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Title: The Tiam1 guanine nucleotide exchange factor is auto-inhibited by its pleckstrin homology coiled-coil extension domain

Abstract

T-cell lymphoma invasion and metastasis 1 (Tiam1) is a Dbl-family guanine nucleotide exchange factor (GEF) that specifically activates the Rho-family GTPase Rac1 in response to upstream signals, thereby regulating cellular processes including cell adhesion and migration. Tiam1 contains multiple domains, including an N-terminal pleckstrin homology coiled-coiled extension (PH n-CC-Ex) and catalytic Dbl homology and C-terminal pleckstrin homology (DH-PH c) domain. Previous studies indicate that larger fragments of Tiam1, such as the region encompassing the N-terminal to C-terminal pleckstrin homology domains (PH n-PH c), are auto-inhibited. However, the domains in this region responsible for inhibition remain unknown. Here, we show that the PH n-CC-Ex domain inhibits Tiam1 GEF activity by directly interacting with the catalytic DH-PH c domain, preventing Rac1 binding and activation. Enzyme kinetics experiments suggested that Tiam1 is auto-inhibited through occlusion of the catalytic site rather than by allostery. Small angle X-ray scattering and ensemble modeling yielded models of the PH n-PH c fragment that indicate it is in equilibrium between “open” and “closed” conformational states. Lastly, single-molecule experiments support a model in which conformational sampling between the open and closed states of Tiam1 contributes to Rac1 dissociation. Our results highlight the role of the PH n-CC-Ex domain inmore » Tiam1 GEF regulation and suggest a combinatorial model for GEF inhibition and activation of the Rac1 signaling pathway.« less

Authors:
 [1];  [1];  [1];  [1]; ORCiD logo [1]
  1. Univ. of Iowa, Iowa City, IA (United States)
Publication Date:
Research Org.:
Bettis Atomic Power Laboratory (BAPL), West Mifflin, PA (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Basic Energy Sciences (BES) (SC-22). Scientific User Facilities Division; National Inst. of Health; National Science Foundation (NSF)
OSTI Identifier:
1432852
Grant/Contract Number:  
[AC02-05CH11231; AC02-06CH11357; R01 GM108617; MCB-0953080; NIH 5T32GM008365]
Resource Type:
Accepted Manuscript
Journal Name:
Journal of Biological Chemistry
Additional Journal Information:
[ Journal Volume: 292; Journal Issue: 43]; Journal ID: ISSN 0021-9258
Publisher:
American Society for Biochemistry and Molecular Biology
Country of Publication:
United States
Language:
ENGLISH
Subject:
59 BASIC BIOLOGICAL SCIENCES; enzyme kinetics; guanine nucleotide exchange factor (GEF); inhibition mechanism; Ras-related C3 botulinum toxin substrate 1 (Rac1); small-angle X-ray scattering (SAXS); single-molecule total internal reflection fluorescence microscopy; Tiam1; auto-inhibition; in vitro GEF assays

Citation Formats

Xu, Zhen, Gakhar, Lokesh, Bain, Fletcher E., Spies, Maria, and Fuentes, Ernesto J. The Tiam1 guanine nucleotide exchange factor is auto-inhibited by its pleckstrin homology coiled-coil extension domain. United States: N. p., 2017. Web. doi:10.1074/jbc.M117.799114.
Xu, Zhen, Gakhar, Lokesh, Bain, Fletcher E., Spies, Maria, & Fuentes, Ernesto J. The Tiam1 guanine nucleotide exchange factor is auto-inhibited by its pleckstrin homology coiled-coil extension domain. United States. doi:10.1074/jbc.M117.799114.
Xu, Zhen, Gakhar, Lokesh, Bain, Fletcher E., Spies, Maria, and Fuentes, Ernesto J. Thu . "The Tiam1 guanine nucleotide exchange factor is auto-inhibited by its pleckstrin homology coiled-coil extension domain". United States. doi:10.1074/jbc.M117.799114. https://www.osti.gov/servlets/purl/1432852.
@article{osti_1432852,
title = {The Tiam1 guanine nucleotide exchange factor is auto-inhibited by its pleckstrin homology coiled-coil extension domain},
author = {Xu, Zhen and Gakhar, Lokesh and Bain, Fletcher E. and Spies, Maria and Fuentes, Ernesto J.},
abstractNote = {T-cell lymphoma invasion and metastasis 1 (Tiam1) is a Dbl-family guanine nucleotide exchange factor (GEF) that specifically activates the Rho-family GTPase Rac1 in response to upstream signals, thereby regulating cellular processes including cell adhesion and migration. Tiam1 contains multiple domains, including an N-terminal pleckstrin homology coiled-coiled extension (PHn-CC-Ex) and catalytic Dbl homology and C-terminal pleckstrin homology (DH-PHc) domain. Previous studies indicate that larger fragments of Tiam1, such as the region encompassing the N-terminal to C-terminal pleckstrin homology domains (PHn-PHc), are auto-inhibited. However, the domains in this region responsible for inhibition remain unknown. Here, we show that the PHn-CC-Ex domain inhibits Tiam1 GEF activity by directly interacting with the catalytic DH-PHc domain, preventing Rac1 binding and activation. Enzyme kinetics experiments suggested that Tiam1 is auto-inhibited through occlusion of the catalytic site rather than by allostery. Small angle X-ray scattering and ensemble modeling yielded models of the PHn-PHc fragment that indicate it is in equilibrium between “open” and “closed” conformational states. Lastly, single-molecule experiments support a model in which conformational sampling between the open and closed states of Tiam1 contributes to Rac1 dissociation. Our results highlight the role of the PHn-CC-Ex domain in Tiam1 GEF regulation and suggest a combinatorial model for GEF inhibition and activation of the Rac1 signaling pathway.},
doi = {10.1074/jbc.M117.799114},
journal = {Journal of Biological Chemistry},
number = [43],
volume = [292],
place = {United States},
year = {2017},
month = {9}
}

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