Kinase Mobility Shift Assay (KiMSA) for Assessing Protein Kinase A Activity

The cAMP-dependent protein kinase (PKA) is one of the most extensively distributed kinases among intracellular signal cascades, with a pivotal role in the regulation of various processes, including the capacitation of sperm cells. Traditional assessments of PKA activity rely on the utilization of [γ-³²P] ATP and the Kemptide peptide as a substrate. This strategy presents several major drawbacks, including high costs and health risks derived from the manipulation of radioactive isotopes. In this work, we introduce an enhanced non-radioactive assay to quantify PKA activity, termed kinase mobility shift assay (KiMSA), based on the use of a fluorescent-labeled Kemptide (Kemptide-FITC). Once the kinase reaction is terminated, the products can be easily resolved through electrophoresis on an agarose gel and quantified by fluorescence densitometry. We show that KiMSA is suitable for isolated PKA as well as for the enzyme in cell extracts. In addition, it enables quantification of PKA activity during the progression of mouse sperm capacitation. Furthermore, the assay enables monitoring the inhibition of PKA with pharmacological inhibitors in live cells. Therefore, the experimental and optimal assay conditions are set so that KiMSA can be used to assess in vitro as well as in vivo PKA activity in sperm cells. Finally, this method allows for measurement of cAMP concentrations, rendering a versatile technique for the study of cAMP/PKA pathways. Key features • KiMSA is a versatile kinase mobility shift assay for measuring PKA activity in sperm physiology, replacing radioactive assays with a fluorescence-labeled substrate for high sensitivity. • This in vitro assay enables evaluation of activation state, drug effects, or PKA kinetics after purification or mutation. • This assay measures PKA activity from cell extracts, reflecting pre-lysis activation status and intracellular signaling, though not a true in vivo readout. • The standard protocol completion time is one day, including sperm preparation, kinase reactions, electrophoresis, and fluorescence quantification.