Arc ubiquitination regulates endoplasmic reticulum-mediated Ca2+ release and CaMKII signaling

Abstract

Synaptic plasticity relies on rapid, yet spatially precise signaling to alter synaptic strength. Arc is a brain enriched protein that is rapidly expressed during learning-related behaviors and is essential for regulating metabotropic glutamate receptor-mediated long-term depression (mGluR-LTD). We previously showed that disrupting the ubiquitination capacity of Arc enhances mGluR-LTD; however, the consequences of Arc ubiquitination on other mGluR-mediated signaling events is poorly characterized. Here we find that pharmacological activation of Group I mGluRs with S-3,5-dihydroxyphenylglycine (DHPG) increases Ca2+ release from the endoplasmic reticulum (ER). Disrupting Arc ubiquitination on key amino acid residues enhances DHPG-induced ER-mediated Ca2+ release. These alterations were observed in all neuronal subregions except secondary branchpoints. Deficits in Arc ubiquitination altered Arc self-assembly and enhanced its interaction with calcium/calmodulin-dependent protein kinase IIb (CaMKIIb) and constitutively active forms of CaMKII in HEK293 cells. Colocalization of Arc and CaMKII was altered in cultured hippocampal neurons, with the notable exception of secondary branchpoints. Finally, disruptions in Arc ubiquitination were found to increase Arc interaction with the integral ER protein Calnexin. These results suggest a previously unknown role for Arc ubiquitination in the fine tuning of ER-mediated Ca2+ signaling that may support mGluR-LTD, which in turn, may regulate CaMKII and its interactions with Arc.

The E3 ubiquitin ligase RNF216 contains a linear ubiquitin chain-determining-like domain that functions to regulate dendritic arborization and dendritic spine type in hippocampal neurons

“…To investigate the role of R2-C on Arc ubiquitination, we co-expressed RNF216 and R2-C domain truncation mutants with Myc-tagged Arc in HEK293 cells. As shown previously, addition of RNF216 led to a robust increase in the ubiquitination of Myc-Arc (19,20). As expected, this enhancement was reduced upon deletion of the R2-C domain in RNF216 (Figure 3C) demonstrating that the R2-C domain of RNF216 also promotes the ubiquitination of a known substrate.…”

“…The nitrocellulose membrane was blocked overnight with Li-COR blocking buffer. Primary antibodies were added and incubated overnight at 4 o C. Secondary antibodies and incubation were conducted as previously described (20).…”

“…Cells were washed three times with DPBS and incubated with Goat anti-Mouse IgG1 Alexa Fluor 647 Secondary Antibody and DAPI (Thermo Fisher Scientific) for 1 hour at room temperature. Following incubation, neurons were washed 3 times with 3% NGS and then mounted on slides as previously described (20).…”

“…Primary antibodies were added and incubated overnight at 4°C. Secondary antibodies and incubation were conducted as previously described (20).…”