Pharmacological agents that raise cAMP and activate protein kinase A (PKA) stimulate 26S proteasome activity, phosphorylation of subunit Rpn6, and intracellular degradation of misfolded proteins. We investigated whether a similar proteasome activation occurs in response to hormones and under various physiological conditions that raise cAMP. Treatment of mouse hepatocytes with glucagon, epinephrine, or forskolin stimulated Rpn6 phosphorylation and the 26S proteasomes’ capacity to degrade ubiquitinated proteins and peptides. These agents promoted the selective degradation of short-lived proteins, which are misfolded and regulatory proteins, but not the bulk of cell proteins or lysosomal proteolysis. Proteasome activities and Rpn6 phosphorylation increased similarly in working hearts upon epinephrine treatment, in skeletal muscles of exercising humans, and in electrically stimulated rat muscles. In WT mouse kidney cells, but not in cells lacking PKA, treatment with antidiuretic hormone (vasopressin) stimulated within 5-minutes proteasomal activity, Rpn6 phosphorylation, and the selective degradation of short-lived cell proteins. In livers and muscles of mice fasted for 12–48 hours cAMP levels, Rpn6 phosphorylation, and proteasomal activities increased without any change in proteasomal content. Thus, in vivo cAMP-PKA–mediated proteasome activation is a common cellular response to diverse endocrine stimuli and rapidly enhances the capacity of target tissues to degrade regulatory and misfolded proteins (e.g., proteins damaged upon exercise). The increased destruction of preexistent regulatory proteins may help cells adapt their protein composition to new physiological conditions.

The ubiquitin proteasome system degrades the great majority of proteins in mammalian cells. Countless studies have described how ubiquitination promotes the selective degradation of different cell proteins. However, there is a small but growing literature that protein half-lives can also be regulated by post-translational modifications of the 26S proteasome. This article reviews the ability of several kinases to alter proteasome function through subunit phosphorylation. For example, PKA and DYRK2 stimulate the proteasome’s ability to degrade ubiquitinated proteins, peptides, and ATP, while one kinase, ASK1, inhibits proteasome function during apoptosis. Proteasome phosphorylation is likely to be important in regulating protein degradation because it occurs downstream of many hormones and neurotransmitters, in conditions that raise cAMP or cGMP levels, after calcium influx following synaptic depolarization, and during phases of the cell cycle. Beyond its physiological importance, pharmacological manipulation of proteasome phosphorylation has the potential to combat various diseases. Inhibitors of phosphodiesterases by activating PKA or PKG can stimulate proteasomal degradation of misfolded proteins that cause neurodegenerative or myocardial diseases and even reduce the associated pathology in mouse models. These observations are promising since in many proteotoxic diseases, aggregation-prone proteins impair proteasome function and disrupt protein homeostasis. Conversely, preventing subunit phosphorylation by DYRK2 slows cell cycle progression and tumor growth. However, further research is essential to determine how phosphorylation of different subunits by these (or other) kinases alter the properties of this complex molecular machine and thus influence protein degradation rates.

Because raising cAMP enhances 26S proteasome activity and the degradation of cell proteins, including the selective breakdown of misfolded proteins, we investigated whether agents that raise cGMP may also regulate protein degradation. Treating various cell lines with inhibitors of phosphodiesterase 5 or stimulators of soluble guanylyl cyclase rapidly enhanced multiple proteasome activities and cellular levels of ubiquitinated proteins by activating protein kinase G (PKG). PKG stimulated purified 26S proteasomes by phosphorylating a different 26S component than is modified by protein kinase A. In cells and cell extracts, raising cGMP also enhanced within minutes ubiquitin conjugation to cell proteins. Raising cGMP, like raising cAMP, stimulated the degradation of short-lived cell proteins, but unlike cAMP, also markedly increased proteasomal degradation of long-lived proteins (the bulk of cell proteins) without affecting lysosomal proteolysis. We also tested if raising cGMP, like cAMP, can promote the degradation of mutant proteins that cause neurodegenerative diseases. Treating zebrafish models of tauopathies or Huntington’s disease with a PDE5 inhibitor reduced the levels of the mutant huntingtin and tau proteins, cell death, and the resulting morphological abnormalities. Thus, PKG rapidly activates cytosolic proteasomes, protein ubiquitination, and overall protein degradation, and agents that raise cGMP may help combat the progression of neurodegenerative diseases.

