Transient activation of fission yeast AMPK is required for cell proliferation during osmotic stress

Abstract

The heterotrimeric kinase AMPK acts as an energy sensor to coordinate cell metabolism with environmental status in species from yeast through humans. Low intracellular ATP leads to AMPK activation through phosphorylation of the activation loop within the catalytic subunit. Other environmental stresses also activate AMPK, but it is unclear whether cellular energy status affects AMPK activation under these conditions. Fission yeast AMPK catalytic subunit Ssp2 is phosphorylated at Thr-189 by the upstream kinase Ssp1 in low-glucose conditions, similar to other systems. Here we find that hyperosmotic stress induces strong phosphorylation of Ssp2-T189 by Ssp1. Ssp2-pT189 during osmotic stress is transient and leads to transient regulation of AMPK targets, unlike sustained activation by low glucose. Cells lacking this activation mechanism fail to proliferate after hyperosmotic stress. Activation during osmotic stress requires energy sensing by AMPK heterotrimer, and osmotic stress leads to decreased intracellular ATP levels. We observed mitochondrial fission during osmotic stress, but blocking fission did not affect AMPK activation. Stress-activated kinases Sty1 and Pmk1 did not promote AMPK activation but contributed to subsequent inactivation. Our results show that osmotic stress induces transient energy stress, and AMPK activation allows cells to manage this energy stress for proliferation in new osmotic states.

FIGURE 1:

Transient activation of Ssp2 by hyperosmotic stress. (A) Western blot showing levels of Ssp2-pT189 in response to the indicated environmental stresses. Treatments were for 15 min. We used α-myc as a loading control for total Ssp2. For α-Ssp2-pT189, asterisks denote background bands, and arrowheads mark the Ssp2-pT189 band. (B) Western blot showing activation kinetics of Ssp2-pT189 in response to 1 M KCl. We used 0.1% glucose treatment as a control for Ssp2-pT189 induction and α-myc as a loading control for total Ssp2. (C) Western blot showing activation kinetics of Ssp2-pT189 in response to 1.2 M sorbitol. We used 0.1% glucose treatment as a control for Ssp2-pT189 induction and α-myc as a loading control for total Ssp2. (D) Western blot showing activation kinetics of Ssp2-pT189 in response to 1 M NaCl. We used 1 M KCl treatment as a control for Ssp2-pT189 induction and α-myc as a loading control for total Ssp2. (E) Localization of Scr1-mEGFP with control EMM4S treatment or EMM4S plus 1 M KCl treatment using a microfluidics device. Images are single focal planes. Preswitch indicates image taken before switch to control or 1 M KCl medium. Scale bar, 8 μm. (F) Quantification of Scr1-mEGFP nuclear localization from microfluidics-based movies as in E. More than 50 cells/condition. Mean ± SD based on three individual biological replicates.

FIGURE 2:

Osmotic stress activation of Ssp2 is dose dependent. (A) Western blot showing activation of Ssp2-pT189 in response to the indicated concentrations of KCl for 15 min. We used α-myc as a loading control for total Ssp2. For α-Ssp2-pT189, asterisks denote background bands, and arrowheads mark Ssp2-pT189 bands. (B) Western blot showing activation kinetics of Ssp2-pT189 in response to 1, 0.8, and 0.6 M KCl osmotic stress. We used α-myc as a loading control for total Ssp2. (C) Quantification of Ssp2-pT189 dynamics for the indicated concentrations of KCl. Mean ± SD based on three individual biological replicates.

FIGURE 3:

Ssp1 activates Ssp2 for cell proliferation in osmotic stress. (A) Western blot showing activation of Ssp2-pT189 in wild-type and ssp1∆ cells in response to 15 min of the indicated treatments. We used α-myc as a loading control for total Ssp2. For α-Ssp2-pT189, asterisks denote background bands, and arrowheads mark Ssp2-pT189 bands. (B) Western blot showing activation kinetics of Ssp1 substrates Ssp2-pT189 and Cdr2-pT166 in response to 1 M KCl osmotic stress. We used α-myc as a loading control for both Ssp2 and Cdr2. (C) Quantification of Ssp2-pT189 and Cdr2-pT166 levels in response to 1 M KCl. Mean ± SD based on three individual biological replicates. (D) Tenfold serial dilutions of the indicated strains were spotted onto control (YE4S) plates or plates containing 0.8 M KCl. Cells were grown at 32°C.

