A paxillin tyrosine phosphorylation switch regulates the assembly and form of cell-matrix adhesions

Abstract

Diverse cellular processes are carried out by distinct integrin-mediated adhesions. Cell spreading and migration are driven by focal complexes; robust adhesion to the extracellular matrix by focal adhesions; and matrix remodeling by fibrillar adhesions. The mechanism(s) regulating the spatio-temporal distribution and dynamics of the three types of adhesion are unknown. Here, we combine live-cell imaging, labeling with phosphospecific-antibodies and overexpression of a novel tyrosine phosphomimetic mutant of paxillin, to demonstrate that the modulation of tyrosine phosphorylation of paxillin regulates both the assembly and turnover of adhesion sites. Moreover, phosphorylated paxillin enhanced lamellipodial protrusions, whereas non-phosphorylated paxillin was essential for fibrillar adhesion formation and for fibronectin fibrillogenesis. We further show that focal adhesion kinase preferentially interacted with the tyrosine phosphomimetic paxillin and its recruitment is implicated in high turnover of focal complexes and translocation of focal adhesions. We created a mathematical model that recapitulates the salient features of the measured dynamics, and conclude that tyrosine phosphorylation of the adaptor protein paxillin functions as a major switch, regulating the adhesive phenotype of cells.

Regulation of focal adhesion turnover by ErbB signalling in invasive breast cancer cells

“…For instance, although Src SH2 interaction with FAK is necessary for appropriate recruitment of Src and FAK to FA in migratory cells, the FAK -Src complex can also regulate the phosphorylation of the adaptor proteins paxillin and p130Cas (Bellis et al, 1995;Cary et al, 1998); the latter can bind independently to both FAK and Src. Phosphorylation of both paxillin and p130Cas creates binding sites for the SH2 domain of the Crk adaptor protein (Iwahara et al, 2004), which can impact on the maturation of FA complexes to stable FAs or turn them rapidly (Webb et al, 2004;Zaidel-Bar et al, 2007). Indeed, cells deficient in p130Cas or paxillin, similar to FAK-and Src-deficient cells, have impaired adhesion disassembly and reduced motility (Webb et al, 2004).…”

Integrins in Cell Migration

“…These studies support a critical role for FAK/Src signaling in adhesion turnover. Although many mechanisms likely contribute, these kinases phosphorylate Rho GTPase signaling scaffolds like paxillin, which in turn bind GEFs for Rac (Brown and Turner 2004;Zaidel-Bar et al 2007), regulate myosin activity, and actin polymerization. Thus, both tyrosine kinases and phosphatases play a role (Yu et al 1998;Angers-Lousteau et al 1999;Sastry et al 2002); it follows therefore that dynamic cycles of phosphorylation-dephosphorylation at adhesion sites are likely essential for adhesion turnover and migration.…”

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

“…Moreover, signalling through immunomodulators such as tumor necrosis factor-α, formyl-methionyl-leucyl-phenylalanine, IgE, T-cell receptor or complement, as well as exposure to the toxic by-products of viral and bacterial infection can also result in paxillin tyrosine phosphorylation [66,86]. Lastly, cellular stress, in particular membrane depolarization, hypertonicity, physical stretch and shear stress can also be attributed with the induction of paxillin tyrosine phosphorylation [86,110].…”

“…A role for paxillin in regulating adhesion dynamics has been previously described [110,139]. Briefly, fibroblasts deficient in paxillin exhibit defects in the cortical cytoskeleton, cell spreading and migration; cell adhesion however, is not affected [96,107,123].…”

“…Specifically, serine phosphorylation of the LIM domain of paxillin has been found to temporally target and/or regulate the integration of paxillin into focal adhesions while tyrosine phosphorylated paxillin has been shown to recruit the focal adhesion proteins FAK and Src into the adhesion complex [99,110,111]. Actively disassembling adhesions, such as early adhesions and the distal part of late adhesions, are preferential zones of microtubule targeting and catastrophe that have been shown to be enriched in phosphorylated paxillin [22,30,110,140].…”