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First published online September 29, 2004
doi: 10.1242/10.1242/jcs.01383

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Progesterone inhibits protein kinase A (PKA) in Xenopus oocytes: demonstration of endogenous PKA activities using an expressed substrate

¹ Ottawa Health Research Institute, Ottawa Hospital Civic Campus, 1053 Carling Avenue, Ottawa, K1Y 4E9, Canada
² Department of Biochemistry, Microbiology and Immunology, University of Ottawa, 550 Cumberland, Ottawa, Ontario, K1N 6N5, Canada
³ Department of Obstetrics and Gynaecology, University of Ottawa, 550 Cumberland, Ottawa, Ontario, K1N 6N5, Canada

^* Author for correspondence (e-mail: jliu{at}ohri.ca)

Accepted 24 June 2004

3'-5' cyclic adenosine monophosphate (cAMP)-dependent protein kinase, PKA, is thought to be a key enzyme that controls prophase arrest in vertebrate oocytes. It has long been established that overexpression of the catalytic subunit of PKA inhibits hormone-induced frog oocyte maturation whereas overexpression of the regulatory subunits induces hormone-independent oocyte maturation. However, the activities of endogenous oocyte PKA, or its regulation by the maturation-inducing hormone progesterone, have never been directly demonstrated in frog oocytes. We have developed a novel expressed substrate for PKA in live oocytes by constructing a fusion protein containing an N-terminal myristylation sequence (derived from the Src tyrosine kinase) followed by an antigenic epitope tag and a substrate motif (the C-terminal cytoplasmic domain of ß₂ adrenergic receptor). Following mRNA injection, the phosphorylation status of the substrate was determined by two-dimensional electrophoresis followed by epitope immunoblotting, or alternatively by SDS-PAGE followed by immunoblotting using antibodies specifically recognizing the PKA-phosphorylated form of the substrate. In prophase oocytes, the expressed protein, myr-HA-ß₂AR-C, was fully phosphorylated on a single PKA site (Ser³⁴⁶ of human ß₂ adrenergic receptor). Within one hour of the addition of progesterone, the PKA site became mostly dephosphorylated. No re-phosphorylation of the PKA site, and therefore no reactivation of PKA, was observed throughout the entire maturation process. To demonstrate the generality of this PKA substrate, we analyzed its phosphorylation status in COS-7 cells following transfection. We show that dibutyryl cAMP rapidly stimulates phosphorylation of the PKA site. These results represent the first biochemical demonstration of regulation of endogenous Xenopus oocyte PKA by progesterone. Furthermore, myr-HA-ß₂AR-C should be widely adaptable as an in vivo PKA activity indicator.

Key words: cAMP-dependent protein kinase, Two-dimensional gel electrophoresis, In vivo substrate, Progesterone, Meiosis, COS-7 cells, C terminus of ß₂ adrenergic receptor

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