A potential SH3 domain-binding site in the Crk SH2 domain

Mordechai Anafi, Michael K. Rosen, Gerald D. Gish, Lewis E. Kay, Tony Pawson

Research output: Contribution to journalArticle

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Abstract

The Src homology 2 (SH2) domain of the mammalian adaptor protein Crk-II contains a proline-rich insert, predicted to lie within an extended DE loop, which is dispensable for phosphopeptide binding. Using the yeast two-hybrid system, this region of the Crk-II SH2 domain was found to interact with a subset of SH3 domains, notably the Abl SH3 domain. Furthermore, this proline- rich insert was found to modify the efficiency with which Crk-II was phosphorylated by the p140(c-abl) tyrosine kinase. In vitro, the interaction of full-length non-phosphorylated Crk-II with a glutathione S-transferase- Abl SH3 domain fusion protein was very weak. However, phosphorylation of Crk- II on Tyr-221 which induces an intramolecular association with the SH2 domain, or addition of a phosphopeptide corresponding to the Crk-II Tyr-221 phosphorylation site, stimulated association of Crk-II with the Abl SH3 domain. NMR spectroscopic analysis showed that binding of the Tyr-221 phosphopeptide to the Crk SH2 domain induced a chemical shift change in Val- 71, located in the proline-rich insert, indicative of a change in the structure of the proline-rich loop in response of Crk SH2-pTyr-221 interaction. These results suggest that the proline-rich insert in the Crk SH2 domain constitutes an SH3 domain-binding site that can be regulated by binding of a phosphopeptide ligand to the Crk SH2 domain.

Original languageEnglish (US)
Pages (from-to)21365-21374
Number of pages10
JournalJournal of Biological Chemistry
Volume271
Issue number35
DOIs
StatePublished - 1996

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src Homology Domains
Phosphopeptides
Proline
Binding Sites
Phosphorylation
Association reactions
Spectroscopic analysis
Chemical shift
Glutathione Transferase
Hybrid systems
Yeast
Protein-Tyrosine Kinases
Proteins
Fusion reactions
Nuclear magnetic resonance
Ligands
Two-Hybrid System Techniques

ASJC Scopus subject areas

  • Biochemistry

Cite this

A potential SH3 domain-binding site in the Crk SH2 domain. / Anafi, Mordechai; Rosen, Michael K.; Gish, Gerald D.; Kay, Lewis E.; Pawson, Tony.

In: Journal of Biological Chemistry, Vol. 271, No. 35, 1996, p. 21365-21374.

Research output: Contribution to journalArticle

Anafi, Mordechai ; Rosen, Michael K. ; Gish, Gerald D. ; Kay, Lewis E. ; Pawson, Tony. / A potential SH3 domain-binding site in the Crk SH2 domain. In: Journal of Biological Chemistry. 1996 ; Vol. 271, No. 35. pp. 21365-21374.
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