Abstract
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-β2AR-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-β2AR-C should be widely adaptable as an in vivo PKA activity indicator.
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© 2006 Humana Press Inc., Totowa, NJ
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Wang, J., Liu, X.J. (2006). Monitoring Protein Kinase A Activities Using Expressed Substrate in Live Cells. In: Liu, X.J. (eds) Xenopus Protocols. Methods in Molecular Biology™, vol 322. Humana Press. https://doi.org/10.1007/978-1-59745-000-3_30
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DOI: https://doi.org/10.1007/978-1-59745-000-3_30
Publisher Name: Humana Press
Print ISBN: 978-1-58829-362-6
Online ISBN: 978-1-59745-000-3
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