Regulation of NMDA receptors by cyclin-dependent kinase-5

Abstract

Members of the N-methyl-d-aspartate (NMDA) class of glutamate receptors (NMDARs) are critical for development, synaptic transmission, learning and memory; they are targets of pathological disorders in the central nervous system. NMDARs are phosphorylated by both serine/threonine and tyrosine kinases. Here, we demonstrate that cyclin dependent kinase-5 (Cdk5) associates with and phosphorylates NR2A subunits at Ser-1232 in vitro and in intact cells. Moreover, we show that roscovitine, a selective Cdk5 inhibitor, blocks both long-term potentiation induction and NMDA-evoked currents in rat CA1 hippocampal neurons. These results suggest that Cdk5 plays a key role in synaptic transmission and plasticity through its up-regulation of NMDARs.

Figure 1

Cdk5 expression and association with NR2A in the central nervous system. (A–D) Rat coronal brain sections were analyzed by in situ hybridization with a digoxigenin-labeled Cdk5 probe. High levels of Cdk5 mRNA expression were observed in the cerebral cortex (Ctx) (A), hippocampal CA3 (B), dentate gyrus (DG) (C), and CA1 region (D). (E) Postembedding immunogold EM labeling of Cdk5 in a representative section from adult rat cerebral cortex showing Cdk5 labeling over presynaptic (Upper) and postsynaptic (Lower) regions. (F) Immunoblot analysis of Cdk5 in rat brain homogenate synaptosomal and postsynaptic density (PSD) fractions. Whole-brain extract (lane 1, 50 μg), synaptosomes (lane 2, 10 μg), and PSD fraction (lane 3, 5 μg) were separated by SDS/PAGE and subjected to immunoblotting with anti-Cdk5, anti-NR2A, and anti-PSD-95 antibodies. (G) Rat brain extracts were solubilized with 1% deoxycholate, and the resulting detergent extract (Input) was used to immunoprecipitate the proteins indicated. The immunoprecipitates then were immunoblotted for Cdk5, NR2A, or control (preimmune IgG) antibodies. The input lane was loaded with 20% of the amount of extract used for IP. (H–J) Colocalization of Cdk5 with NR2A in hippocampal neurons. Rat hippocampal neurons were stained with Cdk5 polyclonal and monoclonal NR2A antibodies; Cdk5 and NR2A staining was visualized with a rhodamine-coupled secondary and FITC-coupled secondary antibody, respectively.

Figure 2

Phosphorylation of NR2A by Cdk5 in vitro. (A) The Cdk5 phosphorylation motif RSPFK is indicated in Mouse and Rat (NR2A). (B) HEK293T cells were transfected with Cdk5/p35 or mutant Cdk5 (K33T)/p35, and lysates were IP by using anti-Cdk5 antibody (C-8). The cells were incubated in a kinase assay system in vitro with histone H1 (Top and Top Middle) or recombinant NR2A (Bottom Middle and Bottom) as substrates. [³²P]-incorporation into substrates is indicated in Top and Bottom Middle, and the amount of substrate stained by Coomassie stain is indicated in Top Middle and Bottom. (C) Recombinant wild-type NR2A and mutant NR2A (S1232A) were purified, and equal amounts were incubated with GST-Cdk5 and GST-p35 (see Materials and Methods). (Top) The incorporation of [³²P] into NR2A. (Middle) The amount of NR2A substrate by Coomassie blue staining of the gel.

Figure 3

Comparison of NR2A phosphorylation with Cdk5 knockout mice and wild-type mice. (A and B) NR2A expression in wild-type and Cdk5^−/− mice was analyzed by Western blotting (A) and reverse transcription–PCR analysis (B). (C) The extracts from ³²P-labeled cultured hippocampal neurons from wild-type and Cdk5^−/− mice were processed for immunoprecipitation by using anti-NR2A antibody and subjected to autoradiography (Middle) or Western blotting (Bottom). Histogram (n = 3) reflects the relative amount of labeled NR2A to the amount of immunoreactive NR2A. (D) ³²P-labeled wild type or Ser mutant (S1232A) of HA-NR2A was IP with anti-HA-tagged antibody from transfected HEK293T cells and subjected to autoradiography (Top Middle) or Western blotting (Bottom Middle). The histogram shows the relative amount of labeled HA-NR2A to the amount of immunoreactive HA-NR2A.

Figure 4

Inhibition of Cdk5 activity in hippocampal CA1 neurons results in reduced LTP- and NMDA-evoked currents. (A–C) Tetanic trains induced LTP in the absence, but not in the presence, of 5 μM roscovitine (20 min preincubation). Representative responses in A and B were recorded before and 40 min after high-frequency stimulation. The data points in C are means ± SEM. (D) The inward current evoked by the local application of NMDA was reduced by roscovitine [compare Right with Left (control)]. (E) Normalized peak currents (I Peak) in the presence of roscovitine (5 μM) compared with those in the absence of roscovitine during the application of NMDA. The responses to NMDA decreased in the presence of roscovitine (n = 6), compared with neurons in the absence of roscovitine (n = 6).