The Nuclear Guanine Nucleotide Exchange Factors Ect2 and Net1 Regulate RhoB-Mediated Cell Death after DNA Damage

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

The Small GTPase RhoA Localizes to the Nucleus and Is Activated by Net1 and DNA Damage Signals

Rho GTPases control many cellular processes, including cell survival, gene expression and migration. Rho proteins reside mainly in the cytosol and are targeted to the plasma membrane (PM) upon specific activation by guanine nucleotide exchange factors (GEFs). Accordingly, most GEFs are also cytosolic or associated with the PM. However, Net1, a RhoA-specific GEF predominantly localizes to the cell nucleus at steady-state. Nuclear localization for Net1 has been seen as a mechanism for sequestering the GEF away from RhoA, effectively rendering the protein inactive. However, considering the prominence of nuclear Net1 and the fact that a biological stimulus that promotes Net1 translocation out the nucleus to the cytosol has yet to be discovered, we hypothesized that Net1 might have a previously unidentified function in the nucleus of cells.Using an affinity precipitation method to pulldown the active form of Rho GEFs from different cellular fractions, we show here that nuclear Net1 does in fact exist in an active form, contrary to previous expectations. We further demonstrate that a fraction of RhoA resides in the nucleus, and can also be found in a GTP-bound active form and that Net1 plays a role in the activation of nuclear RhoA. In addition, we show that ionizing radiation (IR) specifically promotes the activation of the nuclear pool of RhoA in a Net1-dependent manner, while the cytoplasmic activity remains unchanged. Surprisingly, irradiating isolated nuclei alone also increases nuclear RhoA activity via Net1, suggesting that all the signals required for IR-induced nuclear RhoA signaling are contained within the nucleus.These results demonstrate the existence of a functional Net1/RhoA signaling pathway within the nucleus of the cell and implicate them in the DNA damage response

Student-Designed High-Throughput Assays to Assess Effects of Growth Insults in Budding Yeast

Modern high-throughput screening is a versatile method used widely in academic and industrial labs for genetic and pharmacological testing, but is underutilized in undergraduate laboratories.  We designed an open inquiry-based laboratory activity exploiting a small-scale high-throughput screening assay of the budding yeast S. cerevisiae to promote active learning and introduce students to modern data collection and analysis techniques. In this activity, students at both the high school and university levels generate growth curves of S. cerevisiae using a microplate reader after treatment with their insult of choice.  This lab presents an ideal opportunity for both high school and university students to actively learn about the process of science as well as high-throughput data collection and analysis in a cost-effective manner