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

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


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).

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Guanine Nucleotide Exchange Factors / chemistry*
  • Guanine Nucleotide Exchange Factors / genetics
  • Guanine Nucleotide Exchange Factors / metabolism
  • Humans
  • Kinetics
  • Pleckstrin Homology Domains
  • Protein Binding
  • Signal Transduction / physiology
  • T-Lymphoma Invasion and Metastasis-inducing Protein 1
  • X-Ray Diffraction
  • rac1 GTP-Binding Protein / chemistry*
  • rac1 GTP-Binding Protein / genetics
  • rac1 GTP-Binding Protein / metabolism


  • Guanine Nucleotide Exchange Factors
  • RAC1 protein, human
  • T-Lymphoma Invasion and Metastasis-inducing Protein 1
  • TIAM1 protein, human
  • rac1 GTP-Binding Protein

Associated data

  • PDB/3A8N
  • PDB/3KZD
  • PDB/1FOE
  • PDB/4G3X