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IFNγ-induced suppression of β-catenin signaling: evidence for roles of Akt and 14.3.3ζ.

Nava P, Kamekura R, Quirós M, Medina-Contreras O, Hamilton RW, Kolegraff KN, Koch S, Candelario A, Romo-Parra H, Laur O, Hilgarth RS, Denning TL, Parkos CA, Nusrat A - Mol. Biol. Cell (2014)

Bottom Line: Akt1 served as a bimodal switch that promotes or inhibits β-catenin transactivation in response to IFNγ stimulation.IFNγ initially promotes β-catenin transactivation through Akt-dependent C-terminal phosphorylation of β-catenin to promote its association with 14.3.3ζ.These results outline a dual function of Akt1 that suppresses IEC proliferation during intestinal inflammation.

View Article: PubMed Central - PubMed

Affiliation: Epithelial Pathobiology and Mucosal Inflammation Research Unit, Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, GA 30322 Departamento de Fisiología, Biofísica y Neurociencias, Centro de Investigación y de Estudios Avanzados, Instituto Politécnico Nacional, 07360 Mexico City, Mexico.

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Phosphorylation of 14.3.3ζ at serine 58 requires up-regulation of Akt1 protein levels. (A) The effect of increasing concentrations of Akt1 on phosphorylation of 14.3.3ζ was analyzed in cell lysates of SW480 cells. Akt1, 14.3.3ζ, p14.3.3ζ, and pS6 Rib were analyzed by Western blot. Actin was used as a loading control. (B) The expression of Akt1 and Akt2 after IFNγ treatment was analyzed by Western blotting lysates of colonic tissue of mice injected with IFNγ. Western blots were performed using antibodies against pan-Akt, Akt1, Akt2, pAkt308, and pβ-cat552. Actin was used as loading control. (C) The role of Akt1 expression in β-catenin transactivation was evaluated in SW480 cells transfected with 0.01–2 μg/ml vector expressing Akt1. β-Catenin transactivation was evaluated by TOPflash assay. TOP vs. FOP and TOP/FOP activity are plotted. (D) Effects of Akt1 subcellular distribution on β-catenin–mediated transcriptional activity was evaluated by TOPflash assay in SW480 cells. Cells were transfected with 0.75 μg/ml Akt1-myr, Akt1, or Akt-NLS constructs in serum-free medium. (E) The expression of p14.3.3ζ was analyzed in colonic cell lysates of IFNγ-injected animals that were pretreated with dimethyl sulfoxide or Akt inhibitor VIII. Densitometric values were normalized to actin and are presented as a graph (**p < 0.0001).
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Figure 4: Phosphorylation of 14.3.3ζ at serine 58 requires up-regulation of Akt1 protein levels. (A) The effect of increasing concentrations of Akt1 on phosphorylation of 14.3.3ζ was analyzed in cell lysates of SW480 cells. Akt1, 14.3.3ζ, p14.3.3ζ, and pS6 Rib were analyzed by Western blot. Actin was used as a loading control. (B) The expression of Akt1 and Akt2 after IFNγ treatment was analyzed by Western blotting lysates of colonic tissue of mice injected with IFNγ. Western blots were performed using antibodies against pan-Akt, Akt1, Akt2, pAkt308, and pβ-cat552. Actin was used as loading control. (C) The role of Akt1 expression in β-catenin transactivation was evaluated in SW480 cells transfected with 0.01–2 μg/ml vector expressing Akt1. β-Catenin transactivation was evaluated by TOPflash assay. TOP vs. FOP and TOP/FOP activity are plotted. (D) Effects of Akt1 subcellular distribution on β-catenin–mediated transcriptional activity was evaluated by TOPflash assay in SW480 cells. Cells were transfected with 0.75 μg/ml Akt1-myr, Akt1, or Akt-NLS constructs in serum-free medium. (E) The expression of p14.3.3ζ was analyzed in colonic cell lysates of IFNγ-injected animals that were pretreated with dimethyl sulfoxide or Akt inhibitor VIII. Densitometric values were normalized to actin and are presented as a graph (**p < 0.0001).

