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.
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