The molecular mechanisms by which the Abelson (Abl) or Abl-related gene (Arg) kinases interface with the actin polymerization machinery to promote cell edge protrusions during cell–matrix adhesion are unclear. In this study, we show that interactions between Arg and the Arp2/3 complex regulator cortactin are essential to mediate actin-based cell edge protrusion during fibroblast adhesion to fibronectin. Arg-deficient and cortactin knockdown fibroblasts exhibit similar defects in adhesion-dependent cell edge protrusion, which can be restored via reexpression of Arg and cortactin. Arg interacts with cortactin via both binding and catalytic events. The cortactin Src homology (SH) 3 domain binds to a Pro-rich motif in the Arg C terminus. Arg mediates adhesion-dependent phosphorylation of cortactin, creating an additional binding site for the Arg SH2 domain. Mutation of residues that mediate Arg–cortactin interactions abrogate the abilities of both proteins to support protrusions, and the Nck adapter, which binds phosphocortactin, is also required. These results demonstrate that interactions between Arg, cortactin, and Nck1 are critical to promote adhesion-dependent cell edge protrusions.

The nonreceptor tyrosine kinase Pyk2 is highly expressed in invasive breast cancer, but how it potentiates tumor cell invasiveness is unclear. Genna et al. find that Pyk2 and the closely related kinase FAK modulate breast cancer cell invasiveness by distinct mechanisms and coordinate the balance between focal adhesion–mediated migration and invadopodia-dependent extracellular matrix invasion.

Extracellular cues stimulate the Abl family nonreceptor tyrosine kinase Arg to promote actin-based cell edge protrusions. Several Arg-interacting proteins are potential links to the actin cytoskeleton, but exactly how Arg stimulates actin polymerization and cellular protrusion has not yet been fully elucidated. We used affinity purification to identify N-WASp as a novel binding partner of Arg. N-WASp activates the Arp2/3 complex and is an effector of Abl. We find that the Arg SH3 domain binds directly to N-WASp. Arg phosphorylates N-WASp on Y256, modestly increasing the affinity of Arg for N-WASp, an interaction that does not require the Arg SH2 domain. The Arg SH3 domain stimulates N-WASp-dependent actin polymerization in vitro, and Arg phosphorylation of N-WASp weakly stimulates this effect. Arg and N-WASp colocalize to adhesion-dependent cell edge protrusions in vivo. The cell edge protrusion defects of arg−/− fibroblasts can be complemented by re-expression of an Arg-yellow fluorescent protein (YFP) fusion, but not by an N-WASp binding-deficient Arg SH3 domain point mutant. These results suggest that Arg promotes actin-based protrusions in response to extracellular stimuli through phosphorylation of and physical interactions with N-WASp.

Recent advances in proteomic screening approaches have led to the isolation of a wide variety of binding partners to interacting proteins and opened an avenue to analyze and understand signaling pathways. The study of protein-protein interactions is a key component in elucidating and understanding signaling pathways. Despite the importance of these interactions, very few studies are quantitative or report binding affinities. Here we present a simple method for examination and analysis of direct protein-protein binding interactions between two purified proteins. In the quantitative pull-down assay, one protein (the bait protein) is immobilized on beads whereas a second protein (the prey) is kept in solution. The concentration of the bait protein is kept constant, whereas the concentration of the prey protein is increased until binding saturation is achieved. After incubation, the beads are precipitated to separate unbound prey protein in solution from prey protein bound to the bait. The fraction of bound prey protein can then be loaded on a protein gel and the resulting bands can be analyzed with standard software. The quantitative pull-down assay with purified recombinant proteins provides a simple method to obtain dissociation constants (K d ). These quantifications are invaluable to compare relative binding of proteins, to map binding sites, and to show that binding is direct. This assay presents a powerful method to quantitatively analyze protein-protein interactions with tools that are available in most biochemistry laboratories and does not require the use of specialized or expensive equipment.

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