Regulation of local GTP availability controls RAC1 activity and cell invasion

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

doi:10.1038/s41467-021-26324-6

Regulation of local GTP availability controls RAC1 activity and cell invasion

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

Mikhail E. Kandel, Eugene S. Kandel, Klaus M. Hahn, Mikhail A. Nikiforov

Pharmaceutical Sciences

University at Buffalo

Roswell Park Cancer Institute
Wake Forest University
University of North Carolina at Chapel Hill
SUNY Buffalo
University of Houston
University of Texas Health Science Center at San Antonio
Groq Inc.

Research output: Contribution to journal › Article › peer-review

37 Scopus citations

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.

Original language	English
Article number	6091
Journal	Nature Communications
Volume	12
Issue number	1
DOIs	https://doi.org/10.1038/s41467-021-26324-6
State	Published - Dec 1 2021

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Bianchi-Smiraglia, A., Wolff, D. W., Marston, D. J., Deng, Z., Han, Z., Moparthy, S., Wombacher, R. M., Mussell, A. L., Shen, S., Chen, J., Yun, D. H., O’Brien Cox, A., Furdui, C. M., Hurley, E., Feltri, M. L., Qu, J., Hollis, T., Kengne, J. B. N., Fongang, B., ... Nikiforov, M. A. (2021). Regulation of local GTP availability controls RAC1 activity and cell invasion. Nature Communications, 12(1), Article 6091. https://doi.org/10.1038/s41467-021-26324-6

@article{e4374f3e2b25458d952a56ffc54f5865,

title = "Regulation of local GTP availability controls RAC1 activity and cell invasion",

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.",

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

note = "Publisher Copyright: {\textcopyright} 2021, The Author(s).",

year = "2021",

month = dec,

day = "1",

doi = "10.1038/s41467-021-26324-6",

language = "English",

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Bianchi-Smiraglia, A, Wolff, DW, Marston, DJ, Deng, Z, Han, Z, Moparthy, S, Wombacher, RM, Mussell, AL, Shen, S, Chen, J, Yun, DH, O’Brien Cox, A, Furdui, CM, Hurley, E, Feltri, ML, Qu, J, Hollis, T, Kengne, JBN, Fongang, B, Sousa, RJ, Kandel, ME, Kandel, ES, Hahn, KM & Nikiforov, MA 2021, 'Regulation of local GTP availability controls RAC1 activity and cell invasion', Nature Communications, vol. 12, no. 1, 6091. https://doi.org/10.1038/s41467-021-26324-6

TY - JOUR

T1 - Regulation of local GTP availability controls RAC1 activity and cell invasion

AU - Bianchi-Smiraglia, Anna

AU - Wolff, David W.

AU - Marston, Daniel J.

AU - Deng, Zhiyong

AU - Han, Zhannan

AU - Moparthy, Sudha

AU - Wombacher, Rebecca M.

AU - Mussell, Ashley L.

AU - Shen, Shichen

AU - Chen, Jialin

AU - Yun, Dong Hyun

AU - O’Brien Cox, Anderson

AU - Furdui, Cristina M.

AU - Hurley, Edward

AU - Feltri, Maria Laura

AU - Qu, Jun

AU - Hollis, Thomas

AU - Kengne, Jules Berlin Nde

AU - Fongang, Bernard

AU - Sousa, Rui J.

AU - Kandel, Mikhail E.

AU - Kandel, Eugene S.

AU - Hahn, Klaus M.

AU - Nikiforov, Mikhail A.

PY - 2021/12/1

Y1 - 2021/12/1

N2 - 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.

AB - 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.

UR - https://www.scopus.com/pages/publications/85117716858

U2 - 10.1038/s41467-021-26324-6

DO - 10.1038/s41467-021-26324-6

M3 - Article

C2 - 34667203

SN - 2041-1723

VL - 12

JO - Nature Communications

JF - Nature Communications

IS - 1

M1 - 6091

ER -

Regulation of local GTP availability controls RAC1 activity and cell invasion

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