Journal Article

Regulation of local GTP availability controls RAC1 activity and cell invasion

Yun, Dong-Hyun, Sousa, Rui J., O’Brien Cox, Anderson, Feltri, Maria Laura, Kandel, Eugene S., Furdui, Cristina M., Fongang, Bernard, Nikiforov, Mikhail A., Marston, Daniel J., Chen, Jialin, Qu, Jun, Kengne, Jules Berlin Nde, Hollis, Thomas, Hurley, Edward, Bianchi-Smiraglia, Anna, Mussell, Ashley L., Kandel, Mikhail E., Moparthy, Sudha, Wombacher, Rebecca M., Deng, Zhiyong, Shen, Shichen, Hahn, Klaus M., Wolff, David W., Han, Zhannan

2021

Overview

Physiological changes in GTP levels in live cells have never been considered a regulatory step of RAC1 activation because intracellular GTP concentration (determined by chromatography or mass spectrometry) was shown to be substantially higher than the in vitro RAC1 GTP dissociation constant (RAC1-GTP Kd). Here, by combining genetically encoded GTP biosensors and a RAC1 activity biosensor, we demonstrated that GTP levels fluctuating around RAC1-GTP Kd correlated with changes in RAC1 activity in live cells. Furthermore, RAC1 co-localized in protrusions of invading cells with several guanylate metabolism enzymes, including rate-limiting inosine monophosphate dehydrogenase 2 (IMPDH2), which was partially due to direct RAC1-IMPDH2 interaction. Substitution of endogenous IMPDH2 with IMPDH2 mutants incapable of binding RAC1 did not affect total intracellular GTP levels but suppressed RAC1 activity. Targeting IMPDH2 away from the plasma membrane did not alter total intracellular GTP pools but decreased GTP levels in cell protrusions, RAC1 activity, and cell invasion. These data provide a mechanism of regulation of RAC1 activity by local GTP pools in live cells. Changes in intracellular GTP levels are not considered as a regulatory event in RAC1 activation in live cells since total GTP levels are substantially higher than the RAC1 GTP dissociation constant determined in vitro. Here, the authors demonstrate that the availability of free GTP in live cells controls the activity of RAC1 and cell invasion.

• Publisher: Nature Publishing Group UK,Nature Publishing Group,Nature Portfolio
• Subject: 13/106 / 13/89 / 13/95 / 14/33 / 14/34 / 38 / 38/23 / 38/39 / 631/80/86/2341 / 631/92/1643 / Availability / Biosensors / Cell activation / Cell Membrane - metabolism / Cell Movement / Genetic code / Guanosine triphosphate / Guanosine Triphosphate - chemistry / Guanosine Triphosphate - metabolism / HEK293 Cells / Humanities and Social Sciences / Humans / IMP Dehydrogenase - genetics / IMP Dehydrogenase - metabolism / Inosine monophosphate / Intracellular / Kinetics / Mass spectrometry / Mass spectroscopy / multidisciplinary / Protein Binding / rac1 GTP-Binding Protein - chemistry / rac1 GTP-Binding Protein - genetics / rac1 GTP-Binding Protein - metabolism / Rac1 protein / Regulation / Science / Science (multidisciplinary)