title: Phosphatidylinositol phosphate kinase type 1gamma and beta1-integrin cytoplasmic domain bind to the same region in the talin FERM domain.
abstract: Talin is an essential component of focal adhesions that couples beta-integrin cytodomains to F-actin and provides a scaffold for signaling proteins. Recently, the integrin beta3 cytodomain and phosphatidylinositol phosphate (PIP) kinase type 1gamma (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 microm, and the R358A mutation increased the Kd to 35 microm. Comparison of these results with those of the crystal structure of a beta3-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 beta1-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.
authors.affiliation: Department of Biochemistry, University of Leicester, University Road, Leicester LE1 7RH, United Kingdom.
authors.forename: Igor L
authors.initials: IL
authors.lastname: Barsukov
authors.forename: Andrew
authors.initials: A
authors.lastname: Prescot
authors.forename: Neil
authors.initials: N
authors.lastname: Bate
authors.forename: Bipin
authors.initials: B
authors.lastname: Patel
authors.forename: David N
authors.initials: DN
authors.lastname: Floyd
authors.forename: Nina
authors.initials: N
authors.lastname: Bhanji
authors.forename: Clive R
authors.initials: CR
authors.lastname: Bagshaw
authors.forename: Kresimir
authors.initials: K
authors.lastname: Letinic
authors.forename: Gilbert
authors.initials: G
authors.lastname: Di Paolo
authors.forename: Pietro
authors.initials: P
authors.lastname: De Camilli
authors.forename: Gordon C K
authors.initials: GC
authors.lastname: Roberts
authors.forename: David R
authors.initials: DR
authors.lastname: Critchley
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D003461:Crystallography; D003593:Cytoplasm / Q000378:metabolism; D005453:Fluorescence; D019012:Integrin beta1 / Q000737:chemistry / Q000378:metabolism*; D051379:Mice; D008956:Models, Chemical; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D051381:Rats; D016608:Talin / Q000737:chemistry / Q000378:metabolism*
chemical_list: D019012:Integrin beta1; D016608:Talin; D017853:Phosphotransferases (Alcohol Group Acceptor); C020077:1-phosphatidylinositol-4-phosphate 5-kinase