In several neurodegenerative diseases in which misfolded proteins accumulate there is impairment of the ubiquitin proteasome system (UPS). We tested if a similar disruption of proteostasis occurs in hereditary peripheral neuropathies. In sciatic nerves from mouse models of two human neuropathies, Myelin Protein Zero mutation (S63del) and increased copy number (P0 overexpression), polyubiquitinated proteins accumulated, and the overall rates of protein degradation were decreased. 26S proteasomes affinity-purified from sciatic nerves of S63del mice were defective in degradation of peptides and a ubiquitinated protein, unlike proteasomes from P0 overexpression, which appeared normal. Nevertheless, cellular levels of 26S proteasomes were increased in both, through the proteolytic-activation of the transcription factor Nrf1, as occurs in response to proteasome inhibitors. In S63del, increased amounts of the deubiquitinating enzymes USP14, UCH37, and USP5 were associated with proteasomes, the first time this has been reported in a human disease model. Inhibitors of USP14 increased the rate of protein degradation in S63del sciatic nerves and unexpectedly increased the phosphorylation of eIF2α by Perk. Thus, proteasome content, composition and activity are altered in these diseases and USP14 inhibitors have therapeutic potential in S63del neuropathy.

Agents that raise cGMP by activating Protein Kinase G increase 26S proteasome activities, protein ubiquitination, and degradation of misfolded proteins. Therefore, they may be useful in treating neurodegenerative and other diseases caused by an accumulation of misfolded proteins. Mutations in myelin protein zero (MPZ) cause the peripheral neuropathy Charcot Marie Tooth 1B (CMT1B). In peripheral nerves of a mouse model of CMT1B, where the mutant MPZS63del is expressed, proteasome activities are reduced, mutant MPZS63del and polyubiquitinated proteins accumulate, and the Unfolded Protein Response (p-eif2 α) is induced. In HEK293 cells, raising cGMP stimulated ubiquitination and degradation of MPZS63del, but not of MPZWT. Treating S63del mice with the phosphodiesterase 5 inhibitor, sildenafil, to raise cGMP increased proteasome activity in sciatic nerves and reduced the levels of polyubiquitinated proteins, the proteasome reporter ubG76V-GFP, and p-elF2α. Furthermore, sildenafil treatment reduced the number of amyelinated axons, and increased myelin thickness and nerve conduction velocity in sciatic nerves. Thus, agents that raise cGMP, including ones widely used in medicine, may be useful therapies for CMT1B and other proteotoxic diseases.

Rates of degradation by the ubiquitin proteasome system depend not only on rates of ubiquitination but also on the level of proteasome activity, which can be regulated through phosphorylation of proteasome subunits. Many protein kinases have been proposed to influence proteasomal activity. However, for only two is there strong evidence that phosphorylation of a specific 26S subunit enhances the proteasome’s capacity to degrade ubiquitinated proteins and promotes protein breakdown in cells: 1) Protein Kinase A (PKA), which after a rise in cAMP phosphorylates the 19S subunit Rpn6, and 2) Dual Tyrosine Receptor Kinase 2 (DYRK2), which during S through M phases of the cell cycle phosphorylates the 19S ATPase subunit Rpt3. These findings were made possible by the development of methods to rapidly purify 26S proteasomes and to assay their multiple activities. The use of pulse-chase isotopic methods showed that PKA activation in cells enhances the degradation of short-lived cell proteins and misfolded proteins that cause neurodegenerative diseases, while DYRK2 enhances the breakdown of long-lived proteins and promotes progression through the cell cycle. The methods discussed here should be useful in clarifying the roles of other kinases and other post-translational modifications of proteasome subunits.

No abstract

Schwann cells (SCs) are exquisitely sensitive to the elasticity of their environment and their differentiation and capacity to myelinate depend on the transduction of mechanical stimuli by YAP and TAZ. YAP/TAZ, in concert with other transcription factors, regulate several pathways including lipid and sterol biosynthesis as well as extracellular matrix receptor expressions such as integrins and G-proteins. Yet, the characterization of the signaling downstream YAP/TAZ in SCs is incomplete. Myelin sheath production by SC coincides with rapid up-regulation of numerous transcription factors. Here, we show that ablation of YAP/TAZ alters the expression of transcription regulators known to regulate SC myelin gene transcription and differentiation. Furthermore, we link YAP/TAZ to two DNA binding proteins, Cc2d1b and Purβ , which have no described roles in myelinating glial cells. We demonstrate that silencing of either Cc2d1b or Purβ limits the formation of myelin segments. These data provide a deeper insight into the myelin gene transcriptional network and the role of YAP/TAZ in myelinating glial cells.