UC San Diego

Protein degradation via the ubiquitin proteasome system (UPS) has been shown to regulate changes in synaptic strength that underlie multiple forms of synaptic plasticity. It is plausible, therefore, that the UPS 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 neuronal activity. We find that the blockade of action potentials (APs) with tetrodotoxin (TTX) inhibited the activity of the proteasome, while the up-regulation of APs with bicuculline (BIC) 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 (NMDA) receptors and L- type voltage gated calcium channels (VGCCs), 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 19S (PA700) subcomplex of the 26S proteasome. Our data provide a novel mechanism whereby CaMKII may regulate the proteasome in neurons to facilitate remodeling of synaptic connections through protein degradation. We next aimed to further elucidate how phosphorylation of Rpt6 regulates proteasome function in neurons. Using a phospho-specific antibody we showed that Rpt6 is phosphorylated at Serine 120 (S120) in a CaMKII-dependent fashion. While phosphorylation of this site is not sufficient to regulate proteasome activity, it is necessary for CaMKII-dependent stimulation of the proteasome. Interestingly we find that phosphorylation of S120 promotes detergent resistance of Rpt6, presumably by tethering the proteasome to the actin cytoskeleton at postsynaptic compartments. Additionally, electrophysiology experiments suggest that this phosphorylation event is important for proteasome dependent synaptic remodeling. Specifically, a phospho-mimetic Rpt6 S120 mutant leads to a decrease in miniature excitatory postsynaptic current (mEPSC) amplitude, while a phospho-dead mutant causes an increase in mEPSC amplitude. Together this data suggests that CaMKII-dependent phosphorylation of Rpt6 at S120 is an important regulator of proteasome function and UPS dependent synaptic remodeling