Limits...
GATA1 induces epithelial-mesenchymal transition in breast cancer cells through PAK5 oncogenic signaling.

Li Y, Ke Q, Shao Y, Zhu G, Li Y, Geng N, Jin F, Li F - Oncotarget (2015)

Bottom Line: Epithelial-mesenchymal transition (EMT) is a key process in tumor metastatic cascade that is characterized by the loss of cell-cell junctions, resulting in the acquisition of migratory and invasive properties.E-cadherin is a major component of intercellular junctions and the reduction or loss of its expression is a hallmark of EMT.GATA1 recruits HDAC3/4 to E-cadherin promoter, which is reduced by GATA1 S161A S187A mutant.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Key Laboratory of Cell Biology, Ministry of Public Health and Key Laboratory of Medical Cell Biology, Ministry of Education, China Medical University, Shenyang, China.

ABSTRACT
Epithelial-mesenchymal transition (EMT) is a key process in tumor metastatic cascade that is characterized by the loss of cell-cell junctions, resulting in the acquisition of migratory and invasive properties. E-cadherin is a major component of intercellular junctions and the reduction or loss of its expression is a hallmark of EMT. Transcription factor GATA1 has a critical anti-apoptotic role in breast cancer, but its function for metastasis has not been investigated. Here, we found that GATA1, as a novel E-cadherin repressor, promotes EMT in breast cancer cells. GATA1 binds to E-cadherin promoter, down-regulates E-cadherin expression, disrupts intercellular junction and promotes metastasis of breast cancer cell in vivo. Moreover, GATA1 is a new substrate of p21-activated kinase 5 (PAK5), which is phosphorylated on serine 161 and 187 (S161 and S187). GATA1 recruits HDAC3/4 to E-cadherin promoter, which is reduced by GATA1 S161A S187A mutant. These data indicate that phosphorylated GATA1 recruits more HDAC3/4 to promote transcriptional repression of E-cadherin, leading to the EMT of breast cancer cells. Our findings provide insights into the novel function of GATA1, contributing to a better understanding of the EMT, indicating that GATA1 and its phosphorylation may play an important role in the metastasis of breast cancer.

Show MeSH

Related in: MedlinePlus

GATA1 is a physiological substrate of p21-activated kinase 5(A) Western blot analysis demonstrates the protein level of PAK5 and GATA1 in breast cancer tissues and matched adjacent noncancerous tissues. N, matched adjacent noncancerous tissue; T, breast cancer tissue. **p < 0.01. (B) PAK5wt/KM and GATA1 were transfected into MCF-7 cells as indicated for Luciferase Assays. *p < 0.05, **p < 0.01. (C) HEK-293 cells transfected with Myc-PAK5KD (PAK5 kinase domain) were lysed for IP with anti-Myc antibody, the immunoprecipitated PAK5 kinase was incubated with GST or GST-GATA1 for in vitro kinase assay. Histone H3 (HH3) served as a positive control. (D)In vitro kinase assay using commercial PAK5 kinase and GST-GATA1FL (Full length, 1–413aa) or GST-GATA1 deletion mutants (1–160aa, 1–300aa and 241–413aa). Histone H3 (HH3) served as a positive control. (E)In vitro kinase assay using commercial PAK5 kinase and GST-GATA1 wt or GST-GATA1 single-site mutations as indicated. (F) MCF-7 cells transfected with GATA1 wt/S161A S187A and PAK5 wt were used for Ser/Thr phosphoprotein purification. Then concentrated protein was used for western blot. The top lane, the phosphorylated GATA1 from these cells lysates was used for immunoblotting using anti-GFP antibody. Total cells lysates were used for immunoblotting by anti-GFP, GAPDH antibodies.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4414194&req=5

