title: Identification of a phosphatidylinositol 4,5-bisphosphate-binding site in chicken skeletal muscle alpha-actinin.
abstract: We previously reported that phosphatidylinositol 4,5-bisphosphate (PIP2) dramatically increases the gelating activity of smooth muscle alpha-actinin (Fukami, K., Furuhashi, K., Inagaki, M., Endo, T., Hatano, S., and Takenawa, T. (1992) Nature 359, 150-152) and that the hydrolysis of PIP2 on alpha-actinin by tyrosine kinase activation may be important in cytoskeletal reorganization (Fukami, K., Endo, T., Imamura, M., and Takenawa, T. (1994) J. Biol. Chem. 269, 1518-1522). Here we report that a proteolytic fragment with lysylendopeptidase comprising amino acids 168-184 (TAPYRNVNIQNFHLSWK) from striated muscle alpha-actinin contains a PIP2-binding site. A synthetic peptide composed of the 17 amino acids remarkably inhibited the activities of phospholipase C (PLC)-gamma 1 and -delta 1. Furthermore, we detected an interaction between PIP2 and a bacterially expressed alpha-actinin fragment (amino acids 137-259) by PLC inhibition assay. Point mutants in which arginine 172 or lysine 184 of alpha-actinin were replaced by isoleucine reduced the inhibitory effect on PLC activity by nearly half. Direct interactions between PIP2 and the peptide (amino acids 168-184) or the bacterially expressed protein (amino acids 137-259) were confirmed by enzyme-linked immunosorvent assay. We also found this region homologous to the sequence of the PIP2-binding site in spectrin and the pleckstrin homology domains of PLC-delta 1 and Grb7. Synthetic peptides from the homologous regions in spectrin and PLC-delta 1 inhibited PLC activities. These results indicate that residues 168-184 comprise a binding site for PIP2 in alpha-actinin and that similar sequences found in spectrin and PLC-delta 1 may be involved in the interaction with PIP2.
authors.affiliation: Department of Molecular Oncology, University of Tokyo, Japan.
authors.forename: K
authors.initials: K
authors.lastname: Fukami
authors.forename: N
authors.initials: N
authors.lastname: Sawada
authors.forename: T
authors.initials: T
authors.lastname: Endo
authors.forename: T
authors.initials: T
authors.lastname: Takenawa
mesh_terms: D000185:Actinin / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002645:Chickens; D017931:DNA Primers; D008969:Molecular Sequence Data; D018482:Muscle, Skeletal / Q000378:metabolism*; D010455:Peptides / Q000494:pharmacology; D019269:Phosphatidylinositol 4,5-Diphosphate; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism*; D011994:Recombinant Proteins / Q000494:pharmacology; D017386:Sequence Homology, Amino Acid; D010738:Type C Phospholipases / Q000037:antagonists & inhibitors
chemical_list: D017931:DNA Primers; D010455:Peptides; D019269:Phosphatidylinositol 4,5-Diphosphate; D018129:Phosphatidylinositol Phosphates; D011994:Recombinant Proteins; D000185:Actinin; D010738:Type C Phospholipases