doi: 10.1073/pnas.1809254116.
Epub 2019 Feb 19.
26S Proteasomes are rapidly activated by diverse hormones and physiological states that raise cAMP and cause Rpn6 phosphorylation
Affiliations
- PMID: 30782827
- PMCID: PMC6410790
- DOI: 10.1073/pnas.1809254116
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26S Proteasomes are rapidly activated by diverse hormones and physiological states that raise cAMP and cause Rpn6 phosphorylation
Proc Natl Acad Sci U S A.
.
Abstract
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.
Keywords: cAMP; hormones; proteasome phosphorylation; protein degradation; ubiquitin proteasome system.
Conflict of interest statement
The authors declare no conflict of interest.
Figures
Raising cAMP levels with glucagon, epinephrine, or forskolin, enhances 26S proteasome activities and the degradation of short-lived proteins in mouse primary hepatocytes. (A) Mouse primary hepatocytes were incubated with the vehicle (DMSO) control, forskolin (10 μM), epinephrine (1 μg/mL), or glucagon (1 µg/mL) for 1 h and the 26S proteasome’s chymotrypsin-like peptidase activity was measured in cell extracts by following the hydrolysis of suc-LLVY-amc. n = 4, *P < 0.05. In this and subsequent figures, error bars represent the means ± SEM. (B) Raising cAMP levels enhances the peptidase activities of 26S proteasomes purified from mouse primary hepatocytes treated as in A. Proteasomes were affinity-purified by the Ubl-method. The chymotrypsin-like activity was assayed with suc-LLVY-amc, the caspase-like with ac-nLPnLD-amc, and the trypsin-like with boc-LRR-amc. n = 4, *P < 0.05. (C) Degradation of Ub5-DHFR by 26S proteasomes, purified by the Ubl-method from mouse hepatocytes treated as in A. The rates of degradation were measured by following the conversion of radiolabeled protein to TCA-soluble labeled material. n = 4, *P < 0.05. (D) Glucagon and epinephrine increase slightly the amount of assembled 26S proteasomes, both doubly capped and singly capped, in lysates of the mouse primary hepatocytes without increasing proteasome subunit levels. Native PAGE of cell extracts followed by suc-LLVY-amc overlay assay or Western analysis for a 19S subunit (Rpn1) or a 20S subunit (β5). The experiment was performed twice with three samples per condition. (E) Validation of the specificity of the phospho-specific Rpn6-S14 antibody. Plasmids encoding Rpn6-WT, -S14D, and -S14A were transfected into HEK293 cells and after 48 h the cells were treated with forskolin (10 μM) for 5 h and Western blot analysis was performed. There was greater expression of the plasmid-encoded Rpn6 variants, as detected by Western blot for Rpn6, because the CMV promoter on these plasmids contains a cAMP-responsive element, which was induced by the elevation of cAMP by forskolin. (F) Overexpression of constitutively active PKA promotes the phosphorylation of Rpn6-S14 in HEK293 cells. Western blot was performed both on the cell lysates and on 26S proteasomes purified from HEK293 cells or HEK293 cells overexpressing PKA. After 26S proteasomes were purified from PKA-overexpressing cells, they were incubated with PP1. (G) Forskolin, epinephrine, or glucagon increase similarly phosphorylation of Rpn6-S14 in primary mouse hepatocytes. Error bars are SEM for three cells per condition. One-way ANOVA with Bonferroni post hoc against the DMSO control condition. ***P ≤ 0.001. (H) Degradation rates of short-lived proteins increased similarly after treatment with glucagon or forskolin. To follow degradation of short-lived proteins, mouse primary hepatocytes were incubated with [3H]phenylalanine for 10 min and then washed three times with chase medium containing 150 µg/mL cycloheximide and 2 mM nonradioactive phenylalanine. The cells were then resuspended in chase media containing either vehicle control (DMSO), forskolin (10 μM), or glucagon (1 µg/mL), and media samples were collected at the indicated times. The radioactivity released from cell proteins was measured and plotted as a percentage of the total radioactivity incorporated into proteins at time 0. Error bars represent the SEM of four independent samples. (I) PKA activation by forskolin or glucagon does not enhance the degradation of long-lived proteins in hepatocytes. Mouse primary hepatocytes were incubated with [3H]phenylalanine (2 μCi/mL) for 20 h to label cell proteins and then switched to chase medium containing 2 mM nonradioactive phenylalanine for 2 h (15). To dissect the relative rates of lysosomal and proteasomal degradation, cells were pretreated with concanamycin A (100 nM) for 1 h, and then with forskolin, glucagon, or Torin1 for 1 h before collecting medium samples and calculating the rate of proteolysis. The concanamycin A-sensitive portion of total proteolysis was considered lysosomal proteolysis and the concanamycin A-resistant portion of total proteolysis was used as proteasomal proteolysis, as validated previously (15). *P < 0.05.
