Monitoring protein kinase A activities using expressed substrate in live cells.
Protein kinase A (PKA) activity is regulated by intracellular cyclic adenosine monophosphate. 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-beta2AR-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 beta2 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-beta2 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-beta2AR-C should be widely adaptable as an in vivo PKA activity indicator.
Wang
Jing
J
Department of Biochemistry, Microbiology, and Immunology, University of Ottawa, Ontario, Canada.
Liu
X Johné
XJ
D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D005260:Female; D009865:Oocytes / Q000201:enzymology*; D013379:Substrate Specificity; D014981:Xenopus
D017868:Cyclic AMP-Dependent Protein Kinases