The Tsc1–Tsc2 complex homologous to human tuberous sclerosis complex proteins governs amino acid uptake by regulating the expression and intracellular distribution of amino acid transporters in Schizosaccharomyces pombe. Here, we performed a genetic screening for molecules that are involved in amino acid uptake and found Arn1 (also known as Any1). Arn1 is homologous to ART1, an arrestin-related trafficking adaptor (ART) in Saccharomyces cerevisiae, and contains a conserved arrestin motif, a ubiquitination site, and two PY motifs. Overexpression of arn1⁺ confers canavanine resistance on cells, whereas its disruption causes hypersensitivity to canavanine. We also show that Arn1 regulates endocytosis of the Cat1 amino acid transporter. Furthermore, deletion of arn1⁺ suppresses a defect of amino acid uptake and the aberrant Cat1 localization in tsc2Δ. Arn1 interacts with and is ubiquitinated by the Pub1 ubiquitin ligase, which is necessary to regulate Cat1 endocytosis. Cat1 undergoes ubiquitinations on lysine residues within the N-terminus, which are mediated, in part, by Arn1 to determine Cat1 localization. Correctively, Arn1 is an ART in S. pombe and contributes to amino acid uptake through regulating Cat1 endocytosis in which Tsc2 is involved.

Target of rapamycin (TOR) kinase controls cell metabolism and growth in response to environmental cues such as nutrients, growth factors, and stress. TOR kinase is widely conserved across eukaryotes. As in other organisms, the fission yeast Schizosaccharomyces pombe has two types of TOR complex, namely TOR complex 1 (TORC1) and TORC2. It is interesting that the two TOR complexes in S. pombe have opposite roles in sexual differentiation, which is induced by nutrient starvation. TORC1, which contains Tor2 as a catalytic subunit, promotes vegetative growth and represses sexual differentiation in nutrient-rich conditions, while TORC2 is required for the initiation of sexual differentiation. Multiple targets of TORC1 have been identified. Some of these, such as S6 kinase and an autophagy regulator Atg13, are known targets in other organisms. In addition, there is a novel group of TORC1 targets involved in the regulation of sexual differentiation. Here, we review recent findings on phosphorylation targets of TORC1 in S. pombe. Furthermore, we briefly report a novel S. pombe target of TORC1.

Cyclins control cell cycle progression by regulating the activity of cyclin-dependent kinases (Cdks). Cyclin I is a member of the cyclin family because of the presence of a cyclin box motif. It has been suggested that Cyclin I is involved in various biological processes, such as cell survival, angiogenesis, and cell differentiation. However, whether or not Cyclin I has a role in regulating the cell cycle similarly to other cyclins has yet to be clarified. Therefore, we investigated the role for Cyclin I in cell cycle progression. We showed that the protein level of Cyclin I oscillated during the cell cycle, and that Cyclin I was subjected to ubiquitination and degradation in cells. The interaction between Cyclin I and Cdk5 was detected in cells overexpressed with both proteins. Furthermore, depletion of Cyclin I by siRNAs prevented cell proliferation, suggesting the positive role of Cyclin I for the cell cycle progression. In addition, flow cytometric analysis revealed that cells depleted of Cyclin I were accumulated at G2/M phases. By using HeLa.S-Fucci (fluorescent ubiquitination-based cell cycle indicator) cells, we further confirmed that knockdown of Cyclin I induced cell cycle arrest at S/G2/M phases. These results strongly suggest that Cyclin I has the role in the regulation of cell cycle progression.

In Schizosaccharomyces pombe, a general strategy for survival in response to environmental changes is sexual differentiation, which is triggered by TORC1 inactivation. However, mechanisms of TORC1 regulation in fission yeast remain poorly understood. In this study, we found that Pef1, which is an ortholog of mammalian CDK5, regulates the initiation of sexual differentiation through positive regulation of TORC1 activity. Conversely, deletion of pef1 leads to activation of autophagy and subsequent excessive TORC1 reactivation during the early phases of the nitrogen starvation response. This excessive TORC1 reactivation results in the silencing of the Ste11-Mei2 pathway and mating defects. Additionally, we found that pef1 genetically interacts with tsc1/2 in TORC1 regulation, and physically interacts with three types of cyclins, Clg1, Pas1, and Psl1. The double deletion of clg1 and pas1 promotes activation of autophagy and TORC1 during nitrogen starvation, similar to pef1Δ cells. Overall, our work suggests that Pef1-Clg1 and Pef1-Pas1 complexes regulate initiation of sexual differentiation through control of the TSC-TORC1 pathway and autophagy.

Breast cancer is classified into three subtypes by the expression of biomarker receptors such as hormone receptors and human epidermal growth factor receptor 2. Triple-negative breast cancer (TNBC) expresses none of these receptors and has an aggressive phenotype with a poor prognosis, which is insensitive to the drugs that target the hormone receptors and human epidermal growth factor receptor 2. It is, thus, required to develop an effective therapeutic reagent to treat TNBC. The study using a panel of 19 breast cancer cell lines revealed that midostaurin, a multi-target protein kinase inhibitor, suppresses preferentially the growth of TNBC cells comparing with non-TNBC cells. Clustering analysis of the drug activity data for the panel of cancer cell lines predicted that midostaurin shares the target with Aurora kinase inhibitors. Following studies indicated that midostaurin attenuates the phosphorylation reaction mediated by Aurora kinase in the cells and directly inhibits this protein kinase in vitro, and that this reagent induces apoptosis accompanying accumulation of 4N and 8N DNA cells in TNBC cells. Midostaurin suppresses the proliferation of TNBC cells among the breast cancer cell lines presumably through the inhibition of the Aurora kinase family. The precise study of midostaurin on cell growth will contribute to the development of the drug for the treatment of TNBC.

Although senescent cells display various morphological changes including vacuole formation, it is still unclear how these processes are regulated. We have recently identified the gene, lymphocyte antigen 6 complex, locus D (LY6D), to be upregulated specifically in senescent cells. LY6D is a glycosylphosphatidylinositol-anchored cell-surface protein whose function remains unknown. Here, we analyzed the functional relationship between LY6D and the senescence processes. We found that overexpression of LY6D induced vacuole formation and knockdown of LY6D suppressed the senescence-associated vacuole formation. The LY6D-induced vacuoles were derived from macropinocytosis, a distinct form of endocytosis. Furthermore, Src family kinases and Ras were found to be recruited to membrane lipid rafts in an LY6D-dependent manner, and inhibition of their activity impaired the LY6D-induced macropinocytosis. Finally, reduction of senescent-cell survival induced by glutamine deprivation was recovered by albumin supplementation to the culture media in an LY6D-dependent manner. Because macropinocytosis acts as an amino acid supply route, these results suggest that LY6D-mediated macropinocytosis contributes to senescent-cell survival through the incorporation of extracellular nutrients.