Mentions: Experiments were performed to identify the kinase that mediates phosphorylation of 14.3.3ζ. Because Akt1 has been shown to phosphorylate 14.3.3ζ at serine 58 (Powell et al., 2002), we analyzed the role of Akt1 in generation of the p14.3.3ζ. Increasing concentrations of Akt1 were transfected in SW480 cells, and the expression of p14.3.3ζ was quantified. As shown in Figure 4A, whereas low concentrations of Akt1 did not influence p14.3.3ζ, expression of high amounts of the kinase increased levels of p14.3.3ζ. Of interest, we also observed that total levels of 14.3.3ζ and pβ-cat552 were increased when small amounts of Akt1 were expressed, and high levels of Akt1 diminished 14.3.3ζ and pβ-cat552 protein levels (Figure 4A), suggesting a complex relationship between Akt1 protein levels and phosphorylation/stabilization of 14.3.3ζ and pβ-cat552. Of importance, no changes were observed in the phosphorylation of an unrelated protein pS6 rib after overexpression of Akt1 (Figure 4A). To corroborate these findings, we analyzed the levels of Akt protein in the mucosa of IFNγ-treated mice. As shown in Figure 4B, IFNγ treatment resulted in increased Akt1 protein levels in lysates from the intestinal mucosa of mice exposed to IFNγ for 1–3 h. In contrast, the same IFNγ treatment resulted in decreased Akt2 protein levels. Immunofluorescence staining analyses of intestinal mucosa revealed that Akt1 was enriched in the cytosol of epithelial cells in the base of nonproliferative crypt IEC in control and IFNγ-treated conditions, suggesting that increased Akt1 may contribute to inhibition of intestinal epithelial cell proliferation (Supplemental Figure S7).


IFNγ-induced suppression of β-catenin signaling: evidence for roles of Akt and 14.3.3ζ.

Nava P, Kamekura R, Quirós M, Medina-Contreras O, Hamilton RW, Kolegraff KN, Koch S, Candelario A, Romo-Parra H, Laur O, Hilgarth RS, Denning TL, Parkos CA, Nusrat A - Mol. Biol. Cell (2014)

Phosphorylation of 14.3.3ζ at serine 58 requires up-regulation of Akt1 protein levels. (A) The effect of increasing concentrations of Akt1 on phosphorylation of 14.3.3ζ was analyzed in cell lysates of SW480 cells. Akt1, 14.3.3ζ, p14.3.3ζ, and pS6 Rib were analyzed by Western blot. Actin was used as a loading control. (B) The expression of Akt1 and Akt2 after IFNγ treatment was analyzed by Western blotting lysates of colonic tissue of mice injected with IFNγ. Western blots were performed using antibodies against pan-Akt, Akt1, Akt2, pAkt308, and pβ-cat552. Actin was used as loading control. (C) The role of Akt1 expression in β-catenin transactivation was evaluated in SW480 cells transfected with 0.01–2 μg/ml vector expressing Akt1. β-Catenin transactivation was evaluated by TOPflash assay. TOP vs. FOP and TOP/FOP activity are plotted. (D) Effects of Akt1 subcellular distribution on β-catenin–mediated transcriptional activity was evaluated by TOPflash assay in SW480 cells. Cells were transfected with 0.75 μg/ml Akt1-myr, Akt1, or Akt-NLS constructs in serum-free medium. (E) The expression of p14.3.3ζ was analyzed in colonic cell lysates of IFNγ-injected animals that were pretreated with dimethyl sulfoxide or Akt inhibitor VIII. Densitometric values were normalized to actin and are presented as a graph (**p < 0.0001).
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Related In: Results  -  Collection

