Glucose Activates TORC2-Gad8 Protein via Positive Regulation of the cAMP/cAMP-dependent Protein Kinase A (PKA) Pathway and Negative Regulation of the Pmk1 Protein-Mitogen-activated Protein Kinase Pathway

Cohen, Aviv; Kupiec, Martin; Weisman, Ronit

doi:10.1074/jbc.m114.573824

Cited by 38 publications

(43 citation statements)

References 64 publications

(38 reference statements)

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“…61 In contrast, our repeated experiments detected no significant defect in the Gad8 phosphorylation among the cAMP-PKA pathway mutants tested in this study, including Dgit3, Dgpa2 and Dpka1. Because Cohen et al prepared the cellular protein extract under non-denaturing condition without phosphatase inhibitor, it is conceivable that Gad8 was dephosphorylated by phosphatases in the crude extract.…”

Section: Discussioncontrasting

confidence: 42%

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

et al. 2015

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The Target Of Rapamycin (TOR) is an evolutionarily conserved protein kinase that forms 2 distinct protein complexes referred to as TOR complex 1 (TORC1) and 2 (TORC2). Recent extensive studies have demonstrated that TORC1 is under the control of the small GTPases Rheb and Rag that funnel multiple input signals including those derived from nutritional sources; however, information is scarce as to the regulation of TORC2. A previous study using the model system provided by the fission yeast Schizosaccharomyces pombe identified Ryh1, a Rab-family GTPase, as an activator of TORC2. Here, we show that the nucleotide-binding state of Ryh1 is regulated in response to glucose, mediating this major nutrient signal to TORC2. In glucose-rich growth media, the GTP-bound form of Ryh1 induces TORC2-dependent phosphorylation of Gad8, a downstream target of TORC2 in fission yeast. Upon glucose deprivation, Ryh1 becomes inactive, which turns off the TORC2-Gad8 pathway. During glucose starvation, however, Gad8 phosphorylation by TORC2 gradually recovers independently of Ryh1, implying an additional TORC2 activator that is regulated negatively by glucose. The paired positive and negative regulatory mechanisms may allow fine-tuning of the TORC2-Gad8 pathway, which is essential for growth under glucose-limited environment.

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Section: Discussioncontrasting

confidence: 42%

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

et al. 2015

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“…In addition, the TORC2 complex favors de novo Pck2 synthesis during stress, which is necessary to allow Pmk1 activation in response to specific environmental insults, such as cell wall stress or glucose limitation. Although TORC2-Gad8 signaling is required for adequate cellular response to stress, it has been shown recently that the CIP negatively regulates the TORC2-Gad8 pathway (Cohen et al, 2014). Therefore, we envisage a scenario where TORC2 will positively influence CIP signaling but will subsequently undergo CIP-mediated inhibition, thus modulating the stress response in a highly dynamic and flexible fashion.…”

Section: Discussionmentioning

confidence: 99%

Multiple regulatory levels influence cell integrity control by PKC ortholog Pck2 in fission yeast

Madrid

Jiménez

Sánchez-Mir

et al. 2014

Journal of Cell Science

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The fission yeast protein kinase C (PKC) ortholog Pck2 controls cell wall synthesis and is a major upstream activator of the cell integrity pathway (CIP) and its core component, the MAP kinase Pmk1 (also known as Spm1), in response to environmental stimuli. We show that in vivo phosphorylation of Pck2 at the conserved T842 activation loop during growth and in response to different stresses is mediated by the phosphoinositide-dependent kinase (PDK) ortholog Ksg1 and an autophosphorylation mechanism. However, T842 phosphorylation is not essential for Pmk1 activation, and putative phosphorylation at T846 might play an additional role in Pck2 catalytic activation and downstream signaling. These events, together with turn motif autophosphorylation at T984 and binding to small GTPases Rho1 and/or Rho2, stabilize Pck2 and render it competent to exert its biological functions. Remarkably, the target of rapamycin complex 2 (TORC2) does not participate in the catalytic activation of Pck2, but instead contributes to de novo Pck2 synthesis, which is essential to activate the CIP in response to cell wall damage or glucose exhaustion. These results unveil a novel mechanism whereby TOR regulates PKC function at a translational level, and they add a new regulatory layer to MAPK signaling cascades.

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“…Interestingly, most if not all of the cellular functions of S. pombe TORC2 are mediated via phosphorylation and activation of its downstream substrate Gad8, an AGC (PKA, PKG, and PKC kinases)-like kinase with structural and functional similarities to AKT, the most well established substrate kinase of mTORC2 (11,14,15). Although S. pombe TORC1 is responsive to the availability of nitrogen or amino acids (5)(6)(7)(8)16), we (17) and the Shiozaki and co-workers (18) have recently shown that S. pombe TORC2 responds to the availability of glucose. Upon glucose withdrawal or shift from glucose to a less favorable carbon source such as glycerol, there is a reduction of TORC2-dependent phosphorylation of Gad8 at serine 546 (equivalent to serine 473 in AKT) and concomitant reduced Gad8 kinase activity (17,18).…”

