Many in vivo substrates of Src family tyrosine kinases possess sequences conforming to Src homology 2 and 3 (SH2 and SH3) domain-binding motifs. One such substrate is p130Cas, a protein that is hyperphosphorylated in v-Src transformed cells. Cas contains a substrate domain consisting of 15 potential tyrosine phosphorylation sites, C- and N-terminal polyproline regions fitting the consensus sequence for SH3 domain ligands, and a YDYV motif that binds the Src SH2 domain when phosphorylated. In an effort to understand the mechanisms of processive phosphorylation, we have explored the regions of Cas necessary for interaction with Src using the yeast two-hybrid system. Mutations in the SH2 domain-binding region of Cas or the Src SH2 domain have little effect in Cas-Src complex formation or phosphorylation. However, disruption of the C-terminal polyproline region of Cas completely abolishes interaction between the two proteins and results in impaired phosphorylation of Cas. Kinetic analyses using purified proteins indicated that multisite phosphorylation of Cas by Src follows a processive rather than a distributive mechanism. Furthermore, the kinetic studies show that there are two properties of the polyproline region of Cas that are important in enhancing substrate phosphorylation. First, the C-terminal polyproline serves to activate Src kinases through the process of SH3 domain displacement. Second, this region aids in anchoring the kinase to Cas to facilitate processive phosphorylation of the substrate domain. The two processes combine to ensure phosphorylation of Cas with high efficiency.