Epinephrine stimulates 26S proteasome activities in isolated working rat hearts. (A) Epinephrine enhances the peptidase activity of 26S proteasomes purified by the Ubl-method from perfused rat hearts. Working hearts were treated with epinephrine or subjected to increased afterload (IA) or both. Chymotrypsin-like activity of the 26S proteasomes was measured with suc-LLVY-amc. n = 3, *P < 0.05. Error bars represent mean ± SEM. Cardiac work load (power and atrial pressure during perfusion are shown in the SI Appendix, Fig. S3 A and B ). (B) Epinephrine enhances ATP hydrolysis by 26S proteasomes purified from rat hearts treated as in A. Basal ATPase activity was measured by following the production of free phosphate using the malachite green. n = 3. *P < 0.05. (C) Epinephrine stimulates degradation by 26S proteasomes of Ub5-DHFR. Rates of degradation were measured by following the conversion of radiolabeled protein to TCA-soluble labeled peptides. n = 3. *P < 0.05. (D) Epinephrine treatment increased phosphorylation of Rpn6-S14 in 26S proteasomes purified from perfused hearts treated as in A. 26S proteasomes were purified from three hearts per condition. (E) 26S proteasomes purified from rat hearts treated with epinephrine exhibited more phosphorylated bands than from control hearts. Proteasomes were run on SDS/PAGE and stained with ProQ Diamond phospho-stain followed by Coomassie blue. The experiment was performed with three hearts per condition, which gave similar results. Representative gels are shown. (F) The 26S proteasomes from rat hearts were incubated with λ-phosphatase or phosphatase inhibitors. ProQ diamond phospho-stain was used to confirm the phosphorylation status of the proteasomes. (G) Dephosphorylation of 26S proteasomes from rat hearts after treatment with epinephrine reversed the increase in proteasome activity.
Vasopressin enhances proteasomal activity and intracellular proteolysis of only short-lived proteins in mouse kidney collecting duct cells. (A) Desmopressin, like forskolin, stimulates the degradation of short-lived proteins in a PKA-dependent manner in mpkCCD cells. PKA WT and PKA dKO mpkCCD cells (20) were pulsed with [3H]phenylalanine (2.5 μCi/mL) for 20 min and then washed twice with chase medium containing 150 µg/mL cycloheximide and 2 mM nonradioactive phenylalanine. The cells were then resuspended in chase media containing either DMSO, forskolin (5 μM), or desmopressin (100 nM), and media samples were collected at the indicated times. The TCA-soluble radioactivity in the media was plotted as a percentage of the radioactivity initially incorporated into cell proteins. Error bars are the SEM of four samples. (B) Raising cAMP with desmopressin or forskolin did not enhance the degradation of long-lived proteins in mpkCCD cells, unlike Torin1. To label cell proteins, WT mpkCCD cells were incubated with [3H]phenylalanine (1 μCi/mL) for 20 h and then switched to chase medium containing 2 mM nonradioactive phenylalanine for 2 h. New chase media was added containing either forskolin (5 μM), desmopressin (100 nM), or Torin1 (250 nM). Media samples were collected at 1, 2, and 3 h, and the TCA-soluble radioactivity in the media was plotted as a percentage of the radioactivity incorporated into cell proteins over time. Data shown are the slopes calculated from the linear degradation rates. Error bars are the SEM of three samples. One-way ANOVA with a Bonferroni poshoc analysis against DMSO. ***P < 0.001. (C) Desmopressin increases proteasomal peptidase activity in mpkCCD cell lysates and this effect required PKA. PKA WT and PKA dKO mpkCCD cells were treated for indicated times and proteasomal hydrolysis of suc-LLVY-amc was measured in the cell extracts. Error bars are the SEM of three independent samples. One-way ANOVA with a Bonferroni poshoc analysis against time 0. ***P < 0.001, *P < 0.05. (D) Desmopressin stimulates rapid phosphorylation of Rpn6-S14 in mpkCCD cell lysates. The cell lysates from C were analyzed by SDS PAGE and Western blot for pRpn6-S14 and GAPDH. Error bars are the SEM of three samples. One-way ANOVA with a Bonferroni post hoc analysis against time 0. ***P < 0.001, **P < 0.01, *P < 0.05. (E) Desmopressin treatment caused small, but reproducible increases in the amount of doubly capped and singly capped 26S in mpkCCD WT cells, but not in cells lacking PKA. PKA WT and PKA dKO mpkCCD cells were treated for indicated times, lysed, and lysates were analyzed by Native PAGE and Western blot for Rpn1. The same lysates were also analyzed by SDS PAGE and Western blot for Rpn1 to evaluate levels of proteasome subunits. IB, immunoblot.
