Rare cancer-specific mutations in PIK3CA show gain of function

Abstract

Fifteen rare cancer-derived mutants of PIK3CA, the gene coding for the catalytic subunit p110alpha of phosphatidylinositol 3-kinase (PI3K), were examined for their biological and biochemical properties. Fourteen of these mutants show a gain of function: they induce rapamycin-sensitive oncogenic transformation of chicken embryo fibroblasts, constitutively activate Akt and TOR-mediated signaling, and show enhanced lipid kinase activity. Mapping of these mutants on a partial structural model of p110alpha suggests three groups of mutants, defined by their location in distinct functional domains of the protein. We hypothesize that each of these three groups induces a gain of PI3K function by a different molecular mechanism. Mutants in the C2 domain increase the positive surface charge of this domain and therefore may enhance the recruitment of p110alpha to cellular membranes. Mutants in the helical domain map to a contiguous surface of the protein and may affect the interaction with other protein(s). Mutants in the kinase domain are located near the hinge of the activation loop. They may alter the position and mobility of the activation loop. Arbitrarily introduced mutations that have no detectable phenotype map either to the interior of the protein or are positioned on a surface region that lies opposite to the exposed surfaces containing gain-of-function mutants. Engineered mutants that exchange acidic or neutral residues for basic residues on the critical surfaces show a gain of function.

Fig. 1.

A map and frequency distributions of the three hot spot mutations of the 15 rare mutations and of eight arbitrary mutations investigated in this study (catalog of Somatic Mutations in Cancer, www.sanger.ac.uk/genetics/CGP/cosmic). Hot spot mutations are boxed. Mutants that increase the positive charge of the site are marked with ◆. Arbitrary mutants that show transformation are marked with ●.

Fig. 2.

The EOT of primary CEF by mutants of p110α. The EOT (foci per nanogram of DNA) sets the hot spot mutations apart from the rare mutations and suggests the existence of three categories of rare mutations. Shown are transformation induced by a hot spot mutant (H1047R) and the three rare mutants representative of different oncogenic activity: strong (N345K), intermediate (T1025S), and weak (R38H). The H701P mutant has no detectable phenotype, and the wild-type p110α is also nontransforming.

Fig. 3.

Western blots comparing the phosphorylation levels of Akt and S6K that are induced in CEF by mutant and wild-type p110α. Hot spot mutants are marked with ▿, and strongly transforming rare mutants are marked with ◆. ∗ marks phospho p70S6K, whereas the upper band in this doublet marks phospho p85S6K. Phosphorylation of Akt and S6K by the mutant H1047Y was identical to that induced by mutant H1047L.

Fig. 4.

Random mutations without phenotype. (A) Five constructs with mutations in arbitrary sites of p110α (P217K, G912R, D1016G, E52K, and E116K) fail to induce oncogenic transformation. The mutant proteins were expressed with the RCAS vector in primary CEF. Cells were overlaid with nutrient agar on the day after transfection and fixed and stained with crystal violet on day 14. RCAS and wild-type p110α are shown as negative controls of transformation, E545K as positive control. (B) Constructs with mutations in arbitrary sites of p110α (P217K, D1017G, E52K, and E116K) fail to induce enhanced phosphorylation of Akt in the absence of growth factors.

Fig. 5.

Ribbon and molecular surface representation of p110α, showing the locations of gain-of-function mutations on the homology model. The domains of p110α are colored as follows: C2 domain, blue; helical domain, green; N-terminal region of the kinase domain, red; C-terminal region of the kinase domain, yellow; and linker region, white. The gain-of-function mutations are represented as red van der Waals (VDW) spheres. Nontransforming mutation sites that are not solvent-exposed are indicated as blue VDW spheres. The magenta VDW spheres mark nontransforming mutation sites that are solvent-exposed. The activation loop (red) and catalytic loop (green) are also labeled.

Fig. 6.

Ribbon diagram focused on the catalytic domain of p110α mutant residues. Labeling and color code are as in Fig. 5. The model illustrates the position of H1047R hotspot mutation in proximity to the hinge region of the activation loop and suggests that this mutation could affect both the position and the mobility of the activation loop.

Fig. 7.

Mutations targeted to the p110 surface. (A) Mutations targeted to the surface of p110α and involving an increase in positive charge are weakly oncogenic (E579K and D1046K). RCAS and wild-type p110α are shown as negative controls, H1047R as a positive control. These mutations are also depicted in Figs. 5 and 6 as magenta residues. (B) The targeted engineered mutants illustrated in Figs. 5 and 6 do not induce enhanced phosphorylation of Akt.