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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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Reintroduction of wild-type paxillin into paxillinΔLD4 cells rescues the normal spreading phenotype. (A) Western blot analysis using GFP polyclonal antisera was used to confirm the presence of the ectopic GFP–paxillin protein. (B) PaxillinΔLD4 and paxillin wild-type cells were transiently transfected with GFP-paxillinΔLD4 and subjected to spreading assays on fibronectin for 60, 240, and 360 min, and GFP–paxillin transfectants were visualized by GFP fluorescence (a and b), and actin, by RITC-phalloidin (c and d). Arrows in c indicate paxillinΔLD4 cells expressing GFP–paxillin lacking broad lamellipodia, whereas arrowheads in c demonstrate the presence of these structuresin a cell lacking GFP-paxillin. Images of the cells were captured at the 240-min time point and are representative of the differences in cell morphology observed at all time points. (C) The number of cells exhibiting multiple broad lamellipodia was quantified by counting >200 cells per time point. Values are the average of experiments performed in triplicate.
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fig2: Reintroduction of wild-type paxillin into paxillinΔLD4 cells rescues the normal spreading phenotype. (A) Western blot analysis using GFP polyclonal antisera was used to confirm the presence of the ectopic GFP–paxillin protein. (B) PaxillinΔLD4 and paxillin wild-type cells were transiently transfected with GFP-paxillinΔLD4 and subjected to spreading assays on fibronectin for 60, 240, and 360 min, and GFP–paxillin transfectants were visualized by GFP fluorescence (a and b), and actin, by RITC-phalloidin (c and d). Arrows in c indicate paxillinΔLD4 cells expressing GFP–paxillin lacking broad lamellipodia, whereas arrowheads in c demonstrate the presence of these structuresin a cell lacking GFP-paxillin. Images of the cells were captured at the 240-min time point and are representative of the differences in cell morphology observed at all time points. (C) The number of cells exhibiting multiple broad lamellipodia was quantified by counting >200 cells per time point. Values are the average of experiments performed in triplicate.

Mentions: To determine whether this morphological change was attributable solely to the absence of the paxillin LD4 motif, we introduced a full-length wild-type avian GFP–paxillin fusion (GFP–paxillin) into both paxillinΔLD4 and paxillin WT cells and examined the cell's phenotype during spreading on fibronectin. Although GFP–paxillin had no effect on the morphology of paxillin WT cells, its introduction into paxillinΔLD4 cells reduced the generation of multiple broad lamellipodia to ∼20%, as compared with ∼60% in paxillinΔLD4 cells not containing GFP–paxillin (Fig. 2, B , a and c, and C). This effect was entirely due to the reintroduction of wild-type GFP–paxillin since expression of GFP alone did not disrupt the morphological changes observed in paxillinΔLD4 cells (data not shown). The above observations suggest that the LD4 motif of paxillin is involved in the regulation of actin cytoskeletal changes that occur during cell spreading.


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)

Reintroduction of wild-type paxillin into paxillinΔLD4 cells rescues the normal spreading phenotype. (A) Western blot analysis using GFP polyclonal antisera was used to confirm the presence of the ectopic GFP–paxillin protein. (B) PaxillinΔLD4 and paxillin wild-type cells were transiently transfected with GFP-paxillinΔLD4 and subjected to spreading assays on fibronectin for 60, 240, and 360 min, and GFP–paxillin transfectants were visualized by GFP fluorescence (a and b), and actin, by RITC-phalloidin (c and d). Arrows in c indicate paxillinΔLD4 cells expressing GFP–paxillin lacking broad lamellipodia, whereas arrowheads in c demonstrate the presence of these structuresin a cell lacking GFP-paxillin. Images of the cells were captured at the 240-min time point and are representative of the differences in cell morphology observed at all time points. (C) The number of cells exhibiting multiple broad lamellipodia was quantified by counting >200 cells per time point. Values are the average of experiments performed in triplicate.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2196859&req=5

fig2: Reintroduction of wild-type paxillin into paxillinΔLD4 cells rescues the normal spreading phenotype. (A) Western blot analysis using GFP polyclonal antisera was used to confirm the presence of the ectopic GFP–paxillin protein. (B) PaxillinΔLD4 and paxillin wild-type cells were transiently transfected with GFP-paxillinΔLD4 and subjected to spreading assays on fibronectin for 60, 240, and 360 min, and GFP–paxillin transfectants were visualized by GFP fluorescence (a and b), and actin, by RITC-phalloidin (c and d). Arrows in c indicate paxillinΔLD4 cells expressing GFP–paxillin lacking broad lamellipodia, whereas arrowheads in c demonstrate the presence of these structuresin a cell lacking GFP-paxillin. Images of the cells were captured at the 240-min time point and are representative of the differences in cell morphology observed at all time points. (C) The number of cells exhibiting multiple broad lamellipodia was quantified by counting >200 cells per time point. Values are the average of experiments performed in triplicate.
Mentions: To determine whether this morphological change was attributable solely to the absence of the paxillin LD4 motif, we introduced a full-length wild-type avian GFP–paxillin fusion (GFP–paxillin) into both paxillinΔLD4 and paxillin WT cells and examined the cell's phenotype during spreading on fibronectin. Although GFP–paxillin had no effect on the morphology of paxillin WT cells, its introduction into paxillinΔLD4 cells reduced the generation of multiple broad lamellipodia to ∼20%, as compared with ∼60% in paxillinΔLD4 cells not containing GFP–paxillin (Fig. 2, B , a and c, and C). This effect was entirely due to the reintroduction of wild-type GFP–paxillin since expression of GFP alone did not disrupt the morphological changes observed in paxillinΔLD4 cells (data not shown). The above observations suggest that the LD4 motif of paxillin is involved in the regulation of actin cytoskeletal changes that occur during cell spreading.

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