Does Ca 2+ Attenuate CaMKII-Dependent Phosphorylation at an NMDAR Complex?

Abstract

Ca 2+ influx through hippocampal N -methyl-D-aspartate receptors (NMDARs) activates signaling pathways that affect α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor (AMPAR) trafficking, leading to long-lasting changes in synaptic function. Krapivinsky et al. investigated molecular interactions in a surprising pathway involving the synaptic Ras-small guanosine triphosphatase (GTPase)-activating protein (SynGAP). MUPP1, a multi-PDZ domain scaffolding protein that was enriched in postsynaptic densities, coimmunoprecipitated with the NMDAR. A yeast two-hybrid screen of a human brain library indicated that MUPP1's PDZ13 domain interacted with SynGAPα; this was confirmed by glutathione- S -transferase pull-down and coimmunoprecipitation. SynGAP immunoprecipitated from quiescent cultured hippocampal neurons was phosphorylated. Phosphorylation depended on Ca 2+ -calmodulin-dependent kinase II (CamKII) activity and, unexpectedly, synaptic stimulation or exposure to glutamate led to dephosphorylation. Disruption of the SynGAP-MUPP1 complex with membrane-permeant trans-activating transduction (TAT) fusion proteins containing the 111 C-terminal amino acids of SynGAP (TAT-SynGAP111) or PDZ13 (TAT-PDZ13) also promoted SynGAP dephosphorylation. CaMKII associated with the SynGAP-MUPP1 complex through MUPP1's second PDZ domain; however, exposure to Ca 2+ and calmodulin promoted CamKII dissociation. Disruption of SynGAP-MUPP1 interaction with TAT-PDZ13 or TAT-SynGAP111 attenuated p38 mitogen-associated protein kinase (MAPK) phosphorylation (as does synaptic activity). In an in vitro assay, SynGAP stimulated Rap GTPase activity. Finally, patch clamp analysis indicated that exposure to PDZ13 or SynGAP111 increased the amplitude and frequency of AMPAR-mediated miniature excitatory postsynaptic currents, whereas immunofluorescence analysis indicated that TAT-PDZ13 and TAT-SynGAP111 promoted AMPAR clustering. Thus, the authors propose a model in which some effects of Ca 2+ entry through the NMDAR are mediated through dissociation of CaMKII from the MUPP1-SynGAP complex, eliciting a decrease in SynGAP phosphorylation and thereby an increase in its Rap GAP activity and a consequent decrease in p38 MPAK activation. G. Krapivinsky, I. Medina, L. Krapivinsky, S. Gapon, D. E. Clapham, SynGAP-MUPP1-CaMKII synaptic complexes regulate p38 MAP kinase activity and NMDA receptor-dependent synaptic AMPA receptor potentiation. Neuron 43 , 563-574 (2004). [Online Journal]