Christopher E. Turner

FAK–Src signalling through paxillin, ERK and MLCK regulates adhesion disassembly

Cell migration is a complex, highly regulated process that involves the continuous formation and disassembly of adhesions (adhesion turnover). Adhesion formation takes place at the leading edge of protrusions, whereas disassembly occurs both at the cell rear and at the base of protrusions. Despite the importance of these processes in migration, the mechanisms that regulate adhesion formation and disassembly remain largely unknown. Here we develop quantitative assays to measure the rate of incorporation of molecules into adhesions and the departure of these proteins from adhesions. Using these assays, we show that kinases and adaptor molecules, including focal adhesion kinase (FAK), Src, p130CAS, paxillin, extracellular signal-regulated kinase (ERK) and myosin light-chain kinase (MLCK) are critical for adhesion turnover at the cell front, a process central to migration.

Paxillin and focal adhesion signalling

To facilitate a rapid response to environmental change, cells use scaffolding — or adaptor — proteins to recruit key components of their signal-transduction machinery to specific subcellular locations. Paxillin is a multi-domain adaptor found at the interface between the plasma membrane and the actin cytoskeleton. Here it provides a platform for the integration and processing of adhesion- and growth factor-related signals.

Paxillin comes of age

Paxillin is a multi-domain scaffold protein that localizes to the intracellular surface of sites of cell adhesion to the extracellular matrix. Through the interactions of its multiple protein-binding modules, many of which are regulated by phosphorylation, paxillin serves as a platform for the recruitment of numerous regulatory and structural proteins that together control the dynamic changes in cell adhesion, cytoskeletal reorganization and gene expression that are necessary for cell migration and survival. In particular, paxillin plays a central role in coordinating the spatial and temporal action of the Rho family of small GTPases, which regulate the actin cytoskeleton, by recruiting an array of GTPase activator, suppressor and effector proteins to cell adhesions. When paxillin was first described 18 years ago, the amazing complexity of cell-adhesion organization, dynamics and signaling was yet to be realized. Herein we highlight our current understanding of how the multiple protein interactions of paxillin contribute to the coordination of cell-adhesion function.

The Adaptor Protein Paxillin Is Essential for Normal Development in the Mouse and Is a Critical Transducer of Fibronectin Signaling

ABSTRACT The integrin family of cell adhesion receptors are important for a diverse set of biological responses during development. Although many integrins have been shown to engage a similar set of cytoplasmic effector proteins in vitro, the importance of these proteins in the biological events mediated by different integrin receptors and ligands is uncertain. We have examined the role of one of the best-characterized integrin effectors, the focal adhesion protein paxillin, by disruption of the paxillin gene in mice. Paxillin was found to be critically involved in regulating the development of mesodermally derived structures such as heart and somites. The phenotype of the paxillin−/− mice closely resembles that of fibronectin−/− mice, suggesting that paxillin is a critical transducer of signals from fibronectin receptors during early development. Paxillin was also found to play a critical role in fibronectin receptor biology ex vivo since cultured paxillin-null fibroblasts display abnormal focal adhesions, reduced cell migration, inefficient localization of focal adhesion kinase (FAK), and reduced fibronectin-induced phosphorylation of FAK, Cas, and mitogen-activated protein kinase. In addition, we found that paxillin-null fibroblasts show some defects in the cortical cytoskeleton and cell spreading on fibronectin, raising the possibility that paxillin could play a role in structures distinct from focal adhesions. Thus, paxillin and fibronectin regulate some common embryonic developmental events, possibly due to paxillin modulation of fibronectin-regulated focal adhesion dynamics and organization of the membrane cytoskeletal structures that regulate cell migration and spreading.

Vinculin modulation of paxillin–FAK interactions regulates ERK to control survival and motility

Cells lacking vinculin are highly metastatic and motile. The reasons for this finding have remained unclear. Both enhanced survival and motility are critical to metastasis. Here, we show that vinculin null (vin−/−) cells and cells expressing a vinculin Y822F mutant have increased survival due to up-regulated activity of extracellular signal–regulated kinase (ERK). This increase is shown to result from vinculins modulation of paxillin–FAK interactions. A vinculin fragment (amino acids 811–1066) containing the paxillin binding site restored apoptosis and suppressed ERK activity in vin−/− cells. Both vinY822F and vin−/− cells exhibit increased interaction between paxillin and focal adhesion kinase (FAK) and increased paxillin and FAK phosphorylation. Transfection with paxillin Y31FY118F dominant-negative mutant in these cells inhibits ERK activation and restores apoptosis. The enhanced motility of vin−/− and vinY822F cells is also shown to be due to a similar mechanism. Thus, vinculin regulates survival and motility via ERK by controlling the accessibility of paxillin for FAK interaction.

