TY - JOUR
T1 - The interplay of Rac1 activity, ubiquitination and GDI binding and its consequences for endothelial cell spreading
AU - Majolée, Jisca
AU - Podieh, Fabienne
AU - Hordijk, Peter L.
AU - Kovačević, Igor
N1 - Funding Information: FP was funded by NWO grant OCENW. klein.021. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Publisher Copyright: Copyright: © 2021 Majolée et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
PY - 2021/7/1
Y1 - 2021/7/1
N2 - Signaling by the Rho GTPase Rac1 is key to the regulation of cytoskeletal dynamics, cell spreading and adhesion. It is widely accepted that the inactive form of Rac1 is bound by Rho GDI, which prevents Rac1 activation and Rac1-effector interactions. In addition, GDI-bound Rac1 is protected from proteasomal degradation, in line with data showing that Rac1 ubiquitination occurs exclusively when Rac1 is activated. We set out to investigate how Rac1 activity, GDI binding and ubiquitination are linked. We introduced single amino acid mutations in Rac1 which differentially altered Rac1 activity, and compared whether the level of Rac1 activity relates to Rac1 ubiquitination and GDI binding. Results show that Rac1 ubiquitination and the active Rac1 morphology is proportionally increased with Rac1 activity. Similarly, we introduced lysine-to-arginine mutations in constitutively active Rac1 to inhibit site-specific ubiquitination and analyze this effect on Rac1 signaling output and ubiquitination. These data show that the K16R mutation inhibits GTP binding, and consequently Rac1 activation, signaling and-ubiquitination, while the K147R mutation does not block Rac1 signaling, but does inhibits its ubiquitination. In both sets of mutants, no direct correlation was observed between GDI binding and Rac1 activity or -ubiquitination. Taken together, our data show that a strong, positive correlation exists between Rac1 activity and its level of ubiquitination, but also that GDI dissociation does not predispose Rac1 to ubiquitination.
AB - Signaling by the Rho GTPase Rac1 is key to the regulation of cytoskeletal dynamics, cell spreading and adhesion. It is widely accepted that the inactive form of Rac1 is bound by Rho GDI, which prevents Rac1 activation and Rac1-effector interactions. In addition, GDI-bound Rac1 is protected from proteasomal degradation, in line with data showing that Rac1 ubiquitination occurs exclusively when Rac1 is activated. We set out to investigate how Rac1 activity, GDI binding and ubiquitination are linked. We introduced single amino acid mutations in Rac1 which differentially altered Rac1 activity, and compared whether the level of Rac1 activity relates to Rac1 ubiquitination and GDI binding. Results show that Rac1 ubiquitination and the active Rac1 morphology is proportionally increased with Rac1 activity. Similarly, we introduced lysine-to-arginine mutations in constitutively active Rac1 to inhibit site-specific ubiquitination and analyze this effect on Rac1 signaling output and ubiquitination. These data show that the K16R mutation inhibits GTP binding, and consequently Rac1 activation, signaling and-ubiquitination, while the K147R mutation does not block Rac1 signaling, but does inhibits its ubiquitination. In both sets of mutants, no direct correlation was observed between GDI binding and Rac1 activity or -ubiquitination. Taken together, our data show that a strong, positive correlation exists between Rac1 activity and its level of ubiquitination, but also that GDI dissociation does not predispose Rac1 to ubiquitination.
UR - http://www.scopus.com/inward/record.url?scp=85109782746&partnerID=8YFLogxK
U2 - https://doi.org/10.1371/journal.pone.0254386
DO - https://doi.org/10.1371/journal.pone.0254386
M3 - Article
C2 - 34252134
SN - 1932-6203
VL - 16
JO - PLOS ONE
JF - PLOS ONE
IS - 7 July
M1 - e0254386
ER -