Section: Target Of Rapamycin (Tor)mentioning

confidence: 99%

“…Although S. pombe TORC1 is responsive to the availability of nitrogen or amino acids (5)(6)(7)(8)16), we (17) and the Shiozaki and co-workers (18) have recently shown that S. pombe TORC2 responds to the availability of glucose. Upon glucose withdrawal or shift from glucose to a less favorable carbon source such as glycerol, there is a reduction of TORC2-dependent phosphorylation of Gad8 at serine 546 (equivalent to serine 473 in AKT) and concomitant reduced Gad8 kinase activity (17,18). Thus, S. pombe TORC1 and TORC2 play essential roles in regulating growth by responding to two distinct key nutritional cues.…”

Section: Target Of Rapamycin (Tor)mentioning

confidence: 99%

“…Because we have identified several nuclear functions for TORC2-Gad8, such as control of gene expression, telomere length maintenance, and gene silencing (11), we examined the cellular localization of Gad8 using biochemical fractionations. We isolated proteins from cells expressing chromosomally tagged Gad8-HA, a fully active version of the Gad8 protein (14,17), and separated proteins that are localized to the nucleus from those in the non-nucleus fractions. Detection of histone H3 and Rps6 was used as markers for nuclear and cytoplasmic proteins, respectively (Fig.…”

Section: Torc2-gad8 Is Found In the Nucleus And Ismentioning

confidence: 99%

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Gad8 Protein Is Found in the Nucleus Where It Interacts with the MluI Cell Cycle Box-binding Factor (MBF) Transcriptional Complex to Regulate the Response to DNA Replication Stress

Cohen

Kupiec

Weisman

2016

Journal of Biological Chemistry

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The target of rapamycin (TOR) kinase is found at the core of two evolutionarily conserved complexes known as TOR complexes 1 and 2 (TORC1 and TORC2). In fission yeast, TORC2 is dispensable for proliferation under optimal growth conditions but is required for starvation and stress responses. We have previously reported that loss of function of TORC2 renders cells highly sensitive to DNA replication stress; however, the mechanism underlying this sensitivity is unknown. TORC2 has one known direct substrate, the kinase Gad8, which is related to AKT in human cells. Here we show that both TORC2 and its substrate Gad8 are found in the nucleus and are bound to the chromatin. We also demonstrate that Gad8 physically interacts with the MluI cell cycle box-binding factor (MBF) transcription complex that regulates the G 1 /S progression and the response to DNA stress. In mutant cells lacking TORC2 or Gad8, the binding of the MBF complex to its cognate promoters is compromised, and the induction of MBF target genes in response to DNA replication stress is reduced. Consistently, the protein levels of Cdt2 and Cig2, two MBF target genes, are reduced in the absence of TORC2-Gad8 signaling. Taken together, our findings highlight critical functions of TORC2 in the nucleus and suggest a role in surviving DNA replication stress via transcriptional regulation of MBF target genes. Target of rapamycin (TOR)2 is an atypical protein kinase that was isolated as the target of the immunosuppressive and anticancer drug rapamycin. TOR proteins play a central role in growth, proliferation, and survival and can be found in two distinct and evolutionarily conserved complexes, TORC1 and TORC2 (1-3). The two TOR complexes are key regulators of cellular growth; however, they respond to different stimuli and phosphorylate distinct sets of protein substrates (1-3). Human cells contain a single TOR protein, known as mTOR, which functions as the catalytic subunit of both mTORC1 and mTORC2. In the fission yeast, Schizosaccharomyces pombe, there are two genes encoding for TOR proteins. Tor1 interacts with Ste20 (Rictor in human cells) and Sin1 (mSin1 in human cells) to form TORC2, whereas Tor2 interacts with Mip1 (Raptor in human cells) to form TORC1 (4). TORC1 is essential for growth in S. pombe and plays a critical role in regulating the switch between growth and sexual development in response to nitrogen starvation. Consistently, disruption of S. pombe TORC1 results in many features characteristic of nitrogenstarved cells (5-8), and TORC1-dependent phosphorylation of the small ribosomal protein Rps6 is diminished under nitrogen starvation (9).S. pombe TORC2 is not essential for growth but is required for cell survival under a wide variety of stress conditions, including high or low temperatures, oxidative or osmotic stress, and DNA damage or replication stresses (10, 11). TORC2 is also essential for entrance into a stationary (G 0 -like) phase and sexual development, two processes that occur in response to nutritional starvation. Under normal growth ...

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Glucose Activates TORC2-Gad8 Protein via Positive Regulation of the cAMP/cAMP-dependent Protein Kinase A (PKA) Pathway and Negative Regulation of the Pmk1 Protein-Mitogen-activated Protein Kinase Pathway

Cited by 38 publications

References 64 publications

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

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

Multiple regulatory levels influence cell integrity control by PKC ortholog Pck2 in fission yeast

Gad8 Protein Is Found in the Nucleus Where It Interacts with the MluI Cell Cycle Box-binding Factor (MBF) Transcriptional Complex to Regulate the Response to DNA Replication Stress

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