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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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Ajuba colocalized with p130Cas at focal complexes and focal adhesions in lamellipodia, and associated with p130Cas in cells. (A) WT MEFs were cotransfected with GFP-p130Cas (green) and RFP-Ajuba (red) and induced to migrate (toward top of page) by scratch wounding. Arrows indicate localization of p130Cas and Ajuba (colocalization, yellow in merged image). (B) COS-7 cells were transfected with myc-tagged Ajuba plasmids, endogenous p130Cas was immunoprecipitated (right) and bound products Western blotted for the presence of Ajuba (anti-myc; bottom) and p130Cas (top). Left panels are loading controls of cell lysates. (C) COS-7 cells were cotransfected with Flag-tagged Ajuba and various myc-tagged p130Cas mutants. Ajuba was immunoprecipitated (anti-Flag; right) and bound products Western blotted for the presence of p130Cas (anti-myc; top) and Ajuba (anti-Flag; bottom). Loading controls of cell lysates (left). (D) Migration rescue experiments. WT, Ajuba −/−, and p130Cas −/− MEFs were transfected with control empty GFP vector, RFP-Ajuba, GFP-p130Cas, GFP-DOCK180, GFP-paxillin, SuperFAK-IRES.GFP, or myc-Rac1(L61) as indicated. Confluent cell layers were scratch wounded, and videos of wound repair 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.
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fig7: Ajuba colocalized with p130Cas at focal complexes and focal adhesions in lamellipodia, and associated with p130Cas in cells. (A) WT MEFs were cotransfected with GFP-p130Cas (green) and RFP-Ajuba (red) and induced to migrate (toward top of page) by scratch wounding. Arrows indicate localization of p130Cas and Ajuba (colocalization, yellow in merged image). (B) COS-7 cells were transfected with myc-tagged Ajuba plasmids, endogenous p130Cas was immunoprecipitated (right) and bound products Western blotted for the presence of Ajuba (anti-myc; bottom) and p130Cas (top). Left panels are loading controls of cell lysates. (C) COS-7 cells were cotransfected with Flag-tagged Ajuba and various myc-tagged p130Cas mutants. Ajuba was immunoprecipitated (anti-Flag; right) and bound products Western blotted for the presence of p130Cas (anti-myc; top) and Ajuba (anti-Flag; bottom). Loading controls of cell lysates (left). (D) Migration rescue experiments. WT, Ajuba −/−, and p130Cas −/− MEFs were transfected with control empty GFP vector, RFP-Ajuba, GFP-p130Cas, GFP-DOCK180, GFP-paxillin, SuperFAK-IRES.GFP, or myc-Rac1(L61) as indicated. Confluent cell layers were scratch wounded, and videos of wound repair 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.

Mentions: A major signaling pathway leading to Rac activation in migrating cells is the assembly of the p130Cas–Crk–DOCK180–ELMO Rac GEF signaling complex at activated integrin complexes present in nascent adhesive complexes (Kiyokawa et al., 1998b). Lamellipodia of migrating Ajuba MEFs had selective deficiency in p130Cas, Crk, and DOCK180 levels, particularly at nascent adhesive complexes (Fig. 5 and Fig. S2 B). FAK levels were also reduced in Ajuba lamellipodia, and p130Cas is recruited to adhesive complex through an interaction with FAK (Bouton et al., 2001). Furthermore, the tyrosine phosphorylation of p130Cas and FAK was also reduced in Ajuba lamellipodia. These results suggested that Ajuba might affect Rac activity by influencing the recruitment of these proteins into adhesive complexes in the lamellipodia of migrating cells. In a yeast 2-hybrid protein–protein interactive screen we identified HEF1–p130Cas as an Ajuba-interacting protein (unpublished data). When wt MEFs were cotransfected with RFP-Ajuba and GFP-p130Cas, p130Cas and Ajuba were both present at focal adhesions at the base of lamellipodia as well as toward the cell periphery (Fig. 7 A, arrows; Videos 4 and 5, available at http://www.jcb.org/cgi/content/full/jcb.200406083/DC1). Ajuba and p130Cas also colocalized with pY397-FAK at these sites (not depicted).


