Regulation of local GTP availability controls RAC1 activity and cell invasion

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

doi:10.1038/s41467-021-26324-6

Abstract

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.

Highlights

Physiological changes in GTP levels in live cells have never been considered a regulatory step of related C3 botulinum toxin substrate 1 (RAC1) activation because intracellular GTP concentration was shown to be substantially higher than the in vitro RAC1 GTP dissociation constant (RAC1-GTP Kd)
The switch between GTP- and GDP-bound RAC1 is tightly regulated by GTPase-activating proteins (GAPs) that promote GTP hydrolysis and render RAC1 inactive[7,8], and guanine nucleotide exchange factors (GEFs) that promote the release of GDP from RAC17,8
Using GEVAL sensors in combination with sensors for RAC1 activity and conventional biochemical, molecular, and cell biology methods, we provide experimental evidence demonstrating that interactions with inosine monophosphate dehydrogenase 2 (IMPDH2) and generation of local availability of GTP represent an important mechanism of RAC1 activation in live cells

Summary

Introduction

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). 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. 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. The switch between GTP- and GDP-bound RAC1 is tightly regulated by GTPase-activating proteins (GAPs) that promote GTP hydrolysis and render RAC1 inactive[7,8], and guanine nucleotide exchange factors (GEFs) that promote the release of GDP from RAC17,8. Cancer cells often display altered expression of enzymes for the de novo biosynthesis of nucleotides, especially purines, highlighting their dependency on these pathways and substrates[10,11,12,13,14,15,16]

Methods

Results

Conclusion