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

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 (paxillinΔLD4) 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 paxillinΔLD4 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 (PKLΔPBS2) induced phenotypic changes reminiscent of paxillinΔLD4 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.

SLK-mediated phosphorylation of paxillin is required for focal adhesion turnover and cell migration

“…Taken together, these results demonstrate that paxillin S250 is required for efficient cell spreading and migration. shown to reduce focal adhesion disassembly 11-fold when compared to full length, wildtype paxillin [66,123,141]. Subsequent studies have revealed that a phospho-inactivating substitution of an alanine at S274 (human; S273 in chicken), which is contained within the LD4 motif, resulted in a similar, albeit less dramatic phenotype, with adhesions persisting for 25%…”

Src and FAK Kinases Cooperate to Phosphorylate Paxillin Kinase Linker, Stimulate Its Focal Adhesion Localization, and Regulate Cell Spreading and Protrusiveness

“…Images were captured every 60 s for up to 3 h. Cell protrusion was quantitated spanning 1 h at 10-min intervals as reported previously (West et al, 2001;Kinley et al, 2003). To quantitate percentage of change in cell area due to extension of protrusions, two still images representing one 10-min interval were extracted, registered, thresholded, and subtracted to estimate the new area.…”

“…In response to cell attachment and/or growth factor stimulation, PAK is activated and signals to Galisteo et al, 1996;Lu et al, 1997;Sells et al, 1997;Kiosses et al, 2002), the Cdc42/Rac GEF PIX (Bagrodia et al, 1998;Zhao et al, 2000a), the paxillin kinase linker (PKL)/G protein-coupled receptor kinase-interacting protein (GIT) family of ArfGAPs Premont et al, 2000;Brown et al, 2002;Manabe Ri et al, 2002), and the molecular scaffold protein paxillin Zhao et al, 2000b;Hashimoto et al, 2001;West et al, 2001;Brown et al, 2002). A complex of Nck-PAK-PIX-PKL exists within the cytosol Zhao et al, 2000b) and/or endomembranes (Matafora et al, 2001;Paris et al, 2003).…”

“…In response to cell attachment and/or growth factor stimulation, PAK is activated and signals to This article was published online ahead of print in MBC in Press (http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E05-02-0131) on July 6, 2005. sites of cell-matrix interaction called focal adhesions, the cytoskeleton as well as the nucleus to promote appropriate changes in cell function (Manser and Lim, 1999). Several proteins have been shown to contribute to PAK activation and function within the cell through regulation of its subcellular distribution, including the SH3-SH2 adaptor protein Nck (Bokoch et al, 1996;Galisteo et al, 1996;Lu et al, 1997;Sells et al, 1997;Kiosses et al, 2002), the Cdc42/Rac GEF PIX (Bagrodia et al, 1998;Zhao et al, 2000a), the paxillin kinase linker (PKL)/G protein-coupled receptor kinase-interacting protein (GIT) family of ArfGAPs Premont et al, 2000;Brown et al, 2002;Manabe Ri et al, 2002), and the molecular scaffold protein paxillin Zhao et al, 2000b;Hashimoto et al, 2001;West et al, 2001;Brown et al, 2002).…”

“…As a result, the complex is recruited to RhoA-type focal adhesions, where PAK is thought to trigger turnover (Zhao et al, 2000b) and also to nascent Cdc42/Rac adhesions at the leading edge, called focal complexes, where it may mediate maturation and transition to RhoA adhesions (Brown et al, 2002;Manabe Ri et al, 2002). Synchronization of adhesion formation and turnover is critical to the process of cell motility (Webb et al, 2004), and perturbation in the PKL-paxillin link results in enhanced spreading, combined with a significant inhibition of cell polarity, focal adhesion dynamics, and motility Zhao et al, 2000b;West et al, 2001;Brown et al, 2002;Manabe Ri et al, 2002;Webb et al, 2004), in part through the misregulation of Rac (West et al, 2001). Similarly, fibroblasts derived from mice with germline deletions in Src/Yes/Fyn (SYF), FAK, paxillin, and Nck exhibit profound defects in cytoskeletal organization and the capacity to efficiently migrate, providing concrete evidence for the essential nature of these associations and signaling pathways Klinghoffer et al, 1999;Hagel et al, 2002;Bladt et al, 2003;Webb et al, 2004).…”