Requirement of one functional RAS gene and inability of an oncogenic ras variant to mediate the glucose-induced cyclic AMP signal in the yeast Saccharomyces cerevisiae.

Abstract

Addition of glucose to Saccharomyces cerevisiae cells grown on a nonfermentable carbon source triggers a cyclic AMP (cAMP) signal, which induces a protein phosphorylation cascade. In a yeast strain lacking functional RAS] and RAS2 genes and containing a bcy mutation to suppress the lethality of RAS deficiency, the cAMP signal was absent. Addition of dinitrophenol, which stimulates in vivo cAMP synthesis by lowering intracellular pH, also did not enhance the cAMP level. A bcy control strain, with functional RAS genes present, showed cAMP responses similar to those of a wild-type strain. In disruption mutants containing either a functional RAS] gene or a functional RAS2 gene, the cAMP signal was not significantly different from the one in wild-type cells, indicating that RAS function cannot be a limiting factor for cAMP synthesis during induction of the signal. Compared with wild-type cells, the cAMP signal decreased in intensity with increasing temperature in a ras2 disruption mutant. When the mutant RAS2V'-l9, which carries the equivalent of the human H-rasVa~2 oncogene, was grown under conditions in which RAS] expression is repressed, the cAMP signal was absent. The oncogene product is known to be deficient in GTPase activity. However, the amino acid change at position 19 (or 12 in the corresponding human oncogene product) might also have other effects, such as abolishing receptor interaction. Such an additional effect probably provides a better explanation for the lack of signal transmission than the impaired GTPase activity. When the RAS2val-19 mutant was grown under conditions in which RAS] is expressed, the cAMP signal was present but significantly delayed compared with the signal in wild-type cells. This indicates that oncogenic RAS proteins inhibit normal functioning of wild-type RAS proteins in vivo and also that in spite of the presence of the RAS2va'-9 oncogene, adenyl cyclase is not maximally stimulated in vivo. Expression of only the RAS2Va'-l9 gene product also prevented most of the stimulation of cAMP synthesis by dinitrophenol, indicating that lowered intracellular pH does not act directly on adenyl cyclase but on a step earlier in the activation pathway of the enzyme. The results obtained with the control bcy strain, the RAS2va'-I9 strain under conditions in which RAS] is expressed, and with dinitrophenol show that the inability of the oncogene product to mediate the cAMP signal is not due to feedback inhibition by the high protein kinase activity in strains containing the RAS2Va-l9 oncogene. Hence, the present results show that the RAS proteins in S. cerevisiae are involved in the transmission of the glucose-induced cAMP signal and that the oncogenic RAS protein is unable to act as a signal transducer. The RAS proteins in S. cerevisiae apparently act similarly to the G. proteins of mammalian adenyl cyclase, but instead of being involved in hormone signal transmission, they function in a nutrient-induced signal transmission pathway. (23,31) and transient intracellular acidification (10,36,46) we...

Activation State of the Ras2 Protein and Glucose-induced Signaling in Saccharomyces cerevisiae

“…The first evidence for involvement of the Ras proteins in cAMP signaling was provided by Broek et al (20), Toda et al (21), and Mbonyi et al (22). Subsequent work also supported a requirement of CDC25 in glucose-induced cAMP signaling (23)(24)(25)(26).…”

Life in the midst of scarcity: adaptations to nutrient availability in Saccharomyces cerevisiae

“…3). The intracellular glucose-sensing system requires the uptake and phosphorylation of glucose, but no further metabolism of the sugar (Beullens et al 1988;Rolland et al 2000). In S. cerevisiae, there are three glucose phosphorylating enzymes (Hxk1, Hxk2 and Glk1) and any of these proteins can fulfil this phosphorylation requirement to activate the cAMP-PKA pathway.…”

“…Glucose addition to glucose-starved cells triggers a transient increase in the GTP loading of Ras2. This process was shown to require glucose phosphorylation (Mbonyi et al 1988;Colombo et al 2004). The mechanism by which glucose phosphorylation affects Ras-GTP loading remains largely to be elucidated.…”

The glucose‐induced polyphosphoinositides turnover in Saccharomyces cerevisiae is not dependent on the CDC25‐RAS mediated signal transduction pathway

“…and RAS2 genes are elements of this pathway [13]. Thus we measured the stimulation of PI turnover in ras mutants and in cdc25 mutants.…”