Activation of the phosphatidylinositol 3-kinase (PI3K)-AKT/protein kinase B signaling pathway has been associated with multiple human cancers. Recently we showed that AKT is activated in both the thyroid and metastatic lesions of a mouse model of follicular thyroid carcinoma [thyroid hormone beta receptor (TRbeta)(PV/PV) mice]. This TRbeta(PV/PV) mouse harbors a knock-in mutant TRbeta gene (TRbetaPV mutant) that spontaneously develops thyroid cancer and distant metastasis similar to human follicular thyroid cancer. Here we show that in thyroid tumors, PV mutant bound significantly more to the PI3K-regulatory subunit p85alpha, resulting in a greater increase in the kinase activity than did TRbeta1 in wild-type mice. By GST pull-down assays, the ligand-binding domain of TR was identified as the interaction site with p85alpha. By confocal fluorescence microscopy, p85alpha was shown to colocalize with TRbeta1 or PV mainly in the nuclear compartment of cultured tumor cells from TRbeta(PV/PV) mice, but cytoplasmic p85alpha/PV or p85alpha/TRbeta1 complexes were also detectable. Further biochemical analysis revealed that the activation of the PI3K-AKT-mammalian target of the rapamycin-p70(S6K) pathway was observed in both the cytoplasmic and nuclear compartments, whereas the activation of the PI3K-integrin-linked kinase-matrix metalloproteinase 2 pathway was detected mainly in the extranuclear compartments. These results suggest that PV, via the activation of p85alpha, could act to affect PI3K downstream signaling in both the nuclear and extranuclear compartments, thereby contributing to thyroid carcinogenesis. Importantly, the present study unveils a mechanism by which a mutant TR acts to activate PI3K activity via protein-protein interactions.