CDK2 activity crosstalk on the ERK kinase translocation reporter can be resolved computationally.

The mitogen-activated protein kinase (MAPK) pathway integrates growth factor signaling through extracellular signal-regulated kinase (ERK) to control cell proliferation. To study ERK dynamics, many researchers use an ERK activity kinase translocation reporter (KTR). Our study reveals that this ERK KTR also partially senses cyclin-dependent kinase 2 (CDK2) activity, making it appear as if ERK activity rises as cells progress through the cell cycle. Through single-cell time-lapse imaging, we identified a residual ERK KTR signal that was eliminated by selective CDK2 inhibitors, indicating crosstalk from CDK2 onto the ERK KTR. By contrast, EKAREN5, a FRET-based ERK sensor, showed no CDK2 crosstalk. A related p38 KTR is also partly affected by CDK2 activity. To address this, we developed linear and non-linear computational correction methods that subtract CDK2 signal from the ERK and p38 KTRs. These findings will allow for more accurate quantification of MAPK activities, especially for studies of actively cycling cells.