Regulation of the Proteasome by Neuronal Activity and Calcium/Calmodulin-dependent Protein Kinase II

Stevan N Djakovic,Lindsay A Schwarz,Barbara Barylko,George N Demartino,Gentry N Patrick

doi:10.1074/jbc.m109.021956

Abstract

Protein degradation via the ubiquitin proteasome system has been shown to regulate changes in synaptic strength that underlie multiple forms of synaptic plasticity. It is plausible, therefore, that the ubiquitin proteasome system is itself regulated by synaptic activity. By utilizing live-cell imaging strategies we report the rapid and dynamic regulation of the proteasome in hippocampal neurons by synaptic activity. We find that the blockade of action potentials (APs) with tetrodotoxin inhibited the activity of the proteasome, whereas the up-regulation of APs with bicuculline dramatically increased the activity of the proteasome. In addition, the regulation of the proteasome is dependent upon external calcium entry in part through N-methyl-D-aspartate receptors and L-type voltage-gated calcium channels and requires the activity of calcium/calmodulin-dependent protein kinase II (CaMKII). Using in vitro and in vivo assays we find that CaMKII stimulates proteasome activity and directly phosphorylates Rpt6, a subunit of the 19 S (PA700) subcomplex of the 26 S proteasome. Our data provide a novel mechanism whereby CaMKII may regulate the proteasome in neurons to facilitate remodeling of synaptic connections through protein degradation.

Highlights

The ubiquitin proteasome system (UPS)4 is a major pathway for protein turnover in eukaryotic cells
Increased proteasome activity was found to be dependent upon external calcium entry in part through N-methyl-D-aspartate (NMDA) receptors and L-type voltagegated calcium channels (VGCCs) and required the activity of calcium/calmodulin-dependent protein kinase II (CaMKII)
Action Potential Blockade and Up-regulation Produce Rapid and Opposite Effects on Proteasome Activity in Hippocampal Dendrites—To determine the effects of neuronal activity on proteasome function, we utilized a GFP-based reporter imaging strategy to monitor the rate of proteasome activity in the dendrites of cultured hippocampal neurons

Summary

EXPERIMENTAL PROCEDURES

Antibodies and Reagents—␣2 proteasome (MCP21 monoclonal antibody (mAb)), ␣␤ core proteasome (polyclonal antibody), and Rpt (mAb) antibodies were purchased from Biomol. PCMV-IRES-mCherry was created by subcloning mCherry (pRSETB-mCherry; a kind gift from Roger Tsien, University of California San Diego, La Jolla, CA) into IRES2EGFP (Clontech) This was done by ligating the BamHI (blunted)-BsrGI mCherry-containing fragment into BstXI (blunted)BsrGI sites. For Sindbis virus construction, the NheI-XbaI reporter-IRES-mCherry containing fragment from pCMVpaGFP, paGFPu, and paGFP-odc plasmids were subcloned into the XbaI site of SinRep. Live Imaging—For Sindbis virus infection of mature hippocampal neurons (Ͼ21 days in vitro) on glass-bottom 35-mm dishes, paGFPu, paGFP-odc, or paGFP reporter virion were added directly to culture media. For treatment of neurons with APV (50 ␮M), CNQX (40 ␮M), calpeptin (10 ␮M), KN-93 (10 ␮M), AIP (5 ␮M), or Ca2ϩ-free containing HEPES-based saline, cells were preincubated, selected by mCherry expression, photoactivated, and imaged by time-lapse microscopy. After various times of incubation at 30 °C samples were treated with SDS sample buffer and subjected to SDS-PAGE and autoradiography

RESULTS

No imaged value dendrites

DISCUSSION