An alpha-actinin binding site of zyxin is essential for subcellular zyxin localization and alpha-actinin recruitment

The LIM domain protein zyxin is a component of adherens type junctions, str ess fibers, and highly dynamic membrane areas and appears to be involved in microfilament organization. Chicken zyxin and its human counterpart displa y less than 60% sequence identity, raising concern about their functional i dentity. Here, we demonstrate that human zyxin, like the avian protein, spe cifically interacts with alpha-actinin. Furthermore, we map the interaction site to a motif of approximately 22 amino acids, present in the N-terminal domain of human zyxin, This motif is both necessary and sufficient for alp ha-actinin binding, whereas a downstream region, which is related in sequen ce, appears to be dispensable. A synthetic peptide comprising human zyxin r esidues 21-42 specifically binds to alpha-actinin in solid phase binding as says. In contrast to full-length zyxin, constructs lacking this motif do no t interact with alpha-actinin in blot overlays and fail to recruit alpha-ac tinin in living cells. When zyxin lacking the alpha actinin binding site is expressed as a fusion protein with green fluorescent protein, association of the recombinant protein with stress fibers is abolished, and targeting t o focal adhesions is grossly impaired. Our results suggest a crucial role f or the alpha-actinin-zyxin interaction in subcellular zyxin localization an d microfilament organization.