Characterization of the intramolecular interactions and regulatory mechanisms of the scaffold protein tks4

The scaffold protein Tks4 is a member of the p47phox‐related organizer superfamily. It plays a key role in cell motility by being essential for the formation of podosomes and invadopodia. In addition, Tks4 is involved in the epidermal growth factor (EGF) signaling pathway, in which EGF induces the translocation of Tks4 from the cytoplasm to the plasma membrane. The evolutionarily‐related protein p47phox and Tks4 share many similarities in their N‐terminal region: a phosphoinositide‐binding PX domain is followed by two SH3 domains (so called “tandem SH3”) and a proline‐rich region (PRR). In p47phox, the PRR is followed by a relatively short, disordered C‐terminal tail region containing multiple phosphorylation sites. These play a key role in the regulation of the protein. In Tks4, the PRR is followed by a third and a fourth SH3 domain connected by a long (~420 residues) unstructured region. In p47phox, the tandem SH3 domain binds the PRR while the first SH3 domain interacts with the PX domain, thereby preventing its binding to the membrane. Based on the conserved structural features of p47phox and Tks4 and the fact that an intramolecular interaction between the third SH3 and the PX domains of Tks4 has already been reported, we hypothesized that Tks4 is similarly regulated by autoinhibition. In this study, we showed, via fluorescence‐based titrations, MST, ITC, and SAXS measurements, that the tandem SH3 domain of Tks4 binds the PRR and that the PX domain interacts with the third SH3 domain. We also investigated a phosphomimicking Thr‐to‐Glu point mutation in the PRR as a possible regulator of intramolecular interactions. Phosphatidylinositol‐3‐phosphate (PtdIns(3)P) was identified as the main binding partner of the PX domain via lipid‐binding assays. In truncated Tks4 fragments, the presence of the tandem SH3, together with the PRR, reduced PtdIns(3)P binding, while the presence of the third SH3 domain led to complete inhibition. © 2021 by the authors. Licensee MDPI, Basel, Switzerland.