FIGURE 4:

ssp2∆ mutants arrest growth and division during osmotic stress. (A) Differential interference contrast (DIC) images of selected time points for wild-type or ssp2∆ cells growing in a microfluidics device before and after exposure to 1 M KCl. Yellow triangles indicate ssp2∆ cells; unmarked cells are wild type. Time is indicated in hours:minutes. (B) Quantification of total cell number for wild-type vs. ssp2∆ strains after shift to 1 M KCl. Cells were imaged in time lapse using microfluidics, as in A. Cells were manually counted from each time frame, and only cells that were present in the imaging field throughout the entire experiment were counted.

FIGURE 5:

AMPK senses depletion of cellular ATP levels during osmotic stress. (A) Western blot showing activation of Ssp2-pT189 in wild-type, cbs2∆, and amk2∆ cells in response to 1 M KCl. Preswitch (unstressed) and 0.1% glucose are used as a control for Ssp2 activation. Stress treatments were for 15 min. We used α-myc as a loading control for total Ssp2. For α-Ssp2-pT189, asterisks denote background bands, and arrowheads mark Ssp2-pT189 bands. (B) Tenfold serial dilutions of the indicated strains were spotted onto control (YE4S) plates or plates containing 0.8 M KCl. Cells were grown at 32°C. (C) Change in cellular ATP levels by 5-min treatment with the indicated stresses (preswitch and control were unstressed YE4S medium). ATP levels were measured and normalized as described in Materials and Methods. Mean ± SD. An unpaired t test was performed for statistical analyses, and p values were based on two-tailed distributions (*p < 0.05).

FIGURE 6:

Fission of mitochondria during osmotic stress. (A) Representative images of mitochondria in wild-type cells before and after osmotic stress. (B) Quantification of cells expressing fragmented (beads-on-a-string) mitochondrial morphology, shown as a percentage of the total population. Three individual biological replicates were performed and >100 cells counted for each replicate. Mean ± SD. An unpaired t test was performed for statistical analysis with p values shown (*p < 0.05, **p < 0.01) as determined based on a two-tailed distribution. (C) Representative images of mitochondria in ssp2∆ cells before and after osmotic stress. (D) Representative images of mitochondria in dnm1∆ vps1∆ cells before and after osmotic stress. For A, C, and D, images on the left are DIC, and images in middle are corresponding inverted maximum projections for 0.2-μm-spaced z-sections through the entire cell (25 steps for 5 μm total). The red boxes are zoomed in on the right. Arrows denote “beaded” morphology. Scale bars, 5 μm. (E) Western blot showing activation kinetics of Ssp2-pT189 in dnm1∆ vps1∆ cells vs. wild-type cells in response to 1 M KCl osmotic stress. We used α-myc as a loading control for total Ssp2. (F) Tenfold serial dilutions of the indicated strains were spotted onto control (YE4S) plates or YE4S plates containing 0.8 M KCl. Cells were grown at 32°C.

FIGURE 7:

SAPK cascades contribute to deactivation of Ssp2. (A) Activation kinetics of Ssp2-pT189 in wild-type and sty1∆ cells in response to 1 M KCl osmotic stress. Left, Western blot. Right, quantification of three biological replicates. (B) Activation kinetics of Ssp2-pT189 in wild-type and pmk1∆ cells in response to 1 M KCl osmotic stress. Left, Western blot. Right, quantification of three biological replicates. (C) Activation kinetics of Ssp2-pT189 in wild-type and pmk1∆ sty1∆ cells in response to 1 M KCl osmotic stress. Left, Western blot. Right, quantification of three biological replicates. (D) Activation kinetics of Ssp2-pT189 in wild-type and atf1∆ cells in response to 1 M KCl osmotic stress. Left, Western blot. Right, quantification of three biological replicates. For all results, α-myc was used as a loading control for total Ssp2. Values displayed are the mean ± SD based on three biological replicates. An unpaired t test was performed for statistical analyses, and p values were based on two-tailed distributions (*p < 0.05, **p < 0.01).

FIGURE 8:

Glycerol biosynthesis contributes to deactivation of Ssp2. Western blot and quantification showing activation kinetics of Ssp2-pT189 in wild-type and gpd1Δ gpd2Δ cells in response to 1 M KCl osmotic stress. We used α-myc as loading control for total Ssp2. Values displayed are the mean ± SD based on three individual biological replicates. An unpaired t test was performed for statistical analyses, and p values were based on two-tailed distributions (**p < 0.01).