Cyclin B phosphorylation by PKA safeguards the G2-arrest in vertebrate oocytes

Entry into meiotic M-phase in vertebrate oocytes requires activation of Cdk1-Cyclin B, which is restrained by the cAMP-PKA signalling during prophase arrest. Although Cdc25C and ARPP19 are established PKA substrates in this context, they do not fully explain how PKA represses Cdk1 activation. Here we used Turbo-ID approach using catalytic PKA (PKAc) as the bait to identify substrates in Xenopus laevis oocytes. Unexpectedly, this screen identified Cyclin B2, the regulatory subunit of Cdk1, as a PKA-proximal protein. Although Cyclin B2 does not stably associate with PKAc, it harbors a conserved PKA motif around Serine 271 within Cyclin Box 2. We show that PKAc phosphorylates Cyclin B2 at S271 both in vivo and in vitro. Functionally, a phosphomimic Cyclin B2 mutant at this site fails to induce meiotic maturation and Cdk1 activation. S271 phosphorylation neither alters Cyclin B2 stability nor its binding to Cdk1 in vivo. S271 phosphorylation does not impair Cdk1 activity toward a single-site substrate, such as PP1, but slows Cyclin-B-dependent multisite phosphorylation of Cdc25C, a key regulator of the Cdk1 activation network. These findings identify Cyclin B as a direct and conserved PKA target, revealing the mechanistic link between PKA activity and Cdk1-Cyclin B repression that maintains oocyte prophase arrest.