title: Direct demonstration of an intramolecular SH2-phosphotyrosine interaction in the Crk protein.
abstract: Many signal transduction processes are mediated by the binding of Scr-homology-2 (SH2) domains to phosphotyrosine (pTyr)-containing proteins. Although 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, and the protein-binding and transforming activity of Crk-family adaptor proteins, are regulated by intramolecular SH2-pTyr interactions. In addition, the DNA-binding activity of Stat transcription factors seems to be regulated by SH2-mediated homodimerization. Here we examine the phosphorylated and non-phosphorylated forms of murine Crk II (p-mCrk and mCrk, respectively) 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 kinase. Using 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 provide direct demonstration of an intramolecular SH2-pTyr interaction in a signalling molecule.
authors.affiliation: Protein Engineering Network Centres of Excellence, University of Toronto, Ontario, Canada.
authors.forename: M K
authors.initials: MK
authors.lastname: Rosen
authors.forename: T
authors.initials: T
authors.lastname: Yamazaki
authors.forename: G D
authors.initials: GD
authors.lastname: Gish
authors.forename: C M
authors.initials: CM
authors.lastname: Kay
authors.forename: T
authors.initials: T
authors.lastname: Pawson
authors.forename: L E
authors.initials: LE
authors.lastname: Kay
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D009682:Magnetic Resonance Spectroscopy; D051379:Mice; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000378:metabolism; D010766:Phosphorylation; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D016315:Proto-Oncogene Proteins c-abl / Q000378:metabolism*; D050737:Proto-Oncogene Proteins c-crk; D011994:Recombinant Proteins; D014443:Tyrosine / Q000378:metabolism
chemical_list: D010446:Peptide Fragments; D011518:Proto-Oncogene Proteins; D050737:Proto-Oncogene Proteins c-crk; D011994:Recombinant Proteins; D014443:Tyrosine; D016315:Proto-Oncogene Proteins c-abl