Research Paper Volume 14, Issue 2 pp 678—707

Figure 4. 14–3–3ζ regulates ATP synthase 5 alpha/beta dimerization. (A) Multiple sequence alignment shows a conserved motif in 14–3–3ζ across species (human, mouse, rat, sheep, pig). A potential phosphorylation motif in 14–3–3ζ was predicted by using kinase prediction tool (https://services.healthtech.dtu.dk/service.php?NetPhos-3.1). Ser 58 in 14–3–3ζ was predicted as a potential phosphorylation site. (B) Effect of KB1541 on ATP synthase 5 alpha/beta dimerization in vivo. Cells were transfected with pCMV–Myc–14–3–3ζ (WT) or pCMV–Myc–14–3–3ζ (S58A) and treated with DMSO or KB1541. Then, immunoprecipitation was performed with an antibody against Myc-tag. 14–3–3ζ (WT) or 14–3–3ζ (S58A) phosphorylation was detected by a phospho-Ser antibody. 14–3–3ζ (WT) phosphorylation was increased upon KB1541 treatment (blue boxed areas). However, 14–3–3ζ (S58A) phosphorylation did not increase upon KB1541 treatment (red boxed areas). 14–3–3ζ (WT) and ATP synthase 5 alpha interaction increased upon KB1541 treatment. The subsequent interaction of 14–3–3ζ (WT) with ATP synthase 5 beta also increased. However, 14–3–3ζ (S58A) and ATP synthase 5 alpha interaction was not affected by KB1541 treatment, nor was the interaction of 14–3–3ζ (S58A) with ATP synthase 5 beta. (C and D) OXPHOS portion and glycolytic portion of ATP production (^*P < 0.05, ^**P < 0.01, student t-test). Mean ± S.D., n = 3.