Abstract
Using an eGFP-RBD3 probe, which specifically binds Ras-GTP, we recently showed that the fluorescent probe was localized to the plasma membrane and to the nucleus in wild type cells growing exponentially on glucose medium, indicating the presence of active Ras in these cellular compartments. To investigate the nuclear function of Ras-GTP, we generated a strain where Ras2 is fused to the nuclear export signal (NES) from the HIV virus, in order to exclude this protein from the nucleus. Our results show that nuclear active Ras2 is required for invasive growth development in haploid yeast, while the expression of the NES-Ras2 protein does not cause growth defects either on fermentable or non-fermentable carbon sources and does not influence protein kinase A (PKA) activity related phenotypes analysed. Moreover, we show that the cAMP/PKA pathway controls invasive growth influencing the localization of active Ras. In particular, we show that PKA activity plays a role in the localization of active Ras and influences the ability of the cells to invade the agar: high PKA activity leads to a predominant nuclear accumulation of active Ras and induces invasive growth, while low PKA activity leads to plasma membrane localization of active Ras and to a defective invasive growth phenotype.
Highlights
In the yeast Saccharomyces cerevisiae the Ras/cAMP/protein kinase A (PKA) signaling pathway plays an important role in the control of metabolism, stress resistance, proliferation [1,2,3,4] and it affects morphogenesis and development, including pseudohyphal, invasive growth and sporulation [5]
To investigate whether PKA activity plays a role in the localization of active Ras, we introduced the eGFP-RBD3 probe, which binds to Ras-GTP [12,13], into the cyr1D pde2D yak1D strain [22], bearing a deletion in the gene encoding adenylate cyclase
Our results show that addition of KOH to glucose-growing cells triggered a fast increase in the Ras2-GTP level (Figure 3B), suggesting that alkalinization downregulates the cAMP/PKA pathway acting on element(s) downstream Ras and that the increase of Ras2-GTP might be related to a decrease of the feedback inhibition operated by PKA on Ras2 [29]
Summary
In the yeast Saccharomyces cerevisiae the Ras/cAMP/PKA signaling pathway plays an important role in the control of metabolism, stress resistance, proliferation [1,2,3,4] and it affects morphogenesis and development, including pseudohyphal, invasive growth and sporulation [5]. Ras, but not Ras, activates invasive growth using either of two downstream signalling pathways, the filamentation MAPK cascade (Cdc42p/ Ste20p/MAPK) or the cAMP/PKA pathway, indicating a crosstalk between both signalling pathways and this could happen in the nucleus [16,17,18]. This hypothesis is substantiated by the observation that in a strain deleted in GPA2, which has a defect in pseudohyphal growth [19], active Ras does not accumulate in the nucleus, but is mainly localized in mitochondria [13]
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