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, 282 (15), 11221-9

Phosphorylation of Beta-Catenin by AKT Promotes Beta-Catenin Transcriptional Activity

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Phosphorylation of Beta-Catenin by AKT Promotes Beta-Catenin Transcriptional Activity

Dexing Fang et al. J Biol Chem.

Abstract

Increased transcriptional activity of beta-catenin resulting from Wnt/Wingless-dependent or -independent signaling has been detected in many types of human cancer, but the underlying mechanism of Wnt-independent regulation is poorly understood. We have demonstrated that AKT, which is activated downstream from epidermal growth factor receptor signaling, phosphorylates beta-catenin at Ser552 in vitro and in vivo. AKT-mediated phosphorylation of beta-catenin causes its disassociation from cell-cell contacts and accumulation in both the cytosol and the nucleus and enhances its interaction with 14-3-3zeta via a binding motif containing Ser552. Phosphorylation of beta-catenin by AKT increases its transcriptional activity and promotes tumor cell invasion, indicating that AKT-dependent regulation of beta-catenin plays a critical role in tumor invasion and development.

Figures

Fig. 1
Fig. 1. AKT phosphorylates β-catenin at S552 in vitro and in vivo
A, β-catenin immunoprecipated from EGF-stimulated A431 cells was analyzed with mass spectrometry. Mass spectrometric analysis of a tryptic fragment R550–R565 indicates Ser-552 is phosphorylated. The detected b- and y- ions are indicated in red and blue, respectively. The m/z difference between y-14 and y-13 matched with phospho-Ser. BD, Immunoblotting analyses with the indicated antibodies. B, In vitro kinase assays were performed with purified active PKA (left panel), AKT1 (middle panel), or AKT2 (right panel), with purified bacterially expressed His-β-catenin, His-β-catenin S552A, or His-β-catenin S675A with or without AKT inhibitor. C, AKT1 DD or AKT2 DD was co-transfected with FLAG-tagged WT β-catenin, β-catenin S552A, or β-catenin S675A into 293T cells. Anti-FLAG antibody was used for immunoprecipitation. D, Serum-starved A431 cells were pretreated with the AKT inhibitor IV (10 μm) for 30 min before EGF treatment (100ng/ml). Anti-β-catenin antibody was used for immunoprecipitation.
Fig. 2
Fig. 2. β -catenin phosphorylated by AKT disassociates from cell contacts and translocates into the cytosol and nucleus
A, A431 cells transiently expressing HA-AKT1 DD or HA-AKT1 AAA were stained with an anti-β-catenin antibody (green), an anti-HA antibody (red), and Hoechst 33342 (blue). White color indicates overlap of green and blue colors. B, A431 cells stably expressing FLAG-tagged β-catenin, β-catenin S552A, or β-catenin S552D were stained with an anti-FLAG antibody (red) and Hoechst 33342 (blue). Pink color indicates overlap of red and blue colors. C, Nuclear, cytosolic, and membrane fractions of A431 cells stably expressing FLAG-tagged β-catenin, β-catenin S552A, or β-catenin S552D were processed for immunoblotting with anti-FLAG or anti-lamin B antibody (upper group panels). The data were quantified by scanning densitometry (lower panel). D, Immunoprecipitated FLAG-tagged WT β-catenin, β-catenin S552A, and β-catenin S552D, which were transiently expressed in 293T cells, were immnuoblotted with phospho-β-catenin (S33/S37) antibody. E, Pulse-chase analyses of transiently expressed FLAG-tagged β-catenin S552A and β-catenin S552D in 293T cells.
Fig. 3
Fig. 3. β -catenin phosphorylated by AKT increases its association with 14-3-3 ζ
Immunoblotting analyses with the indicated antibodies. A, FLAG-tagged WT β-catenin or pFLAG was co-transfected with or without AKT1 DD or AKT1 AAA into 293T cells, followed by immunoprecipitation with anti-14-3-3ζ antibody. B, FLAG-tagged WT β-catenin, β-catenin S552A, or β-catenin S552D was transfected into 293T cells, followed by immunoprecipitation with anti-14-3-3ζ antibody.
Fig. 4
Fig. 4. Phosphorylation of β-catenin by AKT increases transcriptional activity of β-catenin and enhanced tumor cell invasion
AD, The relative levels of luciferase activity were normalized to the levels of untreated cells and to the levels of luciferase activity of the Renilla control plasmid. Data represent the means ± SD of three independent experiments. A, TOP-FLASH or FOP-FLASH with WT β-catenin or β-catenin S552A mutant was co-transfected with or without constitutively active AKT1 DD into CHO cells. The luciferase activity was determined. B, TOP-FLASH with WT β-catenin or β-catenin S552A mutant was co-transfected with or without constitutively active AKT1 DD together with or without 14-3-3ζ into CHO cells. The luciferase activity was determined. C, TOP-FLASH or FOP-FLASH was co-transfected with EGFR with WT β-catenin or β-catenin S552A mutant into CHO cells. The luciferase activity was determined. D, TOP-FLASH or FOP-FLASH was co-transfected with WT β-catenin, β-catenin S552A, or β-catenin S552D mutant into CHO cells. The luciferase activity was determined. E, A pool of A431 cells stably transfected with a vector, FLAG-tagged WT β-catenin, β-catenin S552A, or β-catenin S552D were processed for immunoblotting with indicated antibodies (upper panel). The cells were plated at the top surface of the Matrigel. One day after plating, cells that migrated to the opposite side of the insert were stained with crystal violet. Representative microphotographs are shown (lower panel). F, The membranes with invaded cells were dissolved in 4% deoxycholic acid and read colorimetrically at 590 nm for quantification of invasion. Data represent the mean ± standard deviation of three independent experiments.

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