Abstract
Protooncogenic protein c-Cbl undergoes tyrosine phosphorylation in response to stimulation through the receptors for antigens, immunoglobulins, cytokines, and growth factors as well as through the integrins. Tyrosine phosphorylation of c-Cbl may play a functional role in signal transduction, since c-Cbl interacts with many crucial signaling molecules including protein-tyrosine kinases, adaptor proteins, and phosphatidylinositol 3'-kinase. Therefore, it is essential for our understanding of the functions of c-Cbl in signal transduction to identify its tyrosine phosphorylation sites, to determine the protein-tyrosine kinases that phosphorylate these sites, and to elucidate the role of these sites in the interactions of c-Cbl with other signaling proteins. In this report, we demonstrate that tyrosines 700, 731, and 774 are the major tyrosine phosphorylation sites of c-Cbl in T cells in response to pervanadate treatment, as well as in response to TcR/CD3 ligation. Coexpression experiments in COS cells demonstrate that among T cell-expressed Src- and Syk-related protein-tyrosine kinases, Fyn, Yes, and Syk appear to play a major role in phosphorylation of c-Cbl, whereas Lck and Zap phosphorylate c-Cbl ineffectively. Fyn, Yes, and Syk phosphorylate the same sites of c-Cbl that become phosphorylated in stimulated T cells. Among these kinases, Fyn and Yes demonstrate strong binding to c-Cbl, which involves both phosphotyrosine-dependent and phosphotyrosine-independent mechanisms.
Highlights
C-Cbl is a protooncogenic protein initially identified as the cellular homologue of a transforming protein expressed by the murine Cas NS-1 retrovirus
Tyrosine phosphorylation of c-Cbl may play a functional role in signal transduction in a variety of cell types, since c-Cbl interacts in an activation- and/or tyrosine phosphorylation-dependent manner with several signaling molecules including receptor and nonreceptor protein-tyrosine kinase (PTK) (4, 9, 11, 13, 15, 16, 24 –26), adaptor proteins [9, 16, 19, 23, 27,28,29,30,31,32,33,34,35], phosphatidylinositol 3Ј-kinase [4, 5, 7,8,9, 16, 21, 32], and 14-3-3 proteins [36, 37]
No information is available regarding the location of tyrosine phosphorylation sites in c-Cbl following external stimulation of T cells, the nature of the PTKs phosphorylating these sites, or the interactions of these phosphotyrosines with proteins that are involved in signal transduction in T cells
Summary
Plasmids—The cDNA of human wild-type c-Cbl and that of its Cterminal truncations containing 357 (v-Cbl), 480, and 655 (HUT) amino acids were described earlier [1, 2, 22, 43] These cDNAs were cloned into the pCEP4 vector for expression in mammalian cells. Proteins of interest were reprecipitated with the corresponding antibodies These immunoprecipitates were incubated with SDS-PAGE sample buffer for 10 min at room temperature and cleared of PANSORBIN by centrifugation. Blots were washed in Tris-buffered saline containing 0.1% Tween 20 and incubated with 1 Ci/ml of 125Ilabeled protein A (ICN, Costa Mesa, CA) or 125I-labeled anti-mouse IgG (NEN Life Science Products) in blocking buffer for an additional 1 h. The intensity of protein bands was determined using Bio-Rad Scanning Densitometer model 1650 and found to be in the linear response range
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