Activation of phosphatidylinositol 3-kinase signaling by a mutant thyroid hormone beta receptor - PubMed (original) (raw)

Activation of phosphatidylinositol 3-kinase signaling by a mutant thyroid hormone beta receptor

Fumihiko Furuya et al. Proc Natl Acad Sci U S A. 2006.

Abstract

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.

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Conflict of interest statement

Conflict of interest statement: No conflicts declared.

Figures

Fig. 1.

Activation of PI3K activity in the thyroid extracts ofTRβPV/PV mice. (A) Increasing concentrations as marked of total thyroid extracts from wild-type mice (solid squares) and TRβPV/PV mice (solid circles) were immunoprecipitated with anti-p85α antibody. PI3K activity of precipitates from each concentration was measured by ELISA, as described in Materials and Methods, and expressed as the relative production of PtdIns(3,4,5)P3 by each sample. (B) One hundred micrograms of proteins derived from the total thyroid extracts of wild-type mice (bars 1–3; three mice) or TRβPV/PV mice (bars 4–8) were immunoprecipitated with 5 μg of anti-TRβ1 (J52, bars 1 and 2, wild-type mice; bars 4 and 5, two TRβPV/PV mice), anti-PV (#302, bars 7 and 8, two mice) antibodies, or an irrelevant monoclonal antibody (MOPC) as control (bars 3 and 6, marked as C). PI3K activities in the immunoprecipitates were measured by ELISA, as described in Materials and Methods. (C) Three hundred, 500, or 1,000 μg of pooled protein lysates from thyroid extracts of six wild-type mice or three TRβPV/PV mice, respectively, was immunoprecipitated with J52 antibody and subjected to immunoblot analysis probed with anti-p85α antibody (catalog no. 06-195). Lane 1 shows Jurkat cell lysate (catalog no. 12-303; Upstate Biotechnology) as a positive control. (D Upper) The abundance of TRβ1 and PV receptor proteins in the thyroids of wild-type mice (lanes 1–3) and TRβPV/PV mice (lanes 4–8), respectively. The loading control (D Lower) used PDI.

Fig. 2.

p85α protein binds to PV more avidly than to TRβ1.(A) GST–p85α fusion protein was incubated with 2, 5, or 10 μl of 35S-labeled TRβ1 (lanes 2, 3, and 4, respectively) or PV (lanes 6, 7, and 8, respectively) prepared by in vitro translation/transcription. Lanes 1 and 5 were from the incubation of GST with 2 μl of TRβ1 and PV, respectively. Lanes 9 and 10 show the input of in vitro translation/transcription samples (0.2 μl). (B) The band intensity was scanned and quantified by using

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software. (C) Ten microliters of 35S-labeled TRβ1 (lanes 1–6) or PV (lanes 7–12) prepared by in vitro translation/transcription was incubated with increasing concentrations of bead-bound GST–p85α as described in Materials and Methods. (D) The band intensity was scanned and quantified by using

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. (E) Identification of the ligand-binding domain of TRβ1 as the interaction site with p85α. Five microliters of 35S-labeled full-length and TRβ1 proteins lacking the A/B domain (ΔA/B) or the A/B/C domains (ΔA/B/C) was synthesized (25) by in vitro transcription/translation and incubated with GST–p85α, as described in Materials and Methods. Lanes 1–3 were the input (0.5 μl of lysates). Lanes are as marked.

Fig. 3.

p85α interacts more avidly with PV than with TRβ1 in the nuclearcompartment. (A and B) The thyroid extracts of 12 wild-type mice or 3 TRβPV/PV mice were pooled and separated into nuclear or cytosolic fractions. The purity of each fraction was monitored by the respective markers, PARP for the nuclear fraction (A) and α-tubulin for the cytosolic fraction (B). (C) An equal amount of the nuclear or cytosolic fraction (100 μg of proteins) was immunoprecipitated with 5 μg of J52 followed by Western blot analysis using anti-p85α antibody. Lanes are as marked. Lanes 5–8 show the corresponding input of the p85α protein.

Fig. 4.

p85α is colocalized with TRβ1 or PV in both the nuclear andextranuclear compartments. The primary cultured cells derived from the thyroids of wild-type mice (I) or TRβPV/PV mice (II) were fixed and incubated with anti-TRβ1 (J52) and anti-p85 antibody (SC423) followed by secondary antibody conjugated with Alexa Fluor 488 (green) or rhodamine (red), respectively, as described in Materials and Methods.

Fig. 5.

Activation of the PI3K–AKT–p70S6K and ILK–MMP2 pathwaysin the thyroid of TRβPV/PV mice. Pooled extracts from thyroids of 12 wild-type mice or 3 TRβPV/PV mice were separated into nuclear and cytosolic fractions, as described in Materials and Methods. Western blot analysis was carried out as described in Materials and Methods to determine cytosolic and nuclear abundance of the following proteins: p85α (Aa), AKT (Ab), p-AKT(S473) (Ac), mTOR (Ad), p-mTOR (Ae), p70S6K (Af), and p-p70S6K (Ag). (B) The expression of PARP (a) or α-tubulin (b) was used for monitoring the quality of nuclear and cytosolic fractions as well as for loading controls for the Western blot analysis shown in A and C. (C) Increased protein abundance of ILK (a) and MMP2 (b) in the cytosolic fraction of thyroid tumors of TRβPV/PV mice.

Fig. 6.

Activation of PI3K signaling by PV. The physical interaction of PV with p85α results in the activation of two PI3K downstream pathways: the AKT–ILK–MMP pathway and the AKT–mTOR–p70S6K pathway. The activation of the former leads to the degradation of the extracellular matrix involved in cell invasion and metastasis, and the activation of the latter results in increased cell proliferation and suppression of apoptosis. The bracket indicates that PV did not have an effect on the expression of PTEN, suggesting that PV-induced activation of PI3K is not mediated by the repression of PTEN.

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Activation of phosphatidylinositol 3-kinase signaling by a mutant thyroid hormone beta receptor - PubMed (original) (raw)