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A bacterial cytotoxin identifies the RhoA exchange factor Net1 as a key effector in the response to DNA damage.

Guerra L, Carr HS, Richter-Dahlfors A, Masucci MG, Thelestam M, Frost JA, Frisan T - PLoS ONE (2008)

Bottom Line: We demonstrate that the nuclear RhoA-specific Guanine nucleotide Exchange Factor (GEF) Net1 becomes dephosphorylated at a critical inhibitory site in cells exposed to CDT or IR.Expression of a dominant negative Net1 or Net1 knock down by iRNA prevented RhoA activation, inhibited the formation of stress fibers, and enhanced cell death, indicating that Net1 activation is required for this RhoA-mediated responses to genotoxic stress.The Net1 and RhoA-dependent signals involved activation of the Mitogen-Activated Protein Kinase p38 and its downstream target MAPK-activated protein kinase 2.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Molecular Biology, Karolinska Institutet, Stockholm, Sweden.

ABSTRACT

Background: Exposure of adherent cells to DNA damaging agents, such as the bacterial cytolethal distending toxin (CDT) or ionizing radiations (IR), activates the small GTPase RhoA, which promotes the formation of actin stress fibers and delays cell death. The signalling intermediates that regulate RhoA activation and promote cell survival are unknown.

Principal findings: We demonstrate that the nuclear RhoA-specific Guanine nucleotide Exchange Factor (GEF) Net1 becomes dephosphorylated at a critical inhibitory site in cells exposed to CDT or IR. Expression of a dominant negative Net1 or Net1 knock down by iRNA prevented RhoA activation, inhibited the formation of stress fibers, and enhanced cell death, indicating that Net1 activation is required for this RhoA-mediated responses to genotoxic stress. The Net1 and RhoA-dependent signals involved activation of the Mitogen-Activated Protein Kinase p38 and its downstream target MAPK-activated protein kinase 2.

Significance: Our data highlight the importance of Net1 in controlling RhoA and p38 MAPK mediated cell survival in cells exposed to DNA damaging agents and illustrate a molecular pathway whereby chronic exposure to a bacterial toxin may promote genomic instability.

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The p38 MAPK effector MK2 is phosphorylated in a Net1- and RhoA-dependent manner in response to genotoxic agents.A) Untreated HeLa cells, or cells pre-treated with the specific p38 MAPK inhibitor SB203580 (20 μM) in complete medium for 30 min, were exposed to CDT (2 μg ml−1) for 4h, or irradiated (20 Gy) and further incubated in complete medium for 4h. Samples were subjected to western blot analysis using antibodies specific for phospho-MK2 (p-MK2) or actin. B) HCT116 cells were left untreated or irradiated (20 Gy) and further incubated in complete medium for 3h. Samples were subjected to western blot analysis as described in Figure 10A. C) HeLa cells transfected with: i) control siRNA; ii) Net1 specific siRNA (left panel); iii) the RhoA specific siRNA1 (right panel) were left untreated or irradiated (20 Gy), and further incubated for 4h in complete medium. MK2 phosphorylation was assessed as in Figure 10A. p-MK2 ratio represents the ratio between the optical density of the phospho-MK2 band in irradiated cells and optical density of the phospho-MK2 band in the untreated cells. D) Quantification of the changes in the levels of MK2 phosphorylation in irradiated HeLa cells: mean of 3 independent experiments performed with the Net1 specific siRNA (left panel) and 2 independent experiment performed with the RhoA specific siRNA (right panel).
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pone-0002254-g010: The p38 MAPK effector MK2 is phosphorylated in a Net1- and RhoA-dependent manner in response to genotoxic agents.A) Untreated HeLa cells, or cells pre-treated with the specific p38 MAPK inhibitor SB203580 (20 μM) in complete medium for 30 min, were exposed to CDT (2 μg ml−1) for 4h, or irradiated (20 Gy) and further incubated in complete medium for 4h. Samples were subjected to western blot analysis using antibodies specific for phospho-MK2 (p-MK2) or actin. B) HCT116 cells were left untreated or irradiated (20 Gy) and further incubated in complete medium for 3h. Samples were subjected to western blot analysis as described in Figure 10A. C) HeLa cells transfected with: i) control siRNA; ii) Net1 specific siRNA (left panel); iii) the RhoA specific siRNA1 (right panel) were left untreated or irradiated (20 Gy), and further incubated for 4h in complete medium. MK2 phosphorylation was assessed as in Figure 10A. p-MK2 ratio represents the ratio between the optical density of the phospho-MK2 band in irradiated cells and optical density of the phospho-MK2 band in the untreated cells. D) Quantification of the changes in the levels of MK2 phosphorylation in irradiated HeLa cells: mean of 3 independent experiments performed with the Net1 specific siRNA (left panel) and 2 independent experiment performed with the RhoA specific siRNA (right panel).

Mentions: The MAPK-activated protein kinase 2 (MK2) is a direct substrate of the p38 MAPK α-and β-isoforms [18]. We asked therefore whether this protein is also activated in a Net1- and RhoA-dependent manner upon induction of DNA damage. As illustrated in Figure 10, a 2- to 4-fold increase in the phosphorylation of MK2 on its activating site Thr334 (p-MK2) was observed in HeLa cells 4h after irradiation or intoxication (Figure 10A), and a similar effect was observed in irradiated HCT116 cells (Figure 10B). As expected, this effect was prevented by pre-treatment with the p38 MAPK specific inhibitor SB203580 (Figure 10A). Importantly, the phosphorylation of MK2 following irradiation was abrogated by knock down of either Net1 or RhoA, indicating that these proteins are required for MK2 activation (Figures 10C and 10D).


