Phosphorylation of Cdc25B3 Ser169 regulates 14-3-3 binding to Ser151 and Cdc25B activity

Cdc25B is a major regulator of G(2) phase progression into mitosis and in recovery from a G(2) phase checkpoint arrest. The activity of Cdc25B is regulated by 14-3-3 dimer binding to a high-affinity phosphosite Ser323 and two lower-affinity phosphosites Ser151 and Ser230, which blocks substrate access to the catalytic site and, thereby, activity. Loss of 14-3-3 binding to any of these sites is sufficient to activate the enzyme. Thus, the mechanisms that disrupt 14-3-3 binding are critical regulators of entry into mitosis. Here, we demonstrate that the mechanism regulating the G(2) phase activation of major cdc25B3 isoform is independent of the dephosphorylation of the 14-3-3 binding sites, instead utilizing phosphorylation of Ser169. Mutation of this site to the phosphomimetic Asp reduced 14-3-3 binding to the same extent as Ser151Ala mutation, increased substrate access to the catalytic site and Cdc25B activity but had no effect on Ser151 or Ser230 phosphorylation. Ser169 phosphorylation was observed in G(2)-phase cells. By contrast, Ser249 phosphorylation, which was associated with cyclin B/Cdk1 activation, had no effect on 14-3-3 binding or Cdc25B activity. This indicates that Ser169 phosphorylation can disrupt 14-3-3 binding to Ser151 activating Cdc25B3, providing a mechanism for regulating Cdc25B3 activation without dephosphorylation of the 14-3-3 binding sites.