Molecular interaction between T-lymphoma invasion and metastasis 2 and the PB1 domain of p67phox

T-lymphoma invasion and metastasis 2 (Tiam2) is a closely related homolog to Tiam1, a guanine nucleotide exchange factor (GEF) that belongs to the Rho family of GTPases. The targeting of these GEFs to plasma membranes is essential for the spatiotemporal activation of Rac GTPases; it is mediated by the N-terminal PHCCEx (pleckstrin homology, coiled-coil, and extra region) domain that interacts with unique acidic peptide regions of approximately 30 amino acids of target proteins, including membrane proteins and scaffolding proteins. Here, we report that the Tiam2 PHCCEx domain binds to the PB1 domain of p67phox, which is a cytoplasmic regulatory subunit of the NADPH oxidase complex, highlighting a novel binding mode of the PHCCEx domain through the recognition of a structural domain fold but not of peptide-binding motifs. Mutation analyses showed that the PHCCEx domain makes contact with the negatively charged surface formed by the β3-β4-α2 region of the p67phox PB1 domain. Moreover, using an in vitro binding assay, we found that the p67phox PB1 domain interfered with the binding of Par3 to the PHCCEx domain, suggesting that the p67phox PB1 domain contributes to the stable association between the mechanosensory complex and the NADPH oxidase complex in shear stress-dependent ROS production. Our study provides a molecular mechanism for the activation of the NADPH oxidase complex during signal transduction via mechanistic stimulation.