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Inhibition of ZAP-70 Kinase Activity Via an Analog Inhibitor-Sensitive Allele Blocks T Cell Receptor Signaling


ZAP-70 is a cytoplasmic protein tyrosine kinase that is required for T cell antigen receptor (TCR) signaling. Both mice and humans deficient in ZAP-70 fail to develop functional T cells, thus demonstrating its necessity for T cell development and function. There is currently no highly specific, cell-permeable small-molecule inhibitor for ZAP-70; therefore, we generated a mutant ZAP-70 allele that retains kinase activity but is sensitive to inhibition by a mutant-specific inhibitor, 3-methylbenzyl-pyrazolopyrimidine (3-MB-PP1). We validated the chemical genetic inhibitor system in Jurkat T cell lines, where the inhibitor blocks ZAP-70 dependent TCR signaling in cells expressing the analog-sensitive allele. Interestingly, the inhibitor also ablates CD28 superagonist signaling, thereby demonstrating the system's utility in dissecting ZAP-70's requirement in alternative mechanisms of T cell activation. We have generated a bacterial artificial chromosome (BAC) transgenic mouse line that expresses the murine homolog of the analog-sensitive ZAP-70 allele. Preliminary studies show that activation of both peripheral naïve and in vitro generated effector T cells can be inhibited by 3-MB-PP1. Thus, we have developed the first specific chemical means for inhibiting ZAP-70, which serves as a valuable tool for studying ZAP-70's function in T cells as well as determining the clinical utility of a ZAP-70 inhibitor.

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