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The nuclear guanine nucleotide exchange factors Ect2 and Net1 regulate RhoB-mediated cell death after DNA damage.

Srougi MC, Burridge K - PLoS ONE (2011)

Bottom Line: The increase in RhoB activity after genotoxic stress is associated with increased activity of the nuclear guanine nucleotide exchange factors (GEFs), Ect2 and Net1, but not the cytoplasmic GEFs p115 RhoGEF or Vav2.Importantly, loss of Ect2 and Net1 via siRNA-mediated protein knock-down inhibited IR-induced increases in RhoB activity, reduced apoptotic signaling events, and protected cells from IR-induced cell death.Collectively, these data suggest a mechanism involving the nuclear GEFs Ect2 and Net1 for activating RhoB after genotoxic stress, thereby facilitating cell death after treatment with DNA damaging agents.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Developmental Biology, University of North Carolina, Chapel Hill, North Carolina, United States of America. melissa_srougi@med.unc.edu

ABSTRACT
Commonly used antitumor treatments, including radiation and chemotherapy, function by damaging the DNA of rapidly proliferating cells. However, resistance to these agents is a predominant clinical problem. A member of the Rho family of small GTPases, RhoB has been shown to be integral in mediating cell death after ionizing radiation (IR) or other DNA damaging agents in Ras-transformed cell lines. In addition, RhoB protein expression increases after genotoxic stress, and loss of RhoB expression causes radio- and chemotherapeutic resistance. However, the signaling pathways that govern RhoB-induced cell death after DNA damage remain enigmatic. Here, we show that RhoB activity increases in human breast and cervical cancer cell lines after treatment with DNA damaging agents. Furthermore, RhoB activity is necessary for DNA damage-induced cell death, as the stable loss of RhoB protein expression using shRNA partially protects cells and prevents the phosphorylation of c-Jun N-terminal kinases (JNKs) and the induction of the pro-apoptotic protein Bim after IR. The increase in RhoB activity after genotoxic stress is associated with increased activity of the nuclear guanine nucleotide exchange factors (GEFs), Ect2 and Net1, but not the cytoplasmic GEFs p115 RhoGEF or Vav2. Importantly, loss of Ect2 and Net1 via siRNA-mediated protein knock-down inhibited IR-induced increases in RhoB activity, reduced apoptotic signaling events, and protected cells from IR-induced cell death. Collectively, these data suggest a mechanism involving the nuclear GEFs Ect2 and Net1 for activating RhoB after genotoxic stress, thereby facilitating cell death after treatment with DNA damaging agents.

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The nuclear GEFs Ect2 and Net1 are activated after IR.(A) MCF-7 cells were mock-treated or irradiated with 5 Gy and pulldowns performed with GST-RhoB(17A) to identify active GEFs. (B) Quantification of blots in (A) as described in Materials and Methods (*, p≤0.05; **, p<0.001). (C) MCF-7 cells were transfected with constructs to the indicated GEFs. 24 h after transfection, GST-RBD pulldowns were performed to detect active RhoB. (D and E) MCF-7 cells were serum starved overnight and treated with (D) 100 ng/mL of EGF for 1 h or (E) treated with serum-containing medium for 1 h and pulldowns performed with GST-RhoB(17A). Immunoblots were probed with an antibody to (D) Vav2 or (E) p115 RhoGEF.
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pone-0017108-g003: The nuclear GEFs Ect2 and Net1 are activated after IR.(A) MCF-7 cells were mock-treated or irradiated with 5 Gy and pulldowns performed with GST-RhoB(17A) to identify active GEFs. (B) Quantification of blots in (A) as described in Materials and Methods (*, p≤0.05; **, p<0.001). (C) MCF-7 cells were transfected with constructs to the indicated GEFs. 24 h after transfection, GST-RBD pulldowns were performed to detect active RhoB. (D and E) MCF-7 cells were serum starved overnight and treated with (D) 100 ng/mL of EGF for 1 h or (E) treated with serum-containing medium for 1 h and pulldowns performed with GST-RhoB(17A). Immunoblots were probed with an antibody to (D) Vav2 or (E) p115 RhoGEF.

Mentions: To determine which GEFs were responsible for the upstream activation of RhoB after DNA damage, we performed precipitation assays with the nucleotide free RhoB mutant, RhoB(17A). Our laboratory has previously validated the use of this assay for the specific isolation of activated GEFs, which bind with high affinity to the nucleotide free state [18], [19]. MCF-7 cells were irradiated with 5 Gy and pulldowns were performed with GST-RhoB(17A) immediately after IR. Immunoblots of the precipitates were then probed with antibodies to various GEFs to determine which were activated based on their association with the nucleotide-free mutant. We narrowed down our search based on GEF subcellular localization. Since RhoB is activated shortly (∼15 min) after DNA damage, we speculated that the signals responsible for its activation were connected directly to DNA damage signaling pathways. We therefore focused our attention on the nuclear GEFs, Ect2 and Net1. For comparison, we also examined two well-characterized cytoplasmic GEFs; Vav2, which has been shown to exchange upon RhoB [20] and p115 RhoGEF. From these studies we found no activation of the cytoplasmic GEFs, Vav2 and p115 RhoGEF either before or after IR as indicated by binding to the nucleotide-free mutant of RhoB (Fig. 3A). In addition, treatment with H2O2 did not significantly increase Vav2 or p115 activity (Fig. S3B and S3C) although it caused an increase in RhoB-GTP levels (Fig. S1C). In contrast, both Net1 and Ect2 were activated ≥5 fold after IR (Fig. 3A and 3B). These increases in Ect2 and Net1 activity were also noted after exposure to 5-FU and H2O2 (Fig. S3B-E).


