Role Of TOR And Sch9 In Regulating Ras-cAMP Signal Pathway In Saccharomyces Cerevisiae

Addition of glucose to glucose-deprived stationary phase Saccharomyces cerevisiae cells triggers a rapid spike in the cAMP level and activation of PKA causing the switch to high PKA phenotype at the same time cells switch to fermentative growth. Similarly, readdition of missing essential nitrogen or phosphate to yeast cells starved on a glucose-containing medium for nitrogen or phosphate, leads to the growth induction and rapid disappearance of PKA-controlled stationary phase characteristics. The cells rapidly switch to high PKA phenotype. Nitrogen or phosphate activate PKA require glucose, glucose activate PKA also require nitrogen or phosphate. There must exist a signal network which accumulate information from different nutrients sensing systems and integrate them into a universal cell response to activate downstream target of PKA. The goal of this work is to systematically investigate the mechanisms of nitrogen regulate glucose induced cAMP signal.Our researches discover that glucose induced cAMP signal disappears under nitrogen starvation condition. It is also found tha疤?pamycin treatment abolishes glucose induced cAMP signal, but expression of TOR1-RR which is insen??ve to rapamycin abolishes the effect of rapamycin treatment on glucose induced cAMP signal. TOR is the target of nitrogen and rapamycin, meanwhile, rapamycin treated cells have similar phenotype with nitrogen starvated cells. These demonstrate that nitrogen regulate glucose induced cAMP signal through TOR. Sch9 is the major target of TORC1. Deletion of SCH9 abolishes glucose induced cAMP signal. These demonstrate that nitrogen regulate glucose induced cAMP signal through TOR-Sch9.Our studies uncover the mechanisms of TOR-Sch9 regulating Ras-cAMP signal pathway. TOR and Sch9 inhibit PKA activity, thus rapamycin treatment or deletion of SCH9 results in high PKA activity which leads to overphosphorylation of Cdc25 by catalytic subunits of PKA. The phosphorylated Cdc25 will isolate from the targets Ras-Cyr1, through which will realize feedback inhibition to Ras-cAMP pathway, so glucose induced cAMP signal disappears.It is found that Sch9 regulate PKA activity through regulating the localization of regulatory subunit Bcy1 and catalytic subunits Tpk1, 2, 3. Deletion of SCH9 results in Bcy1 importing into the nucleus and Tpk1, 2, 3 exporting to the cytoplasm. Isolated from regulatory subunit Bcy1, catalytic subunits Tpk1, 2, 3 turn to active subunits to regulate its downstream targets, so PKA activity increasing. It is also found that Sch9 regulate the phosphorylation of Bcy1, phosphorylation of Bcy1 was markedly weakened in sch9Δmutant.Our studies find that carbon source, nitrogen source and PKA level affect the localization of Bcy1. Bcy1 predominantly localizes in the nucleus in rapidly cells growning on glucose, but it distributes over nucleus and cytoplasm in cells growing on nonfermentable carbon source or in stationary phase cells. Nitrogen regulates Bcy1 localization through Sch9. Bcy1 accumulates in the nucleus under nitrogen starvation or deletion of SCH9 condition. Our studies show that Sch9 acts upstream of Zds1 regulating localization of Bcy1. It is also find that Sch9 regulates phosphorylation of Zds1. Yak1, Rim15, Msn2 and Msn4 also regulate the localization of Bcy1. Bcy1 localizes in the nucleus in yak1Δ, rim15Δand msn2Δmsn4Δmutants. Bcy1 localizes in the nucleus in high PKA cells, but it distributes over nucleus and疤?plasm in low PKA cells.We also find that Sch9 regulates the localization of Yak1. Yak1 mainly local?? in cytoplasm in rapidly glucose grown wild type cells, but it distributes over nucleus and cytoplasm in sch9Δmutant. Yak1 can arrest cell cycle when it localizes in the nucleus. Perhaps this can explain why sch9Δmutant simultaneously has high PKA activity and low PKA activity phenotype.Our studies demonstrate that deletion of RIM15 and GIS1 do not affect glucose induced cAMP signal.