Journal of Cell Science Fission yeast TORC1 prevents eIF2a phosphorylation in response to nitrogen and amino acids via Gcn2 kinase Noelia Valbuena 1 , Ana Elisa Rozale ´n 1,2 and Sergio Moreno 1,2, * 1 Instituto de Biologı ´a Molecular y Celular del Ca ´ ncer, CSIC/University of Salamanca, 37007 Salamanca, Spain 2 Instituto de Biologı ´a Funcional y Geno ´ mica, CSIC/University of Salamanca, 37007 Salamanca, Spain *Author for correspondence (smo@usal.es) Accepted 11 October 2012 Journal of Cell Science 125, 5955–5959 ß 2012. Published by The Company of Biologists Ltd doi: 10.1242/jcs.105395 Summary Serine 51 phosphorylation of the eukaryotic initiation factor-2a (eIF2a) is an important mechanism involved in blocking general protein synthesis in response to diverse types of stress. In fission yeast, three kinases (Hri1, Hri2 and Gcn2) can phosphorylate eIF2a at serine 51. In this study, we show that Tor2, as part of the TORC1 complex, prevents the phosphorylation of eIF2a in cells growing in the presence of nitrogen and amino acids. Inhibition of TORC1, either by rapamycin treatment, mutation of Tor2 or nitrogen deprivation, induces Gcn2-dependent phosphorylation of eIF2a. Key words: S. pombe, TOR, Rapamycin, Gcn2, eIF2a Introduction In response to diverse environmental stresses, the phosphorylation of eukaryotic initiation factor-2 (eIF2a) induces a programme of gene expression that mitigates cellular injury. Several protein kinases phosphorylate eIF2a at serine 51, leading to the inhibition of eIF2B activity, that in turn causes a reduction in general protein synthesis and an enhancement of the translation of specific mRNAs encoding for proteins that remediate the stress. In mammalian cells, four eIF2 kinases (HRI, GCN2, PEK/Perk and PKR) inhibit translation initiation through the phosphorylation of eIF2a in response to different types of cellular stress (Dever, 2002; Proud, 2005). In Saccharomyces cerevisiae, Gcn2p is activated upon nutrient limitation (amino acids, purine and glucose), but also by high concentrations of sodium, rapamycin and methyl methanesulfonate (Cherkasova and Hinnebusch, 2003; Narasimhan et al., 2004; Valenzuela et al., 2001; Yang et al., 2000). In the fission yeast Schizosaccharomyces pombe, besides Gcn2 two additional eIF2a kinases related to mammalian HRI, called Hri1 and Hri2, phosphorylate eIF2a at serine 51 (serine 52 in S. pombe). Hri1 and Hri2 show a differential activation pattern in response to cellular stress, including heat shock, arsenite or cadmium (Zhan et al., 2004; Berlanga et al., 2010). When supplied with enough nutrients, fission yeast cells activate protein translation, promoting cellular growth and proliferation and inhibiting sexual differentiation. The target of the rapamycin (TOR) signaling pathway plays a central role in the regulation of these processes. TOR, a serine/threonine protein kinase conserved from yeasts to mammals, exists in two different complexes, namely TORC1 and TORC2 (Loewith et al., 2002; Sarbassov et al., 2004). TORC1 contains Raptor and regulates cell growth positively by promoting anabolic processes, such as protein synthesis (Fingar et al., 2002; Hay and Sonenberg, 2004), and by inhibiting catabolic processes, such as autophagy (Blommaart et al., 1995; Noda and Ohsumi, 1998; Shigemitsu et al., 1999). In contrast, TORC2, which contains Rictor, regulates Akt and also affects the actin cytoskeleton (Jacinto et al., 2004; Sarbassov et al., 2005). In mammalian cells, mTOR is a critical player in the TSC1– TSC2/Rheb/mTOR signaling pathway, which regulates cell growth in response to growth factors, nutrients and energy conditions. Unlike higher eukaryotes, which contain a single TOR protein, S. pombe and S. cerevisiae have two: Tor1 and Tor2. In contrast to S. cerevisiae, the TSC1–TSC2/Rheb/TOR pathway is conserved in S. pombe, providing an excellent model to study TOR pathway regulation. In S. pombe, Tor2 forms part of the TORC1 complex and is essential for cell growth and the repression of sexual differentiation, meiosis and sporulation (Alvarez and Moreno, 2006; Matsuo et al., 2007; Uritani et al., 2006; Weisman et al., 2007), whereas Tor1 is not essential for growth and is included in the TORC2 complex (Alvarez and Moreno, 2006; Hayashi et al., 2007; Matsuo et al., 2007). The a subunit of the eukaryotic initiation factor-2 (eIF2a) and TOR play important roles in the translation response to nutritional variation. In S. cerevisiae, the inhibition of TOR leads to the activation of Gcn2 kinase, and eIF2a phosphorylation down- regulates general translation initiation (Cherkasova and Hinnebusch, 2003; Kubota et al., 2003). We therefore examined a possible relationship between TOR and eIF2a in S. pombe and obtained evidence that Tor2, as part of the TORC1 complex, prevents the Gcn2-mediated phosphorylation of eIF2a in response to the presence of nitrogen and amino acids in the medium. Results Tor2 impairs the phosphorylation of eIF2a in the presence of nitrogen Fission yeast eIF2a is phosphorylated by three different eIF2a kinases, Gcn2, Hri1 and Hri2, in response to different types of Short Report 5955