Source: Dissertation Abstracts International, Volume: 74-10(E), Section: B.

Adviser: Anthony Koleske.

Arg regulates cortactin binding to F-actin: Cell migration begins with the formation of a branched actin network, which pushes the leading edge of the cell forward. The Arp2/3 complex nucleates actin branches, generating the protrusive force necessary to drive cell edge protrusion. Previous members of our lab identified Arg as an important regulator of cytoskeletal rearrangement and protrusion in fibroblasts. In an effort to understand how it regulates the actin cytoskeleton, we identified cortactin as a substrate and binding partner of Arg. Through studies in fibroblasts, we have shown that Arg interacts extensively with cortactin to promote cell edge protrusion.

Both Arg and cortactin bind F-actin. F-actin cosedimentation assays were used to determine if interactions between Arg and cortactin affect their binding densities and affinities for F-actin. I determined that Arg increases cortactin binding density on F-actin from one cortactin every four actin monomers to a density of one cortactin bound every two actin monomers. Cortactin has no effect on Arg binding to F-actin. Interestingly, cortactin mutations disrupting Arg binding had no effect on Arg's ability to increase cortactin binding on F-actin. I used Arg deletion mutants to show that the C-terminal calponin homology (CH) domain done is necessary and sufficient to increase the binding density between cortactin and F-actin. Previous work from our lab has shown that this CH domain induces a twist in the F-actin filament, which

may provide more space for cortactin to bind. Cortactin does not alter the binding affinity of Arg for F-actin. However, interactions between Arg and cortactin appear to be important to achieve maximum binding affinity between cortactin and F-actin.

Two amino acids regulate binding specificity of Arg and Abl SH2 domains for phosphorylated cortactin: Despite sharing nearly identical sequences, the Arg Src-homology 2 (SH2) domain binds phosphorylated cortactin with significantly higher affinity than the Abl SH2 domain. I used fluorescence anisotropy to identify two non-conserved residues (arginine 161 and serine 187) responsible for this difference in affinity. To determine a structural mechanism for the difference in binding affinity, collaborators in the Boggon lab have generated a crystal structure of the Arg SH2 domain and have modeled in the phosphorylated cortactin peptide. These structural data support our biochemical findings.