Helical domain and kinase domain mutations in p110α of phosphatidylinositol 3-kinase induce gain of function by different mechanisms

Abstract

The phosphatidylinositol 3-kinase (PI3K) signaling pathway is upregulated in cancer. PIK3CA, the gene coding for the catalytic subunit p110␣ of PI3K, is mutated in Ϸ30% 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 p110␣. 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 p110␣ molecule. This synergism also supports the conclusion that the helical and kinase domain mutations operate by two different and independent mechanisms.cancer ͉ molecular mechanisms ͉ p85 ͉ RAS ͉ AKT P hosphatidylinositol 3-kinases (PI3Ks) phosphorylate phosphatidylinositols at the 3Ј position of the inositol ring, generating second messengers that control cellular activities and properties, including proliferation, survival, motility, and cell shape. Mutations that block PI3K function disrupt these processes (1-12). PI3Ks form a family that is divided into three classes, differing in structure, substrate preference, tissue distribution, mechanism of activation, and, ultimately, function (12-16). For the regulation of cell proliferation and in tumorigenesis, the most important PI3K proteins are those of class IA, notably the catalytic subunit p110␣ and its associated regulatory subunits (p85␣, p55␣, p50␣, p85␤, and p55␥).p110␣ contains an N-terminal p85-binding domain (p85BD), a Ras-binding domain (RBD), a protein-kinase-C homology-2 (C2) domain, a helical domain, and a C-terminal kinase domain (17). p110␣ is constitutively associated with regulatory subunits, of which p85␣ is the best studied (herein referred to as p85). p110␣ and p85 exist in the cell as a heterodimeric complex. In quiescent cells, p85 stabilizes p110␣ and inactivates PI3K activity (18). Upon growth factor stimulation, receptor tyrosine kinases (RTKs) undergo autophosphorylation, creating binding sites for Src homology 2 (SH2) domain-containing proteins. The SH2 domains of p8...

Concomitant PI3K–AKT and p53 alterations in endometrial carcinomas are associated with poor prognosis

“…In contrast, mutations in the helical domain (exon 9) cluster on an exposed surface patch of the protein and may change its ability to interact with other regulatory proteins, which may be different for each tissue. [31][32][33] As expected, analyses of the PTEN mutations revealed that these alterations are more common in pure endometrioid adenocarcinomas compared with that in non-endometrioid adenocarcinomas and mixed tumors. Different studies of colorectal and breast carcinomas have claimed that the PIK3CA and PTEN mutations are mutually exclusive and suggest that carcinogenic signaling through this pathway occurs either through the activation of PIK3CA or inactivation of PTEN.…”

The role of PTEN signaling perturbations in cancer and in targeted therapy

“…Conversely, the kinase domain mutant H1047R acts independently of RAS-GTP binding, but requires p85 for activation. The helical and kinase domain double mutants synergistically activate downstream signaling events and increase cellular transformation, further demonstrating that the individual (helical and kinase domain) mutants operate by different mechanisms (Zhao and Vogt, 2008). All told, the recurrent genetic activation of PI3K is another way in which the PTEN pathway can be disturbed in cancer.…”

“…Furthermore, the helical and kinase domain mutants both have increased kinase activity, but are activated by different mechanisms. Studies performed in chicken embryonic fibroblasts demonstrate that the helical domain mutations (E542K and E545K) require interaction with RAS-GTP, but not with p85 (Zhao and Vogt, 2008). In essence, the helical mutants appear to position the kinase in a way such that it is no longer subject to the negative regulation by p85, but the catalytic domain still requires Ras for activation.…”

“…All told, the recurrent genetic activation of PI3K is another way in which the PTEN pathway can be disturbed in cancer. Interestingly, studies show that these mutants are activated by different mechanisms and appear to alter only subsets of AKT signaling Zhao and Vogt, 2008).…”

Recombinant expression of different mutant K-ras gene in pancreatic cancer Bxpc-3 cells and its effects on chemotherapy sensitivity

“…Considering its important role in cell survival and proliferation, K-ras has been extensively studied in recent years. Previous studies revealed that K-ras gene mutations cause excessive activation of K-RAS/ RAF/MEK/ERK and PI3K/AKT/mTOR signaling pathways [4][5][6][7][8][9].…”