Modulation of Functional Phosphorylation Sites by Basic Residues in the Unique Domain of c-Src.

In contrast to the well-studied canonical regulatory mechanisms, the way by which the recently discovered Src N-terminal regulatory element (SNRE) modulates Src activity is not yet well understood. Phosphorylation of serine and threonine residues modulates the charge distribution along the disordered region of the SNRE and may affect a fuzzy complex with the SH3 domain that is believed to act as an information transduction element. The pre-existing positively charged sites can interact with the newly introduced phosphate groups by modulating their acidity, introducing local conformational restrictions, or by coupling various phosphosites into a functional unit. In this paper, we use pH-dependent NMR measurements combined with single point mutations to identify the interactions of basic residues with physiologically important phosphorylated residues and to characterize the effect of these interactions in neighbor residues, thus providing insight into the electrostatic network in the isolated disordered regions and in the entire SNRE. From a methodological point of view, the linear relationships observed between the mutation-induced pKa changes of the phosphate groups of phosphoserine and phosphothreonine and the pH-induced chemical shifts of the NH groups of these residues provide a very convenient alternative to identify interacting phosphate groups without the need to introduce point mutations on specific basic residues.