rdf:type |
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lifeskim:mentions |
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pubmed:dateCreated |
1998-4-16
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pubmed:abstractText |
The Crk proto-oncogene product is an SH2 and SH3 domain-containing adaptor protein. We have previously demonstrated that Crk-II becomes rapidly tyrosine-phosphorylated in response to stimulation with insulin-like growth factor I (IGF-I) and might be involved in the IGF-I receptor signalling pathway. To determine whether this involvement includes the direct interaction of Crk-II with the cytoplasmic region of the receptor, studies were performed in vitro with glutathione S-transferase (GST) fusion proteins containing various domains of Crk-II. The kinase assay in vitro showed that activated IGF-I receptors efficiently phosphorylated the GST-Crk-II fusion protein. This phosphorylation was dependent on the presence of the SH2 domain and Tyr-221 located in the spacer region between the two SH3 domains. Mutation of Tyr-221 not only prevented phosphorylation of GST-Crk in vitro, but also significantly increased the ability of GST-Crk proteins to co-precipitate activated IGF-I receptors from total cell lysates. Additional binding experiments in vitro showed that Crk-II might interact with the phosphorylated IGF-I receptor through its SH2 domain. To elucidate which region of the IGF-I receptor interacts with Crk-II, a peptide association assay was used in vitro. Different domains of the IGF-I receptor were expressed as (His)6-tagged fusion peptides, phosphorylated with activated wheat germ agglutinin-purified IGF-I receptors and tested for association with GST-Crk-II fusion proteins. Using wild-type as well as mutated peptides, we showed that the SH2 domain of Crk-II preferentially binds the peptide encoding the juxtamembrane region of the IGF-I receptor. Phosphorylation of Tyr-950 and Tyr-943 of the receptor is important for this interaction. These findings allow us to propose a model of direct interaction of Crk-II and the IGF-I receptor in vivo. On activation of the IGF-I receptor, Crk-II binds to phosphorylated tyrosine residues, especially in the juxtamembrane region. As a result of this binding, the IGF-I receptor kinase phosphorylates Tyr-221 of Crk-II, resulting in a change in intramolecular folding and binding of the SH2 domain to the phosphorylated Tyr-221, which causes rapid disassociation of the Crk-II-IGF-I receptor complex.
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pubmed:commentsCorrections |
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pubmed:language |
eng
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pubmed:journal |
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pubmed:citationSubset |
IM
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pubmed:chemical |
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pubmed:status |
MEDLINE
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pubmed:month |
Mar
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pubmed:issn |
0264-6021
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pubmed:author |
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pubmed:issnType |
Print
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pubmed:day |
1
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pubmed:volume |
330 ( Pt 2)
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pubmed:owner |
NLM
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pubmed:authorsComplete |
Y
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pubmed:pagination |
923-32
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pubmed:dateRevised |
2009-11-19
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pubmed:meshHeading |
pubmed-meshheading:9480911-3T3 Cells,
pubmed-meshheading:9480911-Animals,
pubmed-meshheading:9480911-Mice,
pubmed-meshheading:9480911-Phosphorylation,
pubmed-meshheading:9480911-Protein Binding,
pubmed-meshheading:9480911-Protein Conformation,
pubmed-meshheading:9480911-Protein Kinases,
pubmed-meshheading:9480911-Proto-Oncogene Proteins,
pubmed-meshheading:9480911-Proto-Oncogene Proteins c-crk,
pubmed-meshheading:9480911-Receptor, IGF Type 1,
pubmed-meshheading:9480911-Recombinant Fusion Proteins,
pubmed-meshheading:9480911-Signal Transduction,
pubmed-meshheading:9480911-Tyrosine,
pubmed-meshheading:9480911-src Homology Domains
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pubmed:year |
1998
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pubmed:articleTitle |
Interaction in vitro of the product of the c-Crk-II proto-oncogene with the insulin-like growth factor I receptor.
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pubmed:affiliation |
Diabetes Branch, National Institute of Diabetes, Digestive and Kidney Diseases, National Institutes of Health, 9000 Rockville Pike, Bethesda, MD 20892-1770, USA.
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pubmed:publicationType |
Journal Article
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