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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PreLIM Ajuba blocks p130Cas localization to adhesive sites and p130 rescue of migration. (A) Migration rescue experiments. WT, Ajuba −/−, and p130Cas −/− MEFs were transfected with control empty GFP vector, RFP-PreLIM, RFP-LIM, both RFP-PreLIM and GFP-p130Cas, or both RFP-LIM and GFP-p130Cas as indicated. Confluent cell layers were scratch wounded, and videos of wound repair were obtained, as described in Fig. 2 B. Shown is the percent wound closure at 12 h. Multiple wounds were analyzed and the data presented as the mean and the SD about the mean. (B and C) Cells were cotransfected with RFP-PreLIM and GFP-p130Cas (B), or RFP-LIM and GFP-p130Cas (C), and immunofluorescence performed. Arrows identify focal adhesions.
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fig8: PreLIM Ajuba blocks p130Cas localization to adhesive sites and p130 rescue of migration. (A) Migration rescue experiments. WT, Ajuba −/−, and p130Cas −/− MEFs were transfected with control empty GFP vector, RFP-PreLIM, RFP-LIM, both RFP-PreLIM and GFP-p130Cas, or both RFP-LIM and GFP-p130Cas as indicated. Confluent cell layers were scratch wounded, and videos of wound repair were obtained, as described in Fig. 2 B. Shown is the percent wound closure at 12 h. Multiple wounds were analyzed and the data presented as the mean and the SD about the mean. (B and C) Cells were cotransfected with RFP-PreLIM and GFP-p130Cas (B), or RFP-LIM and GFP-p130Cas (C), and immunofluorescence performed. Arrows identify focal adhesions.

Mentions: The COOH-terminal region of p130Cas (region that interacts with Ajuba) contains the major focal adhesion targeting sequence in p130Cas (Harte et al., 2000), yet is distinct from other regions in p130Cas that mediate its association with Crk and DOCK180 (Bouton et al., 2001). This suggested the possibility that Ajuba may influence the recruitment of p130Cas to focal adhesion sites. Because the PreLIM region of Ajuba directed its association with p130Cas and does not, itself, localize to adhesive complexes (Fig. 8 B) we asked whether overexpression of the PreLIM region of Ajuba in wt MEFs could act in a “dominant inhibitory” manner to prevent p130Cas recruitment to nascent focal complexes, and thereby inhibit cell migration. When overexpression in wt cells the PreLIM region of Ajuba did indeed inhibit cell migration (Fig. 8 A), but not to the same extent as observed for Ajuba MEFs (Fig. 8 A). PreLIM Ajuba also reduced the amount of p130Cas present at focal complexes in these cells (Fig. 8 B). Overexpression of the Ajuba LIM region (does not interact with p130Cas, yet itself localizes to focal adhesions (Fig. 8 C) did not inhibit either wt MEF migration (Fig. 8 A) or p130Cas localization (Fig. 8 C).


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)

PreLIM Ajuba blocks p130Cas localization to adhesive sites and p130 rescue of migration. (A) Migration rescue experiments. WT, Ajuba −/−, and p130Cas −/− MEFs were transfected with control empty GFP vector, RFP-PreLIM, RFP-LIM, both RFP-PreLIM and GFP-p130Cas, or both RFP-LIM and GFP-p130Cas as indicated. Confluent cell layers were scratch wounded, and videos of wound repair were obtained, as described in Fig. 2 B. Shown is the percent wound closure at 12 h. Multiple wounds were analyzed and the data presented as the mean and the SD about the mean. (B and C) Cells were cotransfected with RFP-PreLIM and GFP-p130Cas (B), or RFP-LIM and GFP-p130Cas (C), and immunofluorescence performed. Arrows identify focal adhesions.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2171823&req=5

fig8: PreLIM Ajuba blocks p130Cas localization to adhesive sites and p130 rescue of migration. (A) Migration rescue experiments. WT, Ajuba −/−, and p130Cas −/− MEFs were transfected with control empty GFP vector, RFP-PreLIM, RFP-LIM, both RFP-PreLIM and GFP-p130Cas, or both RFP-LIM and GFP-p130Cas as indicated. Confluent cell layers were scratch wounded, and videos of wound repair were obtained, as described in Fig. 2 B. Shown is the percent wound closure at 12 h. Multiple wounds were analyzed and the data presented as the mean and the SD about the mean. (B and C) Cells were cotransfected with RFP-PreLIM and GFP-p130Cas (B), or RFP-LIM and GFP-p130Cas (C), and immunofluorescence performed. Arrows identify focal adhesions.
Mentions: The COOH-terminal region of p130Cas (region that interacts with Ajuba) contains the major focal adhesion targeting sequence in p130Cas (Harte et al., 2000), yet is distinct from other regions in p130Cas that mediate its association with Crk and DOCK180 (Bouton et al., 2001). This suggested the possibility that Ajuba may influence the recruitment of p130Cas to focal adhesion sites. Because the PreLIM region of Ajuba directed its association with p130Cas and does not, itself, localize to adhesive complexes (Fig. 8 B) we asked whether overexpression of the PreLIM region of Ajuba in wt MEFs could act in a “dominant inhibitory” manner to prevent p130Cas recruitment to nascent focal complexes, and thereby inhibit cell migration. When overexpression in wt cells the PreLIM region of Ajuba did indeed inhibit cell migration (Fig. 8 A), but not to the same extent as observed for Ajuba MEFs (Fig. 8 A). PreLIM Ajuba also reduced the amount of p130Cas present at focal complexes in these cells (Fig. 8 B). Overexpression of the Ajuba LIM region (does not interact with p130Cas, yet itself localizes to focal adhesions (Fig. 8 C) did not inhibit either wt MEF migration (Fig. 8 A) or p130Cas localization (Fig. 8 C).

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