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.
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 (14-3-3tau) 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 (tau and zeta). 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 RSXSXP, 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.
Liu
Y C
YC
Division of Cell Biology, La Jolla Institute for Allergy and Immunology, San Diego, California 92121, USA.
Liu
Y
Y
Elly
C
C
Yoshida
H
H
Lipkowitz
S
S
Altman
A
A
D048948:14-3-3 Proteins; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D002273:Carcinogens / Q000494:pharmacology; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000378:metabolism*; D006801:Humans; D019169:Jurkat Cells; D051379:Mice; D008969:Molecular Sequence Data; D050722:Oncogene Protein v-cbl; D010449:Peptide Mapping; D010703:Phorbol Esters / Q000494:pharmacology*; D010766:Phosphorylation; D011487:Protein Conformation; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism*; D011817:Rabbits; D017260:Receptor-CD3 Complex, Antigen, T-Cell / Q000378:metabolism; D015682:Retroviridae Proteins, Oncogenic / Q000378:metabolism*; D012694:Serine / Q000494:pharmacology*; D013601:T-Lymphocytes / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014443:Tyrosine / Q000378:metabolism; D014446:Tyrosine 3-Monooxygenase
D048948:14-3-3 Proteins; D002273:Carcinogens; D004791:Enzyme Inhibitors; D050722:Oncogene Protein v-cbl; D010703:Phorbol Esters; D011506:Proteins; D017260:Receptor-CD3 Complex, Antigen, T-Cell; D015682:Retroviridae Proteins, Oncogenic; D014443:Tyrosine; D012694:Serine; D014446:Tyrosine 3-Monooxygenase; D011505:Protein-Tyrosine Kinases; D013755:Tetradecanoylphorbol Acetate