Ser14-Rpn6/PSMD11 Phosphorylation Mediates the Activation of 26S Proteasomes by cAMP and Protects against Cardiac Proteotoxic Stress in Mice.

Means to enhance proteasome (PSM) functioning are highly sought. In cultured cells, cAMP-dependent protein kinase (PKA) was shown to activate 26S PSMs by phosphorylating Ser14 of Rpn6/PSMD11 (a key PSM subunit), but this remains to be established in animals. Hence, we sought to test the hypothesis that Ser14-Rpn6 phosphorylation (pS14-Rpn6) mediates the activation of PSMs by PKA and reduces proteotoxicity in vivo (Figure 1). We generated via CRSPR/Cas9 gene editing two mouse models, in which the codon for Ser14 in the Rpn6 gene was mutated to encode either Alanine (S14A) for blocking pS14-Rpn6 or Aspartic acid (S14D) for mimicking this phosphorylation. Treatment with adenylate cyclase activator forskolin (5mg/kg, i.p.) increased cardiac pS14-Rpn6 (2.52±0.08 vs. 1.0, p<0.001) and PSM activities (292.35±45.75 vs. 100, p<0.005) in wild type mice, but these were abolished by co-treatment of PKA inhibitor H89 (15mg/kg). Forskolin did not show these effects in S14A mice, demonstrating that pS14-Rpn6 is required for PKA to activate the PSM. Cross-breeding transgenic GFPdgn (a proven substrate of the ubiquitin-proteasome system [UPS]) into S14D mice revealed that S14D decreased cardiac GFPdgn proteins (0.81±0.08 vs. 1.0±0.08, p<0.05) without altering GFPdgn mRNA levels, indicating that S14D suffices to enhance UPS performance. Basal myocardial PSM activities were significantly higher in S14D mice compared to WT mice (1.52±0.38 vs. 1.0±0.16, p<0.005). To determine the effect of increasing pS14-Rpn6 on proteotoxicity, we cross-bred S14D into the CryAB-R120G mice (R120G), a classic model of cardiac proteotoxicity. We detected that myocardial total (0.76±0.07 vs. 1.0±0.19, p<0.005) and insoluble (0.80±0.12 vs. 1.0±0.14, p<0.01) CryAB-R120G protein levels were lower in S14D::R120G compared to WT::R120G mice (n=4 males+4 females/group), suggesting that PSM activation by PKA decreases the accumulation and aggregation of misfolded proteins in vivo. Echocardiography further revealed that S14D significantly attenuated the decrease of left ventricular (LV) ejection fraction (51.80±11.65 vs. 40.20±13.88 %, p<0.01), stroke volume (38.44±7.30 vs. 28.65±5.69 μl, p<0.005) and cardiac output (17.45±3.09 vs. 12.77±2.46 ml/min, p<0.005) of R120G mice at 6 months of age, which indicates that genetic mimicry of pS14-Rpn6 ameliorates cardiac proteotoxicity. We have established for the first time in vivo that pS14-Rpn6 is required for activation of 26S PSMs by PKA and provides the first genetic evidence that PKA-induced PSM activation protects against proteotoxicity in animals. © FASEB.

Citation

Liuqing Yang, Nirmal Parajuli, Penglong Wu, Jinbao Liu, Xuejun Wang. Ser14-Rpn6/PSMD11 Phosphorylation Mediates the Activation of 26S Proteasomes by cAMP and Protects against Cardiac Proteotoxic Stress in Mice. FASEB journal : official publication of the Federation of American Societies for Experimental Biology. 2022 May;36 Suppl 1

PMID: 35557166

search → result