Differential and multiple binding of signal transducing molecules to the ITAMs of the TCR-zeta chain.
A biotin-streptavidin-based technique was developed for high affinity, unidirectional, and specific immobilization of synthetic peptides to a solid phase. Biotinylated 23-mer carboxamide peptides corresponding to the three immunoreceptor tyrosine-based activation motifs (ITAMs) of the T cell antigen receptor associated zeta-chain (TCR-zeta) in their bis-, mono-, or unphosphorylated forms were used to study the binding of cellular proteins from human Jurkat T cells to these signal transduction motifs. The protein tyrosine kinase ZAP-70 bound specifically to all bisphosphorylated peptides but not to the mono- or unphosphorylated peptides. In contrast, Shc, phosphatidylinositol 3-kinase (Pl3K), Grb2, and Ras-GTPase activating protein (GAP) bound with different affinities to the bis- or monophosphorylated peptides, while the Src family protein tyrosine kinase (PTK) Fyn did not bind specifically to any of the tested peptides. The different preferences of the studied signaling molecules for distinct ITAMs, and in particular the binding of some of them preferentially to monophosphorylated peptides, suggests that the TCR-zeta may bind multiple signaling molecules with each ITAM binding a unique set of such molecules. In addition, partial phosphorylation of the ITAMs may result in recruitment of different proteins compared to double phosphorylation. This may be crucial for coupling of the TCR to various effector functions under different conditions of receptor triggering.
Hoffmann-La Roche Inc., Nutley, New Jersey 07110, USA.
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Zenner
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Vorherr
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Mustelin
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Burn
D000595:Amino Acid Sequence; D015153:Blotting, Western; D006801:Humans; D019169:Jurkat Cells; D008565:Membrane Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D010766:Phosphorylation; D021382:Protein Sorting Signals / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011948:Receptors, Antigen, T-Cell / Q000378:metabolism*; D013329:Structure-Activity Relationship; D014443:Tyrosine; D051746:ZAP-70 Protein-Tyrosine Kinase
D008565:Membrane Proteins; D021382:Protein Sorting Signals; D011948:Receptors, Antigen, T-Cell; C054979:antigen T cell receptor, zeta chain; D014443:Tyrosine; D011505:Protein-Tyrosine Kinases; D051746:ZAP-70 Protein-Tyrosine Kinase; C497261:ZAP70 protein, human