Abstract
Protein kinases play crucial roles in regulating cellular processes, making real-time visualization of their activity essential for understanding signaling dynamics. While genetically encoded fluorescent biosensors have emerged as powerful tools for studying kinase activity, their development for many kinases remains challenging due to the lack of suitable substrate peptides. Here, we present a novel approach for identifying peptide substrates and demonstrate its effectiveness by developing a biosensor for Protein Kinase N (PKN) activity. Our method identified a new PKN substrate peptide that we optimized for use in a fluorescent biosensor design. The resulting biosensor shows specificity for PKN family kinases and can detect both overexpressed and endogenous PKN activity in live cells. Importantly, our biosensor revealed sustained basal PKN2 activity at the plasma membrane, identifying it as a PKN2 activity hotspot. This work not only provides a valuable tool for studying PKN signaling but also demonstrates a promising strategy for developing biosensors for other understudied kinases, potentially expanding our ability to monitor kinase activity across the human kinome.