Abstract

CDC25B phosphatases must activate cyclin B-CDK1 complexes to restart the cell cycle after an arrest in G2 phase caused by DNA damage. However, little is known about the precise mechanisms involved in this process, which may exert considerable impact on cancer susceptibility and therapeutic responses. Here we report the discovery of novel N-terminally truncated CDC25B isoforms, referred to as ΔN-CDC25B, with an exclusively nuclear and nonredundant function in cell cycle re-initiation after DNA damage. ΔN-CDC25B isoforms are expressed from a distinct promoter not involved in expression of canonical full-length isoforms. Remarkably, in contrast to the high lability and spatial dynamism of the full-length isoforms, ΔN-CDC25B isoforms are highly stable and exclusively nuclear, strongly suggesting the existence of two pools of CDC25B phosphatases in the cell that have functionally distinct properties. Using isoform-specific siRNA, we found that depleting full-length isoforms, but not ΔN-CDC25B isoforms, delays entry into mitosis. Thus, in an unperturbed cell cycle, the full-length isoforms are exclusively responsible for activating cyclin B-CDK1. Strikingly, in the late response to DNA damage, we found a CHK1-dependent shift in accumulation of CDC25B isoforms toward the ΔN-CDC25B species. Under this physiological stress condition, the ΔN-CDC25B isoform was found to play a crucial, nonredundant function in restarting the cell cycle after DNA damage-induced G2 phase arrest. Our findings reveal the existence of a previously unrecognized CDC25B isoform that operates specifically in the nucleus to reinitiate G2/M transition after DNA damage.

Highlights

  • In mammals, there are 3 CDC25 phosphatases—CDC25A, CDC25B, and CDC25C—(1, 2) which are believed to be partially functionally redundant in controlling mitotic entry [3, 4]

  • Medema and colleagues have shown that CDC25B, but not CDC25A or CDC25C, was critically required for the recovery from a prolonged arrest in G2 induced by the activation of the DNA damage checkpoint [9]

  • Whereas our data identified the doublet of higher molecular weight as CDC25B3 and B2, the 2 molecular species of lower apparent molecular weight did not exhibit the molecular weight and reactivity to antibodies features of the known CDC25B molecules suggesting that they correspond to uncharacterized isoforms

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Summary

Introduction

There are 3 CDC25 phosphatases—CDC25A, CDC25B, and CDC25C—(1, 2) which are believed to be partially functionally redundant in controlling mitotic entry [3, 4]. In special physiological conditions in which the cell cycle has to be restarted from an extended block, the functional redundancy between the 3 CDC25s is lost, and CDC25B is the only member of the CDC25 family whose function becomes essential for mitotic entry. This notion stems in part from the fact that in oocyte of female CDC25B-null mice, resumption of the meiotic progression does not occur owing to a defect of activation of cyclin B-CDK1 [7], a phenotype not observed in a CDC25C-null background [8]. The mechanism by which CDC25B is directed to proteolysis involves a DDG motif, located in the N-terminal regulatory domain, which plays a critical role in the recruitment of the E3 ubiquitin ligase SCFßTrCp to the

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