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J Biol Chem. 2017 Oct 27;292(43):17777-17793. doi: 10.1074/jbc.M117.799114. Epub 2017 Sep 7.

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

Author information

1
From the Department of Biochemistry.
2
Protein Crystallography Facility, and.
3
Holden Comprehensive Cancer Center, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, Iowa 52242.
4
From the Department of Biochemistry, ernesto-fuentes@uiowa.edu.

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 (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. Finally, 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.

KEYWORDS:

Ras-related C3 botulinum toxin substrate 1 (Rac1); Tiam1; auto-inhibition; enzyme kinetics; guanine nucleotide exchange factor (GEF); in vitro GEF assays; inhibition mechanism; single-molecule total internal reflection fluorescence microscopy; small-angle X-ray scattering (SAXS)

PMID:
28882897
PMCID:
PMC5663878
[Available on 2018-10-27]
DOI:
10.1074/jbc.M117.799114
[Indexed for MEDLINE]

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