Binding of ZAP-70 to phosphorylated T-cell receptor zeta and eta enhances its autophosphorylation and generates specific binding sites for SH2 domain-containing proteins.
ZAP-70 is a protein tyrosine kinase thought to play a critical role in T-cell receptor (TCR) signal transduction. During T-cell activation, ZAP-70 binds to a conserved signalling motif known as the immune receptor tyrosine activating motif (ITAM) and becomes tyrosine phosphorylated. To determine whether binding of ZAP-70 to the phosphorylated ITAM was able to activate its kinase activity, we measured the kinase activity of ZAP-70 both when it was bound and when it was unbound to phosphorylated TCR subunits. The ability of ZAP-70 to phosphorylate itself, but not exogenous substrates, was enhanced when it was bound to the tyrosine-phosphorylated TCR zeta and eta chains or to a construct that contained duplicated epsilon ITAMs. No enhanced ZAP-70 autophosphorylation was noted when it was bound to tyrosine-phosphorylated CD3 gamma or epsilon. In addition, autophosphorylation of ZAP-70 when bound to zeta or eta resulted in the generation of multiple distinct ZAP-70 phosphorylated tyrosine residues which had the capacity to bind the SH2 domains of fyn, lck, GAP, and abl. As the effect was noted only when ZAP-70 was bound to TCR subunits containing multiple ITAMs, we propose that one of the roles of the tandem ITAMs is to facilitate the autophosphorylation of ZAP-70. Tyrosine-phosphorylated ZAP-70 then mediates downstream signalling by recruiting SH2 domain-containing signalling proteins.
Neumeister
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EN
Center for Immunology, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
Zhu
Y
Y
Richard
S
S
Terhorst
C
C
Chan
A C
AC
Shaw
A S
AS
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D018076:DNA, Complementary; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism*; D011948:Receptors, Antigen, T-Cell / Q000378:metabolism*; D013601:T-Lymphocytes / Q000378:metabolism; D051746:ZAP-70 Protein-Tyrosine Kinase
D018076:DNA, Complementary; D011506:Proteins; D011948:Receptors, Antigen, T-Cell; D011505:Protein-Tyrosine Kinases; D051746:ZAP-70 Protein-Tyrosine Kinase; C497261:ZAP70 protein, human