Functional Mechanism Of Cdc25p In Saccharomyces Cerevisiae In Regulationg Ras-cAMP Pathway

In the yeast Sacchromyces cerevisiae, addition of glucose to cells in nonfermentable carbon source triggers a transient rise in the intracellular level of cyclic AMP that induces a protein phosphorylation cascade. The glucose signal is processed by the Cdc25/Ras/adenylyl cyclase pathway, where the role of Cdc25p is to catalyse the GDP/GTP exchange on Rasp. It has been demonstrated that Cdc25p is a phosphoprotein and that in response to glucose it is hyperphosphorylated by the cyclic AMP-dependent protein kinase. It is also demonstrated that, concomitantly with hyperphosphorylation, Cdc25p partially relocalizes to the cytoplasm, reducing its accessibility to membrane-bound Rasp. All these results suggest that Cdc25p might be a target of PKA in feedback control of cAMP level. However, no direct evidence of reduced GEF activity for phosphorylated Cdc25p has been obtained so far. This work focuses mainly on elucidating the mechanism of yeast Cdc25p in the feedback inhibition of the cAMP pathway by PKA,as well as reduced GEF activity for phosphorylated Cdc25p. We also examined the effect of TPK gene product activities on the feedback inhibition of the Ras-cAMP pathway in yeast cells.Our results indicated that phosphorylation of Cdc25p by PKA is due to the feedback control of Ras-cAMP pathway, which is not dependent on Yak1, Tor1, Rim15, Sch9 kinases.In the yeast Sacchromyces cerevisiae, a single gene, BCY1, encodes the regulatory subunit of PKA. Three other genes, TPK1, TPK2, and TPK3, encode the catalytic subunits. First, we created strains that contained one or two TPK genes with a disrupted BCY1 gene as mutants with high PKA activity, and strains with YAK1 and three TPK genes completely disruption as mutants with low PKA activity. Our results indicated that high PKA activity leads to hyperphosphorylation of Cdc25, a low level of the intracellular Ras2-GTP as well as a low level of Ras2-GTP bound to Cdc25p in vivo. But strains with low PKA activity do not lead to hyperphosphorylation of Cdc25p, has a high level of the intracellular Ras2-GTP as well as a high level of Ras2-GTP bound to Cdc25p. This suggests that phosphorylation of Cdc25p by PKA constitutes a feedback mechanism by which PKA down-regulate cAMP signalling.We also showed that phosphorylation of Cdc25p by PKA can reduce its GEF activity on Ras in vitro, thus indicated the feedback mechanism by which phosphorylation of Cdc25p by PKA down-regulates cAMP signalling. Our results also indicated that with a complete disruption of BCY1, a single TPK gene product can also lead to hyperphosphorylation of Cdc25p, a low level of the intracellular Ras2-GTP and a low level of Ras2-GTP bound to Cdc25p, thus constitutes a feedback mechanism by which phosphorylation of Cdc25p by a single TPK gene product down-regulates cAMP signalling.