Limits...
The LIM protein Ajuba influences p130Cas localization and Rac1 activity during cell migration.

Pratt SJ, Epple H, Ward M, Feng Y, Braga VM, Longmore GD - J. Cell Biol. (2005)

Bottom Line: Lamellipodia production, or function, is defective and there is a selective reduction in the level and tyrosine phosphorylation of FAK, p130Cas, Crk, and Dock180 at nascent focal complexes.In response to migratory cues Rac activation is blunted in Ajuba cells, as detected biochemically and by FRET analysis.Ajuba associates with the focal adhesion-targeting domain of p130Cas, and rescue experiments suggest that Ajuba acts upstream of p130Cas to localize p130Cas to nascent adhesive sites in migrating cells thereby leading to the activation of Rac.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Washington University, St. Louis, MO 63130, USA.

ABSTRACT
Cell migration requires extension of lamellipodia that are stabilized by formation of adhesive complexes at the leading edge. Both processes are regulated by signaling proteins recruited to nascent adhesive sites that lead to activation of Rho GTPases. The Ajuba/Zyxin family of LIM proteins are components of cellular adhesive complexes. We show that cells from Ajuba mice are inhibited in their migration, without associated abnormality in adhesion to extracellular matrix proteins, cell spreading, or integrin activation. Lamellipodia production, or function, is defective and there is a selective reduction in the level and tyrosine phosphorylation of FAK, p130Cas, Crk, and Dock180 at nascent focal complexes. In response to migratory cues Rac activation is blunted in Ajuba cells, as detected biochemically and by FRET analysis. Ajuba associates with the focal adhesion-targeting domain of p130Cas, and rescue experiments suggest that Ajuba acts upstream of p130Cas to localize p130Cas to nascent adhesive sites in migrating cells thereby leading to the activation of Rac.

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Quantitative Western blot analyses of Zyxin/Ajuba family members. WT (+/+, lane 1), Ajuba  (−/−, lane 2), and Ajuba rescued Ajuba  MEFs (−/− Rescue, lane 3). Equal amounts of protein were loaded in each lane. Densitometry was performed and the level of each protein is displayed below each lane. The wt sample is arbitrarily set at 1.
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fig1: Quantitative Western blot analyses of Zyxin/Ajuba family members. WT (+/+, lane 1), Ajuba (−/−, lane 2), and Ajuba rescued Ajuba MEFs (−/− Rescue, lane 3). Equal amounts of protein were loaded in each lane. Densitometry was performed and the level of each protein is displayed below each lane. The wt sample is arbitrarily set at 1.

Mentions: In mouse embryonal stem (ES) cells, exon 1 of the Ajuba gene was replaced with a Neomycin cassette (Fig. S1 A, available at http://www.jcb.org/cgi/content/full/jcb.200406083/DC1). Two different ES clones were used to generate chimeric mice and mice heterozygous for the targeted allele were recovered. Interbreeding between heterozygous mice resulted in the birth of mice of all three genotypes in the expected Mendelian ratios. PCR analysis and Southern blot analysis of genomic DNA (Fig. S1 B) from adult tails revealed that Ajuba mice were viable and reached adulthood without any obvious phenotypes. Ajuba mice were fertile. Western blot analyses of cell lysates from primary fibroblasts (MEFs) derived from E13.5-E15.5 Ajuba or littermate wild-type (wt) control embryos demonstrated that there was no detectable Ajuba protein made in Ajuba cells, and that the expression of related Ajuba/Zyxin family members were not significantly altered (Fig. 1). Stable reintroduction of Ajuba into Ajuba cells also did not affect the levels of related LIM proteins (Fig. 1).


The LIM protein Ajuba influences p130Cas localization and Rac1 activity during cell migration.

Pratt SJ, Epple H, Ward M, Feng Y, Braga VM, Longmore GD - J. Cell Biol. (2005)

Quantitative Western blot analyses of Zyxin/Ajuba family members. WT (+/+, lane 1), Ajuba  (−/−, lane 2), and Ajuba rescued Ajuba  MEFs (−/− Rescue, lane 3). Equal amounts of protein were loaded in each lane. Densitometry was performed and the level of each protein is displayed below each lane. The wt sample is arbitrarily set at 1.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2171823&req=5

fig1: Quantitative Western blot analyses of Zyxin/Ajuba family members. WT (+/+, lane 1), Ajuba (−/−, lane 2), and Ajuba rescued Ajuba MEFs (−/− Rescue, lane 3). Equal amounts of protein were loaded in each lane. Densitometry was performed and the level of each protein is displayed below each lane. The wt sample is arbitrarily set at 1.
Mentions: In mouse embryonal stem (ES) cells, exon 1 of the Ajuba gene was replaced with a Neomycin cassette (Fig. S1 A, available at http://www.jcb.org/cgi/content/full/jcb.200406083/DC1). Two different ES clones were used to generate chimeric mice and mice heterozygous for the targeted allele were recovered. Interbreeding between heterozygous mice resulted in the birth of mice of all three genotypes in the expected Mendelian ratios. PCR analysis and Southern blot analysis of genomic DNA (Fig. S1 B) from adult tails revealed that Ajuba mice were viable and reached adulthood without any obvious phenotypes. Ajuba mice were fertile. Western blot analyses of cell lysates from primary fibroblasts (MEFs) derived from E13.5-E15.5 Ajuba or littermate wild-type (wt) control embryos demonstrated that there was no detectable Ajuba protein made in Ajuba cells, and that the expression of related Ajuba/Zyxin family members were not significantly altered (Fig. 1). Stable reintroduction of Ajuba into Ajuba cells also did not affect the levels of related LIM proteins (Fig. 1).

Bottom Line: Lamellipodia production, or function, is defective and there is a selective reduction in the level and tyrosine phosphorylation of FAK, p130Cas, Crk, and Dock180 at nascent focal complexes.In response to migratory cues Rac activation is blunted in Ajuba cells, as detected biochemically and by FRET analysis.Ajuba associates with the focal adhesion-targeting domain of p130Cas, and rescue experiments suggest that Ajuba acts upstream of p130Cas to localize p130Cas to nascent adhesive sites in migrating cells thereby leading to the activation of Rac.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Washington University, St. Louis, MO 63130, USA.

ABSTRACT
Cell migration requires extension of lamellipodia that are stabilized by formation of adhesive complexes at the leading edge. Both processes are regulated by signaling proteins recruited to nascent adhesive sites that lead to activation of Rho GTPases. The Ajuba/Zyxin family of LIM proteins are components of cellular adhesive complexes. We show that cells from Ajuba mice are inhibited in their migration, without associated abnormality in adhesion to extracellular matrix proteins, cell spreading, or integrin activation. Lamellipodia production, or function, is defective and there is a selective reduction in the level and tyrosine phosphorylation of FAK, p130Cas, Crk, and Dock180 at nascent focal complexes. In response to migratory cues Rac activation is blunted in Ajuba cells, as detected biochemically and by FRET analysis. Ajuba associates with the focal adhesion-targeting domain of p130Cas, and rescue experiments suggest that Ajuba acts upstream of p130Cas to localize p130Cas to nascent adhesive sites in migrating cells thereby leading to the activation of Rac.

Show MeSH
Related in: MedlinePlus