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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Ajuba  MEFs adhere to ECM proteins, spread, and activated integrin receptors normally. (A) Cell adhesion. WT (♦) and Ajuba  (□) MEFs were allowed to adhere for 1 h to plates coated with increasing concentration of the indicated ECM protein. (B) Cell spreading. WT (♦) and Ajuba  (□) MEFs were allowed to adhere and spread on fibronectin-coated coverslips over 2 h. Videos of multiple cells were obtained and data extrapolated at the indicated time points. Results are the mean values plus the SD about the mean. (C) Serum-starved wt (+/+) and Ajuba  (−/−) cells were allowed to adhere to fibronectin-coated plates for 1 h. (D) FAK and p130Cas were immunoprecipitated from cells in suspension (Sus) or bound to fibronectin (Fib) and bound products Western blotted with anti-pY397FAK or anti-phosphotyrosine antibodies, respectively. Actin Western blots are loading controls.
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fig3: Ajuba MEFs adhere to ECM proteins, spread, and activated integrin receptors normally. (A) Cell adhesion. WT (♦) and Ajuba (□) MEFs were allowed to adhere for 1 h to plates coated with increasing concentration of the indicated ECM protein. (B) Cell spreading. WT (♦) and Ajuba (□) MEFs were allowed to adhere and spread on fibronectin-coated coverslips over 2 h. Videos of multiple cells were obtained and data extrapolated at the indicated time points. Results are the mean values plus the SD about the mean. (C) Serum-starved wt (+/+) and Ajuba (−/−) cells were allowed to adhere to fibronectin-coated plates for 1 h. (D) FAK and p130Cas were immunoprecipitated from cells in suspension (Sus) or bound to fibronectin (Fib) and bound products Western blotted with anti-pY397FAK or anti-phosphotyrosine antibodies, respectively. Actin Western blots are loading controls.

Mentions: Cell migration defects are commonly associated with abnormalities in cell–ECM adhesion and spreading (Ilic et al., 1995; Honda et al., 1998; Xu et al., 1998; Monkley et al., 2000; Hagel et al., 2002). Adhesion assays with Ajuba MEFs on collagen, fibronectin, vitronectin, or superfibronectin were therefore performed. The number of Ajuba MEFs adhering to the various ECM protein-coated plates was indistinguishable from wt controls (Fig. 3 A). To determine if Ajuba cells spread normally after adhesion to ECM the rate of cell spreading, on fibronectin, was determined. Both wt and Ajuba MEFs showed the same rate of cell spreading (Fig. 3 B; Video 2, available at http://www.jcb.org/cgi/content/full/jcb.200406083/DC1).


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)

Ajuba  MEFs adhere to ECM proteins, spread, and activated integrin receptors normally. (A) Cell adhesion. WT (♦) and Ajuba  (□) MEFs were allowed to adhere for 1 h to plates coated with increasing concentration of the indicated ECM protein. (B) Cell spreading. WT (♦) and Ajuba  (□) MEFs were allowed to adhere and spread on fibronectin-coated coverslips over 2 h. Videos of multiple cells were obtained and data extrapolated at the indicated time points. Results are the mean values plus the SD about the mean. (C) Serum-starved wt (+/+) and Ajuba  (−/−) cells were allowed to adhere to fibronectin-coated plates for 1 h. (D) FAK and p130Cas were immunoprecipitated from cells in suspension (Sus) or bound to fibronectin (Fib) and bound products Western blotted with anti-pY397FAK or anti-phosphotyrosine antibodies, respectively. Actin Western blots are loading controls.
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Related In: Results  -  Collection

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fig3: Ajuba MEFs adhere to ECM proteins, spread, and activated integrin receptors normally. (A) Cell adhesion. WT (♦) and Ajuba (□) MEFs were allowed to adhere for 1 h to plates coated with increasing concentration of the indicated ECM protein. (B) Cell spreading. WT (♦) and Ajuba (□) MEFs were allowed to adhere and spread on fibronectin-coated coverslips over 2 h. Videos of multiple cells were obtained and data extrapolated at the indicated time points. Results are the mean values plus the SD about the mean. (C) Serum-starved wt (+/+) and Ajuba (−/−) cells were allowed to adhere to fibronectin-coated plates for 1 h. (D) FAK and p130Cas were immunoprecipitated from cells in suspension (Sus) or bound to fibronectin (Fib) and bound products Western blotted with anti-pY397FAK or anti-phosphotyrosine antibodies, respectively. Actin Western blots are loading controls.
Mentions: Cell migration defects are commonly associated with abnormalities in cell–ECM adhesion and spreading (Ilic et al., 1995; Honda et al., 1998; Xu et al., 1998; Monkley et al., 2000; Hagel et al., 2002). Adhesion assays with Ajuba MEFs on collagen, fibronectin, vitronectin, or superfibronectin were therefore performed. The number of Ajuba MEFs adhering to the various ECM protein-coated plates was indistinguishable from wt controls (Fig. 3 A). To determine if Ajuba cells spread normally after adhesion to ECM the rate of cell spreading, on fibronectin, was determined. Both wt and Ajuba MEFs showed the same rate of cell spreading (Fig. 3 B; Video 2, available at http://www.jcb.org/cgi/content/full/jcb.200406083/DC1).

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