An Interaction between Zyxin and aActinin

Zyxin is an 82-kD protein first identified as a component of adhesion plaques and the termini of stress fibers near where they associate with the cytoplasmic face of the adhesive membrane. We report here that zyxin interacts with the actin cross-linking protein a-actinin . Zyxin cosediments with filamentous actin in an a-actinin-dependent manner and an association between zyxin and a-actinin is observed in solution by analytical gel filtration . The specificity of the interaction between zyxin and a-actinin was demonstrated by blot overlay experiments in which 125 I-zyxin recognizes most prominently a-actinin among a complex mixture of proteins extracted from avian smooth muscle . By these blot overlay binding studies, we determined that zyxin interacts with the NH2-terminal CELLS can adhere to extracellular matrix and each other through specializations in the plasma membrane termed adherens junctions . These membraneassociatedjunctional complexes are associated with bundles ofactin filaments that impinge on the cytoplasmic face ofthe plasma membrane (Singer, 1979) . In cultured fibroblasts, these actin arrays are often organized into structures called stress fibers that terminate at adhesion plaques, areas ofclose membrane-substratum apposition that can be visualized directly by interference reflection microscopy (Heath and Dunn, 1978 ; Wehland et al ., 1979) . One of the principal proteins believed to be involved in organizing individual actin filaments into stress fibers is the actin-binding protein, a-actinin. a-Actinin forms antiparallel homodimers (Wallraffetal ., 1986 ; Imamura et al ., 1988) that have the capacity to cross-link actin filaments in vitro (Maruyama and Ebashi, 1965; Podlubnaya et al ., 1975) . By immunofluorescence, «-actinin is found periodically along stress fibers as they traverse the cytoplasm and is also located at the termini ofthese actin filament bundles in the adhesion plaque itself (Lazarides and Burridge, 1975) . Because of its presence at sites where cells contact the extracellular matrix, a-actinin has been postulated to be involved in the attachment of the cytoskeletal framework to the plasma membrane (Lazarides and Burridge, 1975) and the ability of a-actinin to interact with other known adhesion plaque proteins has been investigated extensively. In addition to binding actin, «-actinin has been shown to interact with other proteins involved in membrane-cyto© The Rockefeller University Press, 0021-9525/92/03/1381/13 $2 .00 The Journal of Cell Biology, Volume 116, Number 6, March 1992 1381-1393 27-kD domain of a-actinin, a region that also contains the actin binding site. Solid phase binding assays were performed to evaluate further the specificity of the binding and to determine the affinity of the zyxina-actinin interaction . By these approaches we have demonstrated a specific, saturable, moderate-affinity interaction between zyxin and a-actinin . Furthermore, double-label immunofluorescence reveals that zyxin and a-actinin exhibit extensive overlap in their subcellular distributions in both chicken embryo fibroblasts and pigmented retinal epithelial cells . The significant colocalization of the two proteins is consistent with the possibility that the interaction between zyxin and a-actinin has a biologically relevant role in coordinating membrane-cytoskeletal interactions . skeleton interaction . An association between a-actinin and vinculin, a 116-kD cytoplasmic component of adhesion plaques (Geiger, 1979; Burridge and Feramisco, 1980), was demonstrated by blot overlay techniques using ' 251-aactinin (Belkin and Koteliansky, 1987) and by solution binding assays (Wachsstock et al ., 1987) . Subsequently (Otey et al ., 1990), it was shown by solid phase binding assays that a-actinin can also interact with the cytoplasmic domain of the ß l subunit of integrin, a heterodimeric transmembrane receptor for extracellular matrix molecules found at sites of cell-substratum interaction (Damsky et al ., 1985 ; Chen et al ., 1985) . Thus a-actinin may contribute to membrane-cytoskeletal interactions by more than one mechanism . In one scheme, stress fibers may be tethered directly to the membrane by a-actinin molecules bound to the cytoplasmic domain of ß,-integrins . Alternatively, stress fibers may be attached to the membrane in a less-direct manner through a complex linkage ofproteins assembled at adhesion plaques . Consistent with this multiprotein model, the adhesion plaque protein, talin (Burridge and Connell, 1983 ; Beckerle and Yeh, 1990), has been shown to interact with both integrin (Horwitz et al ., 1986) and vinculin (Burridge and Mangeat, 1984) . Based on a number ofin vitro biochemical studies, it has been proposed that associations among a-actinin, vinculin, talin, and integrin could also contribute to anchoring actin filaments at sites of adhesion (for reviews see Burridge et al ., 1988 ; Crawford and Beckerle, 1990) . Our understanding of the molecular complexity of adhesion plaques continues to increase as more components and pro1381 on F ebuary 1, 2013 jcb.rress.org D ow nladed fom Published March 15, 1992