Regulation of RhoA GTPase signaling by guanine nucleotide exchange factors in response to extracellular matrix adhesion and DNA damage

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  • March 21, 2019
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  • Dubash, Adi Dara
    • Affiliation: School of Medicine, Department of Cell Biology and Physiology
Abstract
  • The Rho family of GTPases are proteins which regulate a large number of biological functions, including cell proliferation, survival, adhesion, and migration. Rho proteins cycle between an inactive GDP-bound form, and an active GTP-bound form. This regulatory cycle is controlled by guanine nucleotide exchange factors (GEFs) which activate Rho proteins by causing exchange of GDP for GTP. While significant advances have been made in understanding how Rho proteins control downstream pathways (specifically cytoskeletal structure), less is known about how GTPase activation is regulated by GEFs in response to specific extracellular signals. Adhesion of cells to components of the the extracellular matrix, such as fibronectin (FN), leads to activation of RhoA. We therefore wanted to determine which GEFs were responsible for activation of RhoA downstream of FN adhesion. Using an affinity pulldown for activated GEFs, we show that the RhoA-specific GEFs Lsc/p115 RhoGEF and LARG are activated when cells are plated onto FN, but not other GEFs such as Ect2 or Dbl. Knockdown of Lsc and LARG together significantly decreases RhoA activation, and stress fiber and focal adhesion formation downstream of FN adhesion. Similarly, overexpression of a catalytically inactive mutant of Lsc/p115 RhoGEF inhibits RhoA activity, stress fiber and focal adhesion formation on FN. These data establish a previously uncharacterized role for the exchange factors Lsc/p115 RhoGEF and LARG in linking FN signals to downstream RhoA activation. RhoA has also been shown to be activated in response to stimuli that causes double stranded DNA breaks, such as ionizing radiation (IR). Activation of RhoA in response to DNA damage causes upregulation of the p38 mitogen activated protein kinase (MAPK) signaling pathway. While the majority of RhoA is present in the cytoplasm of cells, we show for the first time that a small fraction of RhoA localizes to the nucleus, and that activity of the nuclear pool of RhoA is increased in response to IR. IR also causes activation of the nuclear localized RhoA GEF, Net1. These data therefore establish a previously unappreciated role for Net1 and RhoA signaling in the nucleus of cells in response to DNA damage.
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  • In Copyright
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  • Burridge, Keith
Degree granting institution
  • University of North Carolina at Chapel Hill
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