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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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Deletion of paxillin LD4 affects endogenous PKL localization and paxillin–PKL association in vivo, but not FAK or vinculin localization or FAK activity. (A) PaxillinΔLD4, paxillin WT, and parental nontransfected CHO.K1 cells spread on fibronectin were processed for indirect immunofluorescence using antipaxillin antisera (Pax1) (d and e), phosphospecific Y118 paxillin anti-sera (f), and anti-PKL antibodies (a–c), which demonstrates the colocalization of paxillin and endogenous PKL in focal contacts in paxillin WT and parental nontransfected cells (b and e, c and f). In contrast, in paxillinΔLD4 cells, ectopic paxillin was found in focal contacts, whereas the endogenous PKL distribution was cytoplasmic (a and d, respectively). (B) Parental CHO.K1 cells were transiently transfected with either GFP–paxillin or GFP–paxillinΔLD4, detached from tissue culture dishes, and respread on fibronectin for 60 min. Coimmunoprecipitation assays were then performed followed by Western blot analysis and demonstrate that, although GFP–paxillin is capable of precipitating endogenous PKL, GFP–paxillinΔLD4 does not. (C) PaxillinΔLD4 cells spread on fibronectin were processed for indirect immunofluorescence using either anti-FAK (c), or antivinculin antibodies (d) and antipaxillin antisera (Pax1) (a and b). Arrowheads in a and b and arrows in c and d indicate that the deletion of paxillin LD4 does not affect the localization of these other paxillin-binding partners. (D) Parental nontransfected CHO.K1, paxillin WT, and paxillinΔLD4 cells were spread on fibronectin for 0, 20, and 120 min, and FAK was immunoprecipitated from lysates derived from these cells. Immunoblot analysis of cell lysates with antiphosphotyrosine antibody and of FAK immunoprecipitates with phosphospecific Y397 FAK antisera demonstrates equivalent FAK activation in the three cell lines tested.
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fig6: Deletion of paxillin LD4 affects endogenous PKL localization and paxillin–PKL association in vivo, but not FAK or vinculin localization or FAK activity. (A) PaxillinΔLD4, paxillin WT, and parental nontransfected CHO.K1 cells spread on fibronectin were processed for indirect immunofluorescence using antipaxillin antisera (Pax1) (d and e), phosphospecific Y118 paxillin anti-sera (f), and anti-PKL antibodies (a–c), which demonstrates the colocalization of paxillin and endogenous PKL in focal contacts in paxillin WT and parental nontransfected cells (b and e, c and f). In contrast, in paxillinΔLD4 cells, ectopic paxillin was found in focal contacts, whereas the endogenous PKL distribution was cytoplasmic (a and d, respectively). (B) Parental CHO.K1 cells were transiently transfected with either GFP–paxillin or GFP–paxillinΔLD4, detached from tissue culture dishes, and respread on fibronectin for 60 min. Coimmunoprecipitation assays were then performed followed by Western blot analysis and demonstrate that, although GFP–paxillin is capable of precipitating endogenous PKL, GFP–paxillinΔLD4 does not. (C) PaxillinΔLD4 cells spread on fibronectin were processed for indirect immunofluorescence using either anti-FAK (c), or antivinculin antibodies (d) and antipaxillin antisera (Pax1) (a and b). Arrowheads in a and b and arrows in c and d indicate that the deletion of paxillin LD4 does not affect the localization of these other paxillin-binding partners. (D) Parental nontransfected CHO.K1, paxillin WT, and paxillinΔLD4 cells were spread on fibronectin for 0, 20, and 120 min, and FAK was immunoprecipitated from lysates derived from these cells. Immunoblot analysis of cell lysates with antiphosphotyrosine antibody and of FAK immunoprecipitates with phosphospecific Y397 FAK antisera demonstrates equivalent FAK activation in the three cell lines tested.

Mentions: We have previously shown that the LD4 motif of paxillin binds to both FAK and vinculin as well as to the newly characterized putative ARF-GAP protein PKL (Turner and Miller, 1994; Brown et al., 1996; Turner et al., 1999). The association of paxillin with PKL mediates an interaction with a protein complex comprised of PIX/PAK/Nck, which has been implicated in Rac-dependent cytoskeletal rearrangements (Manser et al., 1997; Sells et al., 1997; Obermeier et al., 1998; Zhao et al., 1998). The involvement of the LD4 motif of paxillin in the signaling pathway(s) controlling actin-driven cytoskeletal dynamics during cell spreading and motility led us to investigate a possible role for the interaction between paxillin and PKL in these events. To evaluate the significance of the paxillin–PKL association, the distribution of endogenous PKL was first determined in paxillinΔLD4, paxillin WT, and parental CHO.K1 cells. Although colocalization of paxillin and PKL in focal contacts was observed in paxillin WT and parental CHO.K1 cells (Fig. 6 A, b and e, c and f, respectively), the distribution of PKL in paxillinΔLD4 cells was almost exclusively cytoplasmic (Fig. 6 A, a). In contrast, the ectopic paxillin in paxillinΔLD4 cells was found in peripheral and central focal contacts, as well as the cytoplasm (Fig. 6 A, d).


