G2 arrest in Xenopus oocytes depends on phosphorylation of cdc25 by protein kinase A.
Xenopus oocytes, which are arrested in G(2) 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 G(2) 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 G(2) 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.
Duckworth
Brian C
BC
Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA.
Weaver
Jennifer S
JS
Ruderman
Joan V
JV
D000818:Animals; D000242:Cyclic AMP / Q000502:physiology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000502:physiology*; D004789:Enzyme Activation; D005260:Female; D016195:G2 Phase; D020928:Mitogen-Activated Protein Kinases / Q000502:physiology; D009865:Oocytes / Q000502:physiology*; D010766:Phosphorylation; D011374:Progesterone / Q000494:pharmacology; D014981:Xenopus; D020687:cdc25 Phosphatases / Q000378:metabolism*
D011374:Progesterone; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D020928:Mitogen-Activated Protein Kinases; D020687:cdc25 Phosphatases