The ubiquitin proteasome system (UPS) is a highly conserved and tightly regulated biochemical pathway that degrades the majority of proteins in eukaryotic cells. Importantly, the UPS is responsible for counteracting altered protein homeostasis induced by a variety of proteotoxic stresses. We previously reported that Rpt6, the ATPase subunit of the 19S regulatory particle (RP) of the 26S proteasome, is phosphorylated in mammalian neurons at serine 120 in response to neuronal activity. Furthermore, we found that Rpt6 S120 phosphorylation, which regulates the activity and distribution of proteasomes in neurons, is relevant for proteasome-dependent synaptic remodeling and function. To better understand the role of proteasome phosphorylation, we have constructed models of altered Rpt6 phosphorylation in S. cerevisiae by introducing chromosomal point mutations that prevent or mimic phosphorylation at the conserved serine (S119). We find that mutants which prevent Rpt6 phosphorylation at this site (rpt6-S119A), had increased susceptibility to proteotoxic stress, displayed abnormal morphology and had reduced proteasome activity. Since impaired proteasome function has been linked to the aggregation of toxic proteins including the Huntington’s disease (HD) related huntingtin (Htt) protein with expanded polyglutamine repeats, we evaluated the extent of Htt aggregation in our phospho-dead (rpt6-S119A) and phospho-mimetic (rpt6-S119D) mutants. We showed Htt103Q aggregate size to be significantly larger in rpt6-S119A mutants compared to wild-type or rpt6-S119D strains. Furthermore, we observed that phosphorylation of endogenous Rpt6 at S119 is increased in response to various stress conditions. Together, these data suggest that Rpt6 phosphorylation at S119 may play an important function in proteasome-dependent relief of proteotoxic stress that can be critical in protein aggregation pathologies.

Repeated exposure to cocaine produces structural and functional modifications at synapses from neurons in several brain regions including the nucleus accumbens. These changes are thought to underlie cocaine-induced sensitization. The ubiquitin proteasome system plays a crucial role in the remodeling of synapses and has recently been implicated in addiction-related behavior. The ATPase Rpt6 subunit of the 26S proteasome is phosphorylated by Ca/calmodulin-dependent protein kinases II alpha at ser120 which is thought to regulate proteasome activity and distribution in neurons. Here, we demonstrate that Rpt6 phosphorylation is involved in cocaine-induced locomotor sensitization. Cocaine concomitantly increases proteasome activity and Rpt6 S120 phosphorylation in cultured neurons and in various brain regions of wild type mice including the nucleus accumbens and prefrontal cortex. In contrast, cocaine does not increase proteasome activity in Rpt6 phospho-mimetic (ser120Asp) mice. Strikingly, we found a complete absence of cocaine-induced locomotor sensitization in the Rpt6 ser120Asp mice. Together, these findings suggest a critical role for Rpt6 phosphorylation and proteasome function in the regulation cocaine-induced behavioral plasticity.

Altered phosphorylation of the proteasome subunit Rpt6 has minimal impact on synaptic plasticity and learning.

23Dynamic control of protein degradation via the ubiquitin proteasome system is thought to play a 24 crucial role in neuronal function and synaptic plasticity. The proteasome subunit Rpt6, an AAA 25ATPase subunit of the 19S regulatory particle, has emerged as an important site for regulation of 26 26S proteasome function in neurons. Phosphorylation of Rpt6 on serine 120 (S120) can stimulate 27 the catalytic rate of substrate degradation by the 26S proteasome and this site is targeted by the 28 plasticity-related kinase calcium/calmodulin-dependent kinase II (CaMKII), making it an attractive 29 candidate for regulation of proteasome function in neurons. Several in vitro studies have shown 30 that altered Rpt6 S120 phosphorylation can affect the structure and function of synapses. To 31 evaluate the importance of Rpt6 S120 phosphorylation in vivo, we created two mouse models 32 which feature mutations at S120 that block or mimic phosphorylation at this site. We find that 33 peptidase and ATPase activities are upregulated in the phospho-mimetic mutant and 34 downregulated in the phospho-dead mutant (S120 mutated to aspartic acid (S120D) or alanine 35 (S120A), respectively). Surprisingly, these mutations had no effect on basal synaptic 36 transmission, long-term potentiation, and dendritic spine dynamics and density in the 37 hippocampus. Furthermore, these mutants displayed no deficits in cued and contextual fear 38 memory. Thus, in a mouse model that blocks or mimics phosphorylation at this site, either 39 compensatory mechanisms negate these effects, or small variations in proteasome activity are 40 not enough to induce significant changes in synaptic structure, plasticity, or behavior. 41