Activation of Zap-70 Tyrosine Kinase Due to a Structural Rearrangement Induced by Tyrosine Phosphorylation and/or ITAM Binding
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- 作者：Carlo Visco ; Giovanni Magistrelli ; Roberta Bosotti ; Rita Perego ; Luisa Rusconi ; Salvatore Toma ; Moreno Zamai ; Oreste Acuto ; and Antonella Isacchi
- 刊名：Biochemistry
- 出版年：2000
- 出版时间：March 14, 2000
- 年：2000
- 卷：39
- 期：10
- 页码：2784 - 2791
- 全文大小：116K
- 年卷期：v.39,no.10(March 14, 2000)
- ISSN：1520-4995
文摘
The protein tyrosine kinase ZAP-70 is implicated in the early steps of the T-cell antigen receptor(TCR) signaling. Binding of ZAP-70 to the phosphorylated immunoreceptor tyrosine-based activationmotifs (ITAMs) of the TCR 
chain through its two src-homology 2 (SH2) domains results in its activationcoupled to phosphorylation on multiple tyrosine residues, mediated by Src kinases including Lck as wellas by autophosphorylation. The mechanism of ZAP-70 activation following receptor binding is still notcompletely understood. Here we investigated the effect of intramolecular interactions and autophosphorylation by following the kinetics of recombinant ZAP-70 activation in a spectrophotometric substratephosphorylation assay. Under these conditions, we observed a lag phase of several minutes before fullZAP-70 activation, which was not observed using a truncated form lacking the first 254 residues, suggestingthat it might be due to an intramolecular interaction involving the interdomain A and SH2 region.Accordingly, the lag phase could be reproduced by testing the truncated form in the presence of recombinantSH2 domains and was abolished by the addition of diphosphorylated ITAM peptide. Preincubation withATP or phosphorylation by Lck also abolished the lag phase and resulted in a more active enzyme. Thesame results were obtained using a ZAP-70 mutant lacking the interdomain B tyrosines. These findingsare consistent with a mechanism in which ZAP-70 phosphorylation/autophosphorylation on tyrosine(s)other than 292, 315, and 319, as well as engagement of the SH2 domains by the phosphorylated TCR,can induce a conformational change leading to accelerated enzyme kinetics and higher catalytic efficiency.
chain through its two src-homology 2 (SH2) domains results in its activationcoupled to phosphorylation on multiple tyrosine residues, mediated by Src kinases including Lck as wellas by autophosphorylation. The mechanism of ZAP-70 activation following receptor binding is still notcompletely understood. Here we investigated the effect of intramolecular interactions and autophosphorylation by following the kinetics of recombinant ZAP-70 activation in a spectrophotometric substratephosphorylation assay. Under these conditions, we observed a lag phase of several minutes before fullZAP-70 activation, which was not observed using a truncated form lacking the first 254 residues, suggestingthat it might be due to an intramolecular interaction involving the interdomain A and SH2 region.Accordingly, the lag phase could be reproduced by testing the truncated form in the presence of recombinantSH2 domains and was abolished by the addition of diphosphorylated ITAM peptide. Preincubation withATP or phosphorylation by Lck also abolished the lag phase and resulted in a more active enzyme. Thesame results were obtained using a ZAP-70 mutant lacking the interdomain B tyrosines. These findingsare consistent with a mechanism in which ZAP-70 phosphorylation/autophosphorylation on tyrosine(s)other than 292, 315, and 319, as well as engagement of the SH2 domains by the phosphorylated TCR,can induce a conformational change leading to accelerated enzyme kinetics and higher catalytic efficiency.