Hot-spot mutations in p110alpha of phosphatidylinositol 3-kinase (pI3K): differential interactions with the regulatory subunit p85 and with RAS

Abstract

The phosphatidylinositol 3-kinase (pI3K) signaling pathway is frequently upregulated in cancer. PIK3CA, the gene coding for the catalytic subunit p110alpha of PI3K, is mutated in about 12% of all human cancers. Most of these mutants are single amino acid substitutions that map to three positions (hot spots) in the helical or kinase domains of the enzyme. The mutant proteins show gain of enzymatic function, constitutively activate AKT signaling and induce oncogenic transformation in vitro and in animal model systems. We have shown previously that hot-spot mutations in the helical domain and kinase domain of the avian p110alpha have different requirements for interaction with the regulatory subunit p85 and with RAS-GTP. Here, we have carried out a genetic and biochemical analysis of these "hot-spot" mutations in human p110alpha. The present studies add support to the proposal that helical and kinase domain mutations in p110alpha trigger a gain of function by different molecular mechanisms. The gain of function induced by helical domain mutations requires interaction with RAS-Gtp. In contrast, the kinase domain mutation is active in the absence of RAS-Gtp binding, but depends on the interaction with p85.

Fig 1. Binding to p85 is essential for H1047R-induced cell transformation

(A) Western blots comparing p110α expression levels, p110α and p85 binding, and the phosphorylation of AKT in serum-starved CEF. In the co-immunoprecipitation assay, the bands seen with anti-human p110α antibody in the Δ72 and Δ108 lanes represent the endogenous p110α of CEF. (B) Quantitative analysis of cell transformation induced by full-length or ABD deletion mutants of p110α. CEF were transfected with different p110α constructs. Cells were then cultured under nutrient agar. Foci of transformed cells were counted 14 days post transfection. 545 and 1047 signify the E545K and H1047R mutants respectively. Δp85-545 and Δp85-1047 mark the Δ108-E545K and Δ108-H1047R respectively.

Fig 2. Oncogenic transformation by the helical domain mutation depends on binding to RAS

(A) Western blots comparing p110α expression levels and the phosphorylation of AKT in serum-starved CEF. (B) Quantitative analysis of cell transformation induced by full-length or RBD mutants of p110α. 545 and 1047 signify the E545K and H1047R mutants respectively. ΔRas-545 and ΔRas-1047 mark the E545K/K227A/T208D and H1047R/K227A/T208D mutants respectively.