Helical domain and kinase domain mutations in p110alpha of phosphatidylinositol 3-kinase induce gain of function by different mechanisms

Abstract

The phosphatidylinositol 3-kinase (PI3K) signaling pathway is up-regulated in cancer. PIK3CA, the gene coding for the catalytic subunit p110alpha of PI3K, is mutated in approximately 30% of tumors of the prostate, breast, cervix, and endometrium. The most prominent of these mutants, represented by single amino acid substitutions in the helical or kinase domain, show a gain of enzymatic function, activate AKT signaling, and induce oncogenic transformation. We have carried out a genetic and biochemical analysis of these hot-spot mutations in PIK3CA. The results of this study suggest that the helical and kinase domain mutations trigger gain of function through different mechanisms. They show different requirements for interaction with the PI3K regulatory subunit p85 and with RAS-GTP. The gain of function induced by helical domain mutations is independent of binding to p85 but requires interaction with RAS-GTP. In contrast, the kinase domain mutation is active in the absence of RAS-GTP binding but is highly dependent on the interaction with p85. We speculate that the contrasting roles of p85 and RAS-GTP in helical and kinase domain mutations reflect two distinct states of mutated p110alpha. These two states differ in mutation-induced surface charges and also may differ in conformational properties that are controlled by interactions with p85 and RAS-GTP. The two states do not appear mutually exclusive because the helical and kinase domain mutations act synergistically when present in the same p110alpha molecule. This synergism also supports the conclusion that the helical and kinase domain mutations operate by two different and independent mechanisms.

Fig. 1.

Helical domain and kinase domain mutations act synergically in cell transformation. (A and B) Focus growth curves for CEFs transfected with RCAS vectors encoding the p110α mutants. The focus growth curve of E545K/H1047R is similar to that of E542K/H1047R and is therefore not shown. EOT, number of foci per nanogram of DNA. The means for two experiments are shown. (C) Western blots comparing the protein expression levels of p110α and the phosphorylation levels of AKT and S6K. Cells were starved in basal medium, and lysates were prepared as described in Materials and Methods.

Fig. 2.

Binding to p85 is essential for H1047R-induced cell transformation. (A and B) Cell transformation (focus formation) induced by full-length or p85-binding domain deletion mutants of p110α (δp85BD-p110α). EOT by H1047R is normalized to one. (C) Western blots comparing the p110α expression levels and the phosphorylation levels of AKT and S6K. δp85BD-p110α constructs do not coimmunoprecipitate with endogenous p85α. The assays were carried out as described in Materials and Methods.

Fig. 3.

Helical domain mutations are incapable of rescuing the oncogenic activity of δp85BD-p110α H1047R. (A and B) Cell transformation induced by δp85BD-p110α constructs. EOT by δp85BD-p110α is normalized to one. (C) Western blots comparing the p110α expression levels and the phosphorylation levels of AKT and S6K. The assays were carried out as described in Materials and Methods.

Fig. 4.

Oncogenic transformation by the helical domain mutation depends on binding to RAS. (A and B) Cell transformation induced by full-length or RAS-binding mutants of p110α. EOT by H1047R is normalized to one. (C) Western blots comparing the protein expression levels of p110α and the phosphorylation levels of AKT. The assays were carried out as described in Materials and Methods.

Fig. 5.

RAS-binding mutation of p110α abolishes the activation by HRAS (G12V). CEFs were transfected with the p110α expression vector only or cotransfected with p110α and HRAS (G12V) expression vectors. Cells were maintained in a nutrient medium containing 3% FBS and 1% chicken serum and were then harvested and probed with the indicated antibodies by Western blotting.

Fig. 6.

The H1047R mutation rescues the cell-transformation phenotypes of the p110α mutants K227E/E542K and K227E/E545K from RAS-binding mutation. (A and B) Focus growth curves for CEFs transfected with RCAS vectors encoding the p110α mutants. EOT, number of foci per nanogram of DNA. The means for two experiments are shown. (C) Western blots comparing the protein expression levels of p110α and the phosphorylation levels of AKT. The assays were carried out as described in Materials and Methods.

Fig. 7.

A schematic summary of mutant properties. p85BD, p85-binding domain; RBD, Ras-binding domain; C2, C2 domain. The approximate locations of the point mutations are indicated by inverted triangles, and the deletion in the p85BD is marked by a truncated alias of the p85BD. Cell-transforming activity is qualitatively denoted by + and −, and synergistic activity is denoted by +!.