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.