Processive phosphorylation of p130Cas by Src depends on SH3-polyproline interactions

Many in vivo substrates of Src family tyrosine kinases possess sequences co nforming to Src homology 2 and 3 (SH2 and SH3) domain-binding motifs. One s uch substrate is p130Cas, a protein that is hyperphosphorylated in v-Src tr ansformed cells. Cas contains a substrate domain consisting of 15 potential tyrosine phosphorylation sites, C- and N-terminal polyproline regions fitt ing the consensus sequence for SH3 domain ligands, and a YDYV motif that bi nds the Src SH2 domain when phosphorylated. In an effort to understand the mechanisms of processive phosphorylation, we have explored the regions of C as necessary for interaction with Src using the yeast two-hybrid system. Mu tations in the SH2 domain-binding region of Cas or the Src SH2 domain have little effect in Cas-Src complex formation or phosphorylation. However, dis ruption of the C-terminal polyproline region of Cas completely abolishes in teraction between the two proteins and results in impaired phosphorylation of Cas. Kinetic analyses using purified proteins indicated that multisite p hosphorylation of Cas by Src follows a processive rather than a distributiv e mechanism. Furthermore, the kinetic studies show that there are two prope rties of the polyproline region of Cas that are important in enhancing subs trate phosphorylation. First, the C-terminal polyproline serves to activate Src kinases through the process of SH3 domain displacement. Second, this r egion aids in anchoring the kinase to Cas to facilitate processive phosphor ylation of the substrate domain. The two processes combine to ensure phosph orylation of Cas with high efficiency.