Substrate Localization Creates Specificity in Calcium/Calmodulin-dependent Protein Kinase II Signaling at Synapses

Robert C. Malenka,Jennifer Tsui

doi:10.1074/jbc.m600966200

Abstract

Calcium/calmodulin-dependent protein kinase II (CaMKII), a major component of the postsynaptic density (PSD) of excitatory synapses, plays a key role in the regulation of synaptic function in the mammalian brain. Although many postsynaptic substrates for CaMKII have been characterized in vitro, relatively little is known about their phosphorylation in vivo. By tagging synaptic proteins with a peptide substrate specific for CaMKII and expressing them in cultured neurons, we have visualized substrate phosphorylation by CaMKII at intact synapses. All substrates tested were strongly phosphorylated by CaMKII in HEK293 cells. However, activity-dependent phosphorylation of substrates at synapses was highly selective in that the glutamate receptor subunits NR2B and GluR1 were poorly phosphorylated whereas PSD-95 and Stargazin, proteins implicated in the scaffolding and trafficking of AMPA receptors, were robustly phosphorylated. Phosphatase activity limited phosphorylation of Stargazin but not NR2B and GluR1. These results suggest that the unique molecular architecture of the PSD results in highly selective substrate discrimination by CaMKII.

Highlights

It is unknown whether stimulus-dependent substrate specificity is an inherent property of the postsynaptic density (PSD) and whether the source of the calcium that activates calmodulin-dependent protein kinase II (CaMKII) and protein phosphatase activity influence synaptic substrate phosphorylation
CaMKII interactions with the NMDA receptor (NMDAR) subunit NR2B [10] and the Drosophila homolog of CASK, Cmg [11], a member of the MAGUK family of synaptic scaffolding proteins [12], have both been found to influence its state of activation, with NR2B resulting in calcium-independent activity and Cmg/CASK promoting inhibitory autophosphorylation [10, 11]
When expressed in HEK293 cells only minimal staining with the phosphovimentin antibody was detected (Fig. 1B). Stimulation of these cells with the calcium ionophore ionomycin caused a ϳ2-fold increase in the intensity of pVim staining (2.18 Ϯ 0.09 times control levels, n ϭ 169) and, consistent with previous work in both heterologous cells and neurons [1, 18, 21], this was completely blocked by inhibitors of CaMKII (1.02 Ϯ 0.06, n ϭ 178; Fig. 1, B and C)

Summary

EXPERIMENTAL PROCEDURES

Gene Construction—Vim-CFP was constructed by amplifying the initial 264-bp sequence from the vimentin head by PCR and GFP containing NES from HIV Rev (LPPLERLTL) into BaMHI and EcoRI sites in lentiviral expression vector, FC1.2 under the ␣CaMKII promoter.

CaMKII Signaling at Synapses

RESULTS

DISCUSSION