Dual Specificity Phosphatase Cdc25B Research Articles

CDC25B is required for the metaphase I-metaphase II transition in mouse oocytes.

Mammalian oocytes are arrested at meiotic prophase I. The dual-specificity phosphatase CDC25B is essential for cyclin-dependent kinase 1 (CDK1) activation that drives resumption of meiosis. CDC25B reverses the inhibitory effect of the protein kinases WEE1 and MYT1 on CDK1 activation. Cdc25b-/- female mice are infertile because oocytes cannot activate CDK1. To identify a role for CDC25B following resumption of meiosis, we restored CDK1 activation in Cdc25b-/- oocytes by inhibiting WEE1 and MYT1, or expressing EGFP-CDC25A or constitutively active EGFP-CDK1 from microinjected complementary RNAs. Forced CDK1 activation in Cdc25b-/- oocytes allowed resumption of meiosis, but oocytes mostly arrested at metaphase I (MI) with intact spindles. Similarly, approximately a third of Cdc25b+/- oocytes with a reduced amount of CDC25B arrested in MI. MI-arrested Cdc25b-/- oocytes also displayed a transient decrease in CDK1 activity similar to Cdc25b+/+ oocytes during the MI-MII transition, whereas Cdc25b+/- oocytes exhibited only a partial anaphase-promoting complex/cyclosome activation and anaphase I entry. Thus, CDC25B is necessary for the resumption of meiosis and the MI-MII transition.

Dual-Specificity Phosphatase CDC25B Was Inhibited by Natural Product HB-21 Through Covalently Binding to the Active Site.

Cysteine 473, within the active site of the enzyme, Cdc25B, is catalytically essential for substrate activation. The most well-reported inhibitors of Cdc25 phosphatases, especially quinone-type inhibitors, function by inducing irreversible oxidation at this active site of cysteine. Here, we identified a natural product, HB-21, having a sesquiterpene lactone skeleton that could irreversibly bind to cys473 through the formation of a covalent bond. This compound inhibited recombinant human Cdc25B phosphatase with an IC50 value of 24.25 μM. Molecular modeling predicted that HB-21 not only covalently binds to cys473 of Cdc25B but also forms six hydrogen bonds with residues at the active site. Moreover, HB-21 can dephosphorylate cyclin-dependent kinase (CDK1), the natural substrate of Cdc25b, and inhibit cell cycle progression. In summary, HB-21 is a new type of Cdc25B inhibitor with a novel molecular mechanism.

Pre‐clinical identification of potential molecular diagnostic biomarkers of secondary ischemia in microvascular fasciocutaneous flaps

