Research Paper Volume 7, Issue 2 pp 133—143
Distinct regulation of Maf1 for lifespan extension by Protein kinase A and Sch9
Figure 1. PKA is dispensable for Maf1 phosphorylation and subcellular localization. (A) PKA hyper-activation does not rescue Maf1 phosphorylation in sch9Δ mutant. WT, bcy1Δ, sch9Δ and sch9Δ bcy1Δ cells expressing a Myc-tagged Maf1 (Maf1-Myc) from a low copy centromeric plasmid were cultured to early log phase and protein samples were prepared for western blot analysis. Phosphorylated Maf1 ran slower than dephosphorylated Maf1 on SDS-PAGE. (B) Quantification of the ratio Maf1 phosphorylation vs. de-phosphorylation from 3 independent experiments as shown in A. ns, not significant; * p < 0.01. (C) PKA hyper-activation does not rescue Maf1 cytoplasmic localization in sch9Δ mutant. WT, bcy1Δ, sch9Δ and sch9Δ bcy1Δ cells expressing Maf1-Myc from a low copy centromeric plasmid were cultured to early log phase and cells were fixed for immunofluorescence analysis. Maf1 was distributed in both nucleus and cytoplasm in WT and bcy1Δ cells but was restricted to nucleus when SCH9 was further deleted. (D) Quantification of cells with Maf1 nuclear localization from 3 independent experiments as shown in C. ns, not significant; * p < 0.01.