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Direct demonstration of an intramolecular SH2—phosphotyrosine interaction in the Crk protein

Nature volume 374, pages 477–479 (1995)Cite this article

Abstract

MANY signal transduction processes are mediated by the binding of Src-homology-2 (SH2) domains to phosphotyrosine (pTyr)-containing proteins1. 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 kinases2–4, 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 homodimerization6. Here 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 kinase5. 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 pro-vide direct demonstration of an intramolecular SH2-pTyr inter-action in a signalling molecule.

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Authors and Affiliations

  1. Protein Engineering Network Centres of Excellence and Departments of Medical Genetics, Biochemistry and Chemistry, Medical Sciences Building, University of Toronto, Toronto, Ontario, M5S 1A8, Canada

    Michael K. Rosen, Toshio Yamazaki & Lewis E. Kay

  2. Programme in Molecular Biology and Cancer, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario, M5G 1X5, Canada

    Michael K. Rosen, Gerald D. Gish & Tony Pawson

  3. MRC Group in Protein Structure and Function, Department of Biochemistry, University of Alberta, Edmonton, Alberta, T6G 2H7, Canada

    Cyril M. Kay

Authors
  1. Michael K. Rosen
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  2. Toshio Yamazaki
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  4. Cyril M. Kay
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  5. Tony Pawson
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  6. Lewis E. Kay
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Rosen, M., Yamazaki, T., Gish, G. et al. Direct demonstration of an intramolecular SH2—phosphotyrosine interaction in the Crk protein. Nature 374, 477–479 (1995). https://doi.org/10.1038/374477a0

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