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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.

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From the Department of Biochemistry.
Protein Crystallography Facility, and.
Holden Comprehensive Cancer Center, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, Iowa 52242.
From the Department of Biochemistry,


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.


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)

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