Phosphorylation of gephyrin in hippocampal neurons by cyclin-dependent kinase CDK5 at Ser-270 is dependent on collybistin.
Gephyrin is a scaffold protein essential for the postsynaptic clustering of inhibitory glycine and different subtypes of GABA(A) 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 gephyrin(T276A,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.
Department of Anatomy and Cell Biology, University of Heidelberg, D-69120 Heidelberg, Germany. jochen.kuhse@urz.uni-heidelberg.de
Jochen
J
Kuhse
Heba
H
Kalbouneh
Andrea
A
Schlicksupp
Susanne
S
Mükusch
Ralph
R
Nawrotzki
Joachim
J
Kirsch
D019943:Amino Acid Substitution; D000818:Animals; D016203:CDC2 Protein Kinase / Q000235:genetics / Q000378:metabolism; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D051357:Cyclin-Dependent Kinase 2 / Q000235:genetics / Q000378:metabolism; D051360:Cyclin-Dependent Kinase 5 / Q000235:genetics / Q000378:metabolism*; D020662:Guanine Nucleotide Exchange Factors / Q000235:genetics / Q000378:metabolism*; D006624:Hippocampus / Q000166:cytology / Q000378:metabolism*; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D020125:Mutation, Missense; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism*; D009474:Neurons / Q000166:cytology / Q000378:metabolism*; D010766:Phosphorylation / Q000502:physiology; D051381:Rats; D064067:Rho Guanine Nucleotide Exchange Factors; D013570:Synaptic Membranes / Q000235:genetics / Q000378:metabolism
C577410:ARHGEF9 protein, human; C577404:Arhgef9 protein, rat; D002352:Carrier Proteins; D020662:Guanine Nucleotide Exchange Factors; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D064067:Rho Guanine Nucleotide Exchange Factors; C073441:gephyrin; D051360:Cyclin-Dependent Kinase 5; D016203:CDC2 Protein Kinase; C495894:CDK2 protein, human; C495905:CDK5 protein, human; C495892:Cdk2 protein, rat; C495907:Cdk5 protein, rat; D051357:Cyclin-Dependent Kinase 2