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Filamin A phosphorylation by Akt promotes cell migration in response to arsenic.

Li L, Lu Y, Stemmer PM, Chen F - Oncotarget (2015)

Bottom Line: The results indicate that a cytoskeleton remodeling protein, filamin A, with a molecular weight around 280 kDa, is phosphorylated by Akt in HEK-293 cells treated with As(3+), which was also confirmed in human bronchial epithelial cell line, BEAS-2B cells.To further confirm the importance of Akt-dependent filamin A S2152 phosphorylation in As(3+)-induced cell migration, we over-expressed either wild type filamin A or the mutated filamin A in which the S2152 was substituted with alanine (S2152A).The capability of cell migration was reduced significantly in the cells expressing the mutated filamin A (S2152A).

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmaceutical Sciences, Eugene Applebaum College of Pharmacy and Health Sciences, Wayne State University, Detroit, MI 48201, USA.

ABSTRACT
We had previously reported that trivalent arsenic (As(3+)), a well-known environmental carcinogen, induces phosphorylation of several putative Akt substrates. In the present report, we characterized one of these substrates by immunoprecipitation and proteomics analysis. The results indicate that a cytoskeleton remodeling protein, filamin A, with a molecular weight around 280 kDa, is phosphorylated by Akt in HEK-293 cells treated with As(3+), which was also confirmed in human bronchial epithelial cell line, BEAS-2B cells. Additional biochemical and biological studies revealed that serine 2152 (S2152) of filamin A is phosphorylated by activated Akt in the cells treated with As(3+). To further confirm the importance of Akt-dependent filamin A S2152 phosphorylation in As(3+)-induced cell migration, we over-expressed either wild type filamin A or the mutated filamin A in which the S2152 was substituted with alanine (S2152A). The capability of cell migration was reduced significantly in the cells expressing the mutated filamin A (S2152A). Clinically, we found that increased expression of filamin A predicts poorer overall survival of the lung cancer patients with adenocarcinoma. Thus, these data suggest that Akt dependent filamin A phosphorylation is one of the key events in mediating As(3+)-induced carcinogenesis. Antagonizing Akt signaling can ameliorate As(3+)-induced filamin A phosphorylation and cell migration, which may serve as a molecular targeting strategy for malignancies associated with environmental As(3+) exposure.

No MeSH data available.


Related in: MedlinePlus

Identifying filamin A as an Akt substrateA. HEK-293 cells were treated with 20 μM As3+ in the presence or absence of 10 mM N-acetyl L Cysteine for 2 h. Cell lysates were prepared using non-denature buffer and subjected to immunoprecipitation (IP) using anti-Akt substrate antibody. Block arrow denotes protein band on the PVDF membrane that was retrieved for mass spectrometry analysis. B. Mass Spectrometry result shows the peptide profiling that overlaps sequence of filamin A. Top two panels are representative spectrums of the peptides from filamin A. C. BEAS-2B cell were treated as in (A), and subjected to IP using the indicated antibodies followed by Western blotting (WB). D. BEAS-2B cells were treated with 20 μM As3+ for 2 h, followed by IP using IgG or pAkt antibody as indicated and then WB using phospho-Filamin A (pFilamin A) or pAkt antibody.
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Figure 1: Identifying filamin A as an Akt substrateA. HEK-293 cells were treated with 20 μM As3+ in the presence or absence of 10 mM N-acetyl L Cysteine for 2 h. Cell lysates were prepared using non-denature buffer and subjected to immunoprecipitation (IP) using anti-Akt substrate antibody. Block arrow denotes protein band on the PVDF membrane that was retrieved for mass spectrometry analysis. B. Mass Spectrometry result shows the peptide profiling that overlaps sequence of filamin A. Top two panels are representative spectrums of the peptides from filamin A. C. BEAS-2B cell were treated as in (A), and subjected to IP using the indicated antibodies followed by Western blotting (WB). D. BEAS-2B cells were treated with 20 μM As3+ for 2 h, followed by IP using IgG or pAkt antibody as indicated and then WB using phospho-Filamin A (pFilamin A) or pAkt antibody.

Mentions: We had previously demonstrated several Akt phosphorylation substrates in immunoprecipitation (IP) analyses of the BEAS-2B cells treated with As3+ [10]. To test whether these Akt substrates are also presented in other types of cells in response to As3+, we performed IP analysis using cell lysates from HEK-293 cells cultured in the absence or presence of 20 μM As3+ for 2 h and an antibody specifically recognizing the phosphorylated Akt phosphorylation motif, R-X-R-X-X-S/T. As depicted in Fig. 1A, a band with an estimated molecular weight about 280 kDa was constitutively detected in HEK-293 cells (pointed with a black block arrow). Treatment of the cells with As3+ enhanced the density of this band considerably. Pre-incubation of the cells with an antioxidant, NAC that scavenges reactive oxygen species (ROS), diminished this band completely, suggesting that the occurrence of this band is ROS-dependent. To determine the nature of this protein band that can be recognized by an anti-Akt phosphorylation substrate antibody, we retrieved this band from Western blotting membrane and subjected it to proteomics analysis through tryptic digestion and peptide identification using orbitrap Fusion mass spectrometry as described under Materials and Methods. The peptides identified from this analysis suggested a possible presence of 11 proteins in this protein band as detected in IP, among which only two proteins, filamin A and inositol 1,4,5-trisphosphate receptor type 3 (ITPR3), are within the range of molecular weight between 200 and 300 kDa where the original band was positioned. The filamin A was represented by 10 peptides, whereas ITPR3 was represented by only 4 peptides. Thus, we concluded that the most abundant protein in this IP band is filamin A based on the assumption that the number of peptides identified in mass spectrometry is generally proportional to its abundance or concentration in the sample.


