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Abstract
In a synthetic biology approach using Schneider (S2) cells, we show that SLP-76 is directly phosphorylated at tyrosines Y113 and Y128 by SYK in the presence of ITAM-containing adapters such as CD3ζ, DAP12, or FcεRγ. This phosphorylation was dependent on at least one functional ITAM and a functional SH2 domain within SYK. Inhibition of Src-kinases by inhibitors PP1 and PP2 did not reduce SLP-76 phosphorylation in S2 cells, suggesting an ITAM and SYK dependent, but Src-kinase independent signaling pathway. This direct ITAM/SYK/SLP-76 signaling pathway therefore differs from previously described ITAM signaling. However, the SYK-family kinase ZAP70 required the additional co-expression of the Src-family kinases Fyn or Lck to efficiently phosphorylate SLP-76 in S2 cells. This difference in Src-family kinase dependency of SYK versus ZAP70-mediated ITAM-based signaling was further demonstrated in human lymphocytes. ITAM signaling in ZAP70-expressing T cells was dependent on the activity of Src-family kinases. In contrast, Src-family kinases were partially dispensable for ITAM signaling in SYK-expressing B cells or in natural killer cells, which express SYK and ZAP70. This demonstrates that SYK can signal using a Src-kinase independent ITAM-based signaling pathway, which may be involved in calibrating the threshold for lymphocyte activation.
Highlights
Natural killer (NK) cells are cytotoxic innate lymphoid cells that are important for early and effective immune responses against cancer and virus-infected cells [1,2,3], but they can play a role in tissue homeostasis [4,5,6]
While the typical ITAM signaling pathway requires Src-kinases to phosphorylate ITAM motifs for interaction with the Src-homology domain 2 (SH2) domains of SYK, in our transfected S2-cells SLP-76 phosphorylation by ITAM-activated SYK was independent of Src-family kinases
In our synthetic biology approach, we found SLP-76 phosphorylation upon co-expression of an ITAM-containing receptor, SYK, and SLP-76 in S2-cells
Summary
Natural killer (NK) cells are cytotoxic innate lymphoid cells that are important for early and effective immune responses against cancer and virus-infected cells [1,2,3], but they can play a role in tissue homeostasis [4,5,6]. NK cell effector functions are regulated via signals from activating and inhibitory surface receptors. Many activating NK cell receptors associate with ITAM-containing partner chains such as DAP12, FcεRγ, or CD3ζ. In humans, activating killer cell Ig-like receptors (KIR-S), CD94/NKG2C-E, and NKp44 associate with DAP12, while NKp46, NKp30, and CD16 pair with CD3ζ and FcεRγ. Receptor engagement induces Src-family kinase-dependent phosphorylation of the tyrosines in the ITAM and these in turn form a binding site for the Src-homology domain 2 (SH2) domains of the tyrosine kinases SYK or ZAP70, resulting in downstream signaling. More recent studies on B cell receptor signaling revealed that Syk can phosphorylate and bind to the ITAM tyrosines of Igα and Igβ, which is necessary to open BCR oligomers upon the exposure to multivalent antigens [8]. The Src-family kinase Lyn facilitates the opening, but is not absolutely required for this process,
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