In vivo genetic interrogations establish unequivocally the pathophysiological significance of proteasome phosphoregulation by protein kinase A

Abstract

Protein kinase A (PKA) has been extensively studied in pathophysiology but little is known about its regulation over protein degradation. Rpn6/PSMD11 phosphorylation at Ser14 (pS14-Rpn6), rather than previously reported Rpt6/PSMC5 phosphorylation at Ser120, was recently shown to be fully responsible for PKA-mediated activation of 26S proteasomes in cultured non-cardiac cells. However, the in vivo (patho)physiological relevance of proteasome phosphoregulation by PKA or any kinases has not been established. Hence, we have conducted the following studies. First, PKA activation increased pS14-Rpn6 and shortened the halflife of a surrogate misfolded protein in cardiomyocytes. Second, gene editing was employed to create mice with Ser14 of Rpn6/Psmd11 is replaced with either Ala (S14A) or Asp (S14D) to block or mimic PKA-mediated phosphorylation, respectively. Increases in myocardial pS14-Rpn6 and proteasome activities by PKA activation in wild type (WT) mice were completely lost in S14A mice while S14D mice showed significantly higher basal proteasome activities than WT mice, providing the first in vivo demonstration that Ser14 of Rpn6/Psmd11 is the primary phosphosite for PKA to activate proteasomes. Third, β-adrenergic stimulation with isoproterenol induced significantly greater cardiac hypertrophy in S14A mice compared with WT mice. Lastly, Adult S14A, S14D and WT mice were subject to coronary artery ligation to produce myocardial infarction (MI). Echocardiography at 2-week post-MI revealed no discernible difference in any parameters among the three MI groups; however, the S14D MI group showed significantly less LV chamber dilatation at 4 weeks post-MI and, by 8 weeks, the reduction of fractional shortening and ejection fraction became more severe in the S14A MI group than in the other two MI groups, indicating that proteasome phosphorylation at Ser14-Rpn6 protects against post-MI cardiac remodeling. Taken together, we have unequivocally established that proteasome phosphoregulation by PKA protects hearts against stress.