A better understanding of how proteasome activity is regulated can facilitate the search for proteasome enhancement strategies for disease treatment. A cell culture study shows cAMP-dependent protein kinase (PKA) activates 26S proteasomes by phosphorylating Ser14 of RPN6 (pS14-RPN6), but this discovery and its physiological significance remain to be established in vivo. To test the hypothesis that pS14-RPN6 mediates the activation of proteasomes by PKA and reduces proteotoxicity in animals, two knock-in mouse models with Ser14 of endogenous Rpn6 mutated to either Ala (S14A) or Asp (S14D) to respectively block or mimic pS14-Rpn6 were created. In a PKA-dependent manner, cAMP augmentation increased pS14-Rpn6 and 26S proteasome activities in wild-type (WT) but not S14A embryonic fibroblasts (MEFs), adult cardiomyocytes (AMCMs), and mouse hearts. Basal 26S proteasome activities were significantly greater in the myocardium and AMCMs from S14D mice than those from WT mice. When coupled with transgenic mice expressing GFPdgn (a proven UPS substrate), significantly lower myocardial GFPdgn protein but not mRNA levels were observed in S14D::GFPdgn mice compared with littermate GFPdgn control mice. In CryABR120G mice, a model of cardiac proteotoxicity, basal myocardial pS14-Rpn6 was significantly lower compared with non-transgenic littermates at both 3 and 6 months of age, which was not always associated with reduction of other phosphorylated PKA substrates. Proteasomal degradation of CryABR120G was faster in cultured S14D neonatal mouse cardiomyocytes (NMCMs) than in WT NMCMs. Compared with CryABR120G mice, S14D::CryABR120G mice showed significantly greater myocardial proteasome activities, lower levels of total and K48-linked ubiquitin conjugates and of aberrant CryABR120G protein aggregates, less reactivation of fetal genes and cardiac hypertrophy, and delays in cardiac malfunction and premature death. This study establishes in animals that pS14-Rpn6 is responsible for the activation of 26S proteasomes by PKA and reduced pS14-Rpn6 is a key pathogenic factor in cardiac proteinopathy, thereby identifies a new therapeutic target to reduce cardiac proteotoxicity
