CDC25B Phosphorylation by p38 and MK-2

CDC25B is one of the three human phosphatases that are involved in the control of the activation of cyclin-dependent kinases. CDC25B participates in regulating entry into mitosis and appears to play a key role in the checkpoint response to DNA injury.CDC25B has been reported to be regulated by a number of kinases and controversial evidence suggests that it is phosphorylated by p38SAPK and/or MAPKAP Kinase-2. In this report, we clarify this issue using an approach combining mass spectrometry andthe use of specific antibodies against phosphorylated CDC25B residues. We report that MAPKAP Kinase-2 phosphorylates CDC25B on multiple sites including S169, S323, S353 and S375, while p38 phosphorylates CDC25B on S249. We show that theS323-phosphorylated form of CDC25B is detected at the centrosome during a normal cell cycle. Since most of these sites are also phosphorylated by several other kinases, our observations highlight the difficulty in characterising and understanding in vivo phosphorylation patterns.     

Cdc25 Phosphatases Are Required for Timely Assembly of CDK1-Cyclin B at the G2/M Transition

Progression through mitosis requires the coordinated regulation of Cdk1 kinase activity. Activation of Cdk1 is a multistep process comprising binding of Cdk1 to cyclin B, relocation of cyclin-kinase complexes to the nucleus, activating phosphorylation of Cdk1 on Thr¹⁶¹ by the Cdk-activating kinase (CAK; Cdk7 in metazoans), and removal of inhibitory Thr¹⁴ and Tyr¹⁵ phosphorylations. This dephosphorylation is catalyzed by the dual specific Cdc25 phosphatases, which occur in three isoforms in mammalian cells, Cdc25A, -B, and -C. We find that expression of Cdc25A leads to an accelerated G₂/M phase transition. In Cdc25A-overexpressing cells, Cdk1 exhibits high kinase activity despite being phosphorylated on Tyr¹⁵. In addition, Tyr¹⁵-phosphorylated Cdk1 binds more cyclin B in Cdc25A-overexpressing cells compared with control cells. Consistent with this observation, we demonstrate that in human transformed cells, Cdc25A and Cdc25B, but not Cdc25C phosphatases have an effect on timing and efficiency of cyclin-kinase complex formation. Overexpression of Cdc25A or Cdc25B promotes earlier assembly and activation of Cdk1-cyclin B complexes, whereas repression of these phosphatases by short hairpin RNA has a reverse effect, leading to a substantial decrease in amounts of cyclin B-bound Cdk1 in G₂ and mitosis. Importantly, we find that Cdc25A overexpression leads to an activation of Cdk7 and increase in Thr¹⁶¹ phosphorylation of Cdk1. In conclusion, our data suggest that complex assembly and dephosphorylation of Cdk1 at G₂/M is tightly coupled and regulated by Cdc25 phosphatases.     

Phosphorylation of CDC25A on SER283 in late S/G2 by CDK/cyclin complexes accelerates mitotic entry

The Cdc25A phosphatase is an essential activator of CDK-cyclin complexes at all steps of the eukaryotic cell cycle. The activity of Cdc25A is itself regulated in part by positive and negative feedback regulatory loops performed by its CDK-cyclin substrates that occur in G1 as well as during the G1/S and G2/M transitions. However, the regulation of Cdc25A during G2 phase progression before mitotic entry has not been intensively characterized. Here, we identify by mass spectrometry analysis a new phosphorylation event of Cdc25A on Serine283. Phospho-specific antibodies revealed that the phosphorylation of this residue appears in late S/G2 phase of an unperturbed cell cycle and is performed by CDK-cyclin complexes. Overexpression studies of wild-type and non-phosphorylatable mutant forms of Cdc25A indicated that Ser283 phosphorylation increases the G2/M-promoting activity of the phosphatase without impacting its stability or subcellular localization. Our results therefore identify a new positive regulatory loop between Cdc25A and its CDK-cyclin substrates which contributes to accelerate entry into mitosis through the regulation of Cdc25A activity in G2.     

