Limits...
Actopaxin, a new focal adhesion protein that binds paxillin LD motifs and actin and regulates cell adhesion.

Nikolopoulos SN, Turner CE - J. Cell Biol. (2000)

Bottom Line: 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.Also, actopaxin colocalizes with paxillin to rudimentary focal complexes at the leading edge of migrating cells.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Developmental Biology, State University of New York Upstate Medical University, Syracuse, New York 13210, USA.

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.

Show MeSH

Related in: MedlinePlus

Expression of actopaxin protein in various tissues and cell lines. Organs were dissected from a healthy adult rat, homogenized, and lysed in SDS-PAGE sample buffer. 50 μg of each lysate was loaded per lane. (A) Coomassie blue staining of the gel. (B) Western blotting of an identical gel shown in A probed with the actopaxin antibody. (C) The same blot was stripped and reprobed with an actin antibody to indicate relative loading among lanes. (D) Western blot of total lysates (50 μg) from HISM cells, rat embryo fibroblasts (REF-52), and rat intestinal epithelial cells (IEC-18) probed with the actopaxin antibody.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2150668&req=5

Figure 2: Expression of actopaxin protein in various tissues and cell lines. Organs were dissected from a healthy adult rat, homogenized, and lysed in SDS-PAGE sample buffer. 50 μg of each lysate was loaded per lane. (A) Coomassie blue staining of the gel. (B) Western blotting of an identical gel shown in A probed with the actopaxin antibody. (C) The same blot was stripped and reprobed with an actin antibody to indicate relative loading among lanes. (D) Western blot of total lysates (50 μg) from HISM cells, rat embryo fibroblasts (REF-52), and rat intestinal epithelial cells (IEC-18) probed with the actopaxin antibody.

Mentions: To examine the expression pattern of actopaxin protein, a polyclonal antiserum was produced against a GST fusion protein of full-length actopaxin (aa 1–372). Western blotting of total cell lysates prepared from several rat tissues and cell lines indicated the presence of a major protein migrating just above the 42-kD actin band in all tissues examined (Fig. 2, A–C) and in all cell lines tested (Fig. 2 D). The absence of actopaxin from the skeletal muscle sample is most likely due to the absence in this particular preparation of myotendinous junctions, a specialized region at the end of skeletal muscle fibers enriched in focal adhesion proteins (Tidball et al. 1986; Turner et al. 1991).


Actopaxin, a new focal adhesion protein that binds paxillin LD motifs and actin and regulates cell adhesion.

Nikolopoulos SN, Turner CE - J. Cell Biol. (2000)

Expression of actopaxin protein in various tissues and cell lines. Organs were dissected from a healthy adult rat, homogenized, and lysed in SDS-PAGE sample buffer. 50 μg of each lysate was loaded per lane. (A) Coomassie blue staining of the gel. (B) Western blotting of an identical gel shown in A probed with the actopaxin antibody. (C) The same blot was stripped and reprobed with an actin antibody to indicate relative loading among lanes. (D) Western blot of total lysates (50 μg) from HISM cells, rat embryo fibroblasts (REF-52), and rat intestinal epithelial cells (IEC-18) probed with the actopaxin antibody.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2150668&req=5

Figure 2: Expression of actopaxin protein in various tissues and cell lines. Organs were dissected from a healthy adult rat, homogenized, and lysed in SDS-PAGE sample buffer. 50 μg of each lysate was loaded per lane. (A) Coomassie blue staining of the gel. (B) Western blotting of an identical gel shown in A probed with the actopaxin antibody. (C) The same blot was stripped and reprobed with an actin antibody to indicate relative loading among lanes. (D) Western blot of total lysates (50 μg) from HISM cells, rat embryo fibroblasts (REF-52), and rat intestinal epithelial cells (IEC-18) probed with the actopaxin antibody.
Mentions: To examine the expression pattern of actopaxin protein, a polyclonal antiserum was produced against a GST fusion protein of full-length actopaxin (aa 1–372). Western blotting of total cell lysates prepared from several rat tissues and cell lines indicated the presence of a major protein migrating just above the 42-kD actin band in all tissues examined (Fig. 2, A–C) and in all cell lines tested (Fig. 2 D). The absence of actopaxin from the skeletal muscle sample is most likely due to the absence in this particular preparation of myotendinous junctions, a specialized region at the end of skeletal muscle fibers enriched in focal adhesion proteins (Tidball et al. 1986; Turner et al. 1991).

Bottom Line: 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.Also, actopaxin colocalizes with paxillin to rudimentary focal complexes at the leading edge of migrating cells.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Developmental Biology, State University of New York Upstate Medical University, Syracuse, New York 13210, USA.

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.

Show MeSH
Related in: MedlinePlus