Figure 3: GATA1 is a physiological substrate of p21-activated kinase 5(A) Western blot analysis demonstrates the protein level of PAK5 and GATA1 in breast cancer tissues and matched adjacent noncancerous tissues. N, matched adjacent noncancerous tissue; T, breast cancer tissue. **p < 0.01. (B) PAK5wt/KM and GATA1 were transfected into MCF-7 cells as indicated for Luciferase Assays. *p < 0.05, **p < 0.01. (C) HEK-293 cells transfected with Myc-PAK5KD (PAK5 kinase domain) were lysed for IP with anti-Myc antibody, the immunoprecipitated PAK5 kinase was incubated with GST or GST-GATA1 for in vitro kinase assay. Histone H3 (HH3) served as a positive control. (D)In vitro kinase assay using commercial PAK5 kinase and GST-GATA1FL (Full length, 1–413aa) or GST-GATA1 deletion mutants (1–160aa, 1–300aa and 241–413aa). Histone H3 (HH3) served as a positive control. (E)In vitro kinase assay using commercial PAK5 kinase and GST-GATA1 wt or GST-GATA1 single-site mutations as indicated. (F) MCF-7 cells transfected with GATA1 wt/S161A S187A and PAK5 wt were used for Ser/Thr phosphoprotein purification. Then concentrated protein was used for western blot. The top lane, the phosphorylated GATA1 from these cells lysates was used for immunoblotting using anti-GFP antibody. Total cells lysates were used for immunoblotting by anti-GFP, GAPDH antibodies.

Mentions: It is reported that PAK5 regulates cell adhesion and migration in colorectal carcinoma cells [17]. PAK5 was in higher expression in breast cancer tissues than matched adjacent noncancerous tissues (Figure 3A), which was consistent with the previous report [24]. The high expression rate of the PAK5 and GATA1 were 66.25% (53/80) and 62.50% (50/80) in breast cancer tissues and low expression rate 33.75% (27/80) and 37.50% (30/80) in matched adjacent noncancerous tissues (Figure 3A). A significant statistical difference was found between the two groups. Importantly, the high level of PAK5 expression correlated with the expression of GATA1 in breast cancer tissues (p = 0.033). Since E-cadherin is one of the most important cell-cell adhesion proteins and many kinases are required for the disruption of E-cadherin-based cell-cell junctions, such as PAK1 and CDKL2 [25, 26], we presumed that PAK5 might modulate E-cadherin expression. To test whether the phosphorylation of GATA1 by PAK5 was related to GATA1's inhibitory effect on E-cadherin, luciferase assays were performed. As expected, it was wild type PAK5 (PAK5 wt) rather than kinase dead PAK5 (PAK5 KM) promoted transcriptional repression of E-cadherin by GATA1 (Figure 3B). So, we wondered if GATA1 was a physiological substrate of PAK5. The in vitro kinase assay showed that PAK5 phosphorylated GST-GATA1 (Figure 3C). To identify specific PAK5 phosphorylation sites in GATA1, first, we used GST-GATA1 FL (1–413aa) and GST-GATA1 deletion constructs (1–160aa, 1–300aa and 241–413aa) for in vitro kinase assay. The data indicated that the phosphorylation sites were located between amino acids 160 and 240 (Figure 3D). Next, we created single-site mutations of the predicted sites in GATA1 amino acids 160–240 (S161A, S174A, S178A, S187A, T212A) for kinase assay. As shown in Figure 3E, both mutations of Ser161 to Ala and Ser187 to Ala significantly impaired PAK5 phosphorylation of GATA1. Then, Serine/Threonine phosphoprotein purification kit was used to further test the PAK5-mediated GATA1 phosphorylation in vivo (Figure 3F). Total Ser/Thr phosphorylated protein from cell lysates were analyzed by western blot. The results showed that phosphorylated wild type GATA1 but not GATA1 mutant was increased with overexpressing PAK5 (Figure 3F, the top lane, compare lane 4 with lane 2 and compare lane 5 with lane 3). Since in vitro kinase assay showed Ser161 was main phosphorylation site, phosphor-GATA1 Ser161 antibody was used to further test the PAK5-mediated GATA1 phosphorylation in cells (Figure S1). These results indicate that both GATA1 Ser161 and Ser187 may be the main phosphorylation sites by PAK5.


