Kaiso phosphorylation at threonine 606 leads to its accumulation in the cytoplasm, reducing its transcriptional repression of the tumour suppressor CDH1

Abstract

It is well known that the Kaiso protein (encoded by the ZBTB33 gene) is a transcription factor, and Kaiso-P120ctn [P120 catenin (CTNND1)] interaction increases the translocation of Kaiso from the nucleus into the cytoplasm. However, the regulatory mechanisms of Kaiso compartmentalisation are far from clear. Here, we reported that RAC-alpha serine/threonine-protein kinase (AKT1) could phosphorylate threonine residue 606 (T606) within the RSSTIP motif of Kaiso in the cytoplasm. The T606-phosphorylated Kaiso (pT606-Kaiso) could directly bind to 14-3-3 family proteins, and depletion of T606 phosphorylation by T606A mutation abolished most of the Kaiso-14-3-3 binding. In addition, the Kaiso-P120ctn interaction was essential for pT606-Kaiso accumulation in the cytoplasm. Notably, enforced stratifin (14-3-3σ; SFN) overexpression could increase pT606-Kaiso accumulation in the cytoplasm and de-repress the transcription of Kaiso target gene cadherin 1 (CDH1), which is a tumour suppressor. Decreased amounts of both pT606-Kaiso and CDH1 proteins were frequently observed in human gastric cancer tissues compared to paired normal controls. The mRNA levels of 14-3-3σ and Kaiso target gene CDH1 showed highly significant positive correlations in both human normal tissues and cancer cell lines by bioinformatics analyses. Furthermore, Kaiso T606A mutant (unable to be phosphorylated) significantly increased the migration and invasion of cancer cells in vitro and promoted the growth of these cells in vivo. In conclusion, Kaiso could be phosphorylated at T606 by AKT1 and pT606-Kaiso accumulates in the cytoplasm through binding to 14-3-3/P120ctn, which de-represses the Kaiso target gene CDH1 in normal tissues. Decreased Kaiso phosphorylation might contribute to the development of gastrointestinal cancer. The status of Kaiso phosphorylation is a determinant factor for the role of Kaiso in the development of cancer.

Keywords: 14-3-3 proteins; AKT1; Kaiso; P120ctn; cancer; phosphorylation; transcription factor.

Fig. 1

Different phosphorylation states of Kaiso protein in the cytoplasm and nucleus of cells in vitro and in vivo. (A) The phosphorylation statuses of GFP‐Kaiso stably transfected MGC803 cells and the corresponding xenograft tissues in the Phos‐tag SDS/PAGE analysis. Data are representative of at least two independent experiments. (B) The phosphorylation statuses of endogenous cytoplasmic and nuclear Kaiso in MGC803 cells were validated using anti‐phosphoSer/Thr/Tyr universal antibody. Data are representative of at least two independent experiments.

Fig. 2

AKT1 increases the phosphorylation of Kaiso at T606. (A) A conservative RSXTXP motif within Kaiso of human and other species. (B) After starvation overnight, treatments of insulin (10 ng·mL⁻¹), IL‐6 (1 ng·mL⁻¹) and foetal bovine serum (FBS, 10% v/v) for 15 min increased the pT606‐Kaiso in MGC803 cells. The experiment was performed three times. (C) Effects of AKT inhibitor MK2206 treatment (10 μmol/mL) for 30 min blocked the promotion of insulin induced pT606‐Kaiso and Kaiso phosphorylation as AKT substrate (pAKT‐sub) in MGC803 cells. The experiment was performed two times. (D) AKT1 overexpression at different doses increased the amount of pT606‐Kaiso and phosphorylated AKT substrate (pAKT‐sub) in Kaiso complexes immunoprecipitated by Kaiso antibody in MGC803 cells. The experiment was performed two times. (E) The activity comparison of three kinase candidates to phosphorylate Kaiso at T606 in MGC803 and BGC823 cells. The experiment was performed three times. (F) The T606‐phosphorylation status of endogenous Kaiso in MGC803 and BGC823 with AKT1 overexpression after starvation overnight. The experiment was performed three times.

Fig. 3

Subcellular localisation of pT606‐Kaiso in human cancer cell lines and gastric mucosal tissues. (A) Subcellular location of endogenous pT606‐Kaiso and total Kaiso in cytoplasmic and nuclear proteins (with and without de‐phosphorylation treatment by CIAP for 30 min) extracted from MGC803, BGC823, SGC7901 and RKO cell lines in western blotting. (B) Locations of pT606‐Kaiso and total Kaiso in the cytoplasm and nucleus in confocal microscopy analysis. (C) Locations of pT606‐Kaiso and total Kaiso in human gastric tissues in the confocal microscopy analysis. (D) Locations of pT606‐Kaiso and total Kaiso in representative gastric carcinoma tissues in IHC analysis. Scale bar: 20 or 5 μm in B, 20 μm in C, 25 μm in D. Gastric carcinoma and paired surgical margin samples from four patients were detected.

