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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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Characterization of the paxillin-binding site on PKL. (A) Schematic representations of full-length wild-type (PKL), deletion of PBS1 (PKLΔPBS1), and deletion of PBS2 (PKLΔPBS2) PKL proteins, respectively. (B) Radiolabeled wild-type PKL proteins and PKL protein containing either a deletion of PBS1 or PBS2 were produced by transcription/translation-coupled reactions and used in in vitro binding assays with GST–PIX or GST–LD4 to identify PBS2 as the paxillin-binding site on PKL. PIX binding to PKL was unaffected by either deletion. (C) Parental CHO.K1 cells were transiently transfected with either GFP–PKL or GFP–PKLΔPBS2, 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 GFP–PKL is capable of associating with paxillin in vivo, whereas GFP–PKLΔPBS2 is not. Immunoblot analysis with an antibody to p130cas was used to demonstrate the specificity of PKL–paxillin interaction.
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fig7: Characterization of the paxillin-binding site on PKL. (A) Schematic representations of full-length wild-type (PKL), deletion of PBS1 (PKLΔPBS1), and deletion of PBS2 (PKLΔPBS2) PKL proteins, respectively. (B) Radiolabeled wild-type PKL proteins and PKL protein containing either a deletion of PBS1 or PBS2 were produced by transcription/translation-coupled reactions and used in in vitro binding assays with GST–PIX or GST–LD4 to identify PBS2 as the paxillin-binding site on PKL. PIX binding to PKL was unaffected by either deletion. (C) Parental CHO.K1 cells were transiently transfected with either GFP–PKL or GFP–PKLΔPBS2, 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 GFP–PKL is capable of associating with paxillin in vivo, whereas GFP–PKLΔPBS2 is not. Immunoblot analysis with an antibody to p130cas was used to demonstrate the specificity of PKL–paxillin interaction.

Mentions: We have previously localized the binding site of PIX to the NH2 terminus of PKL, and that of paxillin to the COOH terminus (Turner et al., 1999). However, PKL contains two putative paxillin-binding subdomains (PBSs), based on their homology with the paxillin-binding sites of vinculin and FAK (Wood et al., 1994; Tachibana et al., 1995; Brown et al., 1996). To identify the PKL PBS that mediates paxillin LD4 binding, radiolabeled full-length PKL proteins, as well as PKL proteins containing either a deletion of PBS1 or PBS2 (Fig. 7 A), were produced by transcription/translation-coupled reactions and used in in vitro binding assays with GST-coupled fusion proteins, GST–PIX and GST–LD4. Although GST–PIX was found to bind the wild-type and both mutant PKL proteins, GST–LD4 bound to both full-length PKL and the PBS1 deletion mutant, but no binding was observed to the PBS2 deletion mutant (Fig. 7 B). These results identify the PBS2 region of PKL as the paxillin-binding site.


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)

Characterization of the paxillin-binding site on PKL. (A) Schematic representations of full-length wild-type (PKL), deletion of PBS1 (PKLΔPBS1), and deletion of PBS2 (PKLΔPBS2) PKL proteins, respectively. (B) Radiolabeled wild-type PKL proteins and PKL protein containing either a deletion of PBS1 or PBS2 were produced by transcription/translation-coupled reactions and used in in vitro binding assays with GST–PIX or GST–LD4 to identify PBS2 as the paxillin-binding site on PKL. PIX binding to PKL was unaffected by either deletion. (C) Parental CHO.K1 cells were transiently transfected with either GFP–PKL or GFP–PKLΔPBS2, 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 GFP–PKL is capable of associating with paxillin in vivo, whereas GFP–PKLΔPBS2 is not. Immunoblot analysis with an antibody to p130cas was used to demonstrate the specificity of PKL–paxillin interaction.
© Copyright Policy
Related In: Results  -  Collection

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

fig7: Characterization of the paxillin-binding site on PKL. (A) Schematic representations of full-length wild-type (PKL), deletion of PBS1 (PKLΔPBS1), and deletion of PBS2 (PKLΔPBS2) PKL proteins, respectively. (B) Radiolabeled wild-type PKL proteins and PKL protein containing either a deletion of PBS1 or PBS2 were produced by transcription/translation-coupled reactions and used in in vitro binding assays with GST–PIX or GST–LD4 to identify PBS2 as the paxillin-binding site on PKL. PIX binding to PKL was unaffected by either deletion. (C) Parental CHO.K1 cells were transiently transfected with either GFP–PKL or GFP–PKLΔPBS2, 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 GFP–PKL is capable of associating with paxillin in vivo, whereas GFP–PKLΔPBS2 is not. Immunoblot analysis with an antibody to p130cas was used to demonstrate the specificity of PKL–paxillin interaction.
Mentions: We have previously localized the binding site of PIX to the NH2 terminus of PKL, and that of paxillin to the COOH terminus (Turner et al., 1999). However, PKL contains two putative paxillin-binding subdomains (PBSs), based on their homology with the paxillin-binding sites of vinculin and FAK (Wood et al., 1994; Tachibana et al., 1995; Brown et al., 1996). To identify the PKL PBS that mediates paxillin LD4 binding, radiolabeled full-length PKL proteins, as well as PKL proteins containing either a deletion of PBS1 or PBS2 (Fig. 7 A), were produced by transcription/translation-coupled reactions and used in in vitro binding assays with GST-coupled fusion proteins, GST–PIX and GST–LD4. Although GST–PIX was found to bind the wild-type and both mutant PKL proteins, GST–LD4 bound to both full-length PKL and the PBS1 deletion mutant, but no binding was observed to the PBS2 deletion mutant (Fig. 7 B). These results identify the PBS2 region of PKL as the paxillin-binding site.

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