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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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Introduction of PKL and PKLΔPBS2 mutants does not affect paxillin localization to focal contacts, but PKLΔPBS2 does affect cell protrusiveness. (A) PaxillinΔLD4 and paxillin WT cells were transiently transfected separately with GFP–PKL or GFP–PKLΔPBS2, detached, and subjected to spreading assays on fibronectin for 60, 240, and 360 min. Transfected cells were then visualized by GFP fluorescence (a–d) and ectopic paxillin by Pax1 immunofluorescence (e–h). Immunofluorescence analysis demonstrates that the introduction of GFP–PKL and GFP–PKLΔPBS2 into paxillinΔLD4 and paxillin WT cells does not affect paxillin localization to focal contacts. 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. (B) Quantification of protrusiveness demonstrates an increase in cell area (μm2) of CHO.K1 cells transfected with PKLΔPBS2 (left), as compared with parental nontransfected cells (Fig. 3 B, bottom). This observed increase in cell area is elevated by the cointroduction of paxillin and PKLΔPBS2 (right).
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fig9: Introduction of PKL and PKLΔPBS2 mutants does not affect paxillin localization to focal contacts, but PKLΔPBS2 does affect cell protrusiveness. (A) PaxillinΔLD4 and paxillin WT cells were transiently transfected separately with GFP–PKL or GFP–PKLΔPBS2, detached, and subjected to spreading assays on fibronectin for 60, 240, and 360 min. Transfected cells were then visualized by GFP fluorescence (a–d) and ectopic paxillin by Pax1 immunofluorescence (e–h). Immunofluorescence analysis demonstrates that the introduction of GFP–PKL and GFP–PKLΔPBS2 into paxillinΔLD4 and paxillin WT cells does not affect paxillin localization to focal contacts. 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. (B) Quantification of protrusiveness demonstrates an increase in cell area (μm2) of CHO.K1 cells transfected with PKLΔPBS2 (left), as compared with parental nontransfected cells (Fig. 3 B, bottom). This observed increase in cell area is elevated by the cointroduction of paxillin and PKLΔPBS2 (right).

Mentions: To test whether PKL binding to the paxillin LD4 motif via the PBS2 domain was necessary for normal cell spreading both paxillinΔLD4 and paxillin WT cells were transiently transfected with GFP–PKL or GFP–PKLΔPBS2 and evaluated for spreading on fibronectin. Although the introduction of GFP–PKLΔPBS2 into paxillinΔLD4 cells had no effect on cell morphology, expression of this construct in paxillin WT cells caused the generation of a paxillinΔLD4-like phenotype in ∼50% of cells (Fig. 8, B and C) . Expression of GFP–PKL had no effect on the morphology of either paxillinΔLD4 or paxillin WT cells (Fig. 8, B and C). However, although it can be seen that paxillin and PKL colocalize in paxillin WT cells expressing GFP–PKL (Fig. 9 A, b and f), in paxillinΔLD4 cells expressing either GFP–PKL or GFP–PKLΔPBS2, PKL is cytoplasmic (Fig. 9 A, a and c), whereas paxillin is found in focal contacts (Fig. 9 A, e and g). In paxillin WT cells, GFP–PKLΔPBS2 remained cytoplasmic (Fig. 9 A, d), whereas paxillin was found in focal contacts (Fig. 9 A, h). Thus, the introduction of wild-type PKL or PKL lacking the paxillin-binding site does not affect paxillin localization. Similar effects on morphology and paxillin and PKL subcellular localization were also obtained by the introduction of either GFP–PKL or GFP–PKLΔPBS2 into parental CHO.K1 cells (data not shown).


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)

Introduction of PKL and PKLΔPBS2 mutants does not affect paxillin localization to focal contacts, but PKLΔPBS2 does affect cell protrusiveness. (A) PaxillinΔLD4 and paxillin WT cells were transiently transfected separately with GFP–PKL or GFP–PKLΔPBS2, detached, and subjected to spreading assays on fibronectin for 60, 240, and 360 min. Transfected cells were then visualized by GFP fluorescence (a–d) and ectopic paxillin by Pax1 immunofluorescence (e–h). Immunofluorescence analysis demonstrates that the introduction of GFP–PKL and GFP–PKLΔPBS2 into paxillinΔLD4 and paxillin WT cells does not affect paxillin localization to focal contacts. 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. (B) Quantification of protrusiveness demonstrates an increase in cell area (μm2) of CHO.K1 cells transfected with PKLΔPBS2 (left), as compared with parental nontransfected cells (Fig. 3 B, bottom). This observed increase in cell area is elevated by the cointroduction of paxillin and PKLΔPBS2 (right).
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Related In: Results  -  Collection

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fig9: Introduction of PKL and PKLΔPBS2 mutants does not affect paxillin localization to focal contacts, but PKLΔPBS2 does affect cell protrusiveness. (A) PaxillinΔLD4 and paxillin WT cells were transiently transfected separately with GFP–PKL or GFP–PKLΔPBS2, detached, and subjected to spreading assays on fibronectin for 60, 240, and 360 min. Transfected cells were then visualized by GFP fluorescence (a–d) and ectopic paxillin by Pax1 immunofluorescence (e–h). Immunofluorescence analysis demonstrates that the introduction of GFP–PKL and GFP–PKLΔPBS2 into paxillinΔLD4 and paxillin WT cells does not affect paxillin localization to focal contacts. 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. (B) Quantification of protrusiveness demonstrates an increase in cell area (μm2) of CHO.K1 cells transfected with PKLΔPBS2 (left), as compared with parental nontransfected cells (Fig. 3 B, bottom). This observed increase in cell area is elevated by the cointroduction of paxillin and PKLΔPBS2 (right).
Mentions: To test whether PKL binding to the paxillin LD4 motif via the PBS2 domain was necessary for normal cell spreading both paxillinΔLD4 and paxillin WT cells were transiently transfected with GFP–PKL or GFP–PKLΔPBS2 and evaluated for spreading on fibronectin. Although the introduction of GFP–PKLΔPBS2 into paxillinΔLD4 cells had no effect on cell morphology, expression of this construct in paxillin WT cells caused the generation of a paxillinΔLD4-like phenotype in ∼50% of cells (Fig. 8, B and C) . Expression of GFP–PKL had no effect on the morphology of either paxillinΔLD4 or paxillin WT cells (Fig. 8, B and C). However, although it can be seen that paxillin and PKL colocalize in paxillin WT cells expressing GFP–PKL (Fig. 9 A, b and f), in paxillinΔLD4 cells expressing either GFP–PKL or GFP–PKLΔPBS2, PKL is cytoplasmic (Fig. 9 A, a and c), whereas paxillin is found in focal contacts (Fig. 9 A, e and g). In paxillin WT cells, GFP–PKLΔPBS2 remained cytoplasmic (Fig. 9 A, d), whereas paxillin was found in focal contacts (Fig. 9 A, h). Thus, the introduction of wild-type PKL or PKL lacking the paxillin-binding site does not affect paxillin localization. Similar effects on morphology and paxillin and PKL subcellular localization were also obtained by the introduction of either GFP–PKL or GFP–PKLΔPBS2 into parental CHO.K1 cells (data not shown).

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