Fig 1: Phosphorylation of PRKAR1A inhibits PKA activity(A) Co-immunoprecipitations of V5-tagged PRKAR1A, S77A/S83A PRKAR1A, or S77E/S83 PRKAR1A and immunoblotting for PRKARCA in HEK293T cells stimulated with forskolin (10 µM, 30 min). n = 3 for each condition.(B) PKA activity in HEK293T cells stimulated with forskolin (10 µM, 30 min) and transfected with EV, wild-type PRKAR1A, S77A/S83A PRKAR1A mutant, or S77E/S83E PRKAR1A mutant as indicated.(C) HEK293T cells transfected with the indicated vectors and assessed for growth by Real Time Glo for 5 h after stimulation with forskolin (10 mM). A representative Real Time Glo assay analyzed in sextuplicate ±SEM is shown.(D) PKA activity in HEK293T cells stimulated with forskolin (10 µM, 30 min) and either overexpressing STK25 and/or the siRNA of PRKAR1A.(E) Model of STK25 downregulation of the PKA pathway through phosphorylation of PRKAR1A.For all graphs in this figure, n = 3 for each condition, data are presented as mean ± SD and analyzed in technical triplicates, *p < 0.05, ***p < 0.001, and ****p < 0.0001 by ANOVA with Tukey’s adjustment for multiple comparisons.See also Figure S2.
Fig 2: Stk25 loss increases response to adrenergic stimulation in vivo(A) Immmunoblot of Stk25, Prkaca, Gapdh, phospho-S77, phospho-S83, and total Prkar1a in Stk25+/+ and Stk25-/- whole-heart lysates.(B) Stk25+/+ and Stk25-/- mouse heart lysates were assessed for PKA activity in vitro.(C) Representative m-mode images of Stk25+/+ and Stk25-/- mouse hearts stimulated with either control or isoproterenol.(D) Echocardiographic measurements of ejection fraction (EF) and fractional shortening (FS) at unstimulated baseline and in response to isoproterenol, n = 5 for Stk25+/+ and n = 6 for Stk25-/-.(E) RT-PCR (left) from left ventricular myocardium of normal hearts (n = 6) or heart failure (n = 17) expressed as a ratio of the threshold cycle curve (Ct) of STK25 to GAPDH. Immunoblot (right) of STK25 and PRKAR1A expression and phosphorylation in protein lysates from left ventricular myocardium of normal hearts or failing hearts.Bar graphs presented as mean ± SD and analyzed in technical triplicates, *p < 0.05, **p < 0.01 by Student’s t test in (B), repeated measures two-way ANOVA with Sidak’s correction for multiple comparisons in (D), and Welch’s t test in (E).See also Figures S3 and S4 and Table S1.
Fig 3: STK25 inhibits PKA activity(A) Differential phosphoproteomic spectra of STK25+/+ and STK25-/- cardiomyocytes. Members of the PKA signaling pathway are highlighted.(B) Ingenuity phosphoprotein pathway analysis. Orange indicates pathways upregulated in STK25-/- cardiomyocytes, while blue indicates upregulation in STK25+/+.(C) PKA activity in response to 10 µM forskolin treatment for 30 min in STK25+/+ and STK25-/- cardiomyocytes. n = 3 for each condition.(D) PKA activity in response to 10 µM forskolin treatment for 30 min in HEK293T cells overexpressing either empty vector, wild-type STK2,5 or kinase-dead K49R/T174A. n = 3 for each.Bar graph data are represented as mean ± SD and analyzed in technical triplicates, *p < 0.05, **p < 0.01, ****p < 0.0001 using ANOVA and Tukey’s adjustment for multiple comparisons.
Fig 4: STK25 binds to and phosphorylates PRKAR1A(A) Immunoblot of PRKAR1A phospho-S77 and -S83 in STK25+/+ and STK25-/- cardiomyocyte protein lysates. n = 3 for each condition.(B) Immunoblots of phosphor-S77 and -S83 of PRKAR1A in STK25-/- cardiomyocytes transfected with empty vector (EV), wild-type STK25, and kinase dead K49R/T174A STK25. n = 2 for each condition.(C) Forskolin (10 µM, 30 min)-stimulated STK25+/+ and STK25-/- cardiomyocytes immunoblotted for phosphorylation of PRKAR1A. n = 3 for each condition.(D) Immunoprecipitation of FLAG-STK25 expressed in HEK293T cells and immunoblotted for PRKAR1A, PRKA2A, and GM130 (positive control binding partner). n = 3 for each condition.(E) Co-immunoprecipitation of PRKAR1A-V5 with STK25 and PRKACA in HEK293T cells treated with forskolin (10 µM, 30 min). n = 3 for each condition.(F) In vitro kinase assay of purified STK25 and PRKAR1A, immunoblotted for phosphorylation of S77 and S83 of PRKAR1A. n = 3 for each condition.See also Figure S1.
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