Mechanism and Regulation of Calcium/Calmodulin-dependent Protein Kinase II Targeting to the NR2B Subunit of the N-Methyl-d-aspartate Receptor

Abstract

Calcium influx through the N-methyl-D-aspartate CaMKII1 is a family of ubiquitous, calcium/calmodulin-dependent kinases with broad substrate specificity (1). The ␣ and ␤ isoforms are especially abundant in brain, constituting as much as 2% of total protein in the hippocampus (2). There is now overwhelming evidence that CaMKII is central to the mechanism of hippocampal, NMDA receptor-dependent longterm potentiation (LTP), a widely studied cellular model of learning and memory. Reduction of CaMKII activity by pharmacological or genetic means impairs LTP (3), whereas injecting or overexpressing CaMKII increases synaptic strength, which occludes and is occluded by electrically induced LTP (4, 5). Crucial to its function in LTP and spatial learning (6, 7), CaMKII undergoes rapid autophosphorylation following NMDA receptor-mediated calcium influx at a specific residue in its autoregulatory domain (Thr 286 in the ␣ isoform of CaMKII). This autophosphorylation renders the kinase calcium-independent and has been proposed as a form of molecular memory (8). In support, recent in vitro studies show that CaMKII autophosphorylation permits integration of oscillating calcium signals (9).We have recently demonstrated a second role for Thr 286 autophosphorylation, namely in promoting translocation of CaMKII to postsynaptic densities (PSDs) (10), cytoskeletal scaffolds for the neurotransmitter receptor, ion channels, and their regulators. The search for proteins that target Thr 286 -autophosphorylated CaMKII ([P-T286]CaMKII) to the PSD initially identified a 190-kDa binding activity (11), corresponding in size to the highly PSD-enriched NR2A and NR2B subunits of the NMDA receptor. Indeed, we recently showed that NR2B is a binding protein for [P-T286]CaMKII and isolated a CaMKII⅐NMDA receptor complex from PSDs (12). Subsequently, other laboratories implicated NR2A and NR2B (13,14) and NR1 and NR2B (15) as CaMKII-binding proteins. In this report, we identify amino acids critical for CaMKII binding in NR2B and investigate the regulation of CaMKII targeting to NR2B in vitro and in cells. EXPERIMENTAL PROCEDURES MaterialsThe phospho-Thr 286 -specific CaMKII antibody was a generous gift from Said Goueli (Promega). The recombinant ␥1 isoform of the protein phosphatase 1 catalytic subunit was generously provided by Dr. E. Lee (New York Medical College, Valhalla, NY). Sources of other materials are indicated below. Construction, Mutagenesis, and Expression of cDNAsNR2B Mutants-Fragments of the rat NR2B cDNA were amplified by polymerase chain reaction (PCR) with primers incorporating 5Ј-BamHI and 3Ј-EcoRI sites and subcloned into pGEX-2T (Amersham Pharmacia Biotech) for expression of glutathione S-transferase (GST) fusion proteins. All NR2B mutants were generated in the context of the NR2B-(1260 -1339) sequence, which includes the core CaMKII-binding domain (see Fig. 2) flanked by AlwNI and NdeI sites, allowing for single-step ligation of the mutagenized fragment into the full-length

Excitatory Amino Acid Receptors of the Electrosensory System: The NR1/NR2BN-Methyl-d-Aspartate Receptor

“…Another well-characterized kinase target site is the Ca 2ϩ /calmodulin-dependent protein kinase II (CaMKII) target site at Ser1303 in the murine NR2B sequence (Omkumar et al 1996). The sequence adjacent to Ser1303 forms a binding site for the CaMKII enzyme that targets the kinase to postsynaptic regions of the neuron (Bayer et al 2001;Strack et al 2000). This sequence (boxed in Fig.…”

“…An important property of the NMDA receptor is the relatively slow deactivation time course of the channel after re- (Strack et al 2000) and at Tyr by Fyn (Nakazawa et al 2001). B: phylogenetic comparison of AptNR2B to murine NR1 and NR2 subunits.…”

“…This process is facilitated by a direct interaction of CamKII to the C-terminal domain of the NR2B subunit (Leonard et al 1999;Omkumar et al 1996;Strack and Colbran 1998). A short segment of the C-terminal domain of NR2B, amino acids 1290 -1309 in the murine NR2B sequence, forms the principal site for binding of CaMKII to the receptor (Bayer et al 2001;Strack et al 2000). In addition to its role in binding CamKII, this segment also contains residue Ser1303, which, when phosphorylated by the CaMKII enzyme itself, inhibits the kinasereceptor interaction, thus providing a negative feedback for the kinase-receptor interaction (Bayer et al 2001;Strack et al 2000).…”

“…A short segment of the C-terminal domain of NR2B, amino acids 1290 -1309 in the murine NR2B sequence, forms the principal site for binding of CaMKII to the receptor (Bayer et al 2001;Strack et al 2000). In addition to its role in binding CamKII, this segment also contains residue Ser1303, which, when phosphorylated by the CaMKII enzyme itself, inhibits the kinasereceptor interaction, thus providing a negative feedback for the kinase-receptor interaction (Bayer et al 2001;Strack et al 2000). The sequence of AptNR2B reveals strong conservation of the CamKII binding site residues 1298 -1309 (AptNR2B residues 1404 -1415), including key residues Leu1298, Arg1300, and Ser1303 identified by point mutagenesis (Strack et al 2000).…”

“…In addition to its role in binding CamKII, this segment also contains residue Ser1303, which, when phosphorylated by the CaMKII enzyme itself, inhibits the kinasereceptor interaction, thus providing a negative feedback for the kinase-receptor interaction (Bayer et al 2001;Strack et al 2000). The sequence of AptNR2B reveals strong conservation of the CamKII binding site residues 1298 -1309 (AptNR2B residues 1404 -1415), including key residues Leu1298, Arg1300, and Ser1303 identified by point mutagenesis (Strack et al 2000). Thus the AptNR2B maintains the key elements for both CamKII binding and phosphorylation that have been identified in the rodent NR2B gene.…”