␣-Actinin is an abundant actin-bundling and adhesion protein that directly links actin filaments to integrin receptors. Previously, in platelet-derived growth factortreated fibroblasts, we demonstrated that phosphoinositides bind to ␣-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 ␣-actinin function is further characterized. Phosphoinositide binding specificity, determined using a protein-lipid overlay procedure, suggests that ␣-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 ␣-actinin. Phosphoinositide binding inhibited the bundling activity of ␣-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 ␣-actinin mutant with decreased phosphoinositide affinity. We conclude that the interaction of ␣-actinin with phosphoinositides regulates actin stress fibers in the cell by controlling the extent to which microfilaments are bundled.

Alpha-actinin is a cell-adhesion and cytoskeletal protein that bundles actin microfilaments and links these filaments directly to integrin-adhesion receptors. Phosphoinositides bind to and regulate the interaction of a-actinin with actin filaments and integrin receptors. In the present study, we demonstrate that PtdIns(3,4,5)P3 inhibits and disrupts a-actinin-bundling activity, whereas PtdIns(4,5)P2 can only inhibit activity. In addition, a protease-sensitivity assay was developed to examine the flexibility of the linker region between the actin-binding domain and the spectrin repeats of a-actinin. Both phosphoinositides influenced the extent of proteolysis and the cleavage sites. PtdIns(4,5)P2 binding decreased the proteolysis of a-actinin, suggesting a role in stabilizing the structure of the protein. In contrast, PtdIns(3,4,5)P3 binding enhanced a-actinin proteolysis, indicating an increase in the flexibility of the protein. Furthermore, phosphoinositide binding influenced the proteolysis of the N- and C-terminal domains of a-actinin, indicating regulation of structure within both domains. These results support the hypothesis that PtdIns(4,5)P2 and PtdIns(3,4,5)P3 differentially regulate a-actinin function by modulating the structure and flexibility of the protein.