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

Journal of Cell Science 117, 5107-5116 (2004)
Published by The Company of Biologists 2004
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Progesterone inhibits protein kinase A (PKA) in Xenopus oocytes: demonstration of endogenous PKA activities using an expressed substrate

Jing Wang1,2 and X. Johné Liu1,2,3,*

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



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  		Fig. 1. Schematic representation of the plasmid constructs used in this study. To generate a potential expressed substrate for PKA we added two features to the C-terminus of ß2AR. The first was to add a myristylation sequence to target the truncated protein to the membranes, where ß2AR normally resides. We also included a hemagglutinin (HA) antigenic epitope to facilitate detection by immunoblotting. In addition to myr-HA-ß2AR-C, we also constructed myr-HA-ß2AR-C/PKA- (substituting both Ser345 and Ser346 with Ala), myr-HA-ß2AR-C/GRK- [substituting all 11 GRK sites with either Ala or Gly, as described previously (Hausdorff et al., 1989Go)] and myr-HA-ß2AR-C/PKA-&GRK- (containing neither PKA nor GRK sites). myr, myristylation; HA, hemagglutinin tag.

 


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  		Fig. 3. Progesterone-induced dephosphorylation of myr-HA-ß2AR-C. (A-J) Xenopus oocytes injected with the indicated mRNAs were incubated in OR2 for 24 hours. Groups of at least 20 oocytes were incubated in OR2 or OR2 containing 1 µM progesterone (Pg). Group D received 50 µM forskolin (Fsk) 20 minutes prior to the addition of progesterone. One hour later, oocytes were lysed and analyzed by 2D gel electrophoresis followed by HA immunoblotting. Shown are representative examples of three to ten independent experiments. The long dashed line separates two first dimension mini-tube gels. Shown in the PKA-&GRK- panel (on the right side of panel H) is one example of an extra SDS sample directly loaded on the second-dimension gel. OH- and H+ denote the anion and cation ends, respectively, of the tube gels. Circles represent positions of missing spots (1 or 2). Arrow, myr-HA-ß2AR-C; asterisk, nonspecific protein.

 


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  		Fig. 2. Expression of myr-HA-ß2AR-C in Xenopus oocytes and in COS-7 cells. COS-7 cells mock transfected (-) or transfected with myr-HA-ß2AR-C (+) were lysed and analyzed by immunoblotting with antibodies against HA (left panel). Frog oocytes were injected with water (-) or mRNA for myr-HA-ß2AR-C (+). Oocytes injected with myr-HA-ß2AR-C mRNA were incubated in OR2 (-) or with progesterone for the indicated periods. Oocytes were lysed and analyzed by immunoblotting with antibodies against HA (right panel). Oocytes treated overnight (o/n) with progesterone (Pg) exhibited 100% germinal vesicle breakdown (GVBD). The arrow indicates the position of myr-HA-ß2AR-C and the asterisk indicates a nonspecific protein, that apparently exists in both frog oocytes and in COS-7 cells.

 


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  		Fig. 9. Inhibition of PKA in the absence of de novo protein synthesis. (A) Oocytes were injected with mRNAs for myr-HA-ß2AR-C (S345A) or myr-HA-ß2AR-C(S346A). Following 24 hours incubation, PKA phosphorylation of the two mutants was analyzed by 2D electrophoresis followed by anti-HA immunoblotting. Arrow, myr-HA-ß2AR-C; asterisk, nonspecific protein. (B) Oocytes were injected with myr-HA-ß2ARC/GRK2- (lane 1), myr-HA-ß2AR-C/PKA- (lane 2), myr-HA-ß2AR-C/S346A (lane 3), myr-HA-ß2AR-C/S345A (lane 4) or myr-HA-ß2AR-C (lanes 5-10). After 24 hours of incubation, oocytes in lanes 1-4 were directly lysed and analyzed by SDS-PAGE followed by immunoblotting with anti-phospho-ß2AR-C (S345 346) (upper panel) or anti-HA (lower panel). Oocytes in lane 6 were treated with progesterone for 1 hour. Oocytes in lane 7 were injected with PKAc (0.8 units) 1 hour prior to the progesterone treatment. Oocytes in lanes 9 and 10 were treated with 100 µM H89 for 1 or 2 hours respectively. Representative examples from three to five independent experiments are shown. (C) Oocytes were injected with mRNA for wild-type myr-HA-ß2AR-C. After 24 hours of incubation, half of the oocytes were pre-incubated for 2 hours in OR2 containing 50 µg/ml cycloheximide. Both groups were then incubated with progesterone. At the indicated times following the addition of progesterone, oocytes were scored for GVBD and ten oocytes were randomly withdrawn for lysis. The extracts were analyzed by SDS-PAGE and immunoblotted with the indicated antibodies. Shown are representative examples of five independent experiments.

