The role of tyrosine phosphorylation in the interaction of cellular tyrosine kinases with the T cell receptor zeta chain tyrosine-based activation motif.
Immunoglobulin receptor family tyrosine-based activation motifs (ITAM) define a conserved signaling sequence, EX2YX2L/IX7YX2L/I, that mediates coupling of the T cell antigen receptor (TCR) to protein tyrosine kinases (PTK). In the present study, we explored the role of phosphorylation of the two ITAM tyrosine residues in the interactions of the motif with the PTK ZAP-70 and p59fyn. The data show that the phosphorylation of a single tyrosine within the motif enables binding of p59fyn, whereas phosphorylation of both tyrosines within the motif is required for maximal binding of the PTK ZAP-70. Quantitative binding experiments show that nanomolar concentrations of the doubly phosphorylated zeta 1-ITAM are sufficient for ZAP-70 recruitment, whereas micromolar levels of singly phosphorylated ITAM are necessary for p59fyn binding. ZAP-70 binds with low efficiency to a singly phosphorylated ITAM, but shows preferential binding to the C-terminal phosphotyrosine in the ITAM, whereas p59fyn binds selectively to the N-terminal phosphotyrosine. The present data thus show that there is the potential for a singly phosphorylated ITAM to couple to cellular PTK. Moreover, the data suggest a mechanism for heterogeneity in signal transduction responses by the TCR, since ITAM could differentially couple the TCR to downstream signaling events depending on their phosphorylation state.
Osman
N
N
Lymphocyte Activation Laboratory, Imperial Cancer Research Fund, London, GB.
Lucas
S
S
Cantrell
D
D
D000595:Amino Acid Sequence; D017252:CD3 Complex / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D008213:Lymphocyte Activation; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000378:metabolism; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000502:physiology; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D051076:Proto-Oncogene Proteins c-fyn; D017260:Receptor-CD3 Complex, Antigen, T-Cell / Q000378:metabolism; D011948:Receptors, Antigen, T-Cell / Q000737:chemistry / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology*; D051746:ZAP-70 Protein-Tyrosine Kinase; D018909:src Homology Domains
D017252:CD3 Complex; D008565:Membrane Proteins; D010446:Peptide Fragments; D011518:Proto-Oncogene Proteins; D017260:Receptor-CD3 Complex, Antigen, T-Cell; D011948:Receptors, Antigen, T-Cell; C054979:antigen T cell receptor, zeta chain; D019000:Phosphotyrosine; D011505:Protein-Tyrosine Kinases; C494957:FYN protein, human; D051076:Proto-Oncogene Proteins c-fyn; D051746:ZAP-70 Protein-Tyrosine Kinase; C497261:ZAP70 protein, human