Protocol for reading and imaging live-cell PKA activity using ExRai-AKAR2

Abstract

Summary Fluorescent protein (FP)-based kinase activity biosensors are powerful tools for probing the spatiotemporal dynamics of signaling pathways in living cells. Yet, the limited sensitivity of most kinase biosensors restricts their reliable application in high-throughput detection modalities. Here, we report a protocol for using an ultrasensitive excitation-ratiometric PKA activity reporter, ExRai-AKAR2, to detect live-cell PKA activity via fluorescence microplate reading and epifluorescence microscopy. The high sensitivity of ExRai-AKAR2 is well suited to these high-throughput applications. For complete details on the use and execution of this protocol, please refer to Mehta et al. (2018) and Zhang et al., 2021a ) .

Regulatable assembly of synthetic microtubule architectures using engineered MAP-IDR condensates

“…Next, we tested whether the same design principles governing the FKBP/FRB synMAP circuit could be used to modulate microtubule architectures in response to endogenous cell-signaling activity, using Protein Kinase A (PKA) signaling as a test case. To this end, we replaced the FKBP/FRB heterodimerization module with an FHA/PKAsub interaction pair, in which the FHA domain only binds to PKAsub when it is phosphorylated by PKA 53,54 . Expression of this TPPP-FHA1 / PKAsub-DDX4-PixD/E synMAP circuit led to moderate levels of constitutive bundling in unstimulated cells, consistent with some basal interaction between the FHA1 and PKAsub components 55 (Fig.…”