Phosphorylation of Bcl-2 protein by CDC2 kinase during G2/M phases and its role in cell cycle regulation

Although it has been reported that Bcl-2 phosphorylation is associated with certain types of apoptosis, there is much controversy over the functional significance of and the kinases responsible for the phosphorylation. In this study, we examined whether Bcl-2 is phosphorylated by CDC2 kinase, a master regulator of G(2)/M transition in the eukaryotic cell cycle. When CDC2 was activated by okadaic acid in HL-60 cells, Bcl-2 phosphorylation was readily induced. The phosphorylation was correlated with the accumulation of cells in G(2)/M phases, but was not proportional to the level of apoptosis. Furthermore, we found that Bcl-2 was phosphorylated during G(2)/M phases of normal cell cycle. The ability of CDC2 to phosphorylate Bcl-2 was confirmed by in vitro kinase assay with a highly purified CDC2-cyclin B complex. Using synthetic peptides and mutant cell lines, we identified threonine 56, one of two consensus sites for CDC2 within the Bcl-2 sequence, as a residue phosphorylated by CDC2. Mutation at threonine 56 abrogated the cell cycle inhibitory effect of Bcl-2 without affecting anti-apoptotic function. These results suggest that two distinct functions of Bcl-2 (anti-apoptosis and cell cycle inhibition) are differentially regulated by post-translational mechanisms such as phosphorylation. CDC2-mediated phosphorylation of Bcl-2 may play some physiological roles in the negative regulatory events during mitosis.     

Phosphorylation by Aurora B kinase regulates caspase-2 activity and function

Mitotic catastrophe (MC) is an important oncosuppressive mechanism that serves to eliminate cells that become polyploid or aneuploid due to aberrant mitosis. Previous studies have demonstrated that the activation and catalytic function of caspase-2 are key steps in MC to trigger apoptosis and/or cell cycle arrest of mitotically defective cells. However, the molecular mechanisms that regulate caspase-2 activation and its function are unclear. Here, we identify six new phosphorylation sites in caspase-2 and show that a key mitotic kinase, Aurora B kinase (AURKB), phosphorylates caspase-2 at the highly conserved residue S384. We demonstrate that phosphorylation at S384 blocks caspase-2 catalytic activity and apoptosis function in response to mitotic insults, without affecting caspase-2 dimerisation. Moreover, molecular modelling suggests that phosphorylation at S384 may affect substrate binding by caspase-2. We propose that caspase-2 S384 phosphorylation by AURKB is a key mechanism that controls caspase-2 activation during mitosis.Subject terms: Proteases, Proteolysis     

Regulation of Cdc25C Activity During the Meiotic G2/M Transition

The Cdc25C phosphatase is a key activator of Cdc2/cyclin B that controls M-phase entry in eukaryotic cells. Here we discuss the regulation of Cdc25C by phosphorylation during the meiotic maturation of Xenopus oocytes. In G2 arrested oocytes, Cdc25C is phosphorylated on Ser287 and associated with 14-3-3 proteins. Entry of the oocytes into M-phase of meiosis is triggered by progesterone, which activates a signaling pathway leading to the dephosphorylation of Ser287, probably mediated by the PP1 phosphatase. The activation of Cdc25C during oocyte maturation correlates also with its phosphorylation on multiple sites. These phosphorylations involve several signaling pathways, including Polo kinases and MAP kinases, and might require also the inhibition of the PP2A phosphatase. Finally, Cdc25C is further phosphorylated by its substrate Cdc2/cyclin B, as part of an auto-amplification loop that ensures the high Cdc2/cyclin B activity level required to drive the oocyte through the meiotic cell cycle.     

Human Cdc14A phosphatase modulates the G2/M transition through Cdc25A and Cdc25B

The Cdc14 family of serine-threonine phosphatases antagonizes CDK activity by reversing CDK-dependent phosphorylation events. It is well established that the yeast members of this family bring about the M/G1 transition. Budding yeast Cdc14 is essential for CDK inactivation at the end of mitosis and fission yeast Cdc14 homologue Flp1/Clp1 down-regulates Cdc25 to ensure the inactivation of mitotic CDK complexes to trigger cell division. However, the functions of human Cdc14 homologues remain poorly understood. Here we have tested the hypothesis that Cdc14A might regulate Cdc25 mitotic inducers in human cells. We found that increasing levels of Cdc14A delay entry into mitosis by inhibiting Cdk1-cyclin B1 activity. By contrast, lowering the levels of Cdc14A accelerates mitotic entry. Biochemical analyses revealed that Cdc14A acts through key Cdk1-cyclin B1 regulators. We observed that Cdc14A directly bound to and dephosphorylated Cdc25B, inhibiting its catalytic activity. Cdc14A also regulated the activity of Cdc25A at the G2/M transition. Our results indicate that Cdc14A phosphatase prevents premature activation of Cdk1 regulating Cdc25A and Cdc25B at the entry into mitosis.     