A bacterial cytotoxin identifies the RhoA exchange factor Net1 as a key effector in the response to DNA damage.

Guerra L, Carr HS, Richter-Dahlfors A, Masucci MG, Thelestam M, Frost JA, Frisan T - PLoS ONE (2008)

The p38 MAPK effector MK2 is phosphorylated in a Net1- and RhoA-dependent manner in response to genotoxic agents.A) Untreated HeLa cells, or cells pre-treated with the specific p38 MAPK inhibitor SB203580 (20 μM) in complete medium for 30 min, were exposed to CDT (2 μg ml−1) for 4h, or irradiated (20 Gy) and further incubated in complete medium for 4h. Samples were subjected to western blot analysis using antibodies specific for phospho-MK2 (p-MK2) or actin. B) HCT116 cells were left untreated or irradiated (20 Gy) and further incubated in complete medium for 3h. Samples were subjected to western blot analysis as described in Figure 10A. C) HeLa cells transfected with: i) control siRNA; ii) Net1 specific siRNA (left panel); iii) the RhoA specific siRNA1 (right panel) were left untreated or irradiated (20 Gy), and further incubated for 4h in complete medium. MK2 phosphorylation was assessed as in Figure 10A. p-MK2 ratio represents the ratio between the optical density of the phospho-MK2 band in irradiated cells and optical density of the phospho-MK2 band in the untreated cells. D) Quantification of the changes in the levels of MK2 phosphorylation in irradiated HeLa cells: mean of 3 independent experiments performed with the Net1 specific siRNA (left panel) and 2 independent experiment performed with the RhoA specific siRNA (right panel).
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Related In: Results  -  Collection

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pone-0002254-g010: The p38 MAPK effector MK2 is phosphorylated in a Net1- and RhoA-dependent manner in response to genotoxic agents.A) Untreated HeLa cells, or cells pre-treated with the specific p38 MAPK inhibitor SB203580 (20 μM) in complete medium for 30 min, were exposed to CDT (2 μg ml−1) for 4h, or irradiated (20 Gy) and further incubated in complete medium for 4h. Samples were subjected to western blot analysis using antibodies specific for phospho-MK2 (p-MK2) or actin. B) HCT116 cells were left untreated or irradiated (20 Gy) and further incubated in complete medium for 3h. Samples were subjected to western blot analysis as described in Figure 10A. C) HeLa cells transfected with: i) control siRNA; ii) Net1 specific siRNA (left panel); iii) the RhoA specific siRNA1 (right panel) were left untreated or irradiated (20 Gy), and further incubated for 4h in complete medium. MK2 phosphorylation was assessed as in Figure 10A. p-MK2 ratio represents the ratio between the optical density of the phospho-MK2 band in irradiated cells and optical density of the phospho-MK2 band in the untreated cells. D) Quantification of the changes in the levels of MK2 phosphorylation in irradiated HeLa cells: mean of 3 independent experiments performed with the Net1 specific siRNA (left panel) and 2 independent experiment performed with the RhoA specific siRNA (right panel).
Mentions: The MAPK-activated protein kinase 2 (MK2) is a direct substrate of the p38 MAPK α-and β-isoforms [18]. We asked therefore whether this protein is also activated in a Net1- and RhoA-dependent manner upon induction of DNA damage. As illustrated in Figure 10, a 2- to 4-fold increase in the phosphorylation of MK2 on its activating site Thr334 (p-MK2) was observed in HeLa cells 4h after irradiation or intoxication (Figure 10A), and a similar effect was observed in irradiated HCT116 cells (Figure 10B). As expected, this effect was prevented by pre-treatment with the p38 MAPK specific inhibitor SB203580 (Figure 10A). Importantly, the phosphorylation of MK2 following irradiation was abrogated by knock down of either Net1 or RhoA, indicating that these proteins are required for MK2 activation (Figures 10C and 10D).

Bottom Line: We demonstrate that the nuclear RhoA-specific Guanine nucleotide Exchange Factor (GEF) Net1 becomes dephosphorylated at a critical inhibitory site in cells exposed to CDT or IR.Expression of a dominant negative Net1 or Net1 knock down by iRNA prevented RhoA activation, inhibited the formation of stress fibers, and enhanced cell death, indicating that Net1 activation is required for this RhoA-mediated responses to genotoxic stress.The Net1 and RhoA-dependent signals involved activation of the Mitogen-Activated Protein Kinase p38 and its downstream target MAPK-activated protein kinase 2.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Molecular Biology, Karolinska Institutet, Stockholm, Sweden.

ABSTRACT

Background: Exposure of adherent cells to DNA damaging agents, such as the bacterial cytolethal distending toxin (CDT) or ionizing radiations (IR), activates the small GTPase RhoA, which promotes the formation of actin stress fibers and delays cell death. The signalling intermediates that regulate RhoA activation and promote cell survival are unknown.

Principal findings: We demonstrate that the nuclear RhoA-specific Guanine nucleotide Exchange Factor (GEF) Net1 becomes dephosphorylated at a critical inhibitory site in cells exposed to CDT or IR. Expression of a dominant negative Net1 or Net1 knock down by iRNA prevented RhoA activation, inhibited the formation of stress fibers, and enhanced cell death, indicating that Net1 activation is required for this RhoA-mediated responses to genotoxic stress. The Net1 and RhoA-dependent signals involved activation of the Mitogen-Activated Protein Kinase p38 and its downstream target MAPK-activated protein kinase 2.

Significance: Our data highlight the importance of Net1 in controlling RhoA and p38 MAPK mediated cell survival in cells exposed to DNA damaging agents and illustrate a molecular pathway whereby chronic exposure to a bacterial toxin may promote genomic instability.

Show MeSH
Related in: MedlinePlus