Phosphatidylinositol 3-kinase-dependent activation of mammalian protein kinase B/Akt in Saccharomyces cerevisiae, an in vivo model for the functional study of Akt mutations.
In animal cells, Akt (also called protein kinase B) is activated by stimuli that elevate the level of phosphatidylinositol 3,4,5-trisphosphate and is a major effector for eliciting responses that support cell growth and survival. We have shown previously that co-expression of Akt1 in budding yeast (Saccharomyces cerevisiae) along with hyperactive p110alpha, the catalytic subunit of mammalian phosphatidylinositol 3-kinase, results in Akt1 relocalization to cellular membranes and activation. In the present study, we show that activation of all three mammalian Akt isoforms by wild-type p110alpha causes deleterious effects on yeast cell growth. Toxicity of Akt in S. cerevisiae required its catalytic activity, its pleckstrin homology domain, and phosphorylation of its activation loop, but not phosphorylation of its hydrophobic motif. We demonstrate that expression in yeast of the only purported oncogenic allele, Akt1(E17K), leads to enhanced phenotypes. Ala-scanning mutagenesis of the VL1 region within the phosphatidylinositol 3,4,5-trisphosphate-interacting pocket of the Akt1 pleckstrin homology domain revealed that most residues in this region are essential for Akt1 activity. We found that active Akt leads to enhanced signaling through the yeast cell wall integrity pathway. This effect requires the upstream Rho1 activator Rom2 and involves both phosphorylation of the MAPK Slt2 and expression of its transcriptional targets, thus providing a quantitative reporter system for heterologous Akt activity in vivo. Collectively, our results disclose a heterologous yeast system that allows the functional assessment in vivo of both loss-of-function and tumorigenic Akt alleles.
Rodríguez-Escudero
Isabel
I
Departamento de Microbiología II, Facultad de Farmacia, Universidad Complutense de Madrid, Madrid 28040.
Andrés-Pons
Amparo
A
Centro de Investigación Príncipe Felipe, Valencia 46012, Spain.
Pulido
Rafael
R
Centro de Investigación Príncipe Felipe, Valencia 46012, Spain.
Molina
María
M
Departamento de Microbiología II, Facultad de Farmacia, Universidad Complutense de Madrid, Madrid 28040.
Cid
Víctor J
VJ
Departamento de Microbiología II, Facultad de Farmacia, Universidad Complutense de Madrid, Madrid 28040. Electronic address: vicjcid@farm.ucm.es.
D000818:Animals; D004789:Enzyme Activation / Q000235:genetics; D020662:Guanine Nucleotide Exchange Factors / Q000235:genetics / Q000378:metabolism; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D008322:Mammals; D020928:Mitogen-Activated Protein Kinases / Q000235:genetics / Q000378:metabolism; D008954:Models, Biological; D016296:Mutagenesis; D009154:Mutation; D019869:Phosphatidylinositol 3-Kinases / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary / Q000235:genetics; D051057:Proto-Oncogene Proteins c-akt / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D020741:rho GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism
D020662:Guanine Nucleotide Exchange Factors; D007527:Isoenzymes; C490929:ROM2 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D019869:Phosphatidylinositol 3-Kinases; D051057:Proto-Oncogene Proteins c-akt; D020928:Mitogen-Activated Protein Kinases; C072724:SLT2 protein, S cerevisiae; C051302:RHO1 protein, S cerevisiae; D020741:rho GTP-Binding Proteins