MAPK Pathway Activation Delays G2/M Progression by Destabilizing Cdc25B

Activation of the mitogen-activated protein kinase (MAPK) pathway by growth factors or phorbol esters during G₂ phase delays entry into mitosis; however, the role of the MAPK pathway during G₂/M progression remains controversial. Here, we demonstrate that activation of the MAPK pathway with either epidermal growth factor or 12-O-tetradecanoylphorbol-13-acetate induces a G₂ phase delay independent of known G₂ phase checkpoint pathways but was specifically dependent on MAPK/extracellular signal-regulated kinase kinase (MEK1). Activation of MAPK signaling also blocked exit from a G₂ phase checkpoint arrest. Both the G₂ phase delay and blocked exit from the G₂ checkpoint arrest were mediated by the MEK1-dependent destabilization of the critical G₂/M regulator cdc25B. Reintroduction of cdc25B overcame the MEK1-dependent G₂ phase delay. Thus, we have demonstrated a new function for MEK1 that controls G₂/M progression by regulating the stability of cdc25B. This represents a novel mechanism by which factors that activate MAPK signaling can influence the timing of entry into mitosis, particularly exit from a G₂ phase checkpoint arrest.     

Cdk1 Activity Is Required for Mitotic Activation of Aurora A during G2/M Transition of Human Cells

In mammalian cells entry into and progression through mitosis are regulated by multiple mitotic kinases. How mitotic kinases interact with each other and coordinately regulate mitosis remains to be fully understood. Here we employed a chemical biology approach using selective small molecule kinase inhibitors to dissect the relationship between Cdk1 and Aurora A kinases during G₂/M transition. We find that activation of Aurora A first occurs at centrosomes at late G₂ and is required for centrosome separation independently of Cdk1 activity. Upon entry into mitosis, Aurora A then becomes fully activated downstream of Cdk1 activation. Inactivation of Aurora A or Plk1 individually during a synchronized cell cycle shows no significant effect on Cdk1 activation and entry into mitosis. However, simultaneous inactivation of both Aurora A and Plk1 markedly delays Cdk1 activation and entry into mitosis, suggesting that Aurora A and Plk1 have redundant functions in the feedback activation of Cdk1. Together, our data suggest that Cdk1, Aurora A, and Plk1 mitotic kinases participate in a feedback activation loop and that activation of Cdk1 initiates the feedback loop activity, leading to rapid and timely entry into mitosis in human cells. In addition, live cell imaging reveals that the nuclear cycle of cells becomes uncoupled from cytokinesis upon inactivation of both Aurora A and Aurora B kinases and continues to oscillate in a Cdk1-dependent manner in the absence of cytokinesis, resulting in multinucleated, polyploidy cells.     

The p97-Ufd1-Npl4 ATPase complex ensures robustness of the G2/M checkpoint by facilitating CDC25A degradation

The p97-Ufd1-Npl4 ATPase complex is associated with the response to DNA damage and replication stress, but how its inactivation leads to manifestation of chromosome instability is unclear. Here, we show that p97-Ufd1-Npl4 has an additional direct role in the G₂/M checkpoint. Upon DNA damage, p97-Ufd1-Npl4 binds CDC25A downstream of ubiquitination by the SCF-βTrCP ligase and facilitates its proteasomal degradation. Depletion of Ufd1-Npl4 leads to G₂/M checkpoint failure due to persistent CDC25 activity and propagation of DNA damage into mitosis with deleterious effects on chromosome segregation. Thus, p97-Ufd1-Npl4 is an integral part of G₂/M checkpoint signaling and thereby suppresses chromosome instability.     

