DIRECT DEMONSTRATION OF AN INTRAMOLECULAR SH2-PHOSPHOTYROSINE INTERACTION IN THE CRK PROTEIN

MANY signal transduction processes are mediated by the binding Of Src- homology-2 (SH2) domains to phosphotyrosine (pTyr)-containing proteins (1). Although most SH2-pTyr interactions occur between tao different t ypes of molecules, some appear to involve only a single molecular type . It has been proposed that the enzymatic activity and substrate recog nition of the Src-family kinases(2-4), and the protein-binding and tra nsforming activity of Crk-family adaptor proteins(5), are regulated by intramolecular SH2-pTyr interactions. In addition, the DNA-binding ac tivity of Stat transcription factors seems to be regulated by SH2-medi ated homodimerization(6). Here we examine the phosphorylated and non-p hosphorylated forms of murine Crk II (p-mCrk and mCrk, respectively)(7 -9) using a combination of physical techniques. The Crk protein contai ns a single SH2 domain and two SH3 domains in the order SH2-SH3-SH3. T here is a tyrosine-phosphorylation site between the two SH3 domains at residue 221 which is phosphorylated in vivo by the Abl tyrosine kinas e(5). Using NMR spectroscopic analysis, we show here that the SH2 doma in of purified p-mCrk is bound to pTyr, and by hydrodynamic measuremen ts that the phosphorylated protein is monomeric, These results provide direct demonstration of an intramolecular SH2-pTyr interaction in a s ignalling molecule.