Phosphoinositide Binding Inhibits α-Actinin Bundling Activity

Abstract

␣-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.

Association of Villin with Phosphatidylinositol 4,5-Bisphosphate Regulates the Actin Cytoskeleton

“…For instance PIP 2 has been shown to enhance the actin cross-linking function of ␣-actinin (69) as well as inhibit the bundling activity of ␣-actinin (16). In this regard, studies done with the actin depolymerizing protein cofilin and its association with PIP 2 are relevant (70).…”

“…PIP 2 1 modulates many actin regulatory proteins in vitro and includes the following: actin severing and/or capping proteins (6 -8); monomer-binding proteins (9 -11); the actin-nucleating protein, N-WASP/Arp2/3 (12); and actin cross-linking proteins (13,14). It has been hypothesized that PIP 2 induces actin assembly by dissociating capping proteins from filament ends, releasing actin monomers from actin-sequestering proteins, inhibiting actin severing, stimulating actin nucleation, and increasing or decreasing actin cross-linking (1,13,(15)(16)(17). In general, PIP 2 predisposes the actin cytoskeleton to a "polymerization mode" and assists its remodeling as well as its interaction with the plasma membrane.…”

The V-ATPase Subunit C Binds to Polymeric F-actin as Well as to Monomeric G-actin and Induces Cross-linking of Actin Filaments

“…Low Centrifugal Force Sedimentation of Cross-linked Actin Filaments-Bundling of F-actin was determined by standard sedimentation assays (17)(18)(19). 10 M G-actin was polymerized in buffer F in the presence of 0.4 M human gelsolin (cytoskeleton) to get similar filament sizes and then stabilized with 10 M phalloidin.…”

Integrins in cell migration – the actin connection

“…This regulation could occur at many levels, including phosphorylation of linkage components such as α-actinin (Izaguirre et al, 2001) or interaction with phosphatidylinositol (4,5)-bisphosphate (PIP2), which regulates the affinity of α-actinin and vinculin for actin and talin, respectively (Fraley et al, 2003;Gilmore and Burridge, 1996). Another point of regulation is the modulation of integrin affinity (Kim et al, 2003), which is activated by talin (Tadokoro et al, 2003), probably through Rap1-RIAM (Rap1 interacting adaptor molecule) or kindlin-2 signaling pathways (Han et al, 2006;Montanez et al, 2008).…”