If you treat cancer cells with an ERK1/2 inhibitor and probe a western blot for phospho-ERK, you might expect the signal to drop. In many cases it goes up instead. Does that mean the drug failed? An independent 2026 study from David Engelberg's lab at the Hebrew University of Jerusalem, published in iScience, answers that question directly, and the answer has implications for anyone who uses phosphorylation as a stand-in for kinase activity.
ERK1 and ERK2 sit at the bottom of the RTK–Ras–Raf–MEK cascade, which is constitutively active in many cancers. That makes ERK an attractive drug target. But ERK does two jobs at once: it drives proliferation, and it also restrains the upstream pathway through negative feedback. Block ERK, and that brake comes off.
The researchers tested five clinical-stage ERK1/2 inhibitors in cells transformed by intrinsically active ERK1 variants and in nine cancer-derived cell lines. Every inhibitor produced dramatic, sustained phosphorylation of ERK (and of MEK), consistent with release of feedback inhibition. Read on its own, that western blot would suggest the pathway was more active than before treatment.
To find out what ERK was actually doing, the team measured ERK catalytic activity directly in lysates using the PhosphoSens® ERK1/2 Cell Lysate Kinase Assay, a continuous, fluorescence-based assay that tracks phosphorylation of a selective sensor peptide in real time.
In other words, phosphorylation and catalytic activity told opposite stories. The authors put it plainly: a direct and specific kinase assay "should be the sole readout for the status of Erks' catalytic capabilities."
The activity data were one part of a broader picture. All five inhibitors arrested cells in G1 and activated the stress kinase p38, a response more typical of cytotoxic therapy than targeted drugs. Some cell viability persisted even after 72 hours, and phosphoproteomic analysis of BVD523-treated cells revealed large-scale changes, most prominently in Rho signaling. Because inhibited ERK remains heavily phosphorylated, the authors suggest it may be primed for rapid reactivation once the drug is gone, and they argue that ERK inhibitors should be combined with agents that prevent pathway reactivation.
Phospho-specific antibodies are the default readout for kinase pathway activity in many labs. They are useful, but they report a modification state at a single moment, not catalytic turnover. Whenever feedback loops, inhibitor binding modes, or scaffold interactions decouple phosphorylation from activity, a phospho-blot can point you in the wrong direction. That risk shows up in several common situations:
PhosphoSens-Lysate assays measure endogenous kinase activity in crude cell or tissue lysates with no antibodies and no wash steps. A kinase-selective sensor peptide fluoresces as it is phosphorylated, so each well produces a real-time progress curve on a standard fluorescence plate reader. In addition to ERK1/2, validated lysate assays are available for AKT1/2/3, GSK3α/β, DNA-PK, JNK1/2/3, p38α/β/γ/δ, CDK1/2/3/5, CDK2/Cyclin E1, and ZAP70.
If you are relying on phospho-ERK to judge inhibitor activity, see the ERK1/2 lysate assay or talk to an AssayQuant scientist →
Darash-Yahana M, Soudah N, Baskin A, Levy-Saar I, Levitzki A, Engelberg D. Erk inhibitors intercept Erk-mediated negative feedback while imposing cell-cycle arrest and activating p38. iScience. 2026;29(7):116629. doi:10.1016/j.isci.2026.116629
This study was conducted independently by the Engelberg laboratory using a commercially available AssayQuant kit.