Phosphatidylinositol phosphate kinase type 1γ and β 1-integrin cytoplasmic domain bind to the same region in the talin FERM domain

Abstract

Talin is an essential component of focal adhesions that couples β-integrin cytodomains to F-actin and provides a scaffold for signaling proteins. Recently, the integrin β3 cytodomain and phosphatidylinositol phosphate (PIP) kinase type 1γ (a phosphatidylinositol 4,5-bisphosphate-synthesizing enzyme) were shown to bind to the talin FERM domain (subdomain F3). We have characterized the PIP kinase-binding site by NMR using a 15N-labeled talin F2F3 polypeptide. A PIP kinase peptide containing the minimal talin-binding site formed a 1:1 complex with F2F3, causing a substantial number of chemical shift changes. In particular, two of the three Arg residues (Arg339 and Arg358), four of eight Ile residues, and one of seven Val residues in F3 were affected. Although a R339A mutation did not affect the exchange kinetics, R358A or R358K mutations markedly weakened binding. The Kd for the interaction determined by Trp fluorescence was 6 μM, and the R358A mutation increased the Kd to 35 μM. Comparison of these results with those of the crystal structure of a β3-integrin cytodomain talin F2F3 chimera shows that both PIP kinase and integrins bind to the same surface of the talin F3 subdomain. Indeed, binding of talin present in rat brain extracts to a glutathione S-transferase integrin β 1-cytodomain polypeptide was inhibited by the PIP kinase peptide. The results suggest that ternary complex formation with a single talin FERM domain is unlikely, although both integrins and PIP kinase may bind simultaneously to the talin anti-parallel dimer.