The nuclear guanine nucleotide exchange factors Ect2 and Net1 regulate RhoB-mediated cell death after DNA damage.

Srougi MC, Burridge K - PLoS ONE (2011)

The nuclear GEFs Ect2 and Net1 are activated after IR.(A) MCF-7 cells were mock-treated or irradiated with 5 Gy and pulldowns performed with GST-RhoB(17A) to identify active GEFs. (B) Quantification of blots in (A) as described in Materials and Methods (*, p≤0.05; **, p<0.001). (C) MCF-7 cells were transfected with constructs to the indicated GEFs. 24 h after transfection, GST-RBD pulldowns were performed to detect active RhoB. (D and E) MCF-7 cells were serum starved overnight and treated with (D) 100 ng/mL of EGF for 1 h or (E) treated with serum-containing medium for 1 h and pulldowns performed with GST-RhoB(17A). Immunoblots were probed with an antibody to (D) Vav2 or (E) p115 RhoGEF.
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Related In: Results  -  Collection

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pone-0017108-g003: The nuclear GEFs Ect2 and Net1 are activated after IR.(A) MCF-7 cells were mock-treated or irradiated with 5 Gy and pulldowns performed with GST-RhoB(17A) to identify active GEFs. (B) Quantification of blots in (A) as described in Materials and Methods (*, p≤0.05; **, p<0.001). (C) MCF-7 cells were transfected with constructs to the indicated GEFs. 24 h after transfection, GST-RBD pulldowns were performed to detect active RhoB. (D and E) MCF-7 cells were serum starved overnight and treated with (D) 100 ng/mL of EGF for 1 h or (E) treated with serum-containing medium for 1 h and pulldowns performed with GST-RhoB(17A). Immunoblots were probed with an antibody to (D) Vav2 or (E) p115 RhoGEF.
Mentions: To determine which GEFs were responsible for the upstream activation of RhoB after DNA damage, we performed precipitation assays with the nucleotide free RhoB mutant, RhoB(17A). Our laboratory has previously validated the use of this assay for the specific isolation of activated GEFs, which bind with high affinity to the nucleotide free state [18], [19]. MCF-7 cells were irradiated with 5 Gy and pulldowns were performed with GST-RhoB(17A) immediately after IR. Immunoblots of the precipitates were then probed with antibodies to various GEFs to determine which were activated based on their association with the nucleotide-free mutant. We narrowed down our search based on GEF subcellular localization. Since RhoB is activated shortly (∼15 min) after DNA damage, we speculated that the signals responsible for its activation were connected directly to DNA damage signaling pathways. We therefore focused our attention on the nuclear GEFs, Ect2 and Net1. For comparison, we also examined two well-characterized cytoplasmic GEFs; Vav2, which has been shown to exchange upon RhoB [20] and p115 RhoGEF. From these studies we found no activation of the cytoplasmic GEFs, Vav2 and p115 RhoGEF either before or after IR as indicated by binding to the nucleotide-free mutant of RhoB (Fig. 3A). In addition, treatment with H2O2 did not significantly increase Vav2 or p115 activity (Fig. S3B and S3C) although it caused an increase in RhoB-GTP levels (Fig. S1C). In contrast, both Net1 and Ect2 were activated ≥5 fold after IR (Fig. 3A and 3B). These increases in Ect2 and Net1 activity were also noted after exposure to 5-FU and H2O2 (Fig. S3B-E).

Bottom Line: The increase in RhoB activity after genotoxic stress is associated with increased activity of the nuclear guanine nucleotide exchange factors (GEFs), Ect2 and Net1, but not the cytoplasmic GEFs p115 RhoGEF or Vav2.Importantly, loss of Ect2 and Net1 via siRNA-mediated protein knock-down inhibited IR-induced increases in RhoB activity, reduced apoptotic signaling events, and protected cells from IR-induced cell death.Collectively, these data suggest a mechanism involving the nuclear GEFs Ect2 and Net1 for activating RhoB after genotoxic stress, thereby facilitating cell death after treatment with DNA damaging agents.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Developmental Biology, University of North Carolina, Chapel Hill, North Carolina, United States of America. melissa_srougi@med.unc.edu

ABSTRACT
Commonly used antitumor treatments, including radiation and chemotherapy, function by damaging the DNA of rapidly proliferating cells. However, resistance to these agents is a predominant clinical problem. A member of the Rho family of small GTPases, RhoB has been shown to be integral in mediating cell death after ionizing radiation (IR) or other DNA damaging agents in Ras-transformed cell lines. In addition, RhoB protein expression increases after genotoxic stress, and loss of RhoB expression causes radio- and chemotherapeutic resistance. However, the signaling pathways that govern RhoB-induced cell death after DNA damage remain enigmatic. Here, we show that RhoB activity increases in human breast and cervical cancer cell lines after treatment with DNA damaging agents. Furthermore, RhoB activity is necessary for DNA damage-induced cell death, as the stable loss of RhoB protein expression using shRNA partially protects cells and prevents the phosphorylation of c-Jun N-terminal kinases (JNKs) and the induction of the pro-apoptotic protein Bim after IR. The increase in RhoB activity after genotoxic stress is associated with increased activity of the nuclear guanine nucleotide exchange factors (GEFs), Ect2 and Net1, but not the cytoplasmic GEFs p115 RhoGEF or Vav2. Importantly, loss of Ect2 and Net1 via siRNA-mediated protein knock-down inhibited IR-induced increases in RhoB activity, reduced apoptotic signaling events, and protected cells from IR-induced cell death. Collectively, these data suggest a mechanism involving the nuclear GEFs Ect2 and Net1 for activating RhoB after genotoxic stress, thereby facilitating cell death after treatment with DNA damaging agents.

Show MeSH
Related in: MedlinePlus