G2 arrest in Xenopus oocytes depends on phosphorylation of cdc25 by protein kinase A

BC Duckworth, JS Weaver… - Proceedings of the …, 2002 - National Acad Sciences
BC Duckworth, JS Weaver, JV Ruderman
Proceedings of the National Academy of Sciences, 2002National Acad Sciences
Xenopus oocytes, which are arrested in G2 of meiosis I, contain complexes of cyclin B–cdc2
(M phase-promoting factor) that are kept repressed by inhibitory phosphorylations on cdc2 at
Thr-14 and Tyr-15. Progesterone induces a cytoplasmic signaling pathway that leads to
activation of cdc25, the phosphatase that removes these phosphorylations, catalyzing entry
into M phase. It has been known for 25 years that high levels of cAMP and protein kinase A
(PKA) are required to maintain the G2 arrest and that a drop in PKA activity is required for M …
Xenopus oocytes, which are arrested in G2 of meiosis I, contain complexes of cyclin B–cdc2 (M phase-promoting factor) that are kept repressed by inhibitory phosphorylations on cdc2 at Thr-14 and Tyr-15. Progesterone induces a cytoplasmic signaling pathway that leads to activation of cdc25, the phosphatase that removes these phosphorylations, catalyzing entry into M phase. It has been known for 25 years that high levels of cAMP and protein kinase A (PKA) are required to maintain the G2 arrest and that a drop in PKA activity is required for M phase-promoting factor activation, but no physiological targets of PKA have been identified. We present evidence that cdc25 is a critical target of PKA. (i) In vitro, cdc25 Ser-287 serves as a major site of phosphorylation by PKA, resulting in sequestration by 14-3-3. (ii) Endogenous cdc25 is phosphorylated on Ser-287 in oocytes and dephosphorylated in response to progesterone just before cdc2 dephosphorylation and M-phase entry. (iii) High PKA activity maintains phosphorylation of Ser-287 in vivo, whereas inhibition of PKA by its heat-stable inhibitor (PKI) induces dephosphorylation of Ser-287. (iv) Overexpression of mutant cdc25 (S287A) bypasses the ability of PKA to maintain oocytes in G2 arrest. These findings argue that cdc25 is a physiologically relevant target of PKA in oocytes. In the early embryonic cell cycles, Ser-287 is phosphorylated during interphase and dephosphorylated just before cdc2 activation and mitotic entry. Thus, in addition to its role in checkpoint arrest, cdc25 Ser-287 serves as a site for regulation during normal, unperturbed cell cycles.
National Acad Sciences