GATA1 induces epithelial-mesenchymal transition in breast cancer cells through PAK5 oncogenic signaling.

Li Y, Ke Q, Shao Y, Zhu G, Li Y, Geng N, Jin F, Li F - Oncotarget (2015)

GATA1 is a physiological substrate of p21-activated kinase 5(A) Western blot analysis demonstrates the protein level of PAK5 and GATA1 in breast cancer tissues and matched adjacent noncancerous tissues. N, matched adjacent noncancerous tissue; T, breast cancer tissue. **p < 0.01. (B) PAK5wt/KM and GATA1 were transfected into MCF-7 cells as indicated for Luciferase Assays. *p < 0.05, **p < 0.01. (C) HEK-293 cells transfected with Myc-PAK5KD (PAK5 kinase domain) were lysed for IP with anti-Myc antibody, the immunoprecipitated PAK5 kinase was incubated with GST or GST-GATA1 for in vitro kinase assay. Histone H3 (HH3) served as a positive control. (D)In vitro kinase assay using commercial PAK5 kinase and GST-GATA1FL (Full length, 1–413aa) or GST-GATA1 deletion mutants (1–160aa, 1–300aa and 241–413aa). Histone H3 (HH3) served as a positive control. (E)In vitro kinase assay using commercial PAK5 kinase and GST-GATA1 wt or GST-GATA1 single-site mutations as indicated. (F) MCF-7 cells transfected with GATA1 wt/S161A S187A and PAK5 wt were used for Ser/Thr phosphoprotein purification. Then concentrated protein was used for western blot. The top lane, the phosphorylated GATA1 from these cells lysates was used for immunoblotting using anti-GFP antibody. Total cells lysates were used for immunoblotting by anti-GFP, GAPDH antibodies.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4414194&req=5

Figure 3: GATA1 is a physiological substrate of p21-activated kinase 5(A) Western blot analysis demonstrates the protein level of PAK5 and GATA1 in breast cancer tissues and matched adjacent noncancerous tissues. N, matched adjacent noncancerous tissue; T, breast cancer tissue. **p < 0.01. (B) PAK5wt/KM and GATA1 were transfected into MCF-7 cells as indicated for Luciferase Assays. *p < 0.05, **p < 0.01. (C) HEK-293 cells transfected with Myc-PAK5KD (PAK5 kinase domain) were lysed for IP with anti-Myc antibody, the immunoprecipitated PAK5 kinase was incubated with GST or GST-GATA1 for in vitro kinase assay. Histone H3 (HH3) served as a positive control. (D)In vitro kinase assay using commercial PAK5 kinase and GST-GATA1FL (Full length, 1–413aa) or GST-GATA1 deletion mutants (1–160aa, 1–300aa and 241–413aa). Histone H3 (HH3) served as a positive control. (E)In vitro kinase assay using commercial PAK5 kinase and GST-GATA1 wt or GST-GATA1 single-site mutations as indicated. (F) MCF-7 cells transfected with GATA1 wt/S161A S187A and PAK5 wt were used for Ser/Thr phosphoprotein purification. Then concentrated protein was used for western blot. The top lane, the phosphorylated GATA1 from these cells lysates was used for immunoblotting using anti-GFP antibody. Total cells lysates were used for immunoblotting by anti-GFP, GAPDH antibodies.
Mentions: It is reported that PAK5 regulates cell adhesion and migration in colorectal carcinoma cells [17]. PAK5 was in higher expression in breast cancer tissues than matched adjacent noncancerous tissues (Figure 3A), which was consistent with the previous report [24]. The high expression rate of the PAK5 and GATA1 were 66.25% (53/80) and 62.50% (50/80) in breast cancer tissues and low expression rate 33.75% (27/80) and 37.50% (30/80) in matched adjacent noncancerous tissues (Figure 3A). A significant statistical difference was found between the two groups. Importantly, the high level of PAK5 expression correlated with the expression of GATA1 in breast cancer tissues (p = 0.033). Since E-cadherin is one of the most important cell-cell adhesion proteins and many kinases are required for the disruption of E-cadherin-based cell-cell junctions, such as PAK1 and CDKL2 [25, 26], we presumed that PAK5 might modulate E-cadherin expression. To test whether the phosphorylation of GATA1 by PAK5 was related to GATA1's inhibitory effect on E-cadherin, luciferase assays were performed. As expected, it was wild type PAK5 (PAK5 wt) rather than kinase dead PAK5 (PAK5 KM) promoted transcriptional repression of E-cadherin by GATA1 (Figure 3B). So, we wondered if GATA1 was a physiological substrate of PAK5. The in vitro kinase assay showed that PAK5 phosphorylated GST-GATA1 (Figure 3C). To identify specific PAK5 phosphorylation sites in GATA1, first, we used GST-GATA1 FL (1–413aa) and GST-GATA1 deletion constructs (1–160aa, 1–300aa and 241–413aa) for in vitro kinase assay. The data indicated that the phosphorylation sites were located between amino acids 160 and 240 (Figure 3D). Next, we created single-site mutations of the predicted sites in GATA1 amino acids 160–240 (S161A, S174A, S178A, S187A, T212A) for kinase assay. As shown in Figure 3E, both mutations of Ser161 to Ala and Ser187 to Ala significantly impaired PAK5 phosphorylation of GATA1. Then, Serine/Threonine phosphoprotein purification kit was used to further test the PAK5-mediated GATA1 phosphorylation in vivo (Figure 3F). Total Ser/Thr phosphorylated protein from cell lysates were analyzed by western blot. The results showed that phosphorylated wild type GATA1 but not GATA1 mutant was increased with overexpressing PAK5 (Figure 3F, the top lane, compare lane 4 with lane 2 and compare lane 5 with lane 3). Since in vitro kinase assay showed Ser161 was main phosphorylation site, phosphor-GATA1 Ser161 antibody was used to further test the PAK5-mediated GATA1 phosphorylation in cells (Figure S1). These results indicate that both GATA1 Ser161 and Ser187 may be the main phosphorylation sites by PAK5.

