Delineation of a T-cell activation motif required for binding of protein tyrosine kinases containing tandem SH2 domains.
To define the T-cell receptor signal transduction motif, we have transfected human and murine T-cell lines with a chimeric receptor consisting of the extracellular and transmembrane domains of human CD8 alpha and the membrane-proximal portion of CD3 zeta containing at its C terminus either an 18-amino acid segment (NQLYNELNLGRREEYDVL) or alanine-scanning point mutant derivatives. Crosslinking of the extracellular domain of the chimera is sufficient to initiate Ca2+ flux, interleukin 2 production, and tyrosine phosphorylation of cellular proteins including the chimera. Subsequently, the chimera becomes associated with several tyrosine-phosphorylated proteins, among them the 70-kDa protein tyrosine kinase ZAP70. Mutational data identify the T-cell activation motif as Y(X)2L(X)7Y(X)2L and show that each of the four designated residues is necessary for the above activation events. Recombinant protein containing the two tandem SH2 domains derived from ZAP70 binds to a synthetic peptide corresponding to the above 18-amino acid motif but only when both tyrosines are phosphorylated; in contrast, little or no binding is observed to monophosphorylated or nonphosphorylated analogues. These results imply that after receptor crosslinking in T cells, and by inference also in B cells and mast cells, the motif is phosphorylated on both tyrosine residues, thereafter serving as a docking site for protein tyrosine kinases containing tandem SH2 domains.
Koyasu
S
S
Laboratory of Immunobiology, Dana-Farber Cancer Institute.
Tse
A G
AG
Moingeon
P
P
Hussey
R E
RE
Mildonian
A
A
Hannisian
J
J
Clayton
L K
LK
Reinherz
E L
EL
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D017252:CD3 Complex / Q000096:biosynthesis / Q000378:metabolism*; D016827:CD8 Antigens / Q000096:biosynthesis / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D006801:Humans; D007376:Interleukin-2 / Q000096:biosynthesis; D008213:Lymphocyte Activation; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016376:Oligonucleotides, Antisense; D019000:Phosphotyrosine; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015398:Signal Transduction; D013601:T-Lymphocytes / Q000201:enzymology / Q000276:immunology*; D014162:Transfection; D014443:Tyrosine / Q000031:analogs & derivatives / Q000032:analysis
D017252:CD3 Complex; D016827:CD8 Antigens; D007376:Interleukin-2; D016376:Oligonucleotides, Antisense; D011993:Recombinant Fusion Proteins; D019000:Phosphotyrosine; D014443:Tyrosine; D011505:Protein-Tyrosine Kinases; D002118:Calcium