Phosphoinositide binding inhibits α-actinin bundling activity

Abstract

α-Actinin is an abundant actin-bundling and adhesion protein that directly links actin filaments to integrin receptors. Previously, in platelet-derived growth factor-treated fibroblasts, we demonstrated that phosphoinositides bind to α-actinin, regulating its localization (Greenwood, J. A., Theibert, A. B., Prestwich, G. D., and Murphy-Ullrich, J. E. (2000) J. Cell Biol. 150, 627-642). In this study, phosphoinositide binding and regulation of α-actinin function is further characterized. Phosphoinositide binding specificity, determined using a protein-lipid overlay procedure, suggests that α-actinin interacts with phosphates on the 4th and 5th position of the inositol head group. Binding assays and mutational analyses demonstrate that phosphoinositides bind to the calponin homology domain 2 of α-actinin. Phosphoinositide binding inhibited the bundling activity of α-actinin by blocking the interaction of the actin-binding domain with actin filaments. Consistent with these results, excessive bundling of actin filaments was observed in fibroblasts expressing an α-actinin mutant with decreased phosphoinositide affinity. We conclude that the interaction of α-actinin with phosphoinositides regulates actin stress fibers in the cell by controlling the extent to which microfilaments are bundled.