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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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FAK and p130Cas, but not paxillin, protein levels and tyrosine phosphorylation were decreased at focal complexes in migrating Ajuba  lamellipodia. (A) Indirect immunofluorescence of migrating wt (+/+; a, b, g, h), Ajuba  (−/−; c, d, i, j), and Ajuba rescued Ajuba  MEFs (−/− Rescue; e, f, k, l) at the wound edge. Cells were costained for p130Cas (b, d, f) or pY397-FAK (h, j, l), and paxillin (a, c, e, g, i, k). Secondary antibodies conjugated with FITC (p130Cas and pY397FAK) or Alexa Fluor 568. Arrowheads identify leading edge focal complexes whereas arrows identify focal adhesions present at the base of the lamellipodia. Cells were imaged using a 60× oil immersion lens (NA 1.4). (B) Western blot analysis for the indicated proteins was performed on lysates of cell bodies and lamellipodia from wt (+/+) and Ajuba  (−/−) MEFs. Samples were normalized so that an equal amount of protein was loaded in each lane. (C) Phosphotyrosine-containing proteins were immunoprecipitated from lysates of wt (+/+) and Ajuba  (−/−) cell bodies and lamellipodia and bound products Western blotted for the indicated proteins. Each sample was normalized to equal amounts of protein before immunoprecipitation.
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fig5: FAK and p130Cas, but not paxillin, protein levels and tyrosine phosphorylation were decreased at focal complexes in migrating Ajuba lamellipodia. (A) Indirect immunofluorescence of migrating wt (+/+; a, b, g, h), Ajuba (−/−; c, d, i, j), and Ajuba rescued Ajuba MEFs (−/− Rescue; e, f, k, l) at the wound edge. Cells were costained for p130Cas (b, d, f) or pY397-FAK (h, j, l), and paxillin (a, c, e, g, i, k). Secondary antibodies conjugated with FITC (p130Cas and pY397FAK) or Alexa Fluor 568. Arrowheads identify leading edge focal complexes whereas arrows identify focal adhesions present at the base of the lamellipodia. Cells were imaged using a 60× oil immersion lens (NA 1.4). (B) Western blot analysis for the indicated proteins was performed on lysates of cell bodies and lamellipodia from wt (+/+) and Ajuba (−/−) MEFs. Samples were normalized so that an equal amount of protein was loaded in each lane. (C) Phosphotyrosine-containing proteins were immunoprecipitated from lysates of wt (+/+) and Ajuba (−/−) cell bodies and lamellipodia and bound products Western blotted for the indicated proteins. Each sample was normalized to equal amounts of protein before immunoprecipitation.

Mentions: Because cell adhesion at the leading edge of migrating cells can influence lamellipodia dynamics and cell motility we asked whether cell–ECM adhesive complexes in migrating Ajuba MEFs were altered. To do so cells were induced to migrate, by scratch wounding, then indirect immunofluorescence for select focal adhesion proteins (p130Cas, pY397-FAK, and paxillin) was performed on fixed cells at the wound edge. Compared with wt MEFs, migrating Ajuba cells were found to have reduced staining of pY397-FAK at leading edge nascent focal complexes (Fig. 5 A, arrowheads), whereas staining at mature focal adhesions, present at the base of lamellipodia protrusions, appeared to be unchanged (Fig. 5 A, arrows). p130Cas staining at the leading edge nascent focal complexes in Ajuba lamellipodia was also decreased and disordered (Fig. 5 A, arrowheads), whereas focal adhesion staining at the lamellipodia base was unchanged from wt (Fig. 5 A, arrows). In contrast, paxillin staining at both leading edge nascent focal complexes and focal adhesions was not altered in Ajuba MEFs (Fig. 5 A). There did not appear to be any significant difference in total number of focal complexes and focal adhesions present in migrating Ajuba MEFs compared with wt MEFs. Importantly reintroduction of Ajuba into Ajuba cells normalized the staining patterns for pY397-FAK and p130Cas at nascent focal complexes, whereas paxillin staining remained unchanged (Fig. 5 A). The total cellular level of each of these three proteins was not different between wt and Ajuba MEFs (Fig. S2 A, 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)

