In vivo Functional Analysis of the Counterbalance of Hyperactive Phosphatidylinositol 3-Kinase p110 Catalytic Oncoproteins by the Tumor Suppressor PTEN

Abstract

The signaling pathways involving class I phosphatidylinositol 3-kinases (PI3K) and the phosphatidylinositol-(3,4,5)-trisphosphate phosphatase PTEN regulate cell proliferation and survival. Thus, mutations in the corresponding genes are associated to a wide variety of human tumors. Heterologous expression of hyperactive forms of mammalian p110A and p110B in Saccharomyces cerevisiae leads to growth arrest, which is counterbalanced by coexpression of mammalian PTEN. Using this in vivo yeast-based system, we have done an extensive functional analysis of germ-line and somatic human PTEN mutations, as well as a directed mutational analysis of discrete PTEN functional domains. A distinctive penetrance of the PTEN rescue phenotype was observed depending on the levels of PTEN expression in yeast and on the combinations of the inactivating PTEN mutations and the activating p110A or p110B mutations analyzed, which may reflect pathologic differences found in tumors with distinct alterations at the p110 and PTEN genes or proteins. We also define the minimum length of the PTEN protein required for stability and function in vivo. In addition, a random mutagenesis screen on PTEN based on this system allowed both the reisolation of known clinically relevant PTEN mutants and the identification of novel PTEN loss-of-function mutations, which were validated in mammalian cells. Our results show that the PI3K/PTEN yeast-based system is a sensitive tool to test in vivo the pathologic properties and the functionality of mutations in the human p110 proto-oncogenes and the PTEN tumor suppressor and provide a framework for comprehensive functional studies of these tumor-related enzymes.

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 previous reports, we described that heterologous expression of PI3K, PTEN, and Akt1 in S. cerevisiae reproduces several aspects of their function, including PIP 3 -dependent membrane translocation and phosphorylation of Akt1 at both activation residues by the yeast PDK1 orthologs and an unidentified endogenous kinase (22). We have also reported previously that this heterologous model system could be readily exploited to evaluate oncogenicity of both PI3K and PTEN mutants (23). Here, we show that catalytically active mammalian Akt inhibits yeast cell growth, thus extending the system to perform structure-function relationship studies on Akt in vivo.…”

“…Plasmids pYES2-PTEN, pYES2-GFP-Akt1, pYES-GFP-Akt K179M , pYES2-myr-Akt1, YCpLG-myc-p110␣-CAAX, YCpLG-myc-p110␣-wt, YCpLGmyc-p110␣-E545K, YCpLG-myc-p110␣-H1047R, and pYES3-GFP-Akt1 have been described (22,23). pYES2-SHIP1 was constructed by subcloning human SHIP1 into pYES2 by PCR from pOTB7 SHIP1 (Mammalian Gene Collection, IMAGE ID 6304992, Geneservice, UK).…”

PIK3CA Mutation Associates with Improved Outcome in Breast Cancer

“…This is opposite to the H1047R mutation, where only p85 binding is required. Interestingly, despite appearing stronger in both oncogenic potency and pathway activation in preclinical models (7,38), the H1047R mutation has the strongest association with clinical benefit. This finding, along with the minimal effect seen with rare PIK3CA mutations, strengthens the argument that the observed improvement is a direct effect of mutation activation.…”

Wide-Ranging Effects of the Yeast Ptc1 Protein Phosphatase Acting Through the MAPK Kinase Mkk1

“…Then the BamHI-digested fragments were subcloned into pEG(KG) (Mitchell et al 1993), generating plasmids pEG(KG)-PTC1 and pEG(KG)-NBP2. To overexpress Ptc1 fused to GFP in N-and C-terminal positions, the pYES3-GFP-PTC1 and YCplG-PTC1-GFP plasmids were constructed by subcloning the BamHI-digested PTC1 ORF into pYES3-GFP (Andrés-Pons et al 2007) and YCplG-GFP (Rodriguez-Escudero et al 2006), respectively. The pGEMT-ptc1::HIS3 plasmid was obtained by subcloning the SmaI-digested HIS3 ORF from plasmid p34H-HIS3 into the StuI site of plasmid pGEMT-PTC1, which was made using the same PTC1 PCR product described earlier.…”