Translocation of Autophosphorylated Calcium/Calmodulin-dependent Protein Kinase II to the Postsynaptic Density

Abstract

Calcium/calmodulin-dependent protein kinase II (CaMKII) undergoes calcium-dependent autophosphorylation, generating a calcium-independent form that may serve as a molecular substrate for memory. Here we show that calcium-independent CaMKII specifically binds to isolated postsynaptic densities (PSDs), leading to enhanced phosphorylation of many PSD proteins including the ␣-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA)-type glutamate receptor. Furthermore, binding to PSDs changes CaMKII from a substrate for protein phosphatase 2A to a protein phosphatase 1 substrate. Translocation of CaMKII to PSDs occurs in hippocampal slices following treatments that induce CaMKII autophosphorylation and a form of long term potentiation. Thus, synaptic activation leads to accumulation of autophosphorylated, activated CaMKII in the PSD. This increases substrate phosphorylation and affects regulation of the kinase by protein phosphatases, which may contribute to enhancement of synaptic strength.CaMKII 1 isoforms comprise a family of broad specificity, calcium-activated kinases (1, 2). The ␣ and ␤ isoforms are abundantly expressed in the brain, with ␣ making up as much as 2% of total protein in certain brain regions (3). CaMKII is particularly enriched in PSDs (4, 5), cytoskeletal specializations apposed to the postsynaptic membrane of excitatory synapses that are thought to be scaffolds for neurotransmitter receptors, ion channels, and their postsynaptic modulators and effectors (reviewed in Refs. 6 and 7). Earlier reports suggested that CaMKII␣ constitutes as much as 50% of total PSD protein (8 -10), but PSDs prepared from rapidly homogenized brains are only 2-3-fold enriched in CaMKII␣ compared with whole forebrain extracts (3,11). CaMKII␣ knockout mice show impaired hippocampal long term potentiation, a cellular model for learning and memory (12). Conversely, introduction of CaMKII␣ into neurons augments postsynaptic responses and occludes further electrically induced long term potentiation (13,14).CaMKII␣ undergoes calcium/calmodulin-dependent autophosphorylation on Thr 286 in its regulatory domain, rendering the kinase partially calcium-independent (1, 2). This reaction has been proposed as a "molecular switch," translating transient calcium elevation into prolonged kinase activity (15, 16), which becomes subject to regulation by protein phosphatases. (19).Isolation of PSDs-PSDs were prepared from adult rat forebrains flash frozen within 45 s of euthanasia by detergent lysis of synaptosomes (20) except that 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 1 mM benzamidine, and 10 g/ml leupeptin were included in all buffers. Synaptosomes were lysed in 1% (v/v) Triton X-100 and 150 mM KCl, and a second subsequent sucrose gradient was omitted because it yielded no further purification. PSDs displayed typical "donut" morphology by video-enhanced differential-interference contrast microscopy (21). PSDs prepared in the absence or the presence of the protein phosphatase inhibitor microcystin-LR (1 M) contained ...

αCaMKII autophosphorylation levels differ depending on subcellular localization

“…Several studies have indicated that αCaMKII autophosphorylation of T286 enhances binding of the kinase to the PSD and several of its constituents (Bayer et al, 2001;Strack et al, 1997b;Strack and Colbran, 1998;Gardoni et al, 1999;Leonard et al, 2002;Strack et al, 2000). In addition, T286 phosphorylation has been shown to decrease the dissociation rate of the kinase from the PSD (Shen et al, 2000;Bayer et al, 2006;Yoshimura and Yamauchi, 1997;Dosemeci et al, 2002).…”

“…Localization of CaMKII at the PSD has been proposed to be critical for its role in LTP (Lisman et al, 2002;Barria and Malinow, 2005). Furthermore, several different forms of neuronal activation, including treatments that lead to synaptic potentiation, have been shown to induce translocation of CaMKII to post-synaptic sites (Strack et al, 1997b;Shen and Meyer, 1999;Otmakhov et al, 2004;Dosemeci et al, 2001;Bayer et al, 2001). Accordingly, a more complete knowledge of the molecular mechanisms that regulate CaMKII binding to the PSD is likely to be crucial for our understanding of the biochemical events that occur during LTP.…”

“…Phosphorylation of this residue is thought to enhance CaMKII binding to isolated PSDs (Strack et al, 1997b), densin-180 (Strack et al, 2000), and the Nmethyl-D-aspartate receptor (NMDAR) subunits NR2B (Strack and Colbran, 1998;Bayer et al, 2001;Leonard et al, 2002), NR1 (Leonard et al, 2002), and NR2A (Gardoni et al, 1999). Furthermore, T286 phosphorylation has been reported to decrease the dissociation rate of CaMKII that has been bound to the PSD (Shen et al, 2000;Bayer et al, 2006;Yoshimura and Yamauchi, 1997;Dosemeci et al, 2002) (but see (Strack et al, 1997b)). These studies suggest that phosphorylation of T286 is an important regulatory mechanism controlling αCaMKII association with the PSD, and that PSD-associated αCaMKII is most likely more heavily phosphorylated on T286 than αCaMKII in other parts of the neuron.…”

“…Like T286 phosphorylation, T305/6 phosphorylation has been shown to modulate protein-protein interactions of CaMKII. Phosphorylation of these residues inhibits kinase binding to isolated PSDs (Strack et al, 1997b), α-actinin (Robison et al, 2005), and the NMDAR subunits NR1 and NR2B (Leonard et al, 2002). In addition, T305/6 phosphorylation increases the dissociation rate of CaMKII from the PSD (Shen et al, 2000), and mimicking phosphorylation on these residues prevents while inhibiting phosphorylation enhances PSD association of the kinase (Elgersma et al, 2002).…”

Structural role of zinc ions bound to postsynaptic densities

“…cross-sectional area, thickness and curvature, and the protein content of the PSD (e.g. Greenough et al 1978;Rutledge 1978;Rees et al 1985;Strevaag and Greenough 1985;Desmond and Levy 1986;Strack et al 1997;Hu et al 1998;Dosemeci et al 2001; except for the study of Sorra and Harris 1998). Time-lapse recording of the clusters of green fluorescence protein PSD95 in hippocampal neurons revealed rapid formation and remodeling of the PSD (Marrs et al 2001).…”