cAMP-induced phosphorylation of 26S proteasomes on Rpn6/PSMD11 enhances their activity and the degradation of misfolded proteins

Although rates of protein degradation by the ubiquitin-proteasome pathway (UPS) are determined by their rates of ubiquitination, we show here that the proteasome’s capacity to degrade ubiquitinated proteins is also tightly regulated. We studied the effects of cAMP-dependent protein kinase (PKA) on proteolysis by the UPS in several mammalian cell lines. Various agents that raise intracellular cAMP and activate PKA (activators of adenylate cyclase or inhibitors of phosphodiesterase 4) promoted degradation of short-lived (but not long-lived) cell proteins generally, model UPS substrates having different degrons, and aggregation-prone proteins associated with major neurodegenerative diseases, including mutant FUS (Fused in sarcoma), SOD1 (superoxide dismutase 1), TDP43 (TAR DNA-binding protein 43), and tau. 26S proteasomes purified from these treated cells or from control cells and treated with PKA degraded ubiquitinated proteins, small peptides, and ATP more rapidly than controls, but not when treated with protein phosphatase. Raising cAMP levels also increased amounts of doubly capped 26S proteasomes. Activated PKA phosphorylates the 19S subunit, Rpn6/PSMD11 (regulatory particle non-ATPase 6/proteasome subunit D11) at Ser14. Overexpression of a phosphomimetic Rpn6 mutant activated proteasomes similarly, whereas a nonphosphorylatable mutant decreased activity. Thus, proteasome function and protein degradation are regulated by cAMP through PKA and Rpn6, and activation of proteasomes by this mechanism may be useful in treating proteotoxic diseases.In mammalian cells, the bulk of cell proteins are degraded by the ubiquitin-proteasome system (UPS) (1, 2). Misfolded proteins, which arise from mutations or postsynthetic damage, and normal proteins with regulatory functions tend to be degraded more rapidly than average cell proteins (2). To be degraded by the UPS, proteins are first modified by ubiquitination (2). In this highly selective process, ubiquitin moieties are conjugated to individual proteins by one of the cell’s many ubiquitin ligases (E3s) (3). Protein ubiquitination is generally assumed to be the rate-limiting step in the degradation pathway, and once ubiquitinated, proteins are believed to be efficiently hydrolyzed by the 26S proteasome. This 2.5-MDa proteolytic complex is composed of about 60 subunits (3). Proteins are digested within the core 20S proteasome, a hollow cylindrical particle containing three types of peptidase activities: chymotrypsin-like, trypsin-like, and caspase-like (3). This particle can be associated with one or two 19S regulatory particles forming a 26S proteasome (3). The 19S complex serves multiple key functions: it binds the ubiquitinated substrate, removes the ubiquitin chain, unfolds the protein substrate, and translocates it through a narrow gated channel into the 20S particle (3). This multistep process is coupled to ATP hydrolysis by the hexameric ATPase ring at the base of the 19S complex adjacent to the core particle (3, 5). These various steps are tightly coordinated; for example, gate opening into the 20S particle and ATP hydrolysis are activated upon binding of the ubiquitin (Ub) chain to the deubiquitinating enzymes, Usp14 or Uch37 (5, 6).The development of inhibitors of proteasome function have advanced our knowledge of cell regulation and proven very valuable in treating hematological cancers (7). In principle, agents that enhance proteasome function could be valuable in combating the various diseases resulting from the toxic accumulation of misfolded proteins. In the major neurodegenerative diseases [amyotrophic lateral sclerosis (ALS), Alzheimer’s, Parkinson’s, and Huntington’s diseases (8, 9)], aggregation-prone proteins build up, often as protein inclusions that contain Ub and proteasomes (10). One factor that may contribute to the pathogenesis of these diseases is the progressive impairment of the capacity of the UPS to degrade misfolded proteins (11). In fact, several studies of neurodegenerative disease models have suggested that proteasome function is impaired when these misfolded proteins (e.g., huntingtin aggregates, mutant tau, or PrP^Sc prions) accumulate in cells (11–14).A number of postsynthetic modifications of 26S proteasome subunits have been reported, including O-GlcNAc modification (15), ADP ribosylation (16), and especially phosphorylation (17–19). The subunit phosphorylation may influence the localization (20), activity (17), and formation (18, 21) of the 26S proteasome. For example, phosphorylation of one of the 19S ATPases, Rpt6, in neurons by Ca²⁺/calmodulin-dependent protein kinase II (CaMKII), has been reported to cause proteasome entry into dendrites and promote synaptic plasticity (22, 23). In addition, phosphorylation of Rpt6 by cAMP-dependent proteins kinase (PKA) was reported to increase proteasome activity against small peptides (17, 24, 25). However, the effects of this modification on the proteasome’s capacity to degrade ubiquitin conjugates and on protein degradation in cells were not examined. Although raising cAMP levels and phosphorylation by PKA alter many cellular functions, effects on protein breakdown by the UPS have not been reported, aside from a suppression of overall proteolysis in skeletal muscle (26). Here we demonstrate that PKA directly phosphorylates the 19S subunit Rpn6/PSMD11 (regulatory particle non-ATPase 6/proteasome subunit D11), and that this modification stimulates several key proteasomal processes and enhances its capacity to degrade ubiquitinated proteins. As a result, pharmacological agents that raise cAMP levels and activate PKA promote the breakdown of short-lived cell proteins by the ubiquitin proteasome pathway, and can accelerate the degradation of aggregation-prone proteins that cause major neurodegenerative diseases.