Figure 3: CK2 kinase modulates human HSF1 activity and stability in yeast.
(a) Experimental design of humanized HSF1 yeast screen for kinase inhibitors that promote yeast HSF1-dependent growth. (b) Yeast cells expressing human HSF1 cultivated with ser-thr kinase inhibitors and growth (OD600 nm) monitored over 4 days. Data presented correspond to results at 20 nM concentration. Similar results were obtained at other tested drug concentrations from 2 nM to 20 μM. DMSO was negative control and HSF1A used as positive control. (a=Ser/Thr kinases, b=Try kinases). Error bars represent±s.e.m., (n=4). (c) Yeast CK2 holoenzyme subunit composition and function. (d) Experimental design of humanized HSF1 yeast screen for CKB1 deletion that promote yeast HSF1-dependent growth. (e) WT and CKB1 mutant strain (ckb1Δ) grown in SC-His or 5-FOA medium for 3 days at 30 °C. (f) Protein extracts from WT (CKB1) and mutant strain (ckb1Δ) immunoblotted for human HSF1 using Pgk as loading control. (g) Summary of human HSF1 phosphorylation sites mediated by recombinant CK2α, CK2α′ or CK2 holoenzyme in vitro and analysed by phosphoproteomics, where (+) indicates detection of phosphorylation, DBD; DNA binding domain; LZ1-3 and LZ4; leucine zipper domains. (h) Yeast expressing WT human HSF1, S303A or S303/S307A mutants grown in glucose with DMSO or 10 μM TID43 and OD600 nm monitored over 4 days. Statistical significance was measured 4 days of growth. Error bars represent±s.e.m., (n=3). Unpaired t-test; NS, no significant; *P<0.05, ***P<0.001.
