␣-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.

Background: Cysteine-rich protein 1 (CRP1) is a LIM domain containing protein localized to the nucleus and the actin cytoskeleton. CRP1 has been demonstrated to bind the actin-bundling protein α-actinin and proposed to modulate the actin cytoskeleton; however, specific regulatory mechanisms have not been identified.

The active association-dissociation of dynamic protein-protein interactions is critical for the ability of the actin cytoskeleton to remodel. To determine the influence of phosphoinositide binding on the dynamic interaction of ␣-actinin with actin filaments and integrin adhesion receptors, fluorescence recovery after photobleaching (FRAP) microscopy was carried out comparing wild-type green fluorescent protein (GFP)-␣-actinin and a GFP-␣-actinin mutant with a decreased affinity for phosphoinositides (Fraley, T. S., Tran, T. C., Corgan, A. M., Nash, C. A., Hao, J., Critchley, D. R., and Greenwood, J. A. (2003) J. Biol. Chem. 278, 24039 -24045). In fibroblasts, recovery of the mutant ␣-actinin protein was 2.2 times slower than the wild type along actin stress fibers and 1.5 times slower within focal adhesions. FRAP was also measured in U87MG glioblastoma cells, which have higher levels of 3-phosphorylated phosphoinositides. As expected, ␣-actinin turnover for both the stress fiber and focal adhesion populations was faster in U87MG cells compared with fibroblasts with recovery of the mutant protein slower than the wild type along actin stress fibers. To understand the influence of ␣-actinin turnover on the modulation of the actin cytoskeleton, wild-type or mutant ␣-actinin was co-expressed with constitutively active phosphoinositide (PI) 3-kinase. Co-expression with the ␣-actinin mutant inhibited actin reorganization with the appearance of enlarged ␣-actinin containing focal adhesions. These results demonstrate that the binding of phosphoinositides regulates the association-dissociation rate of ␣-actinin with actin filaments and integrin adhesion receptors and that the dynamics of ␣-actinin is important for PI 3-kinaseinduced reorganization of the actin cytoskeleton. In conclusion, phosphoinositide regulation of ␣-actinin dynamics modulates the plasticity of the actin cytoskeleton influencing remodeling.The dynamic nature of the actin cytoskeleton is essential for various aspects of intracellular physiology and function as well as the ability of the cell to respond to signals stimulating cellular processes such as proliferation, differentiation, and migration. Individual protein molecules exchange between structures and the cytoplasm and/or membrane keeping the actin cytoskeleton pliable and able to quickly respond to signals inducing reorganization. In this report, we will refer to this dynamic exchange of proteins as turnover. Much of what we know about actin cytoskeletal dynamics has focused on the regulation of actin polymerization by specific actin-binding proteins during lamellipodial formation at the leading edge of migrating cells (1). It is largely unknown how actin filament bundling proteins influence actin cytoskeletal dynamics and remodeling in the cell. These proteins have not been thought to play an active role in the modulation of actin organization but have rather been cast in the more passive part as stabilizers of an actin cytoskeleton organized by the proteins regulating actin polymerization. In ...

Calpain-mediated proteolysis regulates cytoskeletal dynamics and is altered during aging and the progression of numerous diseases or pathological conditions. Although several cytoskeletal proteins have been identified as substrates, how localized calpain activity is regulated and the mechanisms controlling substrate recognition are not clear. In this study, we report that phosphoinositide binding regulates the susceptibility of the cytoskeletal adhesion protein ␣-actinin to proteolysis by calpains 1 and 2. At first, ␣-actinin did not appear to be a substrate for calpain 2; however, phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P 3 ) binding to ␣-actinin resulted in nearly complete proteolysis of the full-length protein, producing stable breakdown products. Calpain 1 was able to cleave ␣-actinin in the absence of phosphoinositide binding; however, PtdIns(3,4,5)P 3 binding increased the rate of proteolysis, and phosphatidylinositol 4,5-diphosphate (PtdIns(4,5)P 2 ) binding significantly inhibited cleavage. Phosphoinositide binding appeared to regulate calpain proteolysis of ␣-actinin by modulating the exposure of a highly sensitive cleavage site within the calponin homology 2 domain. In U87MG glioblastoma cells, which contain elevated levels of PtdIns(3,4,5)P 3 , ␣-actinin colocalized with calpain within dynamic actin cytoskeletal structures. Furthermore, proteolysis of ␣-actinin producing stable breakdown products was observed in U87MG cells treated with calcium ionophore to activate the calcium-dependent calpains. Additional evidence of PtdIns(3,4,5)P 3 -mediated calpain proteolysis of ␣-actinin was observed in rat embryonic fibroblasts. These results suggest that PtdIns(3,4,5)P 3 binding is a critical determinant for ␣-actinin proteolysis by calpain. In conclusion, phosphoinositide binding to the substrate is a potential mechanism for regulating susceptibility to proteolysis by calpain.Calpains 1 and 2 are ubiquitous calcium-dependent proteases that play an important role in the signaling of various cellular processes and have been implicated in the degeneration observed in numerous pathological conditions (1). The requirement of calcium concentrations above physiological levels, micromolar for calpain 1 and millimolar for calpain 2, has stimulated much investigation for other factors involved in the activation of calpain. Autolysis lowers the concentration of calcium required for half-maximal activity from 7.1 to 0.6 M for calpain 1 and from 1000 to 180 M for calpain 2 (2). However, there is currently no evidence that autolysis is required for calpain modulation in cells. PtdIns(4,5)P 2 3 binding also lowers the concentration of calcium required for the activation of calpain (3-5) and is a potential mechanism for regulating calpain during cell migration (6). More recently, Glading et al. (7-9) published a series of studies showing that phosphorylation on serine 50 by extracellular signal-regulated kinase activates calpain 2, mediating detachment of the rear of the cell during epidermal growth factor-i...

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