Tyrosine phosphorylation of Cbl upon epidermal growth factor (EGF) stimulation and its association with EGF receptor and downstream signaling proteins.
We and others have shown that Cbl, the protein product of the c-cbl proto-oncogene, is an early target of tyrosine phosphorylation upon stimulation through the immune cell surface receptors, which signal through noncovalently associated cytoplasmic tyrosine kinases. Using human mammary epithelial cells that express a natural epidermal growth factor (EGF) receptor and require EGF as an essential growth factor, we demonstrate here that Cbl is a prominent target of tyrosine phosphorylation upon stimulation through the EGF receptor tyrosine kinase. Phosphorylation of Cbl was EGF dose-dependent, rapid (detectable as early as 5 s and maximal by 2 min), and relatively sustained (detectable even after 1 h). Co-immunoprecipitation studies demonstrated that Cbl became associated with the EGF receptor in an EGF-dependent manner. Cbl was basally associated with the adaptor protein growth factor receptor-binding protein 2 (Grb2), and this interaction was further enhanced by EGF stimulation; however, the interaction was entirely mediated via the Grb2 Src homology 3 (SH3) domains, suggesting that binding of Grb2 SH2 domain to EGF receptor provides one mechanism of Cbl's association with the EGF receptor. EGF stimulation also induced the association of Cbl with Src homology and collagen (Shc) protein, p85 subunit of the phosphatidylinositol 3-kinase and Crk proteins, in particular with the CrkL isoform. Interactions of Cbl with the EGF receptor and multiple downstream signaling proteins suggest a role for this proto-oncogene product in mitogenic signaling through growth factor receptor kinases.
Fukazawa
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Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115, USA.
Miyake
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D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D001940:Breast; D002460:Cell Line; D004815:Epidermal Growth Factor / Q000494:pharmacology*; D004848:Epithelium; D066246:ErbB Receptors / Q000302:isolation & purification / Q000378:metabolism* / Q000502:physiology*; D005260:Female; D051380:GRB2 Adaptor Protein; D005982:Glutathione Transferase; D006801:Humans; D051379:Mice; D019869:Phosphatidylinositol 3-Kinases; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000378:metabolism; D019000:Phosphotyrosine / Q000378:metabolism; D011485:Protein Binding; D011506:Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D000090063:Proto-Oncogene Mas; D011518:Proto-Oncogene Proteins / Q000302:isolation & purification / Q000378:metabolism*; D050721:Proto-Oncogene Proteins c-cbl; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015398:Signal Transduction; D044767:Ubiquitin-Protein Ligases; D018909:src Homology Domains
D048868:Adaptor Proteins, Signal Transducing; D004815:Epidermal Growth Factor; D066246:ErbB Receptors; D051380:GRB2 Adaptor Protein; D005982:Glutathione Transferase; D019869:Phosphatidylinositol 3-Kinases; D017853:Phosphotransferases (Alcohol Group Acceptor); D019000:Phosphotyrosine; D011506:Proteins; D000090063:Proto-Oncogene Mas; D011518:Proto-Oncogene Proteins; D050721:Proto-Oncogene Proteins c-cbl; D011993:Recombinant Fusion Proteins; D044767:Ubiquitin-Protein Ligases; C495948:GRB2 protein, human; C495949:Grb2 protein, mouse; C000715470:MAS1 protein, human; C067419:CBL protein, human; C493849:Cbl protein, mouse