Phosphorylation of Rpt6 Regulates Synaptic Strength in Hippocampal Neurons

It has become increasingly evident that protein degradation via the ubiquitin proteasome system plays a fundamental role in the development, maintenance and remodeling of synaptic connections in the CNS. We and others have recently described the activity-dependent regulation of proteasome activity and recruitment of proteasomes into spine compartments involving the phosphorylation of the 19S ATPase subunit, Rpt6, by the plasticity kinase Ca 2+ /calmodulin-dependent protein kinase II α (CaMKIIα) (Bingol and Schuman, 2006; Djakovic et al., 2009; Bingol et al, 2010). Here, we investigated the role of Rpt6 phosphorylation on proteasome function and synaptic strength. Utilizing a phospho-specific antibody we verified that Rpt6 is phosphorylated at Serine 120 (S120) by CaMKIIα. In addition, we found that Rpt6 is phosphorylated by CaMKIIα in an activity-dependent manner. Furthermore, we showed that a serine 120 to aspartic acid phospho-mimetic mutant of Rpt6 (S120D) increases its resistance to detergent extraction in rat hippocampal dendrites, indicating phosphorylated Rpt6 may promote the tethering of proteasomes to scaffolds and cytoskeletal components. Expression of Rpt6 S120D decreased miniature EPSC (mEPSC) amplitude, while expression of a phospho-dead mutant (S120A) increased mEPSC amplitude. Surprisingly, homeostatic scaling of mEPSC amplitude produced by chronic application of bicuculline or tetrodotoxin is both mimicked and occluded by altered Rpt6 phosphorylation. Together, these data suggest that CaMKII-dependent phosphorylation of Rpt6 at S120 may be an important regulatory mechanism for proteasome-dependent control of synaptic remodeling in slow homeostatic plasticity.