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.