(A, B) Effects of CKA2 deletion on NaCl sensitivity in WT (A), sir2∆ (A), and pde2∆ cells (B). (C, D) Effects of CKA2 deletion on the expression of PMA1 (C) and RPL3 (D) in WT, sir2∆, and pde2∆ cells measured by qRT-PCR (*p < 0.01). (E) Effects of SIR2-S473E or SIR2-S473A on NaCl sensitivity in WT and cka2∆ cells. (F, G) Effects of SIR2-S473E or SIR2-S473A on the expression of PMA1 (F) and RPL3 (G) in WT and cka2∆ cells measured by qRT-PCR (*p < 0.005). (H) Sir2 phosphorylation levels in WT, pde2∆, and pde2∆ cka2∆ cells. Myc-tagged Sir2 proteins were immunoprecipitated (IP) and analyzed by WB as indicated. (I) In vivo Sir2 and Cka2 interaction in WT, pde2∆, and pde2∆ tpk1/2/3∆ cells. Flag-tagged Cka2 proteins (Cka2-Flag) were IP and analyzed by WB. (J) Cka2-Flag enrichment at the PMA1 promoter in WT, pde2∆, and pde2∆ tpk1/2/3∆ cells measured by Flag ChIP (*p < 0.001). Values in (C), (D), (F), (G), and (J) represent the average of at least three independent experiments (±S.D.). NaCl sensitivity of the 121 kinase mutant strains used to identify kinases required for protein kinase A (PKA)-dependent Sir2 phosphorylation is available in the Figure 3—source data 1.