Cyclin-dependent kinase 5 is involved in the phosphorylation of gephyrin and clustering of GABAA receptors at inhibitory synapses of hippocampal neurons.

Heba Kalbouneh,Joachim Kirsch,Andrea Schlicksupp,Jochen Kuhse,William Phillips

doi:10.1371/journal.pone.0104256

Heba Kalbouneh, Joachim Kirsch + Show 3 more

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https://doi.org/10.1371/journal.pone.0104256

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Journal: PloS one	Publication Date: Aug 5, 2014
Citations: 23	License type: CC BY 4.0

Affiliation: Heidelberg University

Abstract

CDK5 has been implicated in neural functions including growth, neuronal migration, synaptic transmission and plasticity of excitatory chemical synapses. Here we report robust effects of CDK5 on phosphorylation of the postsynaptic scaffold protein gephyrin and clustering of inhibitory GABAA receptors in hippocampal neurons. shRNA-mediated knockdown of CDK5 and pharmacological inhibition of cyclin-dependent kinases reduced phosphorylated gephyrin clusters and postsynaptic γ2-containing GABAA receptors. Phosphorylation of S270 is antagonized by PP1/PP2a phosphatase and site-directed mutagenesis and in vitro phosphorylation experiments indicate that S270 is a putative CDK5 phosphorylation site of gephyrin. Our data suggest that CDK5 plays an essential role for the stability of gephyrin-dependent GABAA receptor clusters in hippocampal neurons.

Highlights

Cyclin-dependent kinase 5 is a proline-directed serine/threonine kinase belonging to the class of CDC2 (CDK1) -like kinases [1]
To investigate the functional role of CDK5 for the phosphorylation of gephyrin clusters at GABAergic synapses of cultured hippocampal neurons, we first established a knockdown of CDK5 with three different shRNA expressing lentiviruses
The quantitative analysis of mAb7a-specific immunoreactive puncta of three independent CDK5-specific shRNAs revealed a comparable reduction of gephyrin puncta with kd1: 2.260.77 puncta/30 mm, 1.5860.43 puncta for kd2, and 2.2460.39 puncta for kd3 compared to non-infected neurons: 8.4760.74 puncta, or scrambled control shRNA: 7.860.74 puncta, each per 30 mm dendrite (Fig. 1C)

Summary

Introduction

Cyclin-dependent kinase 5 is a proline-directed serine/threonine kinase belonging to the class of CDC2 (CDK1) -like kinases [1]. Different studies showed a CDK5-dependent change of postsynaptic NMDA receptor clustering depending on subunit phosphorylation [12] Consistent with these reports, Li et al showed that CDK5 inhibition blocks long-term potentiation (LTP) induction in CA1 hippocampal neurons [13]. Another study revealed that CDK5 loss-of-function in hippocampal circuits results in severe impairments in memory formation and retrival associated with LTP deficits in hippocampal CA1 neurons [14]. Whereas these and other studies suggest an involvement of CDK5 in the regulation of glutamatergic neurotransmission, almost nothing is known about a putative functional role of CDK5 at inhibitory synapses. CDK5 knockdown or inhibition results in a reduced number of c2 containing GABAA receptor clusters, suggesting that CDK5 plays an important role at inhibitory synapses

Experimental Procedures

Results

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