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.