Compromise of surgical microvascular flaps due to acute or late post‐operative circulatory complications remains a significant clinical problem worldwide. Timely detection of vessel occlusion may salvage a flap from necrosis, but monitoring of physical flap appearance is not always feasible and the etiology of compromise is hard to identify. Here we used a translational laboratory animal model of abdominal wall repair and Affymetrix gene expression microarray platform to reveal the molecular tissue and wound fluid markers of the arterial (AO), venous (VO) or mixed arterio‐venous (AVO) thrombosis of the pedicled flap in vivo. Bilateral 7×2.5cm epigastric flaps were raised in ~12 month old male and female Sprague‐Dawley retired breeder rats. Baseline fasciocutaneous tissue sample biopsies were obtained from the upper abdominal quadrants of sixteen sex‐matched rats prior to flap elevation. All flaps were then subjected to primary ischemia (PI), which was induced for 4 h by temporary clamping of superficial inferior epigastric artery (SIEA) and vein (SIEV) vessels to mimic free tissue transfer and anastomosis. Normoxic control (NC) samples were obtained from flaps after 24 h of non‐obstructed reperfusion. AO, VO or AVO samples were retrieved from paired flaps undergoing secondary ischemia (SI), in which SIEA, SIEV or both vessels were ligated for 4 h following 20 h of reperfusion. Total RNA was isolated from individual animal samples (n=8/gender group) and then pooled at equal concentrations prior to assessment of RNA quality. We observed 5011 (in males) and 4457 (in females) differentially expressed genes (DEGs) that demonstrated more than 2‐fold change in NC versus baseline conditions. There were 1403, 1643 and 2351 DEGs in male AO, VO and AVO flaps when compared to NC. In females, AO, VO and AVO‐associated DEG numbers were smaller (1074, 828 and 1518, respectively). Independently of gender, downregulation of keratinocyte‐specific small proline‐rich protein 4 (Sprr4) and upregulation of ankyrin repeat domain 37 (Ankrd37), previously identified as a target of hypoxia inducible factor‐1 (HIF‐1) gene, was significantly associated with the arterial SI. Venous SI was characterized by overexpression of major hematopoietic cell proteins (Gypa, Slc4a1, Lgals5, Hbq1b, Hemgn, Add2, Alas2), anti‐inflammatory bioactive lipid mediator arachidonate 15‐lipoxygenase (Alox15), CDK‐activating dual specificity phosphatase Cdc25B and Neuropeptide Y (Npy) ‐ an important neurotransmitter in skin vascular conductance. Upregulation of the latter genes distinguished VO not only from AO, but also from AVO. Finally, we identified 58 “master” DEGs shared by both genders that were present in SI regardless of its type (AO, VO, and AVO). Of these, “first‐tier” proteins included leukemia inhibitory factor (LIF), Depp1 autophagy regulator, adrenomedullin (Adm), growth arrest and DNA‐damage‐inducible, gamma (Gadd45g) and DNA‐damage‐inducible transcript 4 (Ddit4). Change in selected gene expression was further confirmed at protein or mRNA level by Western blotting or RT‐PCR analyses. In summary, our data revealed several SI‐associated diagnostic biomarkers that merit further investigation and, ultimately, validation in the human clinical trial setting.Support or Funding InformationThis work has been funded and supported by the Department of Otolaryngology ‐ Head & Neck Surgery, Thomas Jefferson UniversityThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Synthesis and biological evaluation of 3-aminoisoquinolin-1(2H)-one based inhibitors of the dual-specificity phosphatase Cdc25B

The cell division cycle 25B dual specificity phosphatase (Cdc25B) regulates the normal progression of the mammalian cell cycle by dephosphorylating and activating cyclin-dependent kinase (Cdk) complexes, particularly in response to DNA damage. Elevated Cdc25B levels enable a bypass of normal cell cycle checkpoints, and the overexpression of Cdc25B has been linked to a variety of human cancers. Thus, Cdc25B is an attractive target for the development of anticancer therapeutics. Herein we describe the synthesis and biological evaluation of a series of non-quinoid inhibitors of Cdc25B containing the 3-aminoisoquinolin-1(2H)-one pharmacophore. In addition to several strategies that address specific substitution patterns on isoquinolines, we have applied a regioselective Pd-catalyzed cross-coupling methodology to synthesize a new lead structure, 6-(3-aminophenyl)-3-(phenylamino)isoquinolin-1(2H)-one (13), which proved to be a reversible, competitive Cdc25B inhibitor with a Ki of 1.9μM. Compound 13 prevented human cancer cell growth and blocked Cdc25B-mediated mitotic checkpoint bypass. Molecular docking studies support binding near the catalytic site.

Paracaseolide A, First α-Alkylbutenolide Dimer with an Unusual Tetraquinane Oxa-Cage Bislactone Skeleton from Chinese Mangrove Sonneratia paracaseolaris

A novel α-alkylbutenolide dimer, paracaseolide A (2), characterized by an unusual tetraquinane oxa-cage bislactone skeleton bearing two linear alkyl chains, was isolated from the mangrove plant Sonneratia paracaseolaris. The structure of 2 was elucidated by extensive spectroscopic analysis. A plausible retrosynthetic pathway for paracaseolide A (2) was proposed. Compound 2 exhibited significant inhibitory activity against dual specificity phosphatase CDC25B, a key enzyme for cell cycle progression, with an IC(50) value of 6.44 μM.

