Direct demonstration of an intramolecular SH2Ôphosphotyrosine interaction in the Crk protein

MANY signal transduction processes are mediated by the binding of Src-homology-2 (SH2) domains to phosphotyrosine (pTyr)-containing proteins¹Although most SH2-pTyr interactions occur between two different types of molecules, some appear to involve only a single molecular type. It has been proposed that the enzymatic activity and substrate recognition of the Src-family kinases^2–4, and the protein-binding and transforming activity of Crk-family adaptor proteins⁵, are regulated by intramolecular SH2-pTyr interactions. In addition, the DNA-binding activity of Stat transcription factors seems to be regulated by SH2-mediated homodimerization⁶Here we examine the phosphorylated and non-phosphorylated forms of murine Crk II (p-mCrk and mCrk, respectively)^7–9 using a combination of physical techniques. The Crk protein contains a single SH2 domain and two SH3 domains in the order SH2-SH3-SH3. There is a tyrosine-phosphorylation site between the two SH3 domains at residue 221 which is phosphorylated in vivo by the Abl tyrosine kinase⁵Using NMR spectroscopic analysis, we show here that the SH2 domain of purified p-mCrk is bound to pTyr, and by hydrodynamic measurements that the phosphorylated protein is monomeric. These results pro-vide direct demonstration of an intramolecular SH2-pTyr inter-action in a signalling molecule.