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Akt-signal integration is involved in the differentiation of embryonal carcinoma cells.

Chen B, Xue Z, Yang G, Shi B, Yang B, Yan Y, Wang X, Han D, Huang Y, Dong W - PLoS ONE (2013)

Bottom Line: Meanwhile, Akt phosphorylates Oct4 at threonine 228 and Klf4 at threonine 399, and accelerates their degradation.Moreover, PI3K/Akt signaling enhances the binding of SATB1 to Sox2, thereby probably impairing the formation of Oct4/Sox2 regulatory complexes.Accordingly, Akt-mediated phosphorylation is crucial for the capability of SATB1 to repress Nanog expression and to activate transcription of Bcl2 and Nestin genes.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China.

ABSTRACT
The mechanism by which Akt modulates stem cell homeostasis is still incompletely defined. Here we demonstrate that Akt phosphorylates special AT-rich sequences binding protein 1 (SATB1) at serine 47 and protects SATB1 from apoptotic cleavage. Meanwhile, Akt phosphorylates Oct4 at threonine 228 and Klf4 at threonine 399, and accelerates their degradation. Moreover, PI3K/Akt signaling enhances the binding of SATB1 to Sox2, thereby probably impairing the formation of Oct4/Sox2 regulatory complexes. During retinoic acid (RA)-induced differentiation of mouse F9 embryonal carcinoma cells (ECCs), the Akt activation profile as well as its substrate spectrum is strikingly correlated with the down-regulation of Oct4, Klf4 and Nanog, which suggests Akt activation is coupled to the onset of differentiation. Accordingly, Akt-mediated phosphorylation is crucial for the capability of SATB1 to repress Nanog expression and to activate transcription of Bcl2 and Nestin genes. Taken together, we conclude that Akt is involved in the differentiation of ECCs through coordinated phosphorylations of pluripotency/differentiation factors.

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Akt shields SATB1 from apoptotic cleavage.(A) Evenly distributed signals were observed in the majority of HEK293A cells transfected with GFP-SATB1, whereas punctate structures were detected in a small fraction of cells (white arrowhead). Akt (Myr) decreased the ratio of cells with dot-like signals. (B) In cells with dot-like structures, GFP-SATB1S47A colocalized with Cherry-PML, whereas GFP-SATB1S47D did not. (C) Expression of SATB1 was analyzed in MCF-7 cells stably integrated with empty vector, wild-type SATB1, SATB1S47A or SATB1S47D, respectively. (D) Expression of SATB1 and Akt activation were analyzed in stable SK-BR-3 cell lines. (E) SATB1 stability and Akt activation were documented in SK-BR-3 cells carrying wild-type SATB1 together with Akt (Myr) or Akt (DN). (F) HEK293A cells were transfected with Myc-SATB1, Myc-SATB1S47A or Myc-SATB1S47D, respectively, and treated with CPT for 0, 2, 4, 6, 8 or 10 h. The cell lysates were subjected to immunobloting with anti-SATB1, anti-phospho-SATB1 (S47) and anti-GAPDH. (G) Jurkat cells were treated with CPT as in (F). The cell lysates were subjected to immunoblotting with anti-SATB1, anti-phospho-SATB1 (S47) and anti-GAPDH.
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pone-0064877-g002: Akt shields SATB1 from apoptotic cleavage.(A) Evenly distributed signals were observed in the majority of HEK293A cells transfected with GFP-SATB1, whereas punctate structures were detected in a small fraction of cells (white arrowhead). Akt (Myr) decreased the ratio of cells with dot-like signals. (B) In cells with dot-like structures, GFP-SATB1S47A colocalized with Cherry-PML, whereas GFP-SATB1S47D did not. (C) Expression of SATB1 was analyzed in MCF-7 cells stably integrated with empty vector, wild-type SATB1, SATB1S47A or SATB1S47D, respectively. (D) Expression of SATB1 and Akt activation were analyzed in stable SK-BR-3 cell lines. (E) SATB1 stability and Akt activation were documented in SK-BR-3 cells carrying wild-type SATB1 together with Akt (Myr) or Akt (DN). (F) HEK293A cells were transfected with Myc-SATB1, Myc-SATB1S47A or Myc-SATB1S47D, respectively, and treated with CPT for 0, 2, 4, 6, 8 or 10 h. The cell lysates were subjected to immunobloting with anti-SATB1, anti-phospho-SATB1 (S47) and anti-GAPDH. (G) Jurkat cells were treated with CPT as in (F). The cell lysates were subjected to immunoblotting with anti-SATB1, anti-phospho-SATB1 (S47) and anti-GAPDH.

Mentions: A number of Akt substrates are relocated within cells upon phosphorylation by Akt, for instance, FOXO transcription factors [21] and p27Kip1 [22]. The finding that SATB1 is a nuclear protein and a novel Akt substrate as well leads us to test whether PI3K/Akt signaling is obligate to redirect SATB1 to cytoplasm or sub-nuclear structures. GFP-tagged SATB1 was localized in nucleus in transfected HEK293A cells with less than 10% cells having punctate structures (Figure 2A). Although co-expression of wild-type Akt marginally reduced the ratio of dot-like cells, increased doses of activated Akt gradually and markedly reduced the number of dot-like cells and the number of evenly distributed dots. Intriguingly, with the increased doses of activated Akt, the dots would aggregate to form one or two larger bodies. By contrast, dominant-negative Akt generated a pattern of more, much tinier dots (Figure 2A). This observation suggests that Akt could modulate intra-nuclear aggregation of SATB1.


