Abstract
Dendritic calcium/calmodulin-dependent protein kinase II (CaMKII) is dynamically targeted to the synapse. We show that CaMKIIalpha is associated with the CaMKII-binding proteins densin-180, the N-methyl-D-aspartate receptor NR2B subunit, and alpha-actinin in postsynaptic density-enriched rat brain fractions. Residues 819-894 within the C-terminal domain of alpha-actinin-2 constitute the minimal CaMKII-binding domain. Similar amounts of Thr286-autophosphorylated CaMKIIalpha holoenzyme [P-T286]CaMKII bind to alpha-actinin-2 as bind to NR2B (residues 1260-1339) or to densin-180 (residues 1247-1495) in glutathione-agarose cosedimentation assays, even though the CaMKII-binding domains share no amino acid sequence similarity. Like NR2B, alpha-actinin-2 binds to representative splice variants of each CaMKII gene (alpha, beta, gamma, and delta), whereas densin-180 binds selectively to CaMKIIalpha. In addition, C-terminal truncated CaMKIIalpha monomers can interact with NR2B and alpha-actinin-2, but not with densin-180. Soluble alpha-actinin-2 does not compete for [P-T286]CaMKII binding to immobilized densin-180 or NR2B. However, soluble densin-180, but not soluble NR2B, increases CaMKII binding to immobilized alpha-actinin-2 by approximately 10-fold in a PDZ domain-dependent manner. A His6-tagged NR2B fragment associates with GST-densin or GST-actinin but only in the presence of [P-T286]CaMKII. Similarly, His6-tagged densin-180 or alpha-actinin fragments associate with GST-NR2B in a [P-T286]CaMKII-dependent manner. In addition, GST-NR2B and His6-tagged alpha-actinin can bind simultaneously to monomeric CaMKII subunits. In combination, these data support a model in which [P-T286]CaMKIIalpha can simultaneously interact with multiple dendritic spine proteins, possibly stabilizing the synaptic localization of CaMKII and/or nucleating a multiprotein synaptic signaling complex.
Highlights
Dominate in the brain and are involved in normal regulation of synaptic transmission [1,2,3,4]
calmodulin-dependent protein kinase II (CaMKII) Is Associated with Multiple CaMKII-binding Proteins in PSD-enriched Fractions—PSD-enriched synaptosomal fractions isolated from adult rat forebrain were enriched in CaMKII and three CaMKII-binding proteins, ␣-actinin, NR2B, and densin-180
CaMKII partially colocalizes with NMDA receptor NR2B subunits, densin-180, and ␣-actinin in dendritic spines of cultured neurons [10, 29, 30]
Summary
Dominate in the brain and are involved in normal regulation of synaptic transmission [1,2,3,4]. CaMKII interacts with other proteins that we refer to collectively as CaMKII-associated proteins (CaMKAPs) At postsynaptic sites, these include multiple subunits of the N-methyl-D-aspartate-type glutamate receptor (NMDA receptor) (10 –13), densin-180 [14, 15], ␣-actinin [15, 16], cyclin-dependent protein kinase 5 [16], synGAP [17], and filamentous actin [18, 19]. Prior studies have identified dissimilar high affinity CaMKII-binding domains in the NR2B subunit of the NMDA receptor and in densin-180. These interactions are differentially regulated by calcium/calmodulinbinding, autophosphorylation at Thr286, and phosphorylation of the binding proteins [10, 11, 13,14,15, 20, 21]. The data indicate that CaMKII itself may serve as a structural scaffold for the assembly of a postsynaptic signalosome
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