Molecular mechanisms involved in activation of the Ras proteins by glycolytic flux
Moleculaire mechanismen betrokken in de activatie van de Ras proteïnen door glycolytische flux
Peeters, Ken; S0105462
Saccharomyces cerevisiae cells favour fermentation of sugar over r espiration in the presence of oxygen. In order to obtain a high glycolytic rate to metabolize fermentable sugars generating the required levels of ATP, yeast developed a complex signal transduction system controlling this process. One important protein in this signaling cascade is the small monomeric GTP-binding Ras protein. This essential protein is involved in the regulation of cell proliferation and the establishment of fermentative metabolism. In the presence of glucose, the GTP-bound active Ras stimulates the activity of Cyr1/adenylate cyclase, which in turn, activates the protein kinase PKA. This kinase regulates many proteins by phosphorylation and is the main controller of the transcriptional response to glucose through direct phosphorylation of multiple transcription factors. In this study a direct link between high glycolytic rate and activation of Ras was discovered. This was first demonstrated by use of th e tps1 mutant, which lacks feedback inhibition on hexokinase and as a r esult displays overactive in flux of sugar into glycolysis. We showed that glucose addition to this strain results in a hyperactivation of the Ras protein and in the appearance of typical apoptotical markers and cell death. Our results indicate that the induction of apoptosis in the tps1 mutant is a direct consequence of the hyperactive Ras protein. Reducin g the activity of the Ras/cAMP/PKA pathway in the tps1 background resul ts in the survival of this strain for growth on glucose based medium. By use of wild type permeabilized spheroplasts, where we removed the cell wall enzymatically before chemically creating holes in the plasma membrane through which metabolites but not proteins can difuse, we found the glycolytic intermediate fructose-1,6-bisphosphate (Fru1,6bisP) to be the crucial factor in the activation of Ras. In the tps1 strain this compou nd can accumulate up to 20mM. Inactivation of phosphofructokinase in the tps1 strain abolishes Fru1,6bisP hyperaccumulation as well as hyperact ivation of Ras in vivo.In our search for the mechanism of Fru1,6bisP induced Ras activation, we found the Ras guanine nucleotide exchange factors (GEF), Cdc25 and Sdc25, to be required. Using deletion strains in the Ras GEF and GTPase activating proteins (GAP) and site-directed mutagenesis, we showed that the C-terminus of yeast Cdc25 contains a region with the positively charged residues R1122 and K1491, which is required for Ras activation with Fru1,6bisP, but not with GTP. This region is highly conserved among other Ras GEF proteins in different eukaryotic organisms including the human Ras GEF proteins,Sos1 and Sos2. The positively charged residues R1122 and K1491 in the yeast Cdc25 protein have homologue residues in the human Sos1 protein, namely K602 and K963. These residues are also required to activate the yeast Ras proteins with Fru1,6bisP when CDC25 is replaced by part of the human SOS1 gene, but not with GTP.Mammalian cancer cells also show the unusual characteristic of favouring fermentation over respiration in the presence of oxygen. It remains controversial whether this strong fermentation is a cause or a symptom of cancer since no clear molecular link between glycolysis and proteins controlling cell proliferation has been identified.The here described Fru1,6bisP-dependent activation of Ras may be one such molecular link. Our results therefore suggest that there may be a causal link between high glycolytic activity and rapid cell proliferation in glucose medium in yeast and cancer cells through Fru1,6bisP activation of Ras and mediated by a conserved Fru1,6bisP-binding domain in the C-terminus of Cdc25/Sos.