Atg1, a key regulator of autophagy, functions to promote MAPK activation and cell death upon calcium overload in fission yeast

Abstract

Autophagy promotes or inhibits cell death depending on the environment and cell type. Our previous findings suggested that Atg1 is genetically involved in the regulation of Pmk1 MAPK in fission yeast. Here, we showed that Δatg1 displays lower levels of Pmk1 MAPK phosphorylation than did the wild-type (WT) cells upon treatment with a 1,3-β-D-glucan synthase inhibitor micafungin or CaCl₂, both of which activate Pmk1. Moreover, the overproduction of Atg1, but not that of the kinase inactivating Atg1^D193A activates Pmk1 without any extracellular stimuli, suggesting that Atg1 may promote Pmk1 MAPK signaling activation. Notably, the overproduction of Atg1 induces a toxic effect on the growth of WT cells and the deletion of Pmk1 failed to suppress the cell death induced by Atg1, indicating that the Atg1-mediated cell death requires additional mechanism(s) other than Pmk1 activation. Moreover, atg1 gene deletion induces tolerance to micafungin and CaCl₂, whereas pmk1 deletion induces severe sensitivities to these compounds. The Δatg1Δpmk1 double mutants display intermediate sensitivities to these compounds, showing that atg1 deletion partly suppressed growth inhibition induced by Δpmk1. Thus, Atg1 may act to promote cell death upon micafungin and CaCl₂ stimuli regardless of Pmk1 MAPK activity. Since micafungin and CaCl₂ are intracellular calcium inducers, our data reveal a novel role of the autophagy regulator Atg1 to induce cell death upon calcium overload independent of its role in Pmk1 MAPK activation.

Keywords: Autophagy; Calcium tolerance; Cell death; Fission yeast; Pmk1 MAPK.

Figure 1. FIGURE 1: Atg1 facilitates the phosphorylation of Pmk1.

(A and B) Impact of the deletion of atg1⁺ on Pmk1 phosphorylation. Wild-type (WT) and Δatg1 cells expressing the C-terminal GST-tagged Pmk1 from the endogenous pmk1 promoter were grown in EMM supplemented with 2 µg/ml micafungin (A) or with 200 mM CaCl2 (B) for 60 min at 27°C. Cell lysates bound to glutathione beads were immunoblotted with anti-phospho-ERK antibodies and anti-GST antibodies to detect phosphorylated Pmk1 and Pmk1-GST (loading control), respectively. Upper panel: representative immunoblot. Lower panel: Relative quantification of phosphorylated Pmk1 normalized by Pmk1-GST. Bar graphs show relative values to WT cells grown in the absence of micafungin nor CaCl2 as the mean ± standard error of the mean (SEM) of five independent experiments. N = 5; **p < 0.01 as assessed by a one-way ANOVA followed by the Dunnett's test for multiple comparisons. n.s. not significant. (C) Impact of overexpression of atg1⁺ on the Pmk1 phosphorylation. Δatg1 cells expressing Pmk1-GST under the endogenous pmk1 promoter and harboring pREP1-GFP, pREP1-atg1⁺-GFP, or pREP1-atg1D^193A-GFP were grown in EMM without thiamine for 20 hr at 27°C. Cell lysates bound to glutathione beads were immunoblotted with anti-phospho-Pmk1 and anti-GST antibodies and quantified as described in (B). Bar graphs represent the mean ± SEM (N = 3; *p < 0.05 as assessed by Welch's two sample t-test). The levels of overexpression of WT Atg1 and kinase dead Atg1 were approximately equal (Figure S1).

Figure 2. FIGURE 2: Atg1 influences the cell viability independently on the Pmk1 MAPK pathway.

(A) Influences of overexpression of Atg1 and the kinase-defective variant Atg1^D193A on cell growth. Cells transformed with the thiamine repressible-expression vectors, pREP1-GFP, pREP1-atg1⁺-GFP, or pREP1-atg1^D193A-GFP were streaked onto an EMM plate with or without 4 µM thiamine and incubated for 5 days at 27°C. (B) Measurement of the viability of the cells subjected to Atg1 overexpression. Cells transformed with pREP1-atg1⁺-GFP or its control vector were incubated in liquid EMM in the absence of thiamine for 48 hours at 27°C. The cells were then 10-fold serially diluted as indicated (10⁰ to 10⁻⁴) starting from OD₆₆₀ = 0.5, and 5 µL were spotted onto EMM plates with or without thiamine. Plates were incubated for 4.5 days at 27°C. Two independent transformant clones (#1 and #2) for pREP1-atg1⁺-GFP were tested. (C) Overexpression of Atg1 induces cytotoxicity independently on Pmk1. Δpmk1 cells harboring pREP1-GFP or pREP1-atg1⁺-GFP and wild-type cells harboring pREP1-GFP were streaked onto an EMM plate with or without 4 µM thiamine and incubated for 4 days at 27°C.

Figure 3. FIGURE 3: Δatg1 cells are resistant to micafungin and calcium.

(A) Cells as indicated were serially diluted 10⁰, 10⁻¹, 10⁻², 10⁻³, 10⁻⁴ and 5 µl were spotted onto YPD plates containing the indicated concentrations of micafungin. Plates were incubated at 27°C for 2 days. (B) Minimum Inhibitory Concentration (MIC) of micafungin. Cells as indicated were grown to mid-log phase and adjusted to OD₆₆₀ = 0.5. The cells were then diluted 300-fold with fresh liquid YES and incubated in a 96-well plate with the indicated concentrations of micafungin for 2.5 days at 27°C. The most left wells contain medium only (sterility control). Right panel is a schematic illustration of the result of MIC assay. Open circle (○), closed circle (•), and shaded circle indicate no growth, full growth and a slight growth, respectively. (C) Cells as indicated were serially diluted 10⁰, 10⁻¹, 10⁻², 10⁻³, 10⁻⁴ and 5 µL were spotted onto YPD plates containing the indicated concentrations of CaCl₂. Plates were incubated at 27°C for 3 days.