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)

Deletion of paxillin LD4 affects endogenous PKL localization and paxillin–PKL association in vivo, but not FAK or vinculin localization or FAK activity. (A) PaxillinΔLD4, paxillin WT, and parental nontransfected CHO.K1 cells spread on fibronectin were processed for indirect immunofluorescence using antipaxillin antisera (Pax1) (d and e), phosphospecific Y118 paxillin anti-sera (f), and anti-PKL antibodies (a–c), which demonstrates the colocalization of paxillin and endogenous PKL in focal contacts in paxillin WT and parental nontransfected cells (b and e, c and f). In contrast, in paxillinΔLD4 cells, ectopic paxillin was found in focal contacts, whereas the endogenous PKL distribution was cytoplasmic (a and d, respectively). (B) Parental CHO.K1 cells were transiently transfected with either GFP–paxillin or GFP–paxillinΔLD4, detached from tissue culture dishes, and respread on fibronectin for 60 min. Coimmunoprecipitation assays were then performed followed by Western blot analysis and demonstrate that, although GFP–paxillin is capable of precipitating endogenous PKL, GFP–paxillinΔLD4 does not. (C) PaxillinΔLD4 cells spread on fibronectin were processed for indirect immunofluorescence using either anti-FAK (c), or antivinculin antibodies (d) and antipaxillin antisera (Pax1) (a and b). Arrowheads in a and b and arrows in c and d indicate that the deletion of paxillin LD4 does not affect the localization of these other paxillin-binding partners. (D) Parental nontransfected CHO.K1, paxillin WT, and paxillinΔLD4 cells were spread on fibronectin for 0, 20, and 120 min, and FAK was immunoprecipitated from lysates derived from these cells. Immunoblot analysis of cell lysates with antiphosphotyrosine antibody and of FAK immunoprecipitates with phosphospecific Y397 FAK antisera demonstrates equivalent FAK activation in the three cell lines tested.
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Related In: Results  -  Collection

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fig6: Deletion of paxillin LD4 affects endogenous PKL localization and paxillin–PKL association in vivo, but not FAK or vinculin localization or FAK activity. (A) PaxillinΔLD4, paxillin WT, and parental nontransfected CHO.K1 cells spread on fibronectin were processed for indirect immunofluorescence using antipaxillin antisera (Pax1) (d and e), phosphospecific Y118 paxillin anti-sera (f), and anti-PKL antibodies (a–c), which demonstrates the colocalization of paxillin and endogenous PKL in focal contacts in paxillin WT and parental nontransfected cells (b and e, c and f). In contrast, in paxillinΔLD4 cells, ectopic paxillin was found in focal contacts, whereas the endogenous PKL distribution was cytoplasmic (a and d, respectively). (B) Parental CHO.K1 cells were transiently transfected with either GFP–paxillin or GFP–paxillinΔLD4, detached from tissue culture dishes, and respread on fibronectin for 60 min. Coimmunoprecipitation assays were then performed followed by Western blot analysis and demonstrate that, although GFP–paxillin is capable of precipitating endogenous PKL, GFP–paxillinΔLD4 does not. (C) PaxillinΔLD4 cells spread on fibronectin were processed for indirect immunofluorescence using either anti-FAK (c), or antivinculin antibodies (d) and antipaxillin antisera (Pax1) (a and b). Arrowheads in a and b and arrows in c and d indicate that the deletion of paxillin LD4 does not affect the localization of these other paxillin-binding partners. (D) Parental nontransfected CHO.K1, paxillin WT, and paxillinΔLD4 cells were spread on fibronectin for 0, 20, and 120 min, and FAK was immunoprecipitated from lysates derived from these cells. Immunoblot analysis of cell lysates with antiphosphotyrosine antibody and of FAK immunoprecipitates with phosphospecific Y397 FAK antisera demonstrates equivalent FAK activation in the three cell lines tested.
Mentions: We have previously shown that the LD4 motif of paxillin binds to both FAK and vinculin as well as to the newly characterized putative ARF-GAP protein PKL (Turner and Miller, 1994; Brown et al., 1996; Turner et al., 1999). The association of paxillin with PKL mediates an interaction with a protein complex comprised of PIX/PAK/Nck, which has been implicated in Rac-dependent cytoskeletal rearrangements (Manser et al., 1997; Sells et al., 1997; Obermeier et al., 1998; Zhao et al., 1998). The involvement of the LD4 motif of paxillin in the signaling pathway(s) controlling actin-driven cytoskeletal dynamics during cell spreading and motility led us to investigate a possible role for the interaction between paxillin and PKL in these events. To evaluate the significance of the paxillin–PKL association, the distribution of endogenous PKL was first determined in paxillinΔLD4, paxillin WT, and parental CHO.K1 cells. Although colocalization of paxillin and PKL in focal contacts was observed in paxillin WT and parental CHO.K1 cells (Fig. 6 A, b and e, c and f, respectively), the distribution of PKL in paxillinΔLD4 cells was almost exclusively cytoplasmic (Fig. 6 A, a). In contrast, the ectopic paxillin in paxillinΔLD4 cells was found in peripheral and central focal contacts, as well as the cytoplasm (Fig. 6 A, d).

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