FAK and p130Cas, but not paxillin, protein levels and tyrosine phosphorylation were decreased at focal complexes in migrating Ajuba  lamellipodia. (A) Indirect immunofluorescence of migrating wt (+/+; a, b, g, h), Ajuba  (−/−; c, d, i, j), and Ajuba rescued Ajuba  MEFs (−/− Rescue; e, f, k, l) at the wound edge. Cells were costained for p130Cas (b, d, f) or pY397-FAK (h, j, l), and paxillin (a, c, e, g, i, k). Secondary antibodies conjugated with FITC (p130Cas and pY397FAK) or Alexa Fluor 568. Arrowheads identify leading edge focal complexes whereas arrows identify focal adhesions present at the base of the lamellipodia. Cells were imaged using a 60× oil immersion lens (NA 1.4). (B) Western blot analysis for the indicated proteins was performed on lysates of cell bodies and lamellipodia from wt (+/+) and Ajuba  (−/−) MEFs. Samples were normalized so that an equal amount of protein was loaded in each lane. (C) Phosphotyrosine-containing proteins were immunoprecipitated from lysates of wt (+/+) and Ajuba  (−/−) cell bodies and lamellipodia and bound products Western blotted for the indicated proteins. Each sample was normalized to equal amounts of protein before immunoprecipitation.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2171823&req=5

fig5: FAK and p130Cas, but not paxillin, protein levels and tyrosine phosphorylation were decreased at focal complexes in migrating Ajuba lamellipodia. (A) Indirect immunofluorescence of migrating wt (+/+; a, b, g, h), Ajuba (−/−; c, d, i, j), and Ajuba rescued Ajuba MEFs (−/− Rescue; e, f, k, l) at the wound edge. Cells were costained for p130Cas (b, d, f) or pY397-FAK (h, j, l), and paxillin (a, c, e, g, i, k). Secondary antibodies conjugated with FITC (p130Cas and pY397FAK) or Alexa Fluor 568. Arrowheads identify leading edge focal complexes whereas arrows identify focal adhesions present at the base of the lamellipodia. Cells were imaged using a 60× oil immersion lens (NA 1.4). (B) Western blot analysis for the indicated proteins was performed on lysates of cell bodies and lamellipodia from wt (+/+) and Ajuba (−/−) MEFs. Samples were normalized so that an equal amount of protein was loaded in each lane. (C) Phosphotyrosine-containing proteins were immunoprecipitated from lysates of wt (+/+) and Ajuba (−/−) cell bodies and lamellipodia and bound products Western blotted for the indicated proteins. Each sample was normalized to equal amounts of protein before immunoprecipitation.
Mentions: Because cell adhesion at the leading edge of migrating cells can influence lamellipodia dynamics and cell motility we asked whether cell–ECM adhesive complexes in migrating Ajuba MEFs were altered. To do so cells were induced to migrate, by scratch wounding, then indirect immunofluorescence for select focal adhesion proteins (p130Cas, pY397-FAK, and paxillin) was performed on fixed cells at the wound edge. Compared with wt MEFs, migrating Ajuba cells were found to have reduced staining of pY397-FAK at leading edge nascent focal complexes (Fig. 5 A, arrowheads), whereas staining at mature focal adhesions, present at the base of lamellipodia protrusions, appeared to be unchanged (Fig. 5 A, arrows). p130Cas staining at the leading edge nascent focal complexes in Ajuba lamellipodia was also decreased and disordered (Fig. 5 A, arrowheads), whereas focal adhesion staining at the lamellipodia base was unchanged from wt (Fig. 5 A, arrows). In contrast, paxillin staining at both leading edge nascent focal complexes and focal adhesions was not altered in Ajuba MEFs (Fig. 5 A). There did not appear to be any significant difference in total number of focal complexes and focal adhesions present in migrating Ajuba MEFs compared with wt MEFs. Importantly reintroduction of Ajuba into Ajuba cells normalized the staining patterns for pY397-FAK and p130Cas at nascent focal complexes, whereas paxillin staining remained unchanged (Fig. 5 A). The total cellular level of each of these three proteins was not different between wt and Ajuba MEFs (Fig. S2 A, 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