Integrin-linked Kinase (ILK) Binding to Paxillin LD1 Motif Regulates ILK Localization to Focal Adhesions

Paxillin is a focal adhesion adapter protein involved in integrin signaling. Paxillin LD motifs bind several focal adhesion proteins including the focal adhesion kinase, vinculin, the Arf-GTPase-activating protein paxillin-kinase linker, and the newly identified actin-binding protein actopaxin. Microsequencing of peptides derived from a 50-kDa paxillin LD1 motif-binding protein revealed 100% identity with integrin-linked kinase (ILK)-1, a serine/threonine kinase that has been implicated in integrin, growth factor, and Wnt signaling pathways. Cloning of ILK from rat smooth muscle cells generated a cDNA that exhibited 99.6% identity at the amino acid level with human ILK-1. A monoclonal antibody raised against a region of the carboxyl terminus of ILK, which is identical in rat and human ILK-1 protein, recognized a 50-kDa protein in all cultured cells and tissues examined. Binding experiments showed that ILK binds directly to the paxillin LD1 motif in vitro. Co-immunoprecipitation from fibroblasts confirmed that the association between paxillin and ILK occurs in vivo in both adherent cells and cells in suspension. Immunofluorescence microscopy of fibroblasts demonstrated that endogenous ILK as well as transfected green fluorescent protein-ILK co-localizes with paxillin in focal adhesions. Analysis of the deduced amino acid sequence of ILK identified a paxillin-binding subdomain in the carboxyl terminus of ILK. In contrast to wild-type ILK, paxillin-binding subdomain mutants of ILK were unable to bind to the paxillin LD1 motif in vitro and failed to localize to focal adhesions. Thus, paxillin binding is necessary for efficient focal adhesion targeting of ILK and may therefore impact the role of ILK in integrin-mediated signal transduction events.

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

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.

Paxillin–ARF GAP signaling and the cytoskeleton

Several new families of ARF GTPase activating proteins (ARF GAPs) have been described recently that associate with paxillin and other cytoskeletal and signaling proteins. Important insights have been gained regarding their subcellular distribution, enzymatic specificity and protein scaffold function. Evidence suggests an important role for ARF GAPs in mediating changes in the cells actin cytoskeleton in response to adhesion and growth factor stimulation.

Association of Bovine Papillomavirus Type 1 E6 oncoprotein with the focal adhesion protein paxillin through a conserved protein interaction motif

We have found that the E6 oncoprotein of Bovine Papillomavirus Type 1 (BE6) as well as the E6 protein of the cancer associated HPV-16 (16E6) interact with the focal adhesion protein paxillin. Mutational analysis of paxillin revealed that BE6 binds paxillin through small protein interaction motifs called LD motifs that have been previously identified as important in regulating association of paxillin with vinculin and focal adhesion kinase (FAK), and that BE6 can interact with at least two separate binding sites on paxillin. The LD motifs of paxillin that bind BE6 share homology with the E6 binding site of E6-AP, a ubiquitin ligase that together with 16E6 targets the degradation of the p53 tumor suppressor. Paxillin binding to BE6 excludes simultaneous binding to E6-AP. Mutational analysis of BE6 can distinguish the interaction of BE6 with E6-AP compared to paxillin and revealed that the interaction of BE6 with paxillin may be necessary for the induction of anchorage independent growth of cells by BE6.

Actin-membrane interaction in focal adhesions

Focal adhesions are regions of the plasma membrane where cells in tissue culture adhere strongly to the underlying extracellular matrix, and which at their cytoplasmic face serve to anchor bundles of actin microfilaments. They provide an experimental model for studying the links between the cytoskeleton and the extracellular matrix. Members of the integrin family of extracellular matrix receptors are prominent components, spanning the membrane in focal adhesions, but there is evidence that other membrane components are also needed for these structures to form. A number of proteins are concentrated at the cytoplasmic face of focal adhesions. Recent efforts have sought to determine the links between actin and the integrin cytoplasmic domains. Using in vitro binding assays, two potential bridges between actin and integrin have been identified. One involves talin, which has recently been shown to bind actin directly. The other involves the actin-binding protein, alpha-actinin, which has been found to interact with several integrins. The physiological significance of these two potential bridges between actin and integrin remains to be determined in vivo.