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The LD4 motif of paxillin regulates cell spreading and motility through an interaction with paxillin kinase linker (PKL).

West KA, Zhang H, Brown MC, Nikolopoulos SN, Riedy MC, Horwitz AF, Turner CE - J. Cell Biol. (2001)

Bottom Line: In addition, FAK activity during spreading was not compromised by deletion of the paxillin LD4 motif.Furthermore, overexpression of PKL mutants lacking the paxillin-binding site (PKLDeltaPBS2) induced phenotypic changes reminiscent of paxillinDeltaLD4 mutant cells.These data suggest that the paxillin association with PKL is essential for normal integrin-mediated cell spreading, and locomotion and that this interaction is necessary for the regulation of Rac activity during these events.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Developmental Biology, State University of New York, Upstate Medical University, 750 East Adams St., Syracuse, NY 13210, USA.

ABSTRACT
The small GTPases of the Rho family are intimately involved in integrin-mediated changes in the actin cytoskeleton that accompany cell spreading and motility. The exact means by which the Rho family members elicit these changes is unclear. Here, we demonstrate that the interaction of paxillin via its LD4 motif with the putative ARF-GAP paxillin kinase linker (PKL) (Turner et al., 1999), is critically involved in the regulation of Rac-dependent changes in the actin cytoskeleton that accompany cell spreading and motility. Overexpression of a paxillin LD4 deletion mutant (paxillinDeltaLD4) in CHO.K1 fibroblasts caused the generation of multiple broad lamellipodia. These morphological changes were accompanied by an increase in cell protrusiveness and random motility, which correlated with prolonged activation of Rac. In contrast, directional motility was inhibited. These alterations in morphology and motility were dependent on a paxillin-PKL interaction. In cells overexpressing paxillinDeltaLD4 mutants, PKL localization to focal contacts was disrupted, whereas that of focal adhesion kinase (FAK) and vinculin was not. In addition, FAK activity during spreading was not compromised by deletion of the paxillin LD4 motif. Furthermore, overexpression of PKL mutants lacking the paxillin-binding site (PKLDeltaPBS2) induced phenotypic changes reminiscent of paxillinDeltaLD4 mutant cells. These data suggest that the paxillin association with PKL is essential for normal integrin-mediated cell spreading, and locomotion and that this interaction is necessary for the regulation of Rac activity during these events.

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Expression of paxillin LD4 deletion generates multiple broad lamellipodia during cell spreading. (A) Immunoprecipitation using avian-specific paxillin antisera (Pax1, right) and Western immunoblotting of total cell lysates (left) confirm the relative levels of paxillin, PKL, and p130cas protein, and demonstrates the overexpression of avian paxillin in the paxillinΔLD4 and paxillin WT cells compared with parental nontransfected CHO.K1 cells. (B, a and d) CHO.K1 cells ectopically expressing avian paxillin with the deletion of LD4 (paxillinΔLD4); (b and e) CHO.K1 cells ectopically expressing full-length wild-type avian paxillin (paxillin WT); (c and f) parental nontransfected CHO.K1 cells. CHO.K1 cells were respread on fibronectin-coated (Fn) coverslips (10 μg/ml) for 60, 240, and 360 min, and ectopic paxillin (Pax1, a and b), endogenous paxillin (c), and actin (d–f) were examined. PaxillinΔLD4 cells exhibit a dramatic increase in the generation of broad lamellipodia with ectopic paxillin localizing to the cell periphery in focal contacts (a, arrows). Tail-like retraction fibers were frequently observed (a, arrowhead). Double arrows in a show ectopic paxillin in central focal contacts. Arrows in b and c show ectopic and endogenous paxillin in focal contacts, respectively. Images of the cells were captured at 240 min and are representative of the differences in cell morphology observed at the time points tested; i.e., 60, 240, and 360 min. (C) The number of cells exhibiting multiple broad lamellipodia (as exemplified by the cell pictured in B, a and d) was quantified by counting >200 cells per time point and indicates the dramatic increase in these structures in paxillinΔLD4 cells. Values are the average of experiments performed in triplicate.
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fig1: Expression of paxillin LD4 deletion generates multiple broad lamellipodia during cell spreading. (A) Immunoprecipitation using avian-specific paxillin antisera (Pax1, right) and Western immunoblotting of total cell lysates (left) confirm the relative levels of paxillin, PKL, and p130cas protein, and demonstrates the overexpression of avian paxillin in the paxillinΔLD4 and paxillin WT cells compared with parental nontransfected CHO.K1 cells. (B, a and d) CHO.K1 cells ectopically expressing avian paxillin with the deletion of LD4 (paxillinΔLD4); (b and e) CHO.K1 cells ectopically expressing full-length wild-type avian paxillin (paxillin WT); (c and f) parental nontransfected CHO.K1 cells. CHO.K1 cells were respread on fibronectin-coated (Fn) coverslips (10 μg/ml) for 60, 240, and 360 min, and ectopic paxillin (Pax1, a and b), endogenous paxillin (c), and actin (d–f) were examined. PaxillinΔLD4 cells exhibit a dramatic increase in the generation of broad lamellipodia with ectopic paxillin localizing to the cell periphery in focal contacts (a, arrows). Tail-like retraction fibers were frequently observed (a, arrowhead). Double arrows in a show ectopic paxillin in central focal contacts. Arrows in b and c show ectopic and endogenous paxillin in focal contacts, respectively. Images of the cells were captured at 240 min and are representative of the differences in cell morphology observed at the time points tested; i.e., 60, 240, and 360 min. (C) The number of cells exhibiting multiple broad lamellipodia (as exemplified by the cell pictured in B, a and d) was quantified by counting >200 cells per time point and indicates the dramatic increase in these structures in paxillinΔLD4 cells. Values are the average of experiments performed in triplicate.

