms on the formation of GABAergic synapses in hippocampal neurons.
      ). However, other functional roles like alteration in gephyrin-receptor interactions, ion channel behavior, or “cellular signaling” of gephyrin receptor complexes (
      • Bluem R.
      • Schmidt E.
      • Corvey C.
      • Karas M.
      • Schlicksupp A.
      • Kirsch J.
      • Kuhse J.
      Components of the translational machinery are associated with juvenile glycine receptors and are redistributed to the cytoskeleton upon aging and synaptic activity.
      ) depending on phosphorylation at Ser-270 of gephyrin might be possible.
      The hypothesis that CDKs are involved in the phosphorylation of gephyrin is further supported by the results of in vitro phosphorylation assays. Using purified recombinant gephyrin to perform in vitro assays with recombinant CDK1/cyclinB, CDK2/cyclin A, or CDK5/p25NCK complexes resulted in an increase in mAb7a immunoreactivity as detected by immunoblot analysis. Moreover, a direct incorporation of radioactively labeled phosphate was demonstrated. Using phosphorylation prediction algorithms for the sequence of the C-domain, several sites are indicated to be putative phosphorylation sites of CDK1 or CDK5 (NetPhosK 1.0 Server). Interestingly, the positions Ser-270 as well as Thr-276 and Ser-277 were predicted as putative CDK sites in gephyrin. Using peptide competition experiments, we confirmed that mAb7a binding to peptides phosphorylated at Ser-270 was stronger than binding to the unphosphorylated peptide, thus indicating that mAb7a puncta detected in fluorescence microscopy are indeed composed of gephyrin, which are phosphorylated at position Ser-270. Mass spectrometry analysis identified position Ser-268 to be phosphorylated in addition to five other positions within the C-domain of gephyrin. Future experiments expressing respective gephyrin mutants in neurons with a gephyrin knock-out genotype may allow disclosure of the putative function of these phosphorylation sites.
      Studies in other laboratories have shown that inhibition of phosphatase 1 decreased gephyrin cluster size (
      • Bausen M.
      • Weltzien F.
      • Betz H.
      • O′Sullivan G.A.
      Regulation of postsynaptic gephyrin cluster size by protein phosphatase 1.
      ) and that inhibition of GSK3β kinase with high Li2+ concentrations increased gephyrin clustering (
      • Tyagarajan S.K.
      • Ghosh H.
      • Yévenes G.E.
      • Nikonenko I.
      • Ebeling C.
      • Schwerdel C.
      • Sidler C.
      • Zeilhofer H.U.
      • Gerrits B.
      • Muller D.
      • Fritschy J.M.
      Regulation of GABAergic synapse formation and plasticity by GSK3beta-dependent phosphorylation of gephyrin.
      ), and these findings indicated that dephosphorylation of gephyrin at Ser-270 supports cluster formation, suggesting that mature gephyrin clusters might not be phosphorylated at Ser-270. However, our data indicate that phosphorylation of gephyrin at position Ser-270 is essential for gephyrin cluster detection with mAb7a. As it was shown that the phosphorylation by GSK3β at Ser-270 limits cluster formation (
      • Tyagarajan S.K.
      • Ghosh H.
      • Yévenes G.E.
      • Nikonenko I.
      • Ebeling C.
      • Schwerdel C.
      • Sidler C.
      • Zeilhofer H.U.
      • Gerrits B.
      • Muller D.
      • Fritschy J.M.
      Regulation of GABAergic synapse formation and plasticity by GSK3beta-dependent phosphorylation of gephyrin.
      ) and our study disclosed that mutations at the neighbored position 276/277 induced the formation of giant cluster-like structures in dendrites, one might speculate that this gephyrin domain is involved in restricting the polymerization of gephyrin scaffolds.
      Moreover, we cannot exclude that the gephyrin scaffolds at the postsynaptic membrane are not homogenous in respect of gephyrin modifications. Instead, the assembly from at least two different conformations or states of gephyrin seems possible, as indicated by the presence of two different migrating gephyrin bands from protein extracts of cultured neurons or upon coexpressing gephyrin with Cb2-SH3 in heterologous cells. Thus, one might speculate that gephyrin scaffolds in vivo are composed of a specific mixture of phosphorylated and nonphosphorylated gephyrin at a given site.
      The hypothesis that the long and curving gephyrin structures upon expressing gephyrinS268A,S270A,T276A,S277A reflect an unrestricted oligomerization behavior of gephyrin is in agreement with the description of very similar structures in hippocampal neurons expressing a collybistin impaired in the PH domain (
      • Tyagarajan S.K.
      • Ghosh H.
      • Harvey K.
      • Fritschy J.M.
      Collybistin splice variants differentially interact with gephyrin and Cdc42 to regulate gephyrin clustering at GABAergic synapses.
      ). Interestingly, the gephyrin phenotype in that study was rescued by the coexpression of constitutively active Cdc42. It is, therefore, possible that Cdc42 in those experiments might have activated downstream pathways converging to processes that involve the gephyrin region around Ser-270/Thr-276/Ser-277. The striking “needle” gephyrin phenotype might be related to unrestricted oligomerization behavior dependent on specific gephyrin conformations or interaction with additional proteins.
      Our finding that CDK1 might be a kinase involved in phosphorylation of gephyrin was unexpected. In contrast to CDK5 (
      • Lai K.O.
      • Ip N.Y.
      Recent advances in understanding the roles of Cdk5 in synaptic plasticity.
      ), only few data are available concerning the function of CDK1 in postmitotic neurons. Recently, a study reported the association of CDK1 with the microtubule network and the tubulin-binding protein tau in postmitotic neurons (
      • Schmetsdorf S.
      • Arnold E.
      • Holzer M.
      • Arendt T.
      • Gärtner U.
      A putative role for cell cycle-related proteins in microtubule-based neuroplasticity.
      ). Our data suggest that either CDK1, CDK2, or CDK5 could phosphorylate gephyrin; however, the identification of CDK5 as a gephyrin-binding protein by co-immunoprecipitation experiments suggests that CDK5 is a major candidate as a kinase expressed in postmitotic neurons to phosphorylate gephyrin. Future experiments have to establish which of the identified sites beside Ser-270 are phosphorylated by CDK5 or the other CDKs in vivo. In addition, the functional role of Cb for the phosphorylation of gephyrin and its functional impact in neuronal functions has to be elucidated by further in vitro and in vivo experiments.

      Acknowledgments

      We thank Rita Rossner for excellent technical assistance, Prof. Dr. K. Gorgas for reading the manuscript, M. Kaiser for providing the Cb2-SH3 rescue construct, R. Nonnenmacher for figure lay out, and P. Meller and N. Nierobisch for excellent technical assistance. Mass spectrometry was performed from the Core Facility for Mass Spectrometry and Proteomics, ZMBH, University Heidelberg.

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