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

Actopaxin, a New Focal Adhesion Protein That Binds Paxillin Ld Motifs and Actin and Regulates... 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. actin-binding protein CH domains focal adhesions paxillin hic-5 Footnotes Abbreviations used in this paper: aa, amino acid(s); Arf, ADP-ribosylation factor; CH, calponin homology; FAK, focal adhesion kinase; GST, glutathione S -transferase; HISM, human intestinal smooth muscle; PBS, paxillin-binding subdomain; RT, reverse transcriptase. Submitted: 11 May 2000 Revision requested 31 October 2000 Accepted: 1 November 2000 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The Journal of Cell Biology Rockefeller University Press

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

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References (57)

Publisher
Rockefeller University Press
Copyright
© 2000 The Rockefeller University Press
ISSN
0021-9525
eISSN
1540-8140
DOI
10.1083/jcb.151.7.1435
Publisher site
See Article on Publisher Site

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. actin-binding protein CH domains focal adhesions paxillin hic-5 Footnotes Abbreviations used in this paper: aa, amino acid(s); Arf, ADP-ribosylation factor; CH, calponin homology; FAK, focal adhesion kinase; GST, glutathione S -transferase; HISM, human intestinal smooth muscle; PBS, paxillin-binding subdomain; RT, reverse transcriptase. Submitted: 11 May 2000 Revision requested 31 October 2000 Accepted: 1 November 2000

Journal

The Journal of Cell BiologyRockefeller University Press

Published: Dec 25, 2000

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