Phosphoryl transfer step in the C-terminal Src kinase controls Src recognition

All members of the Src family of nonreceptor protein tyrosine kinases are phosphorylated and subsequently down-regulated by the C-terminal Src kinase, Csk. Although the recognition of Src protein substrates is essential for a diverse set of signaling events linked to cellular growth and differentiation, the factors controlling this critical protein-protein interaction are not well known. To understand how Csk recognizes Src, the chemical/physical events that modulate apparent substrate affinity and turnover were investigated. Src is phosphorylated in a biphasic manner in rapid quench flow experiments, suggesting that the phosphoryl transfer step is fast and highly favorable and does not limit overall turnover. As opposed to other kinase-substrate pairs, turnover is not limited by the physical release of ADP based on stopped-flow fluorescence and catalytic trapping experiments, suggesting that other steps control net phosphorylation. The K-d for Src is considerably larger than the K-m based on single turnover kinetic and equilibrium sedimentation experiments. Taken together, the data are consistent with a mechanism whereby Csk achieves a low K-m for the substrate Src, not by stabilizing protein-protein interactions but rather by facilitating a fast phosphoryl transfer step. In this manner, the phosphoryl transfer step functions as a chemical clamp facilitating substrate recognition.