Phosphoinositides differentially regulate α-actinin flexibility and function

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 alpha-actinin with actin filaments and integrin receptors. In the present study, we demonstrate that PtdIns(3,4,5)P-3 inhibits and disrupts a-actinin-bundling activity, whereas PtdIns(4,5)P-2 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 alpha-actinin. Both phosphoinositides influenced the extent of proteolysis and the cleavage sites. PtdIns(4,5)P-2 binding decreased the proteolysis of alpha-actinin, suggesting a role in stabilizing the structure of the protein. In contrast, PtdIns(3,4,5)P-3 binding enhanced alpha-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)P-2 and PtdIns(3,4,5)P-3 differentially regulate alpha-actinin function by modulating the structure and flexibility of the protein.