Insights Into The Autoinhibition Mechanism Of The Tiam1 Guanine Nucleotide Exchange Factor

The T-cell lymphoma invasion and metastasis 1 (Tiam1) is a Dbl-family guanine nucleotide exchange factor (GEF) specific for the Rho-family GTPase Rac1. Mutations or aberrant regulation of Tiam1 have been implicated in oncogenic transformation of cells and linked to several kinds of invasive and metastatic forms of cancer. In addition to the C-terminal catalytic Dbl-homology (DH) and pleckstrin homology (PH) bi-domain (DH-PHc), Tiam1 contains several N-terminal protein-protein interaction domains (PHn-CC-Ex, RBD and PDZ) that are thought to contribute to the regulation of its GEF activity through inter-domain interactions. The mechanism for how the Tiam1 GEF function is regulated, however, remains poorly understood. Here, we show that truncation of N-terminal domains activates Tiam1 catalytic GEF function, while addition of the PHn-CC-Ex domain inhibited the catalytic activity of the DH-PHc domain in vitro. In addition, enzymatic kinetics experiments revealed that auto-inhibition of Tiam1 GEF function occurs through a competitive inhibition mechanism whereby the N-terminus decreases the substrate (GTPase Rac1) binding affinity, but does not change the maximum velocity of catalytic activity. We will also present ongoing single-molecule total internal reflection fluorescence (TIRF) data that characterize the kinetics of binding between Rac1 and the activated DH-PHc and inhibited PHn-PHc fragments. Finally, we derived a structural model for the Tiam1 PHn-PHc fragment based on small-angle X-ray scattering (SAXS) data. The model reveals an auto-inhibited conformation in which the N-terminal PHn-CC-Ex domain and linker regions block access of Rac1 to the catalytic bi-domain. Together, these data elucidate the biochemical and structural mechanisms for Tiam1 GEF regulation.