Proteomic Analyses Reveal that Sky1 Modulates Apoptosis and Mitophagy in Saccharomyces cerevisiae Cells Exposed to Cisplatin

Abstract

Sky1 is the only member of the SR (Serine–Arginine) protein kinase family in Saccharomyces cerevisiae. When yeast cells are treated with the anti-cancer drug cisplatin, Sky1 kinase activity is necessary to produce the cytotoxic effect. In this study, proteome changes in response to this drug and/or SKY1 deletion have been evaluated in order to understand the role of Sky1 in the response of yeast cells to cisplatin. Results reveal differential expression of proteins previously related to the oxidative stress response, DNA damage, apoptosis and mitophagy. With these precedents, the role of Sky1 in apoptosis, necrosis and mitophagy has been evaluated by flow-cytometry, fluorescence microscopy, biosensors and fluorescence techniques. After cisplatin treatment, an apoptotic-like process diminishes in the ∆sky1 strain in comparison to the wild-type. The treatment does not affect mitophagy in the wild-type strain, while an increase is observed in the ∆sky1 strain. The increased resistance to cisplatin observed in the ∆sky1 strain may be attributable to a decrease of apoptosis and an increase of mitophagy.

Multilayered Control of Protein Turnover by TORC1 and Atg1

“…Within the set of protein kinases exhibiting potential TORC1 sites, we identified several that have previously been linked to autophagic processes: (1) Bck1 mediates signals from Pkc1 to Mkk1/2 within the cell wall integrity MAPK signaling pathway (Krause and Gray, 2002), which is required for the induction of pexophagy in yeast (Manjithaya et al, 2010); (2) Ksp1 inhibits autophagy by antagonizing the dephosphorylation of Atg13 (Umekawa and Klionsky, 2012); and (3) Sky1 modulates mitophagy (Rodríguez-Lombardero et al, 2014). Shared signaling events between organelle-specific autophagy subtypes and bulk autophagy might indicate that selective autophagy contributes to the bulk protein turnover observed in nutrient-starvation conditions.…”

“…Of the ones regulated by TORC1, Sch9 and Ypk3 are bona fide proximal targets (Gonzá lez et al, 2015;Martin et al, 2004;Urban et al, 2007;Yerlikaya et al, 2016), while Npr1 and Gcn2 have been described as distally controlled by TORC1 (Garcia-Barrio et al, 2002;Schmidt et al, 1998). In addition, several of the other TORC1-controlled protein kinases have previously been found to be part of the TORC1associated protein kinase network (Breitkreutz et al, 2010), including Bck1, Ksp1, and Sky1, which were also linked to autophagic processes (Krause and Gray, 2002;Manjithaya et al, 2010;Rodríguez-Lombardero et al, 2014;Umekawa and Klionsky, 2012). Besides precisely pinpointing the phosphorylation events that are likely functionally relevant for processing signals that emanate from TORC1, these findings uncover the existence of multiple regulatory layers by which TORC1 may control auto-phagic processes other than phosphorylating Atg13 (Kamada et al, 2000).…”

Histone H3 and TORC1 prevent organelle dysfunction and cell death by promoting nuclear retention of HMGB proteins

“…To gauge how quickly cell death occurs in H3K37A, and to determine whether it is solely necrotic or whether it may encompass both necrosis and apoptosis at early stages, we mock- or 20 nM rapamycin-treated H3WT and H3K37 for increasing lengths of time. Cells were then stained with YO-PRO-1 (which stains early apoptotic cells) and propidium iodide (PI, which stains necrotic cells) and analyzed by flow cytometry [ 26 ]. No cell death occurred in mock- or rapamycin-treated H3WT or mock-treated H3K37A over the course of the experiment (Fig.…”