Akt-signal integration is involved in the differentiation of embryonal carcinoma cells.

Chen B, Xue Z, Yang G, Shi B, Yang B, Yan Y, Wang X, Han D, Huang Y, Dong W - PLoS ONE (2013)

Akt shields SATB1 from apoptotic cleavage.(A) Evenly distributed signals were observed in the majority of HEK293A cells transfected with GFP-SATB1, whereas punctate structures were detected in a small fraction of cells (white arrowhead). Akt (Myr) decreased the ratio of cells with dot-like signals. (B) In cells with dot-like structures, GFP-SATB1S47A colocalized with Cherry-PML, whereas GFP-SATB1S47D did not. (C) Expression of SATB1 was analyzed in MCF-7 cells stably integrated with empty vector, wild-type SATB1, SATB1S47A or SATB1S47D, respectively. (D) Expression of SATB1 and Akt activation were analyzed in stable SK-BR-3 cell lines. (E) SATB1 stability and Akt activation were documented in SK-BR-3 cells carrying wild-type SATB1 together with Akt (Myr) or Akt (DN). (F) HEK293A cells were transfected with Myc-SATB1, Myc-SATB1S47A or Myc-SATB1S47D, respectively, and treated with CPT for 0, 2, 4, 6, 8 or 10 h. The cell lysates were subjected to immunobloting with anti-SATB1, anti-phospho-SATB1 (S47) and anti-GAPDH. (G) Jurkat cells were treated with CPT as in (F). The cell lysates were subjected to immunoblotting with anti-SATB1, anti-phospho-SATB1 (S47) and anti-GAPDH.
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Related In: Results  -  Collection

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pone-0064877-g002: Akt shields SATB1 from apoptotic cleavage.(A) Evenly distributed signals were observed in the majority of HEK293A cells transfected with GFP-SATB1, whereas punctate structures were detected in a small fraction of cells (white arrowhead). Akt (Myr) decreased the ratio of cells with dot-like signals. (B) In cells with dot-like structures, GFP-SATB1S47A colocalized with Cherry-PML, whereas GFP-SATB1S47D did not. (C) Expression of SATB1 was analyzed in MCF-7 cells stably integrated with empty vector, wild-type SATB1, SATB1S47A or SATB1S47D, respectively. (D) Expression of SATB1 and Akt activation were analyzed in stable SK-BR-3 cell lines. (E) SATB1 stability and Akt activation were documented in SK-BR-3 cells carrying wild-type SATB1 together with Akt (Myr) or Akt (DN). (F) HEK293A cells were transfected with Myc-SATB1, Myc-SATB1S47A or Myc-SATB1S47D, respectively, and treated with CPT for 0, 2, 4, 6, 8 or 10 h. The cell lysates were subjected to immunobloting with anti-SATB1, anti-phospho-SATB1 (S47) and anti-GAPDH. (G) Jurkat cells were treated with CPT as in (F). The cell lysates were subjected to immunoblotting with anti-SATB1, anti-phospho-SATB1 (S47) and anti-GAPDH.
Mentions: A number of Akt substrates are relocated within cells upon phosphorylation by Akt, for instance, FOXO transcription factors [21] and p27Kip1 [22]. The finding that SATB1 is a nuclear protein and a novel Akt substrate as well leads us to test whether PI3K/Akt signaling is obligate to redirect SATB1 to cytoplasm or sub-nuclear structures. GFP-tagged SATB1 was localized in nucleus in transfected HEK293A cells with less than 10% cells having punctate structures (Figure 2A). Although co-expression of wild-type Akt marginally reduced the ratio of dot-like cells, increased doses of activated Akt gradually and markedly reduced the number of dot-like cells and the number of evenly distributed dots. Intriguingly, with the increased doses of activated Akt, the dots would aggregate to form one or two larger bodies. By contrast, dominant-negative Akt generated a pattern of more, much tinier dots (Figure 2A). This observation suggests that Akt could modulate intra-nuclear aggregation of SATB1.

Bottom Line: Meanwhile, Akt phosphorylates Oct4 at threonine 228 and Klf4 at threonine 399, and accelerates their degradation.Moreover, PI3K/Akt signaling enhances the binding of SATB1 to Sox2, thereby probably impairing the formation of Oct4/Sox2 regulatory complexes.Accordingly, Akt-mediated phosphorylation is crucial for the capability of SATB1 to repress Nanog expression and to activate transcription of Bcl2 and Nestin genes.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China.

ABSTRACT
The mechanism by which Akt modulates stem cell homeostasis is still incompletely defined. Here we demonstrate that Akt phosphorylates special AT-rich sequences binding protein 1 (SATB1) at serine 47 and protects SATB1 from apoptotic cleavage. Meanwhile, Akt phosphorylates Oct4 at threonine 228 and Klf4 at threonine 399, and accelerates their degradation. Moreover, PI3K/Akt signaling enhances the binding of SATB1 to Sox2, thereby probably impairing the formation of Oct4/Sox2 regulatory complexes. During retinoic acid (RA)-induced differentiation of mouse F9 embryonal carcinoma cells (ECCs), the Akt activation profile as well as its substrate spectrum is strikingly correlated with the down-regulation of Oct4, Klf4 and Nanog, which suggests Akt activation is coupled to the onset of differentiation. Accordingly, Akt-mediated phosphorylation is crucial for the capability of SATB1 to repress Nanog expression and to activate transcription of Bcl2 and Nestin genes. Taken together, we conclude that Akt is involved in the differentiation of ECCs through coordinated phosphorylations of pluripotency/differentiation factors.

Show MeSH
Related in: MedlinePlus