Journal of Cell Science New roles of the fission yeast eIF2a kinases Hri1 and Gcn2 in response to nutritional stress Ruth Martı ´n, Juan Jose ´ Berlanga* and Ce ´ sar de Haro* Centro de Biologı ´a Molecular Severo Ochoa, CSIC-UAM, Cantoblanco, 28049 Madrid, Spain *Authors for correspondence (cdeharo@cbm.uam.es; jberlanga@cbm.uam.es) Accepted 17 April 2013 Journal of Cell Science 126, 3010–3020 ß 2013. Published by The Company of Biologists Ltd doi: 10.1242/jcs.118067 Summary In fission yeast, three distinct eukaryotic initiation factor 2a (eIF2a) kinases (Hri1, Hri2 and Gcn2), regulate protein synthesis in response to various environmental stresses. Thus, Gcn2 is activated early after exposure to hydrogen peroxide (H 2 O 2 ) and methyl methanesulfonate (MMS), whereas Hri2 is the primary activated eIF2a kinase in response to heat shock. The function of Hri1 is still not completely understood. It is also known that the mitogen-activated protein kinase Sty1 negatively regulates Gcn2 and Hri2 activities under oxidative stress. In this study, we demonstrate that Hri1 is mainly activated, and its expression upregulated, during transition from exponential growth to the stationary phase in response to nutritional limitation. Accordingly, both Hri1 and Gcn2, but not Hri2, are activated upon nitrogen source deprivation. In contrast, Hri2 is stimulated early during glucose starvation. We also found that Gcn2 is implicated in nitrogen starvation-induced growth arrest in the cell cycle G1 phase as well as in the non-selective protein degradation process caused upon this particular cellular stress. Moreover, Gcn2, but not Hri1 or Hri2, is essential for survival of cells growing in minimal medium, upon oxidative stress or glucose limitation. We further show that eIF2a phosphorylation at serine 52 by the eIF2a kinases is necessary for efficient cell cycle arrest in the G1 phase, for the consequent protein degradation and for sexual differentiation, under nitrogen starvation. Therefore, the eIF2a kinase signalling pathway modulates G1 phase cell cycle arrest, cell survival and mating under nutritional stress in the fission yeast Schizosaccharomyces pombe. Key words: eIF2a kinases, Translational control, Stress response, Cell cycle, Schizosaccharomyces pombe Introduction In eukaryotic cells, the reversible phosphorylation of the a- subunit of eukaryotic translation initiation factor 2 (eIF2a) is a well-characterized mechanism of translational control in response to a wide variety of cellular stresses (de Haro et al., 1996; Dever, 2002). Four mammalian protein kinases (HRI, PKR, GCN2 and PERK), which inhibit translation initiation by phosphorylating eIF2a on Ser-51, have been identified. They are regulated independently in response to various different cellular stresses (Dever, 2002; Proud, 2005). In the yeast Saccharomyces cerevisiae, Gcn2 is the sole eIF2a kinase (Dever, 2002), however, in the fission yeast Schizosaccharomyces pombe, besides Gcn2, two additional eIF2a kinases related to mammalian HRI, called Hri1 and Hri2, phosphorylate eIF2a at the same residue (serine 52 in S. pombe). Hri1 and Hri2 showed a differential activation pattern in response to cellular stresses (Zhan et al., 2004). More recently, it has been reported that, upon distinct cellular stresses, phosphorylation of eIF2a constitutes a very early response and that a particular eIF2a kinase is activated in response to each stress stimuli. Thus, Hri2 responds to heat shock, whereas Gcn2 is activated early after exposure to hydrogen peroxide (H 2 O 2 ) and methyl methanesulfonate (MMS) in S. pombe (Berlanga et al., 2010). It is well known that eIF2a phosphorylation can regulate both gene-specific and general translation. Thus, the eIF2a kinases phosphorylate eIF2a, resulting in reduced general translation and increased translation of the transcription factors, Gcn4 in Saccharomyces cerevisiae and ATF4 in mammals, which in turn activate expression of their target genes involved in the stress response (Harding et al., 2000; Hinnebusch, 1997). Despite the finding that phosphorylation of eIF2a is a general response to cellular stress it is well known that, in S. pombe, cellular responses to various environmental stresses are regulated primarily through the stress- and mitogen-activated protein kinase (SAPK/MAPK) Sty1. Sty1, also known as Spc1 and Phh1, is required for, and can be activated by different types of stress conditions, including oxidative stress and glucose or nitrogen starvation (Vivancos et al., 2006). Upon stress activation, Sty1 reversibly accumulates in the nucleus, where it stimulates gene expression via the Atf1 transcription factor. Thus, in response to stress stimuli, Sty1 is required for the transcriptional regulation of a large set of genes which constitute the core environmental stress response (CESR) (Chen et al., 2003). For the majority of these genes, regulation is also dependent on Atf1 (Shiozaki and Russell, 1996; Wilkinson et al., 1996). Atf1, a heterodimeric transcription factor, is bound to stress genes promoters before activation, and only induces transcription of those genes once Atf1 becomes phosphorylated (Chen et al., 2008). In a previous report, it was suggested that downstream components of the Sty1 MAPK pathway can contribute both positively and negatively to the modulation of the eIF2a kinase pathway (Dunand-Sauthier et al., 2005). More recently, it has been reported that, the activated Sty1 negatively regulates Gcn2 and Hri2 activities under oxidative stress, but not upon heat shock or genotoxic stress caused by MMS, in fission yeast (Berlanga et al., 2010). 3010 Research Article