Fig. 4

Kaiso interacted with 14‐3‐3 family members and accumulated in cytoplasm. (A) After the plasmids of 14‐3‐3 family members and GST‐Kaiso were co‐transfected into MGC803 cells, GST‐Kaiso pulled down various isoforms of 14‐3‐3 family, especially 14‐3‐3σ. The experiment was performed three times. (B) Endogenous Kaiso immunoprecipitated several 14‐3‐3 family members in MGC803 cell in Co‐IP analysis. (C) 14‐3‐3σ protein immunoprecipitated endogenous Kaiso in MGC803 cell in Co‐IP analysis. Co‐IP assay for the interaction of Kaiso and 14‐3‐3σ was performed three times. (D) The subcellular location of endogenous Kaiso in MGC803 cells with or without 14‐3‐3γ or 14‐3‐3σ overexpression by indirect immunofluorescence staining assay. As labelled, scale bar for IF = 5 μm. The experiment was performed three times. (E) Proportion of Kaiso in the nucleus and in cytoplasm of MGC803 cells with and without 14‐3‐3γ or 14‐3‐3σ overexpression. N = 2274/1129/476 in the group of Ctrl/14‐3‐3γ/14‐3‐3σ. P‐values in chi‐square tests are displayed. (F) Western blot for detecting the amounts of endogenous Kaiso in cytoplasm and nucleus protein in MGC803 cells with 14‐3‐3σ overexpression. The experiment was performed three times. (G) Comparison of the levels of GFP‐Kaiso (wild‐type) or T606A mutant in the cytoplasm and nucleus in MGC803 cells with and without 14‐3‐3σ overexpression. Red and Green arrows: makeable increased and decreased Kaiso in the cytoplasm and nucleus by 14‐3‐3σ. The experiment was performed three times.

Fig. 5

14‐3‐3σ promotes the interaction of Kaiso and P120ctn in the cytoplasm. (A) More Kaiso‐P120 complex was immunoprecipitated by Kaiso antibody in MGC803 cells with 14‐3‐3σ overexpression in Co‐IP assay. Total protein levels of P120ctn and 14‐3‐3σ in the lysate of MGC803 cells with 14‐3‐3σ overexpression were displayed in the right side. The experiment was performed three times. (B) Alterations of interactions between Kaiso and P120ctn proteins in the cytoplasm and nucleus of these cells with 14‐3‐3σ overexpression in the Co‐IP assay using Kaiso antibody. The experiment was performed three times. (C) The subcellular locations of Kaiso, P120ctn and mCherry‐14‐3‐3σ in MGC803 cells. Scale bar: 5 μm. The experiment was performed two times. (D) Western blotting images for detecting effect of P120ctn knockdown and 14‐3‐3σ overexpression on distribution of Kaiso in the cytoplasm and nucleus of MGC803 cells. The experiment was performed two times. (E) The levels of total P120ctn and Kaiso proteins in the lysate of MGC803 cells with 14‐3‐3σ overexpression and siRNAs knockdown of P120ctn expression. The experiment was performed three times. Red arrow: P120ctn or Kaiso in the cytoplasm with and without makeable change.

Fig. 6

Effects of 14‐3‐3σ on inhibition of Kaiso's target gene CDH1 expression. (A) The Pearson correlation coefficient between mRNA levels of 14‐3‐3 family members and Kaiso target genes in public RNA‐seq datasets from Cancer Cell Line Encyclopaedia (CCLE, 1156 cancer cell lines) and Genotype‐Tissue Expression (GTEx, 11 688 human normal tissues). (B) The level of CDH1 in SGC7901 and MKN45 cells with or without wildtype Kaiso and 14‐3‐3σ overexpression. The experiment was performed three times. (C) The level of the CDH1 mRNA in SGC7901 and MKN45 cells with or without wildtype Kaiso and 14‐3‐3σ overexpression (each experiment was repeated at least three times). Data were presented as the mean ± SD (n = 3). P‐values in Student's t‐test are labelled (**P < 0.01, ns: nonsense). (D) the amounts of pT606‐Kaiso, total Kaiso, 14‐3‐3σ and CDH1 proteins in gastric carcinoma (T) and the paired normal tissues (N) from 12 patients by western blotting. (E) Correlation between ratios of pT606‐Kaiso to total Kaiso and CDH1 to GAPDH proteins based on the density of these proteins in western blot. Spearman's rho coexpression coefficient (r) and P‐value are shown (n = 24).

Fig. 7

Effects of wild‐type Kaiso and its T606A mutant on gastric cancer cell migration and invasion in vitro and growth in vivo. (A) The percentage of open wounds was calculated to determine the migration of gastric cancer (GC) SGC7901 and MKN45 cells overexpressing Kaiso‐WT or its T606A mutant. The distance values are presented as mean ± SD (n = 4). a/b/c: P < 0.05 in Student's t‐test between Ctrl and Kaiso‐WT or Kaiso‐T606A and between Kaiso‐WT and Kaiso‐T606A; The results of western blot were also inserted to illustrate the status of Kaiso overexpression and CDH1 protein level; (B and C) The results of transwell assays to show the migration and invasion of GC cells overexpressing Kaiso‐WT or Kaiso‐T606A mutant respectively. Number of cells are presented as mean ± SD (n = 4); (D) Tumours derived from SGC7901 cells transfected with Kaiso‐WT and its T606A mutant or empty vector control. */**/***: P < 0.05/0.01/0.001 and ns: nonsense in Student's t‐test.