Paxillin comes of age

Abstract

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.

Fig. 1. Focal adhesions provide both a structural and signaling link between the extracellular matrix and the actin cytoskeleton

Cell adhesion to the extracellular matrix, via transmembrane integrin αβ heterodimers, leads to integrin activation and the recruitment of numerous intracellular proteins to the plasma membrane. Focal adhesions now comprise over 125 proteins (only selected examples are depicted), including both structural and regulatory molecules that mediate a physical link to the actin cytoskeleton and also play a major role in regulating actin dynamics necessary for productive cell migration. Molecules such as paxillin serve as scaffold proteins to facilitate the functional integration of these different categories of focal adhesion proteins.

Fig. 2. The LD4 motif of paxillin regulates cell protrusion and retraction

CHO.K1 cells stably transfected with (A) wild-type paxillin or (B.) a mutant of paxillin that lacks the LD4 motif (ΔLD4) were spread on 10μg/ml fibronectin for 2 hours in the presence of serum. Cells were fixed and stained for F-actin (red) and paxillin (green). Deletion of the LD4 domain of paxillin results in extensive peripheral protrusive activity (arrows) and defective tail retraction (arrowhead). It is also of note that the protrusions observed in cells that express paxillin ΔLD4 contain numerous dot-like paxillin-rich focal complexes at their periphery, which are less prevalent in cells that express wild-type paxillin. Scale bar = 20 μm.

Fig. 3. The coordination of Rac signaling by the paxillin LD4 motif

Paxillin is one of the earliest proteins that is recruited to nascent focal adhesions and is necessary for the turnover of focal adhesions during cell migration. The engagement of integrins with the extracellular matrix results in the localized activation of Rac and the Src and FAK tyrosine kinases. Together, Rac, Src and FAK promote the recruitment of the GIT-PIX-PAK-NCK complex to focal adhesions by means of an interaction between the paxillin binding subdomain 2 (PBS2) of GIT2 and the paxillin LD4 motif. Rac mediates this process by binding to and activating its effector PAK, which stimulates a multi-step conformational remodeling of the GIT-PIX-PAK-NCK complex and the PAK-dependent phosphorylation of the paxillin LD4 motif at S273. Src-FAK-dependent phosphorylation of GIT is also necessary for its binding to paxillin. The Rac GEF activity of PIX might function in a feed-forward loop to further promote localized Rac signaling to PAK at the cell’s leading edge. The recruitment of the GIT-PIX-PAK-NCK complex to paxillin also serves as a termination signal for Rac activity. This is also regulated at multiple levels, including the PTP-PEST-dependent tyrosine dephosphorylation of GIT, paxillin and FAK, as well as the GIT-dependent inhibition of Arf6 signaling to Rac (not shown). The serine/threonine phosphatase PP2A might also be recruited to paxillin to dephosphorylate S273.

Fig. 4. Paxillin is enriched in adhesions of fibroblasts migrating on either 2D and 3D fibronectin matrices

Mouse embryonic fibroblasts (MEFs) were allowed to migrate for 16 hours in the presence of serum on (A) a 2D fibronectin-coated surface or (B) a 3D cell-derived fibronectin-rich matrix (CDM) that is more representative of the in vivo environment. Cells were fixed and stained for F-actin (red), paxillin (green) and fibronectin (blue). Although paxillin-rich adhesions are observed in both conditions, MEFs migrating on the 2D fibronectin are well-spread and exhibit robust actin stress fibers, whereas cells migrating within the 3D CDM display long, thin protrusions (arrows) and limited actin organization. Scale bar = 20μm.