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

Abstract

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 β2 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-β2AR-C, was fully phosphorylated on a single PKA site (Ser346 of human β2 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-β2AR-C should be widely adaptable as an in vivo PKA activity indicator.

Oocyte Maturation, Mos and Cyclins—A Matter of Synthesis: Two Functionally Redundant Ways to Induce Meiotic Maturation

“…34,35 Consistently, a complete inactivation of PKA is observed within 30-60 minutes of progesterone treatment. 36 The injection of the regulatory subunit of PKA (PKAr) or of a peptide inhibitor (PKI) causes hormone-independent oocyte maturation (Table 1) whereas injection of the catalytic subunit PKAc prevents progesterone-induced oocyte maturation. 37 Undoubtedly, the rapid and permanent suppression of PKA activity is the most prominent biochemical event, both necessary and sufficient for reinitiation of meiosis, with PKA inhibition occurring several hours before MPF activation.…”

Gβγ signaling reduces intracellular cAMP to promote meiotic progression in mouse oocytes

“…The G proteins regulating the inhibitory signal include Gα s and Gβγ, both of which appear to act together to stimulate adenylyl cyclase and increase intracellular cAMP. Evidence supporting this model is plentiful, including the following: First, steroids trigger a rapid decrease in intracellular cAMP with a concomitant decrease in PKA activity [9][10][11][12]. Second, over-expression of either Gα s or Gβγ inhibits steroid-triggered oocyte maturation, while reduction of Gα s or Gβγ levels or activity leads to enhanced maturation in response to steroids [13][14][15][16].…”

Proteomics of M-phase entry: ‘Omen’ vs. ‘Omre’, the battle for oocyte quality and beyond

“…As long as cAMP level remains high, PKA activity stays high too, and the oocyte is unable to activate MPF. A mixture of steroid hormones (among which progesterone seems to play a major role) delivered physiologically from follicular cells, or via experimental treatment in the case of oocytes isolated from the ovary, triggers a diminution of PKA activity [10][11][12]. The molecular mechanisms involved in the relationship between the inhibition of PKA and the activation of MPF is not fully understood ( Figure 2).…”