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 colocalized with p130Cas at focal complexes and focal adhesions in lamellipodia, and associated with p130Cas in cells. (A) WT MEFs were cotransfected with GFP-p130Cas (green) and RFP-Ajuba (red) and induced to migrate (toward top of page) by scratch wounding. Arrows indicate localization of p130Cas and Ajuba (colocalization, yellow in merged image). (B) COS-7 cells were transfected with myc-tagged Ajuba plasmids, endogenous p130Cas was immunoprecipitated (right) and bound products Western blotted for the presence of Ajuba (anti-myc; bottom) and p130Cas (top). Left panels are loading controls of cell lysates. (C) COS-7 cells were cotransfected with Flag-tagged Ajuba and various myc-tagged p130Cas mutants. Ajuba was immunoprecipitated (anti-Flag; right) and bound products Western blotted for the presence of p130Cas (anti-myc; top) and Ajuba (anti-Flag; bottom). Loading controls of cell lysates (left). (D) Migration rescue experiments. WT, Ajuba −/−, and p130Cas −/− MEFs were transfected with control empty GFP vector, RFP-Ajuba, GFP-p130Cas, GFP-DOCK180, GFP-paxillin, SuperFAK-IRES.GFP, or myc-Rac1(L61) as indicated. Confluent cell layers were scratch wounded, and videos of wound repair 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.
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fig7: Ajuba colocalized with p130Cas at focal complexes and focal adhesions in lamellipodia, and associated with p130Cas in cells. (A) WT MEFs were cotransfected with GFP-p130Cas (green) and RFP-Ajuba (red) and induced to migrate (toward top of page) by scratch wounding. Arrows indicate localization of p130Cas and Ajuba (colocalization, yellow in merged image). (B) COS-7 cells were transfected with myc-tagged Ajuba plasmids, endogenous p130Cas was immunoprecipitated (right) and bound products Western blotted for the presence of Ajuba (anti-myc; bottom) and p130Cas (top). Left panels are loading controls of cell lysates. (C) COS-7 cells were cotransfected with Flag-tagged Ajuba and various myc-tagged p130Cas mutants. Ajuba was immunoprecipitated (anti-Flag; right) and bound products Western blotted for the presence of p130Cas (anti-myc; top) and Ajuba (anti-Flag; bottom). Loading controls of cell lysates (left). (D) Migration rescue experiments. WT, Ajuba −/−, and p130Cas −/− MEFs were transfected with control empty GFP vector, RFP-Ajuba, GFP-p130Cas, GFP-DOCK180, GFP-paxillin, SuperFAK-IRES.GFP, or myc-Rac1(L61) as indicated. Confluent cell layers were scratch wounded, and videos of wound repair 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.
Mentions: A major signaling pathway leading to Rac activation in migrating cells is the assembly of the p130Cas–Crk–DOCK180–ELMO Rac GEF signaling complex at activated integrin complexes present in nascent adhesive complexes (Kiyokawa et al., 1998b). Lamellipodia of migrating Ajuba MEFs had selective deficiency in p130Cas, Crk, and DOCK180 levels, particularly at nascent adhesive complexes (Fig. 5 and Fig. S2 B). FAK levels were also reduced in Ajuba lamellipodia, and p130Cas is recruited to adhesive complex through an interaction with FAK (Bouton et al., 2001). Furthermore, the tyrosine phosphorylation of p130Cas and FAK was also reduced in Ajuba lamellipodia. These results suggested that Ajuba might affect Rac activity by influencing the recruitment of these proteins into adhesive complexes in the lamellipodia of migrating cells. In a yeast 2-hybrid protein–protein interactive screen we identified HEF1–p130Cas as an Ajuba-interacting protein (unpublished data). When wt MEFs were cotransfected with RFP-Ajuba and GFP-p130Cas, p130Cas and Ajuba were both present at focal adhesions at the base of lamellipodia as well as toward the cell periphery (Fig. 7 A, arrows; Videos 4 and 5, available at http://www.jcb.org/cgi/content/full/jcb.200406083/DC1). Ajuba and p130Cas also colocalized with pY397-FAK at these sites (not depicted).

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