 


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  		Fig. 4. Protein kinase A phosphorylated myr-HA-ß2AR-C in Xenopus oocytes. (A-D) Oocytes injected with mRNA for myr-HA-ß2AR-C were incubated in OR2 for 24 hours. One group received a further injection of 10 nl PKAc per oocyte. One hour after the PKAc injection, oocytes were incubated in OR2 or OR2 containing H89 (100 µM) or progesterone (Pg), as indicated. One hour later, 20 oocytes were retrieved from each group for lysis and 2D electrophoresis and immunoblotting with HA antibodies. Circle in (D) represents the position of missing spot 1. Arrow, myr-HA-ß2AR-C; asterisk, nonspecific protein. (E) The remaining oocytes in each group were scored for GVBD at the indicated time following the addition of H89 or progesterone. Only the H89 data are shown here (representative of three independent experiments).

 


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  		Fig. 5. A time course of myr-HA-ß2AR-C dephosphorylation caused by progesterone. (A-D) Xenopus oocytes injected with mRNA for myr-HA-ß2AR-C were incubated for 24 hours. Twenty oocytes were set aside as control (OR2) and the rest were incubated with progesterone (Pg). At each of the indicated times, 20 oocytes were removed and all were then subjected to 2D electrophoresis and HA immunoblotting. (D) Multiple phosphorylated forms of myr-HA-ß2AR-C were seen in oocytes that had undergone GVBD (represented by X). (E) Progesterone-treated oocytes were scored for GVBD. The same lysates were also analyzed for histone H1 kinase (MPF) activities (H1) and for MAP kinase phosphorylation (p-MAPK). Representative examples from three independent experiments are shown.

 


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  		Fig. 6. myr-HA-ß2AR-C is phosphorylated by an unidentified kinase(s) in GVBD oocytes. Xenopus oocytes injected with the indicated mRNAs were incubated for 24 hours before the addition of progesterone. Following progesterone-induced GVBD, oocytes were lysed and analyzed by 2D electrophoresis and immunoblotting with hemagglutinin (HA). Representative examples from two independent experiments are shown. X, multiple phosphorylated forms of myr-HA-ß2ARC.

 


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  		Fig. 7. Progesterone does not alter Xenopus oocyte serine/threonine phosphatase activities. Phosphatase activities were determined in the absence or presence of 100 nM okadaic acid. Means±s.e. of three (with okadaic acid) and five (without okadaic acid) independent experiments are shown. At least 50% of the oocytes at 180 minutes had undergone GVBD. The basal phosphatase activities in G2 oocytes were 76±6.5 nmol phosphate released per oocyte in 30 minutes.

 


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  		Fig. 8. db-cAMP stimulates PKA phosphorylation of myr-HA-ß2AR-C in COS-7 cells. COS-7 cells were transfected with myr-HA-ß2AR-C (A) or myr-HA-ß2AR-C/PKA- (B). The transfected cells were starved for 24 hours in serum-free DMEM and then either left untreated (left panels) or treated with 1.5 mM db-cAMP for 40 minutes (right panels). Cell lysates were prepared and analyzed by 2D electrophoresis and HA immunoblotting. Representative examples from three independent experiments are shown. Circles indicate missing spots. Asterisk indicates position of a nonspecific protein.

 

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© The Company of Biologists Ltd 2004