Monitoring Protein Kinase A Activities Using Expressed Substrate in Live Cells

Protein kinase A (PKA) activity is regulated by intracellular cyclic adenosine mono-phosphate. Conventional protein kinase assays after cell lysis are hence not suitable for analyzing PKA activities. In this chapter, we describe a new method for monitoring PKA activity in live cells. A triparti substrate for PKA (Myr-HA-β₂ AR-C) is constructed that contains an N-terminal myristylation sequence followed by an antigenic hemagglutinin epitope tag and a substrate motif (the C-terminal tail of human β₂ adrenergic receptor). The PKA phosphorylation status of the substrate in frog oocytes is determined either by two-dimensional electrophoresis followed by HA epitope immunoblotting or by direct SDS-PAGE followed by immunoblotting using anti-P-β₂ adrenergic receptor antibodies specifically recognizing the PKA-phosphorylated C-terminus. We also describe the application of this strategy in mammalian somatic cells through DNA transfection. Myr-HA-β₂ AR-C should be widely adaptable as an in vivo PKA activity indicator.