Role of Cationic Groove and Hydrophobic Residues in Phosphatidylinositol-Dependent Membrane-Binding Properties of Tks5-Phox Homology Domain

摘要

Tyrosine kinase substrate with 5 Src homology 3 domains (Tks5) regulates several important cellular functions and is crucial for invadosome formation. The phox homology (PX) domain of Tks5 interacts with phosphatidylinositols (PIPs) and essential for Tks5-induced invadosome formation. However, PIP-binding specificity of the Tks5-PX domain is not clear and its membrane recruitment mechanism also not known. To understand how PX domain deferentially interacts with the PIPs, we quantitatively determined the PIP-binding properties. These measurements revealed that PX domain has strong PIP-binding selectivity for PI(3,4)P-2 and PI(3)P lipids. Mutational studies showed that the presence of hydrophobic residues at the putative membrane binding surface and basic residues within the PIP-binding pocket are essential for its membrane penetration and binding properties. In vitro PIP-selectivity correlates well with the cellular localization patterns of the wild-type and mutant PX domains in A549 cells. The hydrophobic residues play a crucial role in cellular localization and translocation studies. The mutational analysis allowed for understanding these cellular activities and results are in accordance with their membrane binding properties. Based on our findings, we describe characteristic membrane binding properties of Tks5-PX domain, in which the protein first interacts with specific PIP-containing membrane and then its hydrophobic residues partially penetrate into the membrane.