Phosphorylation of Homer3 by Calcium/Calmodulin-Dependent Kinase II Regulates a Coupling State of Its Target Molecules in Purkinje Cells

Abstract

Homer proteins are components of postsynaptic density (PSD) and play a crucial role in coupling diverse target molecules. However, the regulatory aspect of Homer-mediated coupling has been addressed only about a dominant-negative effect of Homer1a, which requires de novo gene expression. Here, we present evidence that Homer-mediated coupling is regulated by its phosphorylation state. We found that Homer3, the predominant isoform in Purkinje cells, is phosphorylated by calcium/calmodulin-dependent protein kinase II (CaMKII) both in vitro and in vivo. Biochemical fractionation with phosphor-specific antibodies revealed the presence of phosphorylated Homer3 in the cytosolic fraction in contrast to high levels of nonphosphorylated Homer3 in PSD. In P/Q-type voltage-gated-Ca 2ϩ channel knock-out mice, in which CaMKII activation was reduced, the levels of Homer3 phosphorylation and the soluble form of Homer 3 were markedly lower. Furthermore, both robust phosphorylation of Homer3 and its dissociation from metabotropic glutamate receptor 1␣ (mGluR1␣) were triggered by depolarization in primary cultured Purkinje cells, and these events were inhibited by CaMKII inhibitor. An in vitro binding kinetic analysis revealed that these phosphorylation-dependent events were attributable to a decrease in the affinity of phosphorylated Homer3 for its ligand. In a heterologous system, the Ca 2ϩ signaling pattern induced by mGluR1␣ activation was modulated by the Homer3 phosphorylation state. Together, these findings suggested that Homer3 in Purkinje cells might function as a reversible coupler regulated by CaMKII phosphorylation and that the phosphorylation is capable of regulating the postsynaptic molecular architecture in response to synaptic activity.

IRBIT regulates CaMKIIα activity and contributes to catecholamine homeostasis through tyrosine hydroxylase phosphorylation

“…In addition, we confirmed the effect of IRBIT on CaMKIIα kinase activity using recombinant Homer 3 protein, which is a substrate for CaMKIIα (31). IRBIT significantly inhibited the phosphorylation of Homer 3 in a concentration-dependent manner ( Fig.…”

Encoding of Contextual Fear Memory Requires De Novo Proteins in the Prelimbic Cortex

“…Recent studies have suggested that Homer 3 interacts with amyloid precursor protein (59–61) and inhibits the production of Aβ (59–61). In Purkinjee neurons, Homer 3 interacts with mGluR1alpha and this interaction is regulated by phosphorylation (62). Our findings (RNAseq, qPCR and Western) revealed that a specific member of the Homer family of proteins, Homer 3, is upregulated in the mPFC within the first hour following CFC training.…”

The Actin-Binding Protein Drebrin Inhibits Neointimal Hyperplasia

“…13-15 Multiple members of the TRPC subfamily of TRP channels have been shown to require the scaffolding protein Homer 1 for proper function 16,17 and several groups have shown that Homer scaffolds associate with Drebrin through an interaction mediated by two Homer-binding sites within the Drebrin C-terminal domain. 18-20 We made the unexpected observation that Drebrin is abundantly expressed in SMCs and is upregulated in response to arterial injury. Because neointimal hyperplasia, a process which involves both SMC migration and proliferation, may involve SMC TRP channels 15 and because TRPC channel function is regulated by the Drebrin-binding protein Homer, 16 we tested whether Drebrin affects SMC proliferation and migration through its interaction with Homer and/or F-actin.…”