Cortactin uses its SH3 domain to bind to the first PXXP region of Arg. (A) Representative Coomassie blue–stained SDS-PAGE gel showing binding of the Arg C-terminal half (Arg 557-C) to cortactin beads but not to ethanolamine-blocked control beads. Arg 557-C binding to cortactin increases as a function of concentration. (B, D, and F) Plots of concentration (x axis) versus the amount bound (y axis) were fitted to a binding curve. Arg 557-C binding to cortactin (K_d = 0.85 ± 0.18 µM; n = 4; B), Arg 557-C binding to the cortactin SH3 domain (K_d = 0.44 ± 0.14 µM; n = 3; D), and Arg 557-C PXXP mut23 binding to the cortactin SH3 domain (K_d = 1.25 ± 0.25 µM; n = 2; F) are shown. (C, E, and G) Affinities of Arg and cortactin binding. Each binding reaction was tested at least twice, with combinations showing positive binding tested at least three times. (C) Affinity of Arg/Arg mutants for cortactin. Arg is composed of an N-terminal SH2 and SH3 domain followed by a kinase domain. This fragment does not bind to cortactin. Arg 557-C containing three conserved PXXP motifs (indicated by vertical lines), two F-actin–binding sites and a microtubule (MT)-binding site binds cortactin, whereas a shorter fragment lacking the Pro-rich stretch (Arg 688-C) does not. (E) Affinity of cortactin/cortactin fragments for Arg 557-C. See Fig. 2 for a description of the cortactin domains. The cortactin SH3 domain is necessary and sufficient to bind Arg 557-C. (G) Affinity of Arg 557-C PXXP mutants for the cortactin SH3 domain. Mutation of PXXP1 in Arg 557-C abrogates binding to the cortactin SH3 domain.