Mitogen-activated protein kinase Hog1 is activated in response to curcumin exposure in the budding yeast Saccharomyces cerevisiae

Abstract

Background: Curcumin (CUR), an active polyphenol derived from the spice turmeric, has been traditionally used for centuries in ancient Indian medicine to treat a number of diseases. The physiological effects of CUR have been shown to be diverse; however, the target molecules and pathways that CUR affects have yet to be fully described.

Results: Here, we demonstrate for the first time that the budding yeast mitogen-activated protein kinase (MAPK) Hog1 is essential for the response to CUR. Moreover, CUR-induced Hog1 phosphorylation was rescued by supplementation of iron to the growth medium. Hog1 was rapidly phosphorylated upon CUR treatment, but unlike the response to hyperosmotic shock (0.8 M NaCl), it remains activated for an extended period of time. A detailed analysis of HOG pathway mutants revealed that Pbs2p, Ptc2p, and Ssk2p are required for optimal CUR-induced Hog1 phosphorylation. We also observed a Hog1 dependent transcriptional response to CUR treatment that involved the up-regulation of glycerol-3-phosphate dehydrogenase 1 (GPD1), a factor that is essential for the hyperosmotic stress response.

Conclusions: Our present finding revealed the role of Hog1 MAPK in regulation of CUR-induced transcriptional response. We anticipate that our finding will enhance the understanding on the molecular mode of action of CUR on S. cerevisiae.

Figure 1

CUR treatment induces growth arrest in yeast cells can be rescued upon iron supplementation. Wild type cells (WT-1588-4C) were cultured in synthetic complete (SC) medium until they reached the exponential phase. Yeast cells were then treated with alpha-factor to synchronize them in the G1 phase. After synchronization, cells were released into media supplemented with (A) DMSO (control), (B) 50 μM CUR, (C) 100 μM CUR and (D) 100 μM CUR supplemented with 100 μM Iron. The cultures were sampled at the indicated time points and their DNA content was then analyzed by FACS.

Figure 2

Hog1 protein is phosphorylated in response to curcumin treatment. (A) A yeast strain expressing GFP-tagged Hog1 (Hog1-GFP) was grown until the exponential phase. Protein was extracted from cells incubated for 1 h with increasing concentrations of CUR (0, 5, 10, 20, 50, and 100 μM). The phosphorylated form of Hog1 was detected using an anti-phospho-p38 antibody (phospho-Hog1). The western blot membranes were probed for total Hog1 using a polyclonal anti-GFP antibody and this served as a loading control. (B) An exponentially growing wild type yeast strain was exposed to 100 μM CUR for 2 h. Samples were taken after 0, 30, 60, and 120 min of incubation. The expression of HOG1 mRNA levels was examined by SYBR Green real-time PCR. The error bars represent the standard deviation (SD) of three independent replicates. (C) Protein was extracted from cells incubated with either 0.8 M NaCl or 100 μM CUR at the indicated time points. The western blot membranes were probed for phospho-Hog1 or total Hog1 (anti-GFP). The anti-GFP signal served as a loading control.

Figure 3

Analysis of Hog1 phosphorylation after addition of iron. (A) A yeast strain expressing GFP-tagged Hog1 (Hog1-GFP) was grown until the exponential phase. Protein was extracted from cells incubated for 1 h with increasing concentrations of iron (50, 100 and 200 μM/1h). The phosphorylated form of Hog1 was detected using an anti-phospho-p38 antibody (phospho-Hog1). The western blot membranes were probed for total Hog1 using a polyclonal anti-GFP antibody and this served as a loading control (B) Cells were treated with BPS for 30 min followed by addition of iron (100 μM) and cells were harvested at indicated time points. Proteins were extracted and phosphorylation of hog1 was detected by phospho-hog1 antibody. The western blot membranes were probed for total Hog1 using a polyclonal anti-GFP antibody and this served as a loading control (C) Protein was extracted from cells incubated with 100 μM CUR at the indicated time points. The western blot membranes were probed for phospho-Hog1 and anti-GFP (Total Hog1p). The intensity of phosphorylated Hog1 was quantified using Image J software and normalized with respect to total Hog1p levels (anti-GFP) and shown in the form of bar diagramme. The error bars represent the standard deviation (SD) of three independent replicates. (D) Yeast strain expressing GFP-tagged Hog1 (Hog1-GFP) was treated with 100 μM CUR for 30 min followed by addition of iron (100 μM). Cells were harvested at indicated time points in figure and protein were extracted. The phosphorylation of hog1 was detected by phospho-hog1 antibody. The western blot membranes were probed for total Hog1 using a polyclonal anti-GFP antibody and this served as a loading control. The intensity of phosphorylated Hog1 was quantified using Image J software and normalized with respect to total Hog1p levels (anti-GFP) and shown in the form of bar diagramme. The error bars represent the standard deviation (SD) of three independent replicates. (E) Hog1Δ cells were treated with alpha-factor to synchronize them in the G1 phase. After synchronization, cells were released into media supplemented with (a) DMSO (control), (b) 100 μM CUR, (c) 100 μM CUR supplemented with 100 μM Iron and (D) 100 μM Iron. The cultures were sampled at the indicated time points and their DNA content was then analyzed by FACS.

Figure 4

Analysis of Hog1 phosphorylation in response to CUR treatment in HOG pathway mutants. (A and B) Wild type and HOG pathway mutant yeast strains were grown in SC media until they reached the exponential phase. Cells were treated with 100 μM CUR for 1 h and protein was extracted as described in the Materials and Methods. The western blot membranes were probed for phospho-Hog1. As a loading control, the same blots were reprobed with anti-TBP antibody. Equal protein loading was further confirmed by Ponceau S staining of the western blot membrane.

Figure 5

CUR-induces Hog1 phosphorylation dependent over-expression GPD1 mRNA. (A) Nuclear-cytoplasmic extracts were made as described in materials and methods. Whole cell extract (WCE), cytoplasmic fraction (CYT) and nuclear fraction (NUC) were loaded on SDS-PAGE and transferred onto nitrocellulose membrane. Western blot with anti-H3K36me3 antibody was performed to ensure the integrity of extract fractionation (H3K36me3 is a nuclear protein). The distribution of phosphorylated Hog1 was analyzed by probing with phospho-hog1 antibody. (B) An exponentially growing wild type or hog1Δ yeast strain was exposed to 100 μM CUR for 2 h. Samples were taken after 0, 30, 60, and 120 min of incubation. The expression of GPD1 mRNA levels in wild type or hog1Δ cells was examined by SYBR Green real-time PCR. The fold-change in GPD1 mRNA levels was normalized to the reference gene ALG9. The error bars represent the standard deviation (SD) of three independent replicates.

Figure 6

Proposed model showing molecular mode of action of CUR on S. cerevisiae. After entering into yeast cells CUR causes activation of osmotic stress. We have shown that CUR-induced phosphorylated Hog1 migrates to nucleus leading to up-regulation of GPD1 mRNA levels.