Actopaxin, a New Focal Adhesion Protein That Binds Paxillin Ld Motifs and Actin and Regulates Cell Adhesion

Abstract

Paxillin is a focal adhesion adapter protein involved in the integration of growth factor– and adhesion-mediated signal transduction pathways. Paxillin LD motifs have been demonstrated to bind to several proteins associated with remodeling of the actin cytoskeleton including the focal adhesion kinase, vinculin, and a complex of proteins comprising p95PKL, PIX, and PAK (Turner, C.E., M.C. Brown, J.A. Perrotta, M.C. Riedy, S.N. Nikolopoulos, A.R. McDonald, S. Bagrodia, S. Thomas, and P.S. Leventhal. 1999. J. Cell Biol. 145:851–863). In this study, we report the cloning and initial characterization of a new paxillin LD motif–binding protein, actopaxin. Analysis of the deduced amino acid sequence of actopaxin reveals a 42-kD protein with two calponin homology domains and a paxillin-binding subdomain (PBS). Western blotting identifies actopaxin as a widely expressed protein. Actopaxin binds directly to both F-actin and paxillin LD1 and LD4 motifs. It exhibits robust focal adhesion localization in several cultured cell types but is not found along the length of the associated actin-rich stress fibers. Similar to paxillin, it is absent from actin-rich cell–cell adherens junctions. Also, actopaxin colocalizes with paxillin to rudimentary focal complexes at the leading edge of migrating cells. An actopaxin PBS mutant incapable of binding paxillin in vitro cannot target to focal adhesions when expressed in fibroblasts. In addition, ectopic expression of the PBS mutant and/or the COOH terminus of actopaxin in HeLa cells resulted in substantial reduction in adhesion to collagen. Together, these results suggest an important role for actopaxin in integrin-dependent remodeling of the actin cytoskeleton during cell motility and cell adhesion.

Paxillin: Adapting to Change

“…In contrast, this structural organization is unlikely in the case of the ILK and actopaxin PBS domains (7,183). Such differences are likely to dictate the optimal binding parameters for each protein and contribute to selection of the "appropriate" paxillin binding partner(s) in response to a particular extracellular cue.…”

“…LD2 binds to vinculin and FAK/PYK2 (24, 278). LD4 binds to actopaxin (183), FAK/PYK2 (24, 265,278), the Arf-GAPs p95PKL/GIT2/ GIT1 (278), and perhaps PAK3 (90), clathrin (278), and PABP1 (307). Thus far no binding partners have been identified for LD5 or the degenerate LD3 motif.…”

“…Paxillin and Nck are critical for the appropriate localization and function of PAK and PIX; thus it will be of interest to further examine the role for these interactions in protrusion (20,28,171). The demonstration that PIX binds to the actin and paxillin-binding protein actopaxin/parvin, which regulates cell adhesion and spreading, provides an additional paxillin connection to the initial events of protrusion (183,217,278).…”

“…Thus a novel protein interaction pair, LD-PBS, was discovered. These data allowed for the creation of an algorithm to search and identify the PBS sequences within the paxillin LD binding partners PKL, ILK, and actopaxin and other potential candidates (23, 183,184,274,278). The general utility of LD-PBS associations has emerged with the discovery of an interaction between gelsolin and PYK2 (294) as well as between the papillomavirus E6 protein and the ubiquitin ligase E6-AP (14, 47), the Rap-GAP E6TP1(67), and the Ca 2ϩ -binding protein ERC-55/ E6BP (38), in addition to paxillin (211).…”

FAK engages multiple pathways to maintain survival of fibroblasts and epithelia – differential roles for paxillin and p130Cas

“…Mutation of tyrosines 31 and 118 to phenylalanine block Crk-mediated fibroblast migration (Petit et al, 2000). Paxillin also interacts with paxillin kinase linker (PKL), integrin-linked kinase (ILK), α-parvin and PTP-PEST (Cote et al, 1999;Nikolopoulos and Turner, 2000;Nikolopoulos and Turner, 2001;Turner et al, 1999). The paxillin-PKL interaction is critical for PAK-mediated cell motility.…”