Abstract
We developed the novel biosensor platform Luciferase Activity Modulated by Phosphorylation (LAMP) to monitor, with unprecedented sensitivity and dynamic range, reversible protein phosphorylation in cells. Based on NanoLuc luciferase complementation (NanoBiT), LAMP sensors are small (22 kD) and provide stable and bright light output that decreases upon phosphorylation and increases upon dephosphorylation. In this report, we designed LAMP biosensors to report spatial and temporal dynamics of cAMP-dependent protein kinase A (PKA) signaling. By incorporating both PKA phosphorylation and protein phosphatase 2A (PP2A) dephosphorylation motifs into the small component of split NanoLuc (generating PKABiT/pBiT), we achieved a sevenfold dynamic range. LAMP sensors are modular and flexible, allowing the two components, LgBiT and pBiT, to be expressed as part of the same polypeptide (cis) or as part of separate, but interacting polypeptides (trans). With trans LAMP, we show that RIα and RIβ form heterodimers with activities indistinguishable from homodimers of the two PKA regulatory subunit isoforms. Cis PKA LAMP sensors revealed different activation and inactivation kinetics of endogenous, membrane anchored PKA/RI and PKA/RII holoenzymes. They also allowed us to measure kinetics of cAMP diffusion and PKA catalytic subunit translocation to the nucleus. Finally, we used a regeneratively phosphorylated PKA LAMP sensor to identify an autoinhibitory sequence in the PP2A regulatory subunit B56δ. By tailoring the sequence of pBiT, the LAMP platform can be extended to track the activity of other protein kinases and phosphatases and second messengers they respond to, thus providing new tools for cell signaling research and drug discovery.