Filamin A phosphorylation by Akt promotes cell migration in response to arsenic.

Li L, Lu Y, Stemmer PM, Chen F - Oncotarget (2015)

Identifying filamin A as an Akt substrateA. HEK-293 cells were treated with 20 μM As3+ in the presence or absence of 10 mM N-acetyl L Cysteine for 2 h. Cell lysates were prepared using non-denature buffer and subjected to immunoprecipitation (IP) using anti-Akt substrate antibody. Block arrow denotes protein band on the PVDF membrane that was retrieved for mass spectrometry analysis. B. Mass Spectrometry result shows the peptide profiling that overlaps sequence of filamin A. Top two panels are representative spectrums of the peptides from filamin A. C. BEAS-2B cell were treated as in (A), and subjected to IP using the indicated antibodies followed by Western blotting (WB). D. BEAS-2B cells were treated with 20 μM As3+ for 2 h, followed by IP using IgG or pAkt antibody as indicated and then WB using phospho-Filamin A (pFilamin A) or pAkt antibody.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
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Figure 1: Identifying filamin A as an Akt substrateA. HEK-293 cells were treated with 20 μM As3+ in the presence or absence of 10 mM N-acetyl L Cysteine for 2 h. Cell lysates were prepared using non-denature buffer and subjected to immunoprecipitation (IP) using anti-Akt substrate antibody. Block arrow denotes protein band on the PVDF membrane that was retrieved for mass spectrometry analysis. B. Mass Spectrometry result shows the peptide profiling that overlaps sequence of filamin A. Top two panels are representative spectrums of the peptides from filamin A. C. BEAS-2B cell were treated as in (A), and subjected to IP using the indicated antibodies followed by Western blotting (WB). D. BEAS-2B cells were treated with 20 μM As3+ for 2 h, followed by IP using IgG or pAkt antibody as indicated and then WB using phospho-Filamin A (pFilamin A) or pAkt antibody.
Mentions: We had previously demonstrated several Akt phosphorylation substrates in immunoprecipitation (IP) analyses of the BEAS-2B cells treated with As3+ [10]. To test whether these Akt substrates are also presented in other types of cells in response to As3+, we performed IP analysis using cell lysates from HEK-293 cells cultured in the absence or presence of 20 μM As3+ for 2 h and an antibody specifically recognizing the phosphorylated Akt phosphorylation motif, R-X-R-X-X-S/T. As depicted in Fig. 1A, a band with an estimated molecular weight about 280 kDa was constitutively detected in HEK-293 cells (pointed with a black block arrow). Treatment of the cells with As3+ enhanced the density of this band considerably. Pre-incubation of the cells with an antioxidant, NAC that scavenges reactive oxygen species (ROS), diminished this band completely, suggesting that the occurrence of this band is ROS-dependent. To determine the nature of this protein band that can be recognized by an anti-Akt phosphorylation substrate antibody, we retrieved this band from Western blotting membrane and subjected it to proteomics analysis through tryptic digestion and peptide identification using orbitrap Fusion mass spectrometry as described under Materials and Methods. The peptides identified from this analysis suggested a possible presence of 11 proteins in this protein band as detected in IP, among which only two proteins, filamin A and inositol 1,4,5-trisphosphate receptor type 3 (ITPR3), are within the range of molecular weight between 200 and 300 kDa where the original band was positioned. The filamin A was represented by 10 peptides, whereas ITPR3 was represented by only 4 peptides. Thus, we concluded that the most abundant protein in this IP band is filamin A based on the assumption that the number of peptides identified in mass spectrometry is generally proportional to its abundance or concentration in the sample.

Bottom Line: The results indicate that a cytoskeleton remodeling protein, filamin A, with a molecular weight around 280 kDa, is phosphorylated by Akt in HEK-293 cells treated with As(3+), which was also confirmed in human bronchial epithelial cell line, BEAS-2B cells.To further confirm the importance of Akt-dependent filamin A S2152 phosphorylation in As(3+)-induced cell migration, we over-expressed either wild type filamin A or the mutated filamin A in which the S2152 was substituted with alanine (S2152A).The capability of cell migration was reduced significantly in the cells expressing the mutated filamin A (S2152A).

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmaceutical Sciences, Eugene Applebaum College of Pharmacy and Health Sciences, Wayne State University, Detroit, MI 48201, USA.

ABSTRACT
We had previously reported that trivalent arsenic (As(3+)), a well-known environmental carcinogen, induces phosphorylation of several putative Akt substrates. In the present report, we characterized one of these substrates by immunoprecipitation and proteomics analysis. The results indicate that a cytoskeleton remodeling protein, filamin A, with a molecular weight around 280 kDa, is phosphorylated by Akt in HEK-293 cells treated with As(3+), which was also confirmed in human bronchial epithelial cell line, BEAS-2B cells. Additional biochemical and biological studies revealed that serine 2152 (S2152) of filamin A is phosphorylated by activated Akt in the cells treated with As(3+). To further confirm the importance of Akt-dependent filamin A S2152 phosphorylation in As(3+)-induced cell migration, we over-expressed either wild type filamin A or the mutated filamin A in which the S2152 was substituted with alanine (S2152A). The capability of cell migration was reduced significantly in the cells expressing the mutated filamin A (S2152A). Clinically, we found that increased expression of filamin A predicts poorer overall survival of the lung cancer patients with adenocarcinoma. Thus, these data suggest that Akt dependent filamin A phosphorylation is one of the key events in mediating As(3+)-induced carcinogenesis. Antagonizing Akt signaling can ameliorate As(3+)-induced filamin A phosphorylation and cell migration, which may serve as a molecular targeting strategy for malignancies associated with environmental As(3+) exposure.

No MeSH data available.


Related in: MedlinePlus