A potential SH3 domain-binding site in the Crk SH2 domain.
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.
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Programme in Molecular Biology and Cancer, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, 600 University Ave., Toronto, Ontario, M5G 1X5 Canada.
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Gish
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Kay
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Pawson
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D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D003001:Cloning, Molecular; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000378:metabolism; D011392:Proline / Q000378:metabolism; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism*; D011518:Proto-Oncogene Proteins; D050737:Proto-Oncogene Proteins c-crk; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D018909:src Homology Domains
D011518:Proto-Oncogene Proteins; D050737:Proto-Oncogene Proteins c-crk; D019000:Phosphotyrosine; D011392:Proline; D011494:Protein Kinases