ZAP70 is the kinase that turns T-cell receptor (TCR) engagement into downstream signaling. When the TCR is stimulated, ZAP70 is recruited to the receptor complex, activated, and goes on to phosphorylate adaptor proteins such as LAT and SLP-76. That central role has made ZAP70 a target of interest in autoimmune and inflammatory disease and a marker of interest in some lymphoid malignancies.
Measuring what ZAP70 is actually doing in cells has been harder than it sounds. This post explains why, and how the PhosphoSens® ZAP70 lysate assay measures endogenous ZAP70 activity directly in T-cell lysates.
The usual approach is to blot for phosphorylated ZAP70 or its substrates. Those readouts tell you a modification is present at one moment, but not how much catalytic work the enzyme is doing. Feedback, phosphatase activity, and inhibitor binding can all change phosphorylation without a matching change in activity. (A recent independent study of ERK inhibitors showed just how far apart the two can drift; see pERK Is Not ERK Activity.)
ZAP70 adds a second problem: SYK. The two kinases are close relatives with overlapping substrate preferences, and many inhibitors hit both. An activity assay is only useful if you can show which kinase is generating the signal.
PhosphoSens-Lysate assays use a kinase-selective sensor peptide that fluoresces as it is phosphorylated, giving a continuous readout of catalytic activity with no antibodies and no wash steps. The ZAP70 substrate, AQT1450, was profiled against 411 wild-type kinases. Except for SYK, every off-target kinase showed less than 10% of ZAP70's activity on the substrate.
To test the assay in a native setting, Jurkat T cells were serum-starved and then stimulated through the TCR with anti-CD3 (OKT3) and anti-CD28 antibodies plus a crosslinker. Stimulated lysates showed up to a 1.9-fold increase in phosphorylation rate over unstimulated controls. Key performance characteristics from the validation:
The clearest test of which kinase drives the lysate signal is to compare inhibitor potency in lysate with potency against each recombinant enzyme. Three inhibitors with different ZAP70/SYK preferences were used:
| Inhibitor | Recombinant ZAP70 IC50 | Recombinant SYK IC50 | Jurkat lysate IC50 |
|---|---|---|---|
| RDN2150 | 267 nM | 9,200 nM | 183 nM |
| PRT062607 | 53 nM | 2 nM | 48 nM |
| RO9021 | 411 nM | 12 nM | 670 nM |
In every case, the lysate IC50 tracks the ZAP70 value, not the SYK value. The two SYK-preferring inhibitors would have shut down a SYK-driven signal at low-nanomolar concentrations; instead they inhibited the lysate signal only at concentrations matching their ZAP70 potency. The conclusion: AQT1450 phosphorylation in stimulated Jurkat lysate is driven primarily by ZAP70.
The ZAP70 lysate assay (substrate AQT1450) is part of the PhosphoSens-Lysate portfolio, alongside assays for ERK1/2, AKT1/2/3, GSK3α/β, DNA-PK, JNK1/2/3, p38α/β/γ/δ, CDK1/2/3/5, and CDK2/Cyclin E1. You can download the ZAP70 validation report, and for broader selectivity work, ZAP70 and SYK are both available in our KinSight™ profiling panels. Questions about your T-cell model? Talk to an AssayQuant scientist →