Tyrosine-phosphorylated Cbl binds to Crk after T cell activation.
Crk is a Src homology 2 (SH2)/Src homology 3 (SH3)-containing adapter protein that has been implicated in intracellular signaling in fibroblasts and PC12 pheochromocytoma cells. Crk has been shown to bind to a tyrosine-phosphorylated protein of 116 kDa after TCR-mediated T cell activation. Here we demonstrate that the Crk-associated p116 ... phosphoprotein is not the Crk-associated substrate (Cas) but, rather, is a protein product of the c-cbl proto-oncogene. Whereas Cas was not tyrosine-phosphorylated after T cell activation, Cbl became highly phosphorylated. Crk immunoprecipitates from activated T cell lysates contain tyrosine-phosphorylated Cbl. This association is mediated by the SH2 domain of Crk, as evidenced by the interaction between Cbl and the fusion protein product of a glutathione S-transferase (GST) expression construct encoding the Crk-SH2 domain in vitro. Furthermore, phosphopeptide-binding studies revealed that the GST-Crk SH2 domain binds to a tyrosine-phosphorylated peptide corresponding to amino acids 770-781 of Cbl with high affinity. Cbl is a protein tyrosine kinase (PTK) substrate that becomes phosphorylated after engagement of numerous cell surface receptors including the TCR. Data revealed by genetic studies in the nematode, Caenorhabditis elegans, implicates a Cbl-like molecule, Sli-1, as a negative regulator of the Let-23-signaling pathway. Because the signal from the Let-23 pathway affects the activation status of the Let-60 (Ras homologue in C. elegans) pathway, the activation-dependent association between Crk and Cbl may represent another TCR-generated signal leading to Ras-related pathways.
Mesh Terms:
Amino Acid Sequence, Animals, Antibodies, Monoclonal, Hybridomas, Lymphocyte Activation, Mice, Molecular Sequence Data, Phosphopeptides, Phosphorylation, Precipitin Tests, Protein Binding, Proto-Oncogene Proteins, Proto-Oncogene Proteins c-cbl, Proto-Oncogene Proteins c-crk, T-Lymphocytes, Tyrosine, Ubiquitin-Protein Ligases, src Homology Domains
Amino Acid Sequence, Animals, Antibodies, Monoclonal, Hybridomas, Lymphocyte Activation, Mice, Molecular Sequence Data, Phosphopeptides, Phosphorylation, Precipitin Tests, Protein Binding, Proto-Oncogene Proteins, Proto-Oncogene Proteins c-cbl, Proto-Oncogene Proteins c-crk, T-Lymphocytes, Tyrosine, Ubiquitin-Protein Ligases, src Homology Domains
J. Immunol.
Date: Jul. 01, 1996
PubMed ID: 8683103
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