Bottom Line: Epithelial-mesenchymal transition (EMT) is a key process in tumor metastatic cascade that is characterized by the loss of cell-cell junctions, resulting in the acquisition of migratory and invasive properties.E-cadherin is a major component of intercellular junctions and the reduction or loss of its expression is a hallmark of EMT.GATA1 recruits HDAC3/4 to E-cadherin promoter, which is reduced by GATA1 S161A S187A mutant.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Key Laboratory of Cell Biology, Ministry of Public Health and Key Laboratory of Medical Cell Biology, Ministry of Education, China Medical University, Shenyang, China.

ABSTRACT
Epithelial-mesenchymal transition (EMT) is a key process in tumor metastatic cascade that is characterized by the loss of cell-cell junctions, resulting in the acquisition of migratory and invasive properties. E-cadherin is a major component of intercellular junctions and the reduction or loss of its expression is a hallmark of EMT. Transcription factor GATA1 has a critical anti-apoptotic role in breast cancer, but its function for metastasis has not been investigated. Here, we found that GATA1, as a novel E-cadherin repressor, promotes EMT in breast cancer cells. GATA1 binds to E-cadherin promoter, down-regulates E-cadherin expression, disrupts intercellular junction and promotes metastasis of breast cancer cell in vivo. Moreover, GATA1 is a new substrate of p21-activated kinase 5 (PAK5), which is phosphorylated on serine 161 and 187 (S161 and S187). GATA1 recruits HDAC3/4 to E-cadherin promoter, which is reduced by GATA1 S161A S187A mutant. These data indicate that phosphorylated GATA1 recruits more HDAC3/4 to promote transcriptional repression of E-cadherin, leading to the EMT of breast cancer cells. Our findings provide insights into the novel function of GATA1, contributing to a better understanding of the EMT, indicating that GATA1 and its phosphorylation may play an important role in the metastasis of breast cancer.

Show MeSH
Related in: MedlinePlus