Methionine Sulfoximine Treatment and Carbon Starvation Elicit Snf1-independent Phosphorylation of the Transcription Activator Gln3 in Saccharomyces cerevisiae

Abstract

Tor proteins are global regulators situated at the top of a signal transduction pathway conserved from yeast to humans. Specific inhibition of the two Saccharomyces cerevisiae Tor proteins by rapamycin alters many cellular processes and the expression of hundreds of genes. Among the regulated genes are those whose expression is activated by the GATA family transcription activator, Gln3. The extent of Gln3 phosphorylation has been thought to determine its intracellular localization, with phosphorylated and dephosphorylated forms accumulating in the cytoplasm and nucleus, respectively. Data presented here demonstrate that rapamycin and the glutamine synthetase inhibitor, methionine sulfoximine (MSX), although eliciting the same outcomes with respect to Gln3-Myc 13 nuclear accumulation and nitrogen catabolite repression-sensitive transcription, generate diametrically opposite effects on Gln3-Myc 13 phosphorylation. MSX increases Gln3-Myc 13 phosphorylation and rapamycin decreases it. Gln3-Myc 13 phosphorylation levels are regulated by at least three mechanisms as follows: (i) depends on Snf1 kinase as observed during carbon starvation, (ii) is Snf1-independent as observed during both carbon starvation and MSX treatment, and (iii) is rapamycin-induced dephosphorylation. MSX and rapamycin act additively on Gln3-Myc 13 phosphorylation, but MSX clearly predominates. These results suggest that MSX-and rapamycin-inhibited proteins are more likely to function in separate regulatory pathways than they are to function tandemly in a single pathway as thought previously. Furthermore, as we and others have detected thus far, Gln3 phosphorylation/dephosphorylation is not a demonstrably required step in achieving Gln3 nuclear localization and nitrogen catabolite repression-sensitive transcription in response to MSX or rapamycin treatment.

Yeast RNA-Binding Protein Nab3 Regulates Genes Involved in Nitrogen Metabolism

“…The approximately 90 nitrogen catabolite-repressed genes are controlled through a set of four GATA transcription factors that cross-regulate each other in a manner that depends on the source of nitrogen. Although the TORC1 signaling pathway plays a role in GATA factor-mediated NCR-responsive gene transcription (59), other pathways have also been implicated (58,(60)(61)(62)(63)(64). In fact, regulation of NCR genes is largely independent of TOR signaling, but details of how different nitrogen sources lead to regulation of NCR genes is not well understood.…”

Distinct Phosphatase Requirements and GATA Factor Responses to Nitrogen Catabolite Repression and Rapamycin Treatment in Saccharomyces cerevisiae

“…GATA Factor-specific Responses to Methionine Sulfoximine Inhibition of Glutamine Synthetase-The final set of observations pointing to the distinct regulation of Gln3 and Gat1 localization derive from experiments with Msx (43,44,57). Treating cells with Msx results in rapid and complete nuclear Gln3-Myc 13 and Gln3-GFP localization (Ref.…”

“…Treating cells with Msx results in rapid and complete nuclear Gln3-Myc 13 and Gln3-GFP localization (Ref. 43 and Fig. 7).…”

“…Gln3-Myc 13 is hyperphosphorylated in nitrogen-starved, proline-grown, or methionine sulfoximine-treated cells but hypophosphorylated in rapamycin-treated cells. Yet Gln3-Myc 13 is nuclear in all four cases (43)(44)(45). (iii) Sit4-dependent Gln3-Myc 13 dephosphorylation is greater in glutamine-than proline-grown cells (46); i.e.…”

“…It is important to emphasize that this is a rough qualitative rather than quantitative representation. Data for the construction of this panel were taken from the present work (Gat1) and that appearing earlier for Gln3 (40,41,43,46,47,48 …”