Serine Phosphorylation of Cbl Induced by Phorbol Ester Enhances Its Association with 14-3-3 Proteins in T Cells via a Novel Serine-rich 14-3-3-binding Motif

• Published in: The Journal of biological chemistry 1997-04, Vol.272 (15), p.9979-9985
• Main Authors: Liu, Y C, Liu, Y, Elly, C, Yoshida, H, Lipkowitz, S, Altman, A
• Format: Article
• Language: English
• Subjects: 14-3-3 Proteins; Amino Acid Sequence; Animals; Binding Sites; Carcinogens - pharmacology; COS Cells; Enzyme Activation; Enzyme Inhibitors - metabolism; Humans; Jurkat Cells; Mice; Molecular Sequence Data; Oncogene Protein v-cbl; Peptide Mapping; Phorbol Esters - pharmacology; Phosphorylation; Protein Conformation; Protein-Tyrosine Kinases - metabolism; Proteins - metabolism; Rabbits; Receptor-CD3 Complex, Antigen, T-Cell - metabolism; Retroviridae Proteins, Oncogenic - metabolism; Serine - pharmacology; T-Lymphocytes - metabolism; Tetradecanoylphorbol Acetate - pharmacology; Tyrosine - metabolism; Tyrosine 3-Monooxygenase
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.272.15.9979

Stimulation of the T cell antigen receptor (TCR)·CD3 complex induces rapid tyrosine phosphorylation of Cbl, a protooncogene product which has been implicated in intracellular signaling pathways via its interaction with several signaling molecules. We found recently that Cbl associates directly with a member of the 14-3-3 protein family (14-3-3τ) in T cells and that the association is increased as a consequence of anti-CD3-mediated T cell activation. We report here that phorbol 12-myristate 13-acetate stimulation of T cells also enhanced the interaction between Cbl and two 14-3-3 isoforms (τ and ζ). Tyrosine phosphorylation of Cbl was not sufficient or required for this increased interaction. Thus, cotransfection of COS cells with Cbl plus Lck and/or Syk family protein-tyrosine kinases caused a marked increase in the phosphotyrosine content of Cbl without a concomitant enhancement of its association with 14-3-3. Phorbol 12-myristate 13-acetate stimulation induced serine phosphorylation of Cbl, and dephosphorylation of immunoprecipitated Cbl by a Ser/Thr phosphatase disrupted its interaction with 14-3-3. By using successive carboxyl-terminal deletion mutants of Cbl, the 14-3-3-binding domain was mapped to a serine-rich 30-amino acid region (residues 615-644) of Cbl. Mutation of serine residues in this region further defined a binding motif distinct from the consensus sequence RS X S X P, which was recently identified as a 14-3-3-binding motif. These results suggest that TCR stimulation induces both tyrosine and serine phosphorylation of Cbl. These phosphorylation events allow Cbl to recruit distinct signaling elements that participate in TCR-mediated signal transduction pathways.