Mitotic Phosphorylation of Cdc25B Ser321 Disrupts 14-3-3 Binding to the High Affinity Ser323 Site

Puji Astuti,Rose Boutros,Bernard Ducommun,Brian Gabrielli

doi:10.1074/jbc.m110.138412

Abstract

Cdc25B is a key regulator of entry into mitosis, and its activity and localization are regulated by binding of the 14-3-3 dimer. There are three 14-3-3 binding sites on Cdc25B, with Ser(323) being the highest affinity binding and is highly homologous to the Ser(216) 14-3-3 binding site on Cdc25C. Loss of 14-3-3 binding to Ser(323) increases cyclin/Cdk substrate access to the catalytic site, thereby increasing its activity. It also affects the localization of Cdc25B. Thus, phosphorylation and 14-3-3 binding to this site is essential for down-regulating Cdc25B activity, blocking its mitosis promoting function. The question of how this inhibitory signal is relieved to allow Cdc25B activation and entry into mitosis is yet to be resolved. Here, we show that Ser(323) phosphorylation is maintained into mitosis, but phosphorylation of Ser(321) disrupts 14-3-3 binding to Ser(323), mimicking the effect of inhibiting Ser(323) phosphorylation on both Cdc25B activity and localization. The unphosphorylated Ser(321) appears to have a role in stabilizing 14-3-3 binding to Ser(323), and loss of the Ser hydroxyl group appears to be sufficient to significantly reduce 14-3-3 binding. A consequence of loss of 14-3-3 binding is dephosphorylation of Ser(323). Ser(321) is phosphorylated in mitosis by Cdk1. The mitotic phosphorylation of Ser(321) acts to maintain full activation of Cdc25B by disrupting 14-3-3 binding to Ser(323) and enhancing the dephosphorylation of Ser(323) to block 14-3-3 binding to this site.

Highlights

Cdc25B is regulated by multiple mechanisms, including its expression, stability, localization, and its interaction with 14-3-3 adapter proteins
We have proposed previously that the 14-3-3 dimer forms an intramolecular bridge connecting the N- and C-terminal domains of Cdc25B, regulating cyclin/Cdk substrate access to the catalytic site, thereby regulating Cdc25B. 14-3-3 binding to the Ser323 site appears to affect access to the proximal nuclear localization sequence, whereas binding to the Ser151 and Ser230 site affects access to the proximal nuclear export sequence (12)
The mutant Cdc25B had severely reduced substrate binding. This was clearly demonstrated with the substrate trapping mutant of Cdc25B3, where the substrate binding Y511A mutation was introduced, Cdk1 phosphorylated at Tyr15 and its major cyclin partner cyclin B1 was reduced by Ͼ90% (Fig. 1A)

Summary

Introduction

Cdc25B is regulated by multiple mechanisms, including its expression, stability, localization, and its interaction with 14-3-3 adapter proteins. The effect of expression of this mutation on cell cycle progression, interaction with substrate cyclin/ Cdks, and its regulation by 14-3-3 proteins and localization was examined.

Results

Conclusion