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Figure 4: Phosphorylation of 14.3.3ζ at serine 58 requires up-regulation of Akt1 protein levels. (A) The effect of increasing concentrations of Akt1 on phosphorylation of 14.3.3ζ was analyzed in cell lysates of SW480 cells. Akt1, 14.3.3ζ, p14.3.3ζ, and pS6 Rib were analyzed by Western blot. Actin was used as a loading control. (B) The expression of Akt1 and Akt2 after IFNγ treatment was analyzed by Western blotting lysates of colonic tissue of mice injected with IFNγ. Western blots were performed using antibodies against pan-Akt, Akt1, Akt2, pAkt308, and pβ-cat552. Actin was used as loading control. (C) The role of Akt1 expression in β-catenin transactivation was evaluated in SW480 cells transfected with 0.01–2 μg/ml vector expressing Akt1. β-Catenin transactivation was evaluated by TOPflash assay. TOP vs. FOP and TOP/FOP activity are plotted. (D) Effects of Akt1 subcellular distribution on β-catenin–mediated transcriptional activity was evaluated by TOPflash assay in SW480 cells. Cells were transfected with 0.75 μg/ml Akt1-myr, Akt1, or Akt-NLS constructs in serum-free medium. (E) The expression of p14.3.3ζ was analyzed in colonic cell lysates of IFNγ-injected animals that were pretreated with dimethyl sulfoxide or Akt inhibitor VIII. Densitometric values were normalized to actin and are presented as a graph (**p < 0.0001).
Mentions: Experiments were performed to identify the kinase that mediates phosphorylation of 14.3.3ζ. Because Akt1 has been shown to phosphorylate 14.3.3ζ at serine 58 (Powell et al., 2002), we analyzed the role of Akt1 in generation of the p14.3.3ζ. Increasing concentrations of Akt1 were transfected in SW480 cells, and the expression of p14.3.3ζ was quantified. As shown in Figure 4A, whereas low concentrations of Akt1 did not influence p14.3.3ζ, expression of high amounts of the kinase increased levels of p14.3.3ζ. Of interest, we also observed that total levels of 14.3.3ζ and pβ-cat552 were increased when small amounts of Akt1 were expressed, and high levels of Akt1 diminished 14.3.3ζ and pβ-cat552 protein levels (Figure 4A), suggesting a complex relationship between Akt1 protein levels and phosphorylation/stabilization of 14.3.3ζ and pβ-cat552. Of importance, no changes were observed in the phosphorylation of an unrelated protein pS6 rib after overexpression of Akt1 (Figure 4A). To corroborate these findings, we analyzed the levels of Akt protein in the mucosa of IFNγ-treated mice. As shown in Figure 4B, IFNγ treatment resulted in increased Akt1 protein levels in lysates from the intestinal mucosa of mice exposed to IFNγ for 1–3 h. In contrast, the same IFNγ treatment resulted in decreased Akt2 protein levels. Immunofluorescence staining analyses of intestinal mucosa revealed that Akt1 was enriched in the cytosol of epithelial cells in the base of nonproliferative crypt IEC in control and IFNγ-treated conditions, suggesting that increased Akt1 may contribute to inhibition of intestinal epithelial cell proliferation (Supplemental Figure S7).

Bottom Line: Akt1 served as a bimodal switch that promotes or inhibits β-catenin transactivation in response to IFNγ stimulation.IFNγ initially promotes β-catenin transactivation through Akt-dependent C-terminal phosphorylation of β-catenin to promote its association with 14.3.3ζ.These results outline a dual function of Akt1 that suppresses IEC proliferation during intestinal inflammation.

View Article: PubMed Central - PubMed

Affiliation: Epithelial Pathobiology and Mucosal Inflammation Research Unit, Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, GA 30322 Departamento de Fisiología, Biofísica y Neurociencias, Centro de Investigación y de Estudios Avanzados, Instituto Politécnico Nacional, 07360 Mexico City, Mexico.

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Related in: MedlinePlus