Tissue cells respond to changes in tensional forces with proliferation or death through the control of RhoA. However, the response coupling mechanisms that link force with RhoA activation are poorly understood. We found that tension applied to fibronectin-coated microbeads caused recruitment of all three isoforms of the Shc adapter (p66 Shc , p52 Shc , and p46 Shc ) to adhesion complexes. The Shc PTB domain was necessary and sufficient for this recruitment, and screening studies revealed the direct interactions with the FERM domain of focal adhesion kinase (FAK) that were required for Shc translocation to adhesion complexes. The FAK/p66Shc complex specifically bound and activated the Rho guanyl exchange factors (GEFs) p115-RhoGEF and GEF-H1, leading to tension-induced RhoA activation. In contrast, the FAK/p52Shc complex bound SOS1 but not the Rho GEFs to mediate tension-induced Ras activation. Nuclear translocation and activation of the YAP/TAZ transcription factors on firm substrates required the FAK/p66Shc /Rho GEF complex, and both proliferation on firm substrates and anoikis in suspension required signaling through p66Shc and its associated Rho GEFs. These studies reveal the binary and exclusive assignment of p66 Shc and p52Shc to tension-induced Rho or Ras signals, respectively, and suggest an integrated role for the two Shc isoforms in coordinating the cellular response to mechanical stimuli.

Objectives In response to ER stress, endothelial cells initiate corrective pathways such as the unfolded protein response (UPR). Recent studies suggest that reactive oxidant species (ROS) produced on the ER may participate in homeostatic signaling through Ras in response to ER stress. We sought to identify mechanisms responsible for this focal signaling pathway. Approach and Results In endothelial cells we found that ER stress induced by tunicamycin activates the NADPH oxidase Nox4 focally on the ER surface but not on the plasma membrane. Ras activation is also restricted to the ER, occurs downstream of Nox4, and is required for activation of the UPR. In contrast, treatment with the growth factor VEGF results in Ras activation and ROS production confined instead to the plasma membrane and not the ER, demonstrating local coupling of ROS-Ras signals. We further identify the calcium-responsive, ER resident guanyl exchange factors RasGRF1 and RasGRF2 as novel upstream mediators linking Nox4 with Ras activation in response to ER stress. Oxidation of the calcium pump SERCA and increases in cytosolic calcium caused by ER stress are blocked by Nox4 knockdown, and reduction in cytosolic free calcium prevents both Ras activation and the UPR. Conclusions ER stress triggers a localized signaling module on the ER surface involving Nox4-dependent calcium mobilization, which directs local Ras activation through ER-associated, calcium-responsive RasGRF.