Crosstalk between the catalytic and regulatory domains allows bidirectional regulation of Src.

The catalytic activity of Src family tyrosine kinases is inhibited by intramolecular interactions between the regulatory SH3 and SH2 domains and the catalytic domain. In the inactive state, the critical alphaC-helix in the catalytic domain is positioned such that the formation of the Glu 310-Lys 295 salt bridge is precluded, Tyr 416 in the activation loop is unphosphorylated, and the SH2 and SH3 domains are unavailable for interactions with other proteins. We found that phosphorylation of the activation loop or mutation of the loop preceding the alphaC-helix activates Src and increases the accessibility of the SH3 domain for ligands.

The role of the linker between the SH2 domain and catalytic domain in the regulation and function of Src.

The crystal structures of the regulated Src and Hck tyrosine kinases show intramolecular interactions between the phosphorylated tail and the SH2 domain as well as between the SH3 domain, the SH2-catalytic domain linker (SH2-CD linker) and the catalytic domain. The relative contribution of these interactions to regulation of activity is poorly understood. Mutational analysis of Src and Lck revealed that interaction of the SH2-CD linker with the SH3 domain is crucial for regulation.

The 2.35 A crystal structure of the inactivated form of chicken Src: a dynamic molecule with multiple regulatory interactions.

The Src protein tyrosine kinase plays a critical role in a variety of signal transduction pathways. Strict regulation of its activity is necessary for proper signalling. We present here the crystal structure of chicken Src which is phosphorylated at Tyr527 and represents its least active form.

Src regulated by C-terminal phosphorylation is monomeric.

The activity of the c-Src protein tyrosine kinase is regulated by phosphorylation of a tyrosine residue (Tyr-527) in the C-terminal tail of the molecule. Phosphorylation of Tyr-527 promotes association of the tail with the SH2 domain and a concomitant reduction of the enzymatic activity of Src. We asked the question whether regulation by C-terminal phosphorylation was accompanied by a change in the quaternary structure of the enzyme or if it occurred within a monomeric form of Src.

The purification and characterization of the catalytic domain of Src expressed in Schizosaccharomyces pombe. Comparison of unphosphorylated and tyrosine phosphorylated species.

The catalytic domain of chicken Src including the C-terminal tail (Src-CD), has been expressed in Schizosaccharomyces pombe and purified to homogeneity. The expressed protein is a mixture of unphosphorylated (80%) and mono-phosphorylated (20%) species, that can be separated from each other by Mono Q chromatography. By a novel mass spectrometric method that utilizes parent ion scans of unseparated peptide mixtures, we found that the mono-phosphorylated form is phosphorylated either at Tyr416 or at Tyr436.