MUTATIONAL ANALYSIS OF THE STAT6 SH2 DOMAIN

The SH2 domain of the STAT family of transcription factors is essentia l for STAT binding to phosphorylated cytoplasmic domains of activated cytokine receptors, Furthermore, the same domain mediates dimerization of activated STAT monomers, a prerequisite for DNA binding by this fa mily of proteins. To identify amino acid residues within the STAT prot ein that mediate these various interactions, we have carried out an ex tensive mutational analysis of the Stat6 SH2 domain. Recombinant prote ins carrying C-terminal deletions or double alanine substitutions were expressed in mammalian and insect cells and assayed for DNA binding, transcription activation, tyrosine phosphorylation, and the ability to interact with a tyrosine-phosphorylated peptide derived from the inte rleukin-4 receptor signaling chain. From these studies, we have identi fied amino acids that are required for both DNA binding and interleuki n-4 receptor interaction, as well as residues that when mutated impair only one of the two functions. Our results suggest that the structura l homology between the SH2 domain of Stat6 and that of the distantly r elated Src protein may be higher than predicted on the basis of primar y amino acid sequence comparisons. However, the two types of SH2 domai ns may differ at their C-terminal ends.