Studies of GATA-family transcription factors Gln3 and Gat1 in the model organism S. cerevisiae are important to further our understanding of the molecular events in two high priority clinical areas: (i) human GATA- family transcription factor regulation, increasingly found associated with human diseases, such as myeloproliferative disorder and acute megakaryoblastic leukemia, and (ii) the cellular and molecular biology of the global regulatory protein kinase, mTor, the in vivo target of the drugs, rapamycin and its analogues, sirolimus, everolimus and CCI-779. These drugs are currently in phase II and III clinical trials evaluating their use in stents to treat coronary artery disease, as antineoplastic agents in the treatment of estrogen-induced breast cancer and renal cell carcinoma, and as immunosuppressants following organ transplant surgery. In S. cerevisiae, Gln3 and Gat1 are the transcription factors responsible for selective nitrogen source utilization. They are cytoplasmic and non-functional when cells are cultured in excess nitrogen, and accumulate in the nucleus and activate transcription during nitrogen starvation or growth in limiting nitrogen. Treating cells with the Tor inhibitor, rapamycin, induces Gln3 dephosphorylation and nuclear accumulation even when excess nitrogen is available. These characteristics have resulted in the use of Gln3 localization and phosphorylation as a principal assay of Tor function, hence the importance of studying Tor regulation of Gln3. An engaging model posits that the Tor signal transduction pathway regulates Gln3 localization through the action of type- 2A-related phosphatase, Sit4. When Tor is active, Sit4 is inactive, Gln3 is phosphorylated, complexed with Ure2 and localized to the cytoplasm. When Tor is inhibited by rapamycin or nitrogen starvation, Sit4 becomes active, Gln3 is dephosphorylated, dissociates from Ure2, and accumulates in the nucleus. Data generated in the past grant period clearly demonstrate the above current model for Tor1, 2 control of Gln3 requires significant revision. For example, Tor regulation was thought to occur via Mks1, which was posited to be a negative regulator of Ure2 and positive regulator of Gln3. We demonstrated Mks1 affects Gln3 localization only indirectly through its negative regulation of a-ketoglutarate production required to assimilate the nitrogen source (ammonia), and that Sit4 actively dephosphorylates Gln3 irrespective of nitrogen source availability. Experiments in this application identify where additional alterations are required, explain instances in which predictions generated by the current model are not fulfilled, and demonstrate how that segment of the Tor pathway is regulated. This new information will generate a more accurate understanding of the mechanisms through which Gln3 is regulated by Tor and by which it responds to its external environment. More importantly it will serve as an efficient model system that generates information, much of which will be directly applicable to mammalian cells because the Tor pathway is so well conserved between these organisms.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM035642-22
Application #
7784534
Study Section
Cellular Signaling and Dynamics Study Section (CSD)
Program Officer
Maas, Stefan
Project Start
1985-02-01
Project End
2012-03-31
Budget Start
2010-04-01
Budget End
2012-03-31
Support Year
22
Fiscal Year
2010
Total Cost
$346,896
Indirect Cost
Name
University of Tennessee Health Science Center
Department
Other Basic Sciences
Type
Schools of Medicine
DUNS #
941884009
City
Memphis
State
TN
Country
United States
Zip Code
38163
Tate, Jennifer J; Buford, David; Rai, Rajendra et al. (2017) General Amino Acid Control and 14-3-3 Proteins Bmh1/2 Are Required for Nitrogen Catabolite Repression-Sensitive Regulation of Gln3 and Gat1 Localization. Genetics 205:633-655
Cooper, Terrance G (2017) What do the pictures say-snapshots of a career. FEMS Yeast Res 17:
Cooper, Terrance G (2017) Editorial: Saccharomyces riding the waves of technology and transition. FEMS Yeast Res 17:
Cooper, Terrance G (2016) Editorial: Retrospectives - lives behind the science. FEMS Yeast Res 16:fow005
Rai, Rajendra; Tate, Jennifer J; Cooper, Terrance G (2016) Multiple Targets on the Gln3 Transcription Activator Are Cumulatively Required for Control of Its Cytoplasmic Sequestration. G3 (Bethesda) 6:1391-408
Slonimski, Piotr P; Cooper, Terrance G; von Borstel, Robert C Jack (2016) Piotr P. Slonimski - The Warrior Pope: The discovery of mitochondrial (petite) mutants and split genes. FEMS Yeast Res 16:fow004
Rai, Rajendra; Tate, Jennifer J; Shanmuganatham, Karthik et al. (2015) Nuclear Gln3 Import Is Regulated by Nitrogen Catabolite Repression Whereas Export Is Specifically Regulated by Glutamine. Genetics 201:989-1016
Georis, Isabelle; Isabelle, Georis; Tate, Jennifer J et al. (2015) Premature termination of GAT1 transcription explains paradoxical negative correlation between nitrogen-responsive mRNA, but constitutive low-level protein production. RNA Biol 12:824-37
Tate, Jennifer J; Georis, Isabelle; Rai, Rajendra et al. (2015) GATA Factor Regulation in Excess Nitrogen Occurs Independently of Gtr-Ego Complex-Dependent TorC1 Activation. G3 (Bethesda) 5:1625-38
Tate, Jennifer J; Rai, Rajendra; Cooper, Terrance G (2015) Nitrogen starvation and TorC1 inhibition differentially affect nuclear localization of the Gln3 and Gat1 transcription factors through the rare glutamine tRNACUG in Saccharomyces cerevisiae. Genetics 199:455-74

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