Phosphorylation of Human CDC25B Phosphatase by CDK1-Cyclin A Triggers Its Proteasome-dependent Degradation

Abstract

In eukaryotes the activity of CDK1 (CDC2), a cyclindependent kinase that initiates the structural changes that culminate in the segregation of chromosomes at mitosis, is regulated by the synergistic and opposing activities of a cascade of kinases and phosphatases. Dephosphorylation of threonine 14 and tyrosine 15 of CDK1 by the CDC25 phosphatases is a key step in the activation of the CDK1-cyclin B protein kinase. Little is currently known about the role and the regulation of CDC25B. Here we report in vitro and in vivo data that indicate that CDC25B is degraded by the proteasome. This degradation is dependent upon phosphorylation by the CDK1-cyclin A complex but not by CDK1-cyclin B. These results indicate that CDK1-cyclin A phosphorylation targets CDC25B for degradation and that this might be an important component of cell cycle regulation at the G 2 /M transition.The eukaryotic cell cycle is controlled by a family of cyclindependent kinases that regulate its key transitions. The precise timing of the activation of these enzymes is a central issue of cell cycle regulation. A major regulatory mechanism is provided by the wee1/mik1-and myt1-dependent phosphorylation on threonine 14 and tyrosine 15 of CDK1 (cdc2). This inhibitory phosphorylation keeps the kinase inactive until it is dephosphorylated by the dual specificity CDC25 phosphatase at the G 2 /M transition (1). In human cells, three homologues of CDC25 called CDC25A, CDC25B, and CDC25C have been identified (2-4). In HeLa cells, expression of CDC25B is low throughout the cell cycle with an increase in G 2 , CDC25C is also predominantly expressed in G 2 (2, 3), and CDC25A is abundant both at the mRNA and protein levels in late G 1 (5). Phosphorylation of CDC25C by CDK1-cyclin B was shown and proposed to be part of the self-amplification mechanism of CDK1-cyclin B at mitosis (6). CDK2-cyclin E-dependent phosphorylation of CDC25A was also demonstrated, indicating that a similar feedback loop might regulate the progression in S phase (7). The mechanisms that regulate CDC25B activity and the precise role of that phosphatase and its splicing variants (8) in the control of entry into mitosis remain unclear.Here we report in vitro and in vivo evidences indicating that CDC25B is degraded by the proteasome and that this process is dependent on the phosphorylation by the CDK1-cyclin A kinase. We propose that the rapid degradation of CDC25B is an important regulatory mechanism that ensures the timely coordinated activation of CDK-cyclin complexes. MATERIALS AND METHODSProduction of Recombinant Proteins-The CDC25B coding sequence corresponding to the B1 splicing variant (8) was cloned in the pET14b (Novagen) vector. In vitro transcription and translation were performed using the TNT system (Promega) in the presence or the absence of [ 35 S]methionine. Sf9 insect cells were co-infected with recombinant baculovirus encoding for human CDK1 (CDC2) or CDK2 and cyclin B or cyclin A. Insect cell extracts were prepared as follows: cells were lysed with lysis buffer (10 mM Tris...

Differential mitotic degradation of the CDC25B phosphatase variants

“…In line with this view, we propose a model to account for the regulation of CDC25B protein stability ( Figure 7). As we demonstrated previously, CDC25B remains very unstable before mitosis by phosphorylation by CDK1-cyclin A that targets it for proteasomedependent degradation (Baldin et al, 1997a). We also showed that prior phosphorylation of CDC25B by CDK1-cyclin B was able to prevent CDK1-cyclin A-induced degradation (Cans et al, 1999).…”

“…CDC25B was shown to be a very unstable protein in interphase cells, with a half-life of less than 30 min in BHK cells arrested with hydroxyurea (Nishijima et al, 1997). We also reported that CDC25B is degraded in a proteasome-dependent manner on phosphorylation by the CDK1-cyclin A complex (Baldin et al, 1997a). These data suggest that the fine tuning of CDC25B levels might be an important aspect of the control of entry into mitosis.…”

“…CDC25B phosphatase activity appears to be highly regulated by phosphorylation mechanisms. A number of regulatory kinases that activate or inhibit CDC25B have been identified (Baldin et al, 1997a(Baldin et al, , 2003Bulavin et al, 2002;Theis-Febvre et al, 2003;Dutertre et al, 2004;Cazales et al 2005;Manke et al, 2005;Mirey et al, 2005;Lemaire et al, 2006;Schmitt et al, 2006) (our unpublished data). These phosphorylation events modulate the catalytic activity, the interactions with regulatory partners such as 14-3-3 proteins, the localization and probably also the stability of CDC25B.…”

Cdc25B is negatively regulated by p53 through Sp1 and NF-Y transcription factors

“…Cdc25B is an unstable protein degraded in a proteasomedependent manner (Baldin et al, 1997a). Treatment of HCT116 WT or p53 null cells with the translational inhibitor, cycloheximide, or the proteasomal inhibitor, MG132, at various times showed that the higher Cdc25B protein level observed in HCT116 p53 null cells was not the result of deregulation of protein degradation or synthesis (Supplementary Figure S1).…”

Cdc25A stability is controlled by the ubiquitin-proteasome pathway during cell cycle progression and terminal differentiation

“…It had been previously reported that yeast cdc25 and human cdc25B are regulated by the ubiquitin-proteasome pathway (Nefsky and Beach, 1996;Baldin et al, 1997). The ®nding that cdc25A is also subject to regulation by the ubiquitin-proteasome machinery extends the involvement of this pathway to yet another family member.…”