Cellular growth control by TOR signaling 
 

Cellular growth, proliferation and differentiation are key aspects of cellular biology. The TOR signaling pathway is most well-known for its role in promoting cellular growth programs, while inhibiting starvation responses. Other distinguishing features of the TOR pathway are its unique role in regulating cell, organs and whole body size, and its role in regulating aging. More recently, TOR has been shown to regulate certain aspects of genomic and epigenomic stability, further extending the scope of the cellular functions of the TOR proteins. Deregulation of TOR signaling is observed in many different pathological conditions, including cancer, as well as neurodegenerative and metabolic diseases. TOR specific inhibitors have already been approved for medical use.

TOR is a large, a-typical protein kinase that belongs to the family of phosphatidylinositol 3-kinase-related kinases and is conserved in eukaryotes. The first TOR genes were isolated in the budding yeast Saccharomyces cerevisiae in a genetic screen for rapamycin resistant mutant cells, hence the name TOR, for Target of Rapamycin. Critical aspects of TOR signaling and the mode of action of rapamycin were also first discovered in S. cerevisiae, demonstrating the importance of using yeast for biomedical research.

Rapamycin, which specifically inhibits the activity of TOR, is a small molecule that is produced by a soil bacterium. It has several important clinical implications, including in immunosuppressive and cancer therapy. An intriguing property of rapamycin is the expansion of life span in model organisms, an effect that is likely associated with that of calorie restriction. Since rapamycin only inhibits a sub-set of TOR-dependent functions, next-generation TOR inhibitors have been developed. The search for additional TOR inhibitors that will modulate TOR signaling is one of the important challenges that researchers face in the TOR field. 

TOR kinases serve as the catalytic subunit of two protein complexes, TOR complex 1 (TORC1) and TOR complex 2 (TORC2). TORC1 is a pro-growth complex that promotes anabolic processes such as, translation, ribosome biogenesis, nucleotides and lipid synthesis, while inhibiting catabolic processes such as autophagy. TORC2 regulates growth and cell survival via the phosphorylation and activation of distinct protein kinases, such as AKT, SGK1 and PKC in human cells, YPK1/2 in S. cerevisiae or Gad8 in S. pombe. Deregulation of TORC2 is closely associated with cancer development, thus, further understanding of TORC2 signaling is much sought-after.

We employ the yeast Schizosaccharomyces pombe to study TOR signaling. The benefits of studying TOR in S. pombe include all the benefits of studying a complex signaling pathway in a genetic tractable organism, as well as the advantages unique to S. pombe, such as a notable evolutionarily conservation to human cells with respect to certain aspects of TOR signaling. Our long-term goal is to understand how growth is coordinated with cell divisions, development, genomic and epigenomic stability. Current goals in the lab include isolation of proximal and distal effectors of TORC1 and TORC2 and use of genetically engineered yeast cells to screen for novel anti-TOR inhibitors

Selected findings from our studies:

The two TOR complexes are conserved in S. pombe (Fig. 1). The catalytic subunit of SpTORC1 is designated Tor2 and the catalytic subunit of SpTORC2 is designated Tor1. This somewhat confusing designation is the result of naming the S. pombe Tor1 and Tor2 genes according the chronological order by which they were identified (Weisman and Choder, 2001), several years before the isolation and naming of the TOR complexes in S. cerevisiae. Several labs, including ours, have shown that while SpTORC1 is activated in response to the nitrogen quantity and quality, SpTORC2 is responsive to glucose levels (Laor et al., 2013; Laor et al., 2015, Cohen et al., 2014).

Figure 1: TORC1 and TORC2 in S. pombe – representative cellular activities

Upon reduction in the quantity or quality of the nitrogen source, SpTORC1 is inactivated, leading to growth arrest and induction of starvation responses, such as sexual development and autophagy. SpTORC1 plays a major role in regulating transcription in response to nitrogen availability (Laor et al., 2015, Reidman et al., 2019). We demonstrated that SpTORC1 regulates the nuclear localization of the GATA transcription factor Gaf1 in a manner dependent on type 2A-related serine/threonine phosphatase (Laor et al., 2015). By regulating Gaf1 cellular localization, SpTORC1 affects sexual development and amino acid uptake in response to nitrogen availability (Fig. 2). 

