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Phosphorylation of SLP-76 by the ZAP-70 protein-tyrosine kinase is required for T-cell receptor function
J Bubeck Wardenburg1, C Fu, J K Jackman
1Center for Immunology, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
Abstract:
Two families of tyrosine kinases, the Src and Syk families, are required for T-cell receptor activation. While the Src kinases are responsible for phosphorylation of receptor-encoded signaling motifs and for up-regulation of ZAP-70 activity, the downstream substrates of ZAP-70 are unknown. Evidence is presented herein that the Src homology 2 (SH2) domain-containing leukocyte protein of 76 kDa (SLP-76) is a substrate of ZAP-70. Phosphorylation of SLP-76 is diminished in T cells that express a catalytically inactive ZAP-70. Moreover, SLP-76 is preferentially phosphorylated by ZAP-70 in vitro and in heterologous cellular systems. In T cells, overexpression of wild-type SLP-76 results in a hyperactive receptor, while expression of a SLP-76 molecule that is unable to be tyrosine-phosphorylated attenuates receptor function. In addition, the SH2 domain of SLP-76 is required for T-cell receptor function, although its role is independent of the ability of SLP-76 to undergo tyrosine phosphorylation. As SLP-76 interacts with both Grb2 and phospholipase C-gamma1, these data indicate that phosphorylation of SLP-76 by ZAP-70 provides an important functional link between the T-cell receptor and activation of ras and calcium pathways.
Insights
The Src homology 2 (SH2) domain-containing leukocyte protein of 76 kDa (SLP-76) is phosphorylated by ZAP-70, linking T-cell receptor signaling to downstream pathways. This phosphorylation is crucial for T-cell activation and function.
Area of Science:
- Immunology
- Cell signaling
- Molecular biology
Background:
- T-cell receptor (TCR) activation relies on Src and Syk tyrosine kinases.
- Src kinases phosphorylate signaling motifs and regulate ZAP-70 activity.
- Downstream targets of ZAP-70 in TCR signaling remain largely unidentified.
Purpose of the Study:
- To identify downstream substrates of ZAP-70 in T-cell activation.
- To investigate the role of SLP-76 phosphorylation by ZAP-70 in TCR signaling.
Main Methods:
- Investigated SLP-76 phosphorylation in T cells with catalytically inactive ZAP-70.
- Performed in vitro and heterologous system phosphorylation assays with ZAP-70 and SLP-76.
- Analyzed the impact of SLP-76 overexpression and tyrosine phosphorylation-deficient mutants on TCR function.
- Examined the role of SLP-76's SH2 domain in TCR signaling.
Main Results:
- SLP-76 is identified as a direct substrate of ZAP-70.
- SLP-76 phosphorylation by ZAP-70 is essential for TCR-mediated signaling.
- Overexpression of wild-type SLP-76 enhances TCR signaling, while non-phosphorylatable mutants attenuate it.
- The SH2 domain of SLP-76 is critical for TCR function, independent of its phosphorylation status.
Conclusions:
- ZAP-70 directly phosphorylates SLP-76, establishing a key link in TCR signaling.
- Phosphorylation of SLP-76 by ZAP-70 connects the TCR to downstream Ras and calcium signaling pathways.
- SLP-76 acts as a crucial signaling hub downstream of ZAP-70 in T-cell activation.