Fission yeast TOR complex 2 activates the AGC-family Gad8 kinase essential for stress resistance and cell cycle control

Abstract

Members of the mitogen-activated protein kinase (MAPK) subfamily responsive to environmental stress stimuli are known as SAPKs (stress-activated protein kinases), which are conserved from yeast to humans. In the fission yeast Schizosaccharomyces pombe, Spc1/Sty1 SAPK is activated by diverse forms of stress, such as osmostress, oxidative stress and heat shock, and induces gene expression through the Atf1 transcription factor. Sin1 (SAPK interacting protein 1) was originally isolated as a protein that interacts with Spc1, and its orthologs were also found in diverse eukaryotes. Here we report that Sin1 is not required for the stress gene expression regulated by Spc1 and Atf1, and that Sin1 is an essential component of TOR (target of rapamycin) complex 2 (TORC2). TORC2 is not essential for cell viability in S. pombe but plays important roles in cellular survival of stress conditions through phosphorylation and activation of an AGC-family protein kinase, Gad8. In addition, inactivation of Gad8 results in a synthetic growth defect with cdc25-22, a temperature-sensitive mutation of the Cdc25 phosphatase that activates Cdc2 kinase at G 2 /M. Gad8 also positively regulates expression of the CDK inhibitor gene rum1 + , which is essential for cell cycle arrest in G 1 after nitrogen starvation. These results strongly suggest that the TORC2-Gad8 pathway has multiple physiological functions in cellular stress resistance and cell cycle progression at both G 1 /S and G 2 /M transitions. IntroductionStress-activated protein kinases (SAPKs) form an evolutionarily conserved subfamily of the MAP kinase (MAPK), members of which are stimulated in response to environmental stress. The prototype of SAPK was first identified in budding yeast as Hog1, which is mainly activated by high osmolarity stress. 1 On the other hand, human JNK (c-Jun N-terminal Kinase) and p38 SAPKs 2 as well as Spc1/Sty1 in the fission yeast Schizosaccharomyces pombe 3 are activated in response to diverse forms of stress, including osmostress, heat shock, oxidative stress and nutritional starvation. In S. pombe, activated Spc1 induces expression of a set of stress resistance genes through the Atf1 transcription factor. [4][5][6] Cells lacking the functional Spc1-Atf1 pathway are hypersensitive to diverse forms of stress conditions, indicating the essential role of the SAPK signaling in cellular survival of environmental changes. In addition, Spc1 positively regulates the initiation of mitosis independently of the Atf1-regulated gene expression. [7][8][9] Sin1 (SAPK interacting protein 1) was isolated by a yeast twohybrid screen as a protein that interacts with Spc1 MAPK. 10 It was reported that a sin1 mutant was hypersensitive to various stress conditions and defective in the stress gene expression regulated by the Spc1-Atf1 pathway and that by Pap1, an AP-1 family transcription factor. 10 Sin1 is widely conserved among eukaryotic species, 11,12 and its mammalian ortholog also interacts with JNK SAPK 13 and MEKK2 MAPK kinase kinase. 14 More recently, however, Si...

Fission yeast Ryh1 GTPase activates TOR Complex 2 in response to glucose

“…17,19,22 The key downstream target of TORC2 in fission yeast is the AGCfamily kinase Gad8, as evidenced by the observation that the gad8 deletion mutant phenocopies the TORC2-defective mutants. 16,19 Like Akt activation by mammalian TORC2, Gad8 is phosphorylated within its hydrophobic motif by TORC2, resulting in increased Gad8 kinase activity essential for its cellular functions. 16,19 On the other hand, a major upstream regulator of fission yeast TORC2 has been found as the Rab small GTPase Ryh1.…”

“…22,[32][33][34] In this organism, TORC2 is also implicated in cellular nutritional responses, including the G1 arrest upon nitrogen starvation and the mitotic control in response to glucose. 16,19,35 Little is known, however, about the physiological stimuli that regulate fission yeast TORC2, whereas insulin and growth factors activate mammalian TORC2 (mTORC2) in a PI3K-dependent manner. 7,8,[36][37][38] In this report, we show that TORC2 in fission yeast is responsive to glucose and that the functional TORC2-Gad8 pathway is essential for cell proliferation in glucose-limited environment.…”

“…16,19 Like Akt activation by mammalian TORC2, Gad8 is phosphorylated within its hydrophobic motif by TORC2, resulting in increased Gad8 kinase activity essential for its cellular functions. 16,19 On the other hand, a major upstream regulator of fission yeast TORC2 has been found as the Rab small GTPase Ryh1. 21 The GTP-bound, active form of Ryh1 physically interacts with TORC2 and promotes TORC2-dependent phosphorylation/activation of the Gad8 kinase.…”

“…Among those is the fission yeast Schizosaccharomyces pombe, a genetically amenable eukaryote with nonessential TORC2, which eases mutational analysis of this particular TOR complex. [14][15][16][17][18][19][20][21] Fission yeast TORC2 is composed of the catalytic subunit Tor1 kinase and the regulatory subunits named Ste20, Sin1, Wat1 and Bit61. 17,19,22 The key downstream target of TORC2 in fission yeast is the AGCfamily kinase Gad8, as evidenced by the observation that the gad8 deletion mutant phenocopies the TORC2-defective mutants.…”

“…[14][15][16][17][18][19][20][21] Fission yeast TORC2 is composed of the catalytic subunit Tor1 kinase and the regulatory subunits named Ste20, Sin1, Wat1 and Bit61. 17,19,22 The key downstream target of TORC2 in fission yeast is the AGCfamily kinase Gad8, as evidenced by the observation that the gad8 deletion mutant phenocopies the TORC2-defective mutants. 16,19 Like Akt activation by mammalian TORC2, Gad8 is phosphorylated within its hydrophobic motif by TORC2, resulting in increased Gad8 kinase activity essential for its cellular functions.…”