Figure 2: SpTORC1 regulates the nitrogen starvation response via localization of Gaf1 into the nucleus

TORC2 in S. pombe

SpTORC2 is not essential under normal growth conditions, but is required for timely regulation of cell cycleprogression, starvation responses, and survival under a wide variety of cellular stresses, including temperature, osmotic nutritional, DNA replication and DNA damage stresses (Weisman and Choder 2001, Weisman et al., 2007, Schonbrun et al. 2009, Schonbrun et al., 2013, Cohen et al., 2016). Our transcriptional profile analyses of cells lacking the catalytic subunit of SpTORC2 or its downstream effector, the Gad8 kinase, indicated an extensive similarity with chromatin structure mutants (Schonbrun et al. 2009, Cohen et al., 2018). This finding has led us to unravel roles for SpTORC2 in DNA damage response, telomere length maintenance and gene silencing. SpTORC2 affects genome stability via suppressing accumulation of DNA damage sites (Fig. 3) and by preventing chromosomal loss. The mechanisms underlying the genome instability in SpTORC2 mutant cells are still unclear and are currently under investigation. A first clue as to the possible mechanisms by which SpTORC2 affects tolerance to DNA-damaging conditions is our finding showing that the catalytic subunit of SpTORC2, as well as Gad8 interact with, and are required for the loading onto chromatin of the Mlu-binding factor (MBF) transcription complex (Cohen et al., 2016). Remarkably, SpTORC2-Gad8 also affects epigenomic stability as it is required for gene silencing and H3 lysine-9 methylation (see Fig. 4) in a manner antagonistic to the action of the RNA polymerase II -associated Paf1C complex (Choen et al., 2018; Oya et al., 2019). 

Figure 3: Elevated levels of DNA damage (Rad52-YFP) foci in SpTORC2 mutant cell.

Rad52-YFP foci are observed as small dots within the blue-stained nuclei.

Figure 4: Loss of TORC2 leads to a decrease in heterochromatic markers at the subtelomeric region. Genome browser view showing ChIP-Seq analysis of H3K9me2 levels in WT (green), tor1 (red), and gad8 (orange) in log2 scale.

Understanding how TORC2 relays its activation signals is critical for discovering novel drugs that would selectively inhibit TORC2 signaling. Such drugs could potentially be beneficial for treating cancer. Immediate targets of TOR complexes are members of the AGC kinase family. These kinases share a common mechanism of activation that involves phosphorylation at three major conserved sites: one is located at the activation loop and two are located C- terminally to the kinase domain at the so called turn motif (TM) and hydrophobic motif (HM). Tor1 is responsible for phosphorylation of Gad8 at S546 in the HM and S527 in the TM. We isolated point mutations in the PIF pocket of Gad8 that render its kinase activity independent of TORC2 (Fig. 5), suggesting structural roles for specific amino acids in keeping Gad8 activity in check in a manner dependent on TORC2 (Pataki et al., 2020).

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Figure 5: The gad8-K263C mutation is located at the PIF pocket and induces higher flexibility at the PIF pocket and activation loop. 

The protein is shown as a ribbon diagram (grey), the ATP is shown as sticks, and the Mg ion is shown as a green sphere (ATP is colored according to atom types). The activation loop (A-loop) is shown in red. The highly conserved structures in AGC kinases, the DFG-motif and the Gly-rich loop are colored in blue and magenta, respectively. The C-terminal extension in cyan. The residues K263, T387, S527, and S546 are shown in balls and sticks.

 Research Goals

Our long-term goal is to understand how growth is regulated in eukaryotes and coordinated with cell divisions, development, genomic and epigenomic stability.

Current goals in the lab:

• Isolation of proximal and distal effectors of TORC1 and TORC2
 
• Understanding the mode of activation of TORC1 and TORC2

• Use of genetically engineered yeast cells to screen for novel anti-TOR inhibitors