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Cloning and Mutational Analysis of the Shc-Phosphotyrosine Interaction/Phosphotyrosine-Binding Domain

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Transmembrane Signaling Protocols

Part of the book series: Methods In Molecular Biology™ ((MIMB,volume 84))

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Abstract

The interaction between tyrosme phosphorylated growth-factor receptors and Src Homology 2 (SH2)-domain-containing proteins plays a critical role in growth-factor mediated signal transduction (1). The SH2 domains bind to receptors at phosphotyrosine residues with specificity dictated by ammo acids that lie carboxy-terminal to the phosphotyrosme. Identification of new SH2-domain proteins has provided great insight into signaling by growth-factor receptors. Our work has focused on cloning new SH2-domain proteins by screening bacterial-expression libraries with tyrosine phosphorylated epidermal growth-factor receptor (EGF-receptor). We call this method CORT for cloning of receptor targets and the proteins isolated Grbs for growth-factor receptor bound (2–6) The clomng of Grb2 with this technique helped to elucldate the signaling pathway that leads from growth-factor receptors to Ras (7),

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References

  1. Pawson T (1995) Protein modules and signalling networks. Nature 373, 573–580

    Article  PubMed  CAS  Google Scholar 

  2. Skolink E. Y, Margobs B, Mohammadi M, Lowenstem E., Fischer R, Drepps A., Ullrich A, and Schlessinger J. (1991) Cloning of PI3 kinase-assoct-ated p85 utilizing a novel method for expression/cloning of target proteins for receptor tyrosme kinases Cell 65, 83–90.

    Article  Google Scholar 

  3. Lowenstem E J., Daly R J, Batzer A G., Lt W., Margohs B., Lammers R, Ullrich A, Skolink E Y, Bar-Sagl D, and Schlessinger J (1992) The SH2 and SH3 domain-containing protein GRB2 lurks receptor tyrosine kinases to ras signaling Cell 70, 43l–442

    Google Scholar 

  4. Margolts B, Stivennomen O., Comoglto F, Roonprapunt C., Skolnik E, Ullrich A., and Schlessinger J (1992) High-efficiency expresston/clomng of epidermal growth factor-receptor-binding proteins with Src homology 2 domains. Proc Natl Acad Sci USA 89, 8894–8898

    Article  Google Scholar 

  5. Blaikte P., Immanuel D, Wu J., Li N., Yajmk V., and Margohs B. (1994) A region in She distinct from the SH2 domain can bind tyrosme phosphorylated growth factor receptors. J Biol Chem 269, 32,03l–32,034.

    Google Scholar 

  6. Ooi J., Yajmk V., Immanuel D., Gordon M, Moskow J J., Buchberg A. M, and Margohs B. (1995) The cloning of Grb l0 reveals a new family of SH2 domain proteins. Oncogene 10, 1621–1630

    PubMed  CAS  Google Scholar 

  7. Schlessinger J. (1993) How receptor tyrosme kinases activate Ras. Trends Biochem Sci 18, 213–275.

    Article  Google Scholar 

  8. Margohs B., Skolink E Y, and Schlessinger J. (1995) The use of tyrosme-phosphorylated proteins to screen bactertal expression libraries for SH2 domains Methods Enzymol 255, 360–369.

    Article  Google Scholar 

  9. Margohs B. and Young R. A. (1995) Screening lambda expresston libraries with antibody and protein probes in DNA Cloning 2 (Glover D. M. and Hames B. D., eds), IRL, Oxford, UK, pp. 1–14

    Google Scholar 

  10. Cadwell R C and Joyce G F. (1992) Randomization of genes by PCR mutagenesis. PCR Methods Appl 2, 28–33.

    PubMed  CAS  Google Scholar 

  11. Yajnik V., Blaikte P., Bork P., and Margolis B (1996) Identification of residues within the She phosphotyrosme binding/phosphotyrosme interaction domain crucial for phosphopeptide interaction. J Biol Chem. 271, 1813–1816

    Article  PubMed  CAS  Google Scholar 

  12. Ausubel F. M., Brent R, Kingston R. E., Moore D. D, Seidman J. G., Smith J. A., and Struhl K (1996) Current protocols in molecular biology. Wiley Interscience, New York.

    Google Scholar 

  13. Zhou M. M., Ravihandran K. S., Olejniczak E. F., Petros A. M., Meadows R P., Satiler M., Harlan J E., Wade W. S., Burakoff S J, and Fesik S. W (1995) Structure and ligand recogninon of the phosphotyrosme binding domain of She. Nature 378, 584–592.

    Article  PubMed  CAS  Google Scholar 

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Author information

Authors and Affiliations

  1. New York University School of Medicine, New York

    Vijay Yajnik & Pamela Blaikie

  2. Howard Hughes Medical Institute, Department of Internal Medicine and Biological Chemistry, University of Michigan Medical School, Ann Arbor, MI

    Ben Margolis

Authors
  1. Vijay Yajnik
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  2. Pamela Blaikie
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  3. Ben Margolis
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Editor information

Editors and Affiliations

  1. State University of New York at Stony Brook, NY

    Dafna Bar-Sagi

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© 1998 Humana Press Inc.

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Yajnik, V., Blaikie, P., Margolis, B. (1998). Cloning and Mutational Analysis of the Shc-Phosphotyrosine Interaction/Phosphotyrosine-Binding Domain. In: Bar-Sagi, D. (eds) Transmembrane Signaling Protocols. Methods In Molecular Biology™, vol 84. Humana Press. https://doi.org/10.1385/0-89603-488-7:223

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  • DOI: https://doi.org/10.1385/0-89603-488-7:223

  • Publisher Name: Humana Press

  • Print ISBN: 978-0-89603-488-4

  • Online ISBN: 978-1-59259-568-6

  • eBook Packages: Springer Protocols

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