Mentions: The focal adhesion adapter protein paxillin participates in the assembly of a complex of proteins involved in Rac regulation of the cytoskeleton during cell spreading and migration (Turner et al., 1999). To further characterize the role of paxillin, and specifically the LD4 motif, in controlling these Rac-dependent processes, clonal cell lines expressing full-length avian paxillin engineered with a deletion of the LD4 motif (paxillinΔLD4) and full-length wild-type avian paxillin (paxillin WT) were generated in CHO.K1 fibroblasts. Fig. 1 A demonstrates the level of overexpression of both the ectopic paxillinΔLD4 and paxillin WT in these clonal cell lines, as compared with the level of endogenous paxillin in the parental CHO.K1 cells.


The LD4 motif of paxillin regulates cell spreading and motility through an interaction with paxillin kinase linker (PKL).

West KA, Zhang H, Brown MC, Nikolopoulos SN, Riedy MC, Horwitz AF, Turner CE - J. Cell Biol. (2001)

Expression of paxillin LD4 deletion generates multiple broad lamellipodia during cell spreading. (A) Immunoprecipitation using avian-specific paxillin antisera (Pax1, right) and Western immunoblotting of total cell lysates (left) confirm the relative levels of paxillin, PKL, and p130cas protein, and demonstrates the overexpression of avian paxillin in the paxillinΔLD4 and paxillin WT cells compared with parental nontransfected CHO.K1 cells. (B, a and d) CHO.K1 cells ectopically expressing avian paxillin with the deletion of LD4 (paxillinΔLD4); (b and e) CHO.K1 cells ectopically expressing full-length wild-type avian paxillin (paxillin WT); (c and f) parental nontransfected CHO.K1 cells. CHO.K1 cells were respread on fibronectin-coated (Fn) coverslips (10 μg/ml) for 60, 240, and 360 min, and ectopic paxillin (Pax1, a and b), endogenous paxillin (c), and actin (d–f) were examined. PaxillinΔLD4 cells exhibit a dramatic increase in the generation of broad lamellipodia with ectopic paxillin localizing to the cell periphery in focal contacts (a, arrows). Tail-like retraction fibers were frequently observed (a, arrowhead). Double arrows in a show ectopic paxillin in central focal contacts. Arrows in b and c show ectopic and endogenous paxillin in focal contacts, respectively. Images of the cells were captured at 240 min and are representative of the differences in cell morphology observed at the time points tested; i.e., 60, 240, and 360 min. (C) The number of cells exhibiting multiple broad lamellipodia (as exemplified by the cell pictured in B, a and d) was quantified by counting >200 cells per time point and indicates the dramatic increase in these structures in paxillinΔLD4 cells. Values are the average of experiments performed in triplicate.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2196859&req=5

