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RESEARCH PRODUCT

Phosphorylation of an Overexpressed Yeast Ras2 Protein During the G1 Phase of the Cell Cycle

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RAS proteins regulate growth and differentiation in evolutionarily distant systems such as vertebrates and yeast (for reviews, see Tamanoi, 1988; Gibbs and Marshall, 1989; Broach and Deschenes, 1990). At the moleular level, a key function of the yeast RAS1 and RAS2 proteins (collectively referred to as RAS) is to positively regulate the production of cyclic AMP at the onset of the G1 phase of the cell cycle (Toda et al., 1985; De Vendittis et al., 1986). At this stage, RAS proteins are transiently activated by the noncovalent binding of a GTP molecule. Reversal of the effect occurs by the hydrolytic splitting of the ’γ-phosphate of GTP, that leaves a functionally inactive RASGDP complex, thus terminating cyclic AMP synthesis. While the mechanism and functional role of the binding of either GTP or GDP to RAS has been investigated in detail, less is known about the physiological role of covalent modifications of RAS involving phosphorylation of serine residues. A major obstacle to the analysis of the functional relevance of this covalent modification is represented by the very low concentration of RAS proteins into the cell. To bypass this difficulty, we have taken advantage of a mutated form of the RAS2 gene encoding a protein that can be overexpressed at ligh levels. This gene, which is called ras2-ts1, was previously isolated as an attenuated form of the wild-type RAS2 gene (Fasano et al., 1988). The corresponding ras2-tsl protein, that is functional at 30 °C while becoming nonfunctional at 37 °C, was easily labelled metabolically with radioactive 32P under the form of orthophosphate, and was immunoprecipitated with anti-RAS specific antibodies. We have used this protein to explore RAS2 phosphorylation in cells at different stages of the cell cycle.