A Mitotic Phosphorylation Feedback Network Connects Cdk1, Plk1, 53BP1, and Chk2 to Inactivate the G2/M DNA Damage Checkpoint

DNA damage checkpoints arrest cell cycle progression to facilitate DNA repair. The ability to survive genotoxic insults depends not only on the initiation of cell cycle checkpoints but also on checkpoint maintenance. While activation of DNA damage checkpoints has been studied extensively, molecular mechanisms involved in sustaining and ultimately inactivating cell cycle checkpoints are largely unknown. Here, we explored feedback mechanisms that control the maintenance and termination of checkpoint function by computationally identifying an evolutionary conserved mitotic phosphorylation network within the DNA damage response. We demonstrate that the non-enzymatic checkpoint adaptor protein 53BP1 is an in vivo target of the cell cycle kinases Cyclin-dependent kinase-1 and Polo-like kinase-1 (Plk1). We show that Plk1 binds 53BP1 during mitosis and that this interaction is required for proper inactivation of the DNA damage checkpoint. 53BP1 mutants that are unable to bind Plk1 fail to restart the cell cycle after ionizing radiation-mediated cell cycle arrest. Importantly, we show that Plk1 also phosphorylates the 53BP1-binding checkpoint kinase Chk2 to inactivate its FHA domain and inhibit its kinase activity in mammalian cells. Thus, a mitotic kinase-mediated negative feedback loop regulates the ATM-Chk2 branch of the DNA damage signaling network by phosphorylating conserved sites in 53BP1 and Chk2 to inactivate checkpoint signaling and control checkpoint duration.     

Ataxia Telangiectasia-mutated- and Rad3-related Protein Regulates the DNA Damage-induced G2/M Checkpoint through the Aurora A Cofactor Bora Protein

Polo-like kinase1 (Plk1) activation is inhibited in response to DNA damage, and this inhibition contributes to the activation of the G₂/M checkpoint, although the molecular mechanism by which Plk1 is inhibited is not clear. Here we report that the DNA damage signaling pathway inhibits Plk1 activity through Bora. Following UV irradiation, ataxia telangiectasia-mutated- and Rad3-related protein phosphorylates Bora at Thr-501. The phosphorylated Thr-501 is subsequently recognized by the E3 ubiquitin ligase SCF-β-TRCP, which targets Bora for degradation. The degradation of Bora compromises Plk1 activation and contributes to DNA damage-induced G₂ arrest. These findings shed new light on Plk1 regulation by the DNA damage response pathway.     

SNHG16 as the miRNA let-7b-5p sponge facilitates the G2/M and epithelial-mesenchymal transition by regulating CDC25B and HMGA2 expression in hepatocellular carcinoma

Long noncoding RNAs (lncRNAs) play crucial roles in hepatocellular carcinoma (HCC). However, the underlying molecular mechanisms of small nucleolar RNA host gene 16 (SNHG16) for regulating the cell cycle and epithelial to mesenchymal transition (EMT) remain elusive. In this study, SNHG16 expression profiles of HCC tissues or cell lines were compared with those of normal tissues or hepatocyte cell line. The effect of SNHG16 knockdown in HCC cell lines was investigated by using in vitro loss-of-function experiments and in vivo nude mouse experiments. The potential molecular regulatory mechanism of SNHG16 in HCC progression was investigated by using mechanistic experiments and rescue assays. The results revealed that SNHG16 was highly expressed in HCC tissues and cell lines, which predicted poor prognosis of HCC patients. On one hand, the downregulation of SNHG16 induced G2/M cell cycle arrest, inducing cell apoptosis and suppression of cell proliferation. On the other hand, it inhibited cell metastasis and EMT progression demonstrated by in vitro loss-of-function cell experiments. Besides, knockdown of SNHG16 increased the sensitivity of HCC cells to cisplatin. For the detailed mechanism, SNHG16 was demonstrated to act as a let-7b-5p sponge in HCC. SNHG16 facilitated the G2/M cell cycle transition by directly acting on the let-7b-5p/CDC25B/CDK1 axis, and promoted cell metastasis and EMT progression by regulating the let-7b-5p/HMGA2 axis in HCC. In addition, the mechanism of SNHG16 for regulating HCC cell proliferation and metastasis was further confirmed in vivo by mouse experiments. Furthermore, these results can provide new insights into HCC treatment and its molecular pathogenesis, which may enlighten the further research of the molecular pathogenesis of HCC.