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

The small GTPases of the Rho family are intimately involved in integrin-med iated changes in the actin cytoskeleton that accompany cell spreading and m otility. The exact means by which the Rho family members elicit these chang es is unclear. Here, we demonstrate that the interaction of paxillin via it s LD4 motif with the putative ARF-CAP paxillin kinase linker (PKL) (Turner et at., 1999), is critically involved in the regulation of Rac-dependent ch anges in the actin cytoskeleton that accompany cell spreading and motility. Overexpression of a paxillin LD4 deletion mutant (paxillin Delta LD4) in C HO.K1 fibroblasts caused the generation of multiple broad lamellipodia. The se morphological changes were accompanied by an increase in cell protrusive ness and random motility, which correlated with prolonged activation of Rac . In contrast, directional motility was inhibited. These alterations in mor phology and motility were dependent on a paxillin-PKL interaction. In cells overexpressing paxillin Delta LD4 mutants, PKL localization to focal conta cts 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 mut ants lacking the paxillin-binding site (PKL Delta PBS2) induced phenotypic changes reminiscent of paxillin Delta LD4 mutant cells. These data suggest that the paxillin association with PKL is essential for normal integrin-med iated cell spreading, and locomotion and that this interaction is necessary for the regulation of Rac activity during these events.