fig1: Expression of paxillin LD4 deletion generates multiple broad lamellipodia during cell spreading. (A) Immunoprecipitation using avian-specific paxillin antisera (Pax1, right) and Western immunoblotting of total cell lysates (left) confirm the relative levels of paxillin, PKL, and p130cas protein, and demonstrates the overexpression of avian paxillin in the paxillinΔLD4 and paxillin WT cells compared with parental nontransfected CHO.K1 cells. (B, a and d) CHO.K1 cells ectopically expressing avian paxillin with the deletion of LD4 (paxillinΔLD4); (b and e) CHO.K1 cells ectopically expressing full-length wild-type avian paxillin (paxillin WT); (c and f) parental nontransfected CHO.K1 cells. CHO.K1 cells were respread on fibronectin-coated (Fn) coverslips (10 μg/ml) for 60, 240, and 360 min, and ectopic paxillin (Pax1, a and b), endogenous paxillin (c), and actin (d–f) were examined. PaxillinΔLD4 cells exhibit a dramatic increase in the generation of broad lamellipodia with ectopic paxillin localizing to the cell periphery in focal contacts (a, arrows). Tail-like retraction fibers were frequently observed (a, arrowhead). Double arrows in a show ectopic paxillin in central focal contacts. Arrows in b and c show ectopic and endogenous paxillin in focal contacts, respectively. Images of the cells were captured at 240 min and are representative of the differences in cell morphology observed at the time points tested; i.e., 60, 240, and 360 min. (C) The number of cells exhibiting multiple broad lamellipodia (as exemplified by the cell pictured in B, a and d) was quantified by counting >200 cells per time point and indicates the dramatic increase in these structures in paxillinΔLD4 cells. Values are the average of experiments performed in triplicate.
Mentions: The focal adhesion adapter protein paxillin participates in the assembly of a complex of proteins involved in Rac regulation of the cytoskeleton during cell spreading and migration (Turner et al., 1999). To further characterize the role of paxillin, and specifically the LD4 motif, in controlling these Rac-dependent processes, clonal cell lines expressing full-length avian paxillin engineered with a deletion of the LD4 motif (paxillinΔLD4) and full-length wild-type avian paxillin (paxillin WT) were generated in CHO.K1 fibroblasts. Fig. 1 A demonstrates the level of overexpression of both the ectopic paxillinΔLD4 and paxillin WT in these clonal cell lines, as compared with the level of endogenous paxillin in the parental CHO.K1 cells.

Bottom Line: In addition, FAK activity during spreading was not compromised by deletion of the paxillin LD4 motif.Furthermore, overexpression of PKL mutants lacking the paxillin-binding site (PKLDeltaPBS2) induced phenotypic changes reminiscent of paxillinDeltaLD4 mutant cells.These data suggest that the paxillin association with PKL is essential for normal integrin-mediated cell spreading, and locomotion and that this interaction is necessary for the regulation of Rac activity during these events.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Developmental Biology, State University of New York, Upstate Medical University, 750 East Adams St., Syracuse, NY 13210, USA.

ABSTRACT
The small GTPases of the Rho family are intimately involved in integrin-mediated changes in the actin cytoskeleton that accompany cell spreading and motility. The exact means by which the Rho family members elicit these changes is unclear. Here, we demonstrate that the interaction of paxillin via its LD4 motif with the putative ARF-GAP paxillin kinase linker (PKL) (Turner et al., 1999), is critically involved in the regulation of Rac-dependent changes in the actin cytoskeleton that accompany cell spreading and motility. Overexpression of a paxillin LD4 deletion mutant (paxillinDeltaLD4) in CHO.K1 fibroblasts caused the generation of multiple broad lamellipodia. These morphological changes were accompanied by an increase in cell protrusiveness and random motility, which correlated with prolonged activation of Rac. In contrast, directional motility was inhibited. These alterations in morphology and motility were dependent on a paxillin-PKL interaction. In cells overexpressing paxillinDeltaLD4 mutants, PKL localization to focal contacts was disrupted, whereas that of focal adhesion kinase (FAK) and vinculin was not. In addition, FAK activity during spreading was not compromised by deletion of the paxillin LD4 motif. Furthermore, overexpression of PKL mutants lacking the paxillin-binding site (PKLDeltaPBS2) induced phenotypic changes reminiscent of paxillinDeltaLD4 mutant cells. These data suggest that the paxillin association with PKL is essential for normal integrin-mediated cell spreading, and locomotion and that this interaction is necessary for the regulation of Rac activity during these events.

Show MeSH
Related in: MedlinePlus