MUTATIONAL ANALYSIS OF THE SRC SH3 DOMAIN - THE SAME RESIDUES OF THE LIGAND-BINDING SURFACE ARE IMPORTANT FOR INTRAMOLECULAR AND INTERMOLECULAR INTERACTIONS

The protein tyrosine kinase c-Src is negatively regulated by phosphory lation of Tyr527 in its C-terminal tail. The repressed state is achiev ed through intramolecular interactions involving the phosphorylated ta il, the Src homology 2 (SH2) domain and the SH3 domain. Both the SH2 a nd SH3 domains have also been shown to mediate the intermolecular inte raction of Src with several proteins. To test which amino acids of the Src SH3 domain are important for these interactions, and whether the intra- and intermolecular associations involve the same residues, we c arried out a detailed mutational analysis of the presumptive interacti on surface. All mutations of conserved hydrophobic residues had an eff ect on both inter- and intramolecular interactions of the Src SH3 doma in, although not all amino acids were equally important. Chimeric mole cules in which the Src SH3 domain was replaced with those of spectrin or Lck showed derepressed kinase activity, whereas a chimera containin g the Fyn SH3 domain was fully regulated. Since spectrin and Lck SH3 d omains share the conserved hydrophobic residues characteristic of SH3 domains, other amino acids must be important for specificity. Mutation al analysis of non- or semi-conserved residues in the RT and n-Src loo ps showed that some of these were also involved in inter- and intramol ecular interactions. Stable transfection of selected SH3 domain mutant s into NIH-3T3 cells showed that despite elevated levels of phosphotyr osine, the cells were morphologically normal, indicating that the SH3 domain was required for efficient transformation of NIH-3T3 cells by S rc.