Proteomic analysis identifies that 14-3-3zeta interacts with beta-catenin and facilitates its activation by Akt

Abstract

beta-Catenin is a central effector of Wnt signaling in embryonic and stem cell development and in tumorigenesis. Here, through a mass spectrometric analysis of a beta-catenin protein complex, we identified 12 proteins as putative beta-catenin interactors. We show that one of them, 14-3-3zeta, enhances beta-catenin-dependent transcription by maintaining a high level of beta-catenin protein in the cytoplasm. More importantly, 14-3-3zeta facilitates activation of beta-catenin by the survival kinase Akt and colocalizes with activated Akt in intestinal stem cells. We propose that Akt phosphorylates beta-catenin, which results in 14-3-3zeta binding and stabilization of beta-catenin, and these interactions may be involved in stem cell development.

Fig. 1.

14-3-3ζ interacts with β-catenin in vivo. 293T cells were transfected with myc-tagged β-catenin plasmid. Twenty-four hours later, the whole-cell lysates were immunoprecipitated (IP) with either anti-myc (a) or anti-14-3-3ζ (b) antibodies and immunoblotted with indicated antibodies. (c) 293T cells were cotransfected with myc-β-catenin and FLAG-14-3-3ζ. The whole-cell lysates were immunoprecipitated (IP) with anti-myc antibody and blotted with anti-FLAG antibody.

Fig. 2.

14-3-3ζ increases β-catenin-dependent transcription and steady-state level of β-catenin in the cytoplasm. Three different cell lines, CHO (a), 293T (b), and DLD1 colon adenocarcinoma (c) cells, were transfected with pTOPFLASH and pFOPFLASH reporter and increasing amounts of 14-3-3ζ in the absence or presence of β-catenin. The Renilla luciferase reporter pRL-TK was cotransfected as internal control for transfection efficiency. The experiments were repeated in duplicate at least three times. Shown are means ± SD from one representative experiment. Whole-cell lysates from CHO cells transfected with β-catenin and 14-3-3ζ (d) or fractionated cytosolic and nuclear extract (e) were immunoblotted with β-catenin antibody. (f) Transfected CHO cells were fixed in methanol and stained with FITC-conjugated anti-β-catenin antibody.

Fig. 3.

14-3-3ζ enhances activation of β-catenin by Akt. (a) Whole-cell lysates from transfected CHO cells were immunoblotted with antibodies that specifically recognize either Ser-33/37- or Ser-45-phosphorylated β-catenin. (b) HeLa cells were transfected with14-3-3ζ-specific small interference RNA (siRNA), and whole-cell lysates were blotted with the indicated antibodies. (c) CHO cells were transfected with pTOPFLASH or pFOPFLASH reporter in the presence of constitutive Akt (AKT-ca) or the kinase domain mutated Akt (Akt-km) together with β-catenin and 14-3-3ζ. (d) CHO cell transfectants were fractionated into either nuclear or cytosolic fractions and blotted with β-catenin and 14-3-3ζ antibodies. (e) Purified human β-catenin protein was incubated with increasing amounts of Akt kinase in the presence of radioactive ATP. The resolved protein gel was exposed to x-ray film (Upper) after staining with Coomassie blue (Lower).

Fig. 4.

Colocalization of phophorylated Akt with 14-3-3ζ in ISCs. (a) The crypt-villus of intestine from BMP receptor type 1A knockout mouse were stained with antiphosphorylated Akt-specific (red) (Upper Left) or anti-14-3-3ζ-specific (green) (Upper Right) antibodies. Counterstaining with 4′,6-diamidino-2-phenylindole reveals the cell nuclei (blue). (b) A model for the stabilization mechanism of β-catenin by Akt and 14-3-3ζ.