Intense exercise in humans and repetitive contractions in rat hindlimbs enhances Rpn6-S14 phosphorylation and proteasome activity in skeletal muscles. (A) High-intensity bicycling by human volunteers caused phosphorylation of Rpn6-S14 and reduced the levels of K48-linked ubiquitin conjugates in biopsies of quadricep muscles from four male volunteers. Biopsies are the same ones analyzed previously (21). Both preexercise and postexercise muscle samples were subjected to immunoblot analysis for pRpn6-S14, Rpn6, β5, LC3, and K48-Ub. GAPDH was used as loading control. Line graphs represent the levels of K48-linked polyubiquitinated proteins and pRpn6-S14 determined by densitometry. n = 4. *P < 0.05. Error bars here and below represent mean ± SEM. (B) High-intensity cycling exercise in humans promotes peptidase activity of 26S proteasomes in muscle lysates. Biopsies studied in A were lysed and chymotrypsin-like peptidase activity was measured in muscle extracts using suc-LLVY-amc as the substrate. n = 4, *P < 0.05. (C) High-intensity repetitive contractions of rat anterior tibialis muscles by repetitive stimulation for 5 min of the sciatic nerves in anesthetized rats (Stim) enhances 26S proteasome activity. Chymotrypsin-like peptidase activity (the hydrolysis of suc-LLVY-amc) was measured in muscle extracts. n = 5, *P < 0.05. (D) Repetitive contractions of rat anterior tibialis muscles by stimulation of the sciatic nerves (Stim) as described in C increased phosphorylation of Rpn6-S14 and reduced the levels of K48-linked polyubiquitinated proteins conjugates in muscle lysates. Both sham and stimulated muscle samples were subjected to immunoblot analysis for pRpn6-S14, Rpn6, and K48-Ub. GAPDH was used as the loading control. Bar graphs represent the levels of K48-Ub and pRpn6-S14 determined by densitometry. n = 5, *P < 0.05.
Food deprivation of mice increases cAMP levels, proteasomal activities, and Rpn6-S14 phosphorylation in skeletal muscle and liver. (A) Depriving mice of food for the indicated times increased at 12 and 24 h the amount of cAMP in skeletal muscles. cAMP content was measured in TA muscle extracts using a cAMP-ELISA kit. Error bars here and below represent mean ± SEM. n = 4, *P < 0.05. (B) Fasting stimulates the peptidase activity of 26S proteasomes purified from hindlimb muscles. Food was removed from the mice at the indicated times, the muscles were homogenized, and 26S proteasomes were affinity-purified from the muscle extracts of the fasted mice and fed controls. The chymotrypsin-like activity was measured with suc-LLVY-amc. n = 4 mice per time point, *P < 0.05. (C) Fasting increases the capacity of muscle 26S proteasomes to degrade Ub5-DHFR. Proteasome preparations were the same as studied in B. Rates of degradation were measured by following the conversion of radiolabeled protein to TCA-soluble 32P-labeled peptides. n = 4 mice per time point, *P < 0.05. (D) Food deprivation enhances phosphorylation of Rpn6 but not the phosphorylation of Rpt6. 26S proteasomes were purified from hindlimb muscles of mice fasted for indicated times as described in A. Samples were subjected to Zn2+-Phos-tag SDS/PAGE and followed by immunoblot analysis for Rpn6 and Rpt6. The same samples were also analyzed by SDS/PAGE and Western blot for Rpn2, Rpt5, and MCP. *P ≤ 0.05, **P ≤ 0.01. (E) After food deprivation of mice levels of cAMP in liver extracts increased. Levels of cAMP were measured by cAMP-ELISA kit. n = 4 mice per time point, *P < 0.05. (F) Peptidase activity of hepatic 26S proteasomes increased after 12–48 h of food deprivation. At each time of food deprivation, livers were removed from the mice studied in A, homogenized, and 26S proteasomes purified via the Ubl-method. Chymotrypsin-like activity was measured as in B. *P < 0.05. (G) Food deprivation of mice stimulates the phosphorylation of Rpn6-S14 in liver. 26S proteasomes purified from livers were subjected to immunoblot analysis for pRpn6-S14 and Rpn6. n = 4 mice per time point, *P < 0.05.
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