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. 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

Zhen Xu  1 , Lokesh Gakhar  1   2 , Fletcher E Bain  1 , Maria Spies  1   3 , Ernesto J Fuentes  4   3
Affiliations

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

Zhen Xu et al. J Biol Chem. .

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).

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Conflict of interest statement

The authors declare that they have no conflicts of interest with the contents of this article

Figures

Figure 1.
Figure 1.
A schematic of Tiam1 domain architecture. A, myristoylation site is indicated by the twisting line at the N terminus. P, PEST region; PDZ, PSD-95/DlgA/ZO-1 domain. The N-terminal 50 amino acids (N50) are auto-inhibitory. B, SDS-PAGE gel of purified Rac1 and Tiam1 fragments used in this study.
Figure 2.
Figure 2.
Tiam1 was auto-inhibited by structured domains located N-terminal of the catalytic DH-PHc domain. A, the exchange of MANT-GDP from Rac1 was measured in the presence of Tiam1 DH-PHc, PDZ-PHc, or PHn-PHc fragments. Rac1 alone and EDTA were negative and positive controls, respectively. Data represent the average of three independent reactions for each trace. B, quantification of Tiam1 catalyzed exchange reactions. *, determined by a pairwise t test assuming equal variances.
Figure 3.
Figure 3.
The Tiam1 PHn-CC-Ex domain directly inhibited the GEF activity of the catalytic DH-PHc domain. A, the exchange of MANT-GDP from Rac1, as monitored by the fluorescence intensity, was measured in the presence of Tiam1 DH-PHc alone or mixed with increasing concentrations of the PHn-CC-Ex fragment. The Tiam1 DH-PHc protein in the presence of the PHn-CC-Ex fragment showed reduced activity compared with Tiam1 DH-PHc domain alone. B, quantification of Tiam1 catalyzed exchange reactions. *, determined by a pairwise t test assuming equal variances.
Figure 4.
Figure 4.
Enzyme kinetics of inhibited and active Tiam1 proteins. A and C, progress of Tiam1 DH-PHc or PHn-PHc (0.5 μm) catalyzed exchange reactions in the presence of Rac1 (1–32 μm for DH-PHc or 1–64 μm for PHn-PHc). The changes in fluorescence intensity corresponding to Tiam1-mediated exchange of MANT-GDP were monitored over time. a.u., arbitrary fluorescence units. B and D, plot of specific activity of Tiam1 DH-PHc or PHn-PHc as a function of Rac1 concentration. The data were fit to the Michaelis-Menten equation to determine the apparent Km and Vmax kinetic parameters.
Figure 5.
Figure 5.
SAXS analysis of Tiam1 proteins. A, experimental SAXS profiles (logarithmic scale) for Tiam1 PDZ-PHc was collected at ALS, whereas the Tiam1 DH-PHc and PHn-PHc datasets were collected at the APS (see “Experimental procedures” for details). B, comparison of a Guinier plot at low q-range (q < 1.3/Rg). The intensity is represented by the relationship I(q) = I(0)exp(−(q × Rg)2/3). The curves were arbitrarily scaled for clarity. The calculated reciprocal space Rg values are presented in Table 3. C, normalized pair distribution function, P(r), for Tiam1 DH-PHc, PDZ-PHc, and PHn-PHc calculated in AutoGNOM. D, normalized Kratky plots for Tiam1 DH-PHc, PDZ-PHc, and PHn-PHc.
Figure 6.
Figure 6.
SAXS-based structural models of Tiam1 PHn-PHc. A, ab initio model of Tiam1 PHn-PHc protein from SAXS data using DAMMIN and rigid-body modeling in CORAL. B, ensemble structural models for Tiam1 PHn-PHc determined by the program BILBOMD. C, ensemble structural models for Tiam1 PHn-PHc determined by the program EOM2.0. D, comparison of experimental scattering curve and the fitted scattering curves for Tiam1 PHn-PHc based on the rigid-body model (CORAL) and ensemble models (BILBOMD and EOM2.0).
Figure 7.
Figure 7.
Single-molecule TIRF experiments of Tiam1/Rac1 kinetic interactions. A, schematic of the single-molecule TIRF experiment. N-terminally biotinylated Tiam1 DH-PHc (gray) or PHn-PHc constructs were tethered to the PEG-passivated slide surface through a biotin/neutravidin interaction. Cy3-labeled Rac1 was introduced to the chamber, and the Cy3 fluorescence monitored as Rac1-Cy3 entered the evanescent wave. Persistence of the fluorescence signal for at least three frames within a diffraction limited spot was indicative of Rac1 binding to the Tiam1 DH-PHc or PHn-PHc construct. B and C, representative traces of the Tiam1 DH-PHc/Rac1 and Tiam1 PHn-PHc/Rac1 interaction. The raw fluorescence signal is shown as a green line. The black line represents the idealized fit of the data using hidden Markov modeling, from which bound and unbound durations were determined. D and E, histogram of binned data for each Rac1 protein concentration and the global fit of the data to a double exponential decay function. Insets are the residuals of the globally fit data. AU, arbitrary fluorescence units.
Figure 8.
Figure 8.
Combinatorial model of Tiam1 auto-inhibition and activation. Full-length Tiam1 is in equilibrium between inactive and fully auto-inhibited forms, where the N50 and the PHn-CC-Ex domain combine to prevent Rac1 from accessing the catalytic DH-PHc domain. Full Tiam1 activation likely follows multiple steps: 1) phosphorylation of Ser-29 and Ser-33 by aPKCγ releases the PHn-CC-Ex/N50 interaction; 2) phosphorylation of Tyr-829 (by protein kinases TrkB etc.) and/or protein/protein interactions between of the PHn-CC-Ex and/or RBD domains with partner proteins (e.g. Par3 and Ras, respectively) relieving the PHn-CC-Ex/DH-PHc interaction. Once fully activated, Tiam1 interacts with Rac1 to promote GDP to GTP nucleotide exchange and downstream signaling.
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