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.
Sawasdikosol
S
S
Division of Pediatric Oncology, Dana-Farber Cancer Institute, Boston, MA 02115, USA.
Chang
J H
JH
Pratt
J C
JC
Wolf
G
G
Shoelson
S E
SE
Burakoff
S J
SJ
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D006825:Hybridomas; D008213:Lymphocyte Activation; D051379:Mice; D008969:Molecular Sequence Data; D010748:Phosphopeptides / Q000378:metabolism; D010766:Phosphorylation; D011233:Precipitin Tests; D011485:Protein Binding / Q000276:immunology; D011518:Proto-Oncogene Proteins / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D050721:Proto-Oncogene Proteins c-cbl; D050737:Proto-Oncogene Proteins c-crk; D013601:T-Lymphocytes / Q000276:immunology*; D014443:Tyrosine / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases; D018909:src Homology Domains / Q000276:immunology
D000911:Antibodies, Monoclonal; D010748:Phosphopeptides; D011518:Proto-Oncogene Proteins; D050737:Proto-Oncogene Proteins c-crk; D014443:Tyrosine; D050721:Proto-Oncogene Proteins c-cbl; D044767:Ubiquitin-Protein Ligases; C493849:Cbl protein, mouse