The target of rapamycin complex 1 (TORC1) plays a prominent role in a ubiquitously conserved signal transduction cascade that responds to nutrients and growth factor cues to control cell growth and proliferation. Dysregulation of the TORC1 cascade results in multiple types of human malignancies. The central TORC1 members are the Tor protein kinases, which were discovered in the yeast Saccharomyces cerevisiae, a model system that has been crucial in elucidating the TORC1 signaling cascade. Based on its potent antiproliferative activity, rapamycin is in use in several clinical areas including immunosuppression, cancer chemotherapy, and interventional cardiology. The TORC1 pathway remains partially characterized despite much study. We uncovered an unexpected role for the endomembrane vesicular trafficking system in regulating TORC1 signaling in yeast;mutations in protein complexes with roles in vesicular trafficking and protein sorting in combination with mutation of the nonessential Tor1 kinase, render cells inviable or severely growth impaired. We demonstrated that the HOPS complex is required to provide amino acid homeostasis for efficient TORC1 signaling and for normal expression of TORC1-governed genes. These studies also revealed a novel facet in the rapamycin mechanism of action: rapamycin bypasses vesicular trafficking events to activate TORC1- controlled transactivators. Moreover, we showed that mutations in the Ego complex (EGOC) compromise TORC1 signaling and both EGOC and TORC1 activity are required for optimal cell growth. The EGOC possesses evolutionary conserved amino acid-sensitive GTPase subunits and orthologs of these (Gtr1,2 and RagA-D proteins) mediate TORC1 activation in yeast, insect, and mammalian cells. However, the identity of the amino acid sensors and the underlying mechanisms by which amino acids activate EGOC GTPase are unknown. Intriguingly, TORC1 and the EGOC colocalize to endomembranes including those of endosomes and vacuoles, along with vacuolar amino acid transporters.
The aims of this proposal are: 1) to elucidate the roles and underlying mechanism by which the endomembrane system enables TORC1 signaling and 2) to determine whether the vacuolar amino acid transporters, which are conserved in mammals, are integrated into the molecular cascade that conveys amino acid signals to evoke TORC1 activation. Our working model is that the endomembrane network provides a platform to facilitate molecular interactions that activate and enable TORC1 signaling. Similar to yeast TORC1, mammalian TORC1 is also localized to endomembranes, in particular lysosomes, which are the counterparts of yeast vacuoles. Thus, we submit that our research will continue to uncover fundamental, conserved TORC1 signaling aspects that could be directly extrapolated to mammalian models and guide pharmacological intervention in select TORC1 pathway defects in human cancer and other diseases.

Public Health Relevance

The rapamycin sensitive-TORC1 is the central component of an evolutionary-conserved signaling cascade that in response to nutrients and growth factors regulates cell growth and proliferation. Defects in mTORC1 signaling lead to cancer and other human diseases. Rapamycin and its analogs are being developed as therapeutic agents for the treatment of a wide range of malignances and could also find indication in cognitive- and aging-related diseases. The model yeast S. cerevisiae has been crucial in the discovery of the TORC1 pathway and elucidation of rapamycin mechanism of action. Characterization of this pathway in yeast is continuing to reveal basic aspects of TORC1 signaling that could foster analogous studies in mammals and lead to improved anticancer therapies.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA154499-04
Application #
8602069
Study Section
Basic Mechanisms of Cancer Therapeutics Study Section (BMCT)
Program Officer
Forry, Suzanne L
Project Start
2011-01-01
Project End
2015-12-31
Budget Start
2014-01-01
Budget End
2014-12-31
Support Year
4
Fiscal Year
2014
Total Cost
$293,198
Indirect Cost
$106,448
Name
Duke University
Department
Genetics
Type
Schools of Medicine
DUNS #
044387793
City
Durham
State
NC
Country
United States
Zip Code
27705
Xu, Baoshan; Sowa, Nenja; Cardenas, Maria E et al. (2015) L-leucine partially rescues translational and developmental defects associated with zebrafish models of Cornelia de Lange syndrome. Hum Mol Genet 24:1540-55
Calo, Silvia; Shertz-Wall, Cecelia; Lee, Soo Chan et al. (2014) Antifungal drug resistance evoked via RNAi-dependent epimutations. Nature 513:555-8
Kingsbury, Joanne M; Sen, Neelam D; Maeda, Tatsuya et al. (2014) Endolysosomal membrane trafficking complexes drive nutrient-dependent TORC1 signaling to control cell growth in Saccharomyces cerevisiae. Genetics 196:1077-89
Bastidas, Robert J; Shertz, Cecelia A; Lee, Soo Chan et al. (2012) Rapamycin exerts antifungal activity in vitro and in vivo against Mucor circinelloides via FKBP12-dependent inhibition of Tor. Eukaryot Cell 11:270-81
Kozubowski, Lukasz; Thompson, J Will; Cardenas, Maria E et al. (2011) Association of calcineurin with the COPI protein Sec28 and the COPII protein Sec13 revealed by quantitative proteomics. PLoS One 6:e25280
Kozubowski, Lukasz; Aboobakar, Eanas F; Cardenas, Maria E et al. (2011) Calcineurin colocalizes with P-bodies and stress granules during thermal stress in Cryptococcus neoformans. Eukaryot Cell 10:1396-402
Shertz, Cecelia A; Cardenas, Maria E (2011) Exploiting and subverting Tor signaling in the pathogenesis of fungi, parasites, and viruses. PLoS Pathog 7:e1002269