βTrCP-dependent degradation of CDC25B phosphatase at the metaphase-anaphase transition is a pre-requisite for correct mitotic exit

The dual-specificity phosphatase CDC25B, a key regulator of CDK/Cyclin complexes, is considered as the starter of mitosis. It is an unstable protein, degraded by the proteasome, but often over-expressed in various human cancers. Based on experiments carried out in Xenopus eggs, and on video microscopy studies in mammalian cells, it has been proposed that human CDC25B degradation is dependent of the F-box protein bTrCP, but the involvement of this latter protein was not formally demonstrated yet. Here, we show that indeed, in mammalian cells, bTrCP participates to CDC25B turn-over, and is required for the complete degradation of CDC25B at the metaphase-anaphase transition. Using a stabilized mutant of CDC25B, which cannot interact anymore with bTrCP, we further show that, during late phases of mitosis, reduced degradation of CDC25B leads to an extended window of expression of the protein, which in turn induces a delay in mitosis exit and entails mitotic defects such as chromosomes missegregation. These findings show that a dysfunction in the rapid and precisely controlled degradation of CDC25B at the metaphase-anaphase transition is sufficient to cause genomic instability and suggest that, in human tissues, pathologic stabilization or untimed expression of CDC25B could contribute to tumorigenesis.

Inappropriate Activation of Cyclin-dependent Kinases by the Phosphatase Cdc25b Results in Premature Mitotic Entry and Triggers a p53-dependent Checkpoint

The dual specificity phosphatase Cdc25B is capable of inhibiting cellular proliferation, and this occurs in a manner dependent upon its catalytic activity. Here it is shown that this is accompanied by inappropriate cyclin-dependent kinase activation and premature mitotic entry, leading to both p53-dependent and independent checkpoints. Forced expression of Cdc25B inappropriately up-regulated the activity of Cdk1 and Cdk2, by reducing levels of inhibitory phosphorylation. In cells lacking p14ARF, p53 is induced, and components of the ATM and ATR pathways are activated. Cdc25B triggers cell cycle arrest in the G(1) and G(2) phases that is p53- and p21-dependent and is inhibited by caffeine. Cdc25B also causes cells with an S phase DNA content to enter mitosis prematurely in a p53-independent manner. Synchronization of cells with aphidicolin results in these cells undergoing apoptosis. Thus, inappropriate cell cycle progression and premature mitotic entry via dysregulation of cyclin-dependent kinases results in activation of both p53-dependent and independent responses. Because Cdc25B is known to have oncogenic activity, this provides insight into the multistep nature of cancer development and why there is p53 loss during tumorigenesis.

Computational design, synthesis and biological evaluation of para-quinone-based inhibitors for redox regulation of the dual-specificity phosphatase Cdc25B

Quinoid inhibitors of Cdc25B were designed based on the Linear Combination of Atomic Potentials (LCAP) methodology. In contrast to a published hypothesis, the biological activities and hydrogen peroxide generation in reducing media of three synthetic models did not correlate with the quinone half-wave potential, E(1/2).

The Dual Specificity Phosphatase Cdc25B, but Not the Closely Related Cdc25C, Is Capable of Inhibiting Cellular Proliferation in a Manner Dependent upon Its Catalytic Activity

Cdc25B and Cdc25C are closely related dual specificity phosphatases that activate cyclin-dependent kinases by removal of inhibitory phosphorylations, thereby triggering entry into mitosis. Cdc25B, but not Cdc25C, has been implicated as an oncogene and been shown to be overexpressed in a variety of human tumors. Surprisingly, ectopic expression of Cdc25B, but not Cdc25C, inhibits cell proliferation in long term assays. Chimeric proteins generated from the two phosphatases show that the anti-proliferative activity is associated with the C-terminal end of Cdc25B. Indeed, the catalytic domain of Cdc25B is sufficient to suppress cell viability in a manner partially dependent upon its C-terminal 26 amino acids that is shown to influence substrate binding. Mutation analysis demonstrates that both the phosphatase activity of Cdc25B as well as its ability to interact with its substrates contribute to the inhibition of cell proliferation. These results demonstrate key differences in the biological activities of Cdc25B and Cdc25C caused by differential substrate affinity and recognition. This also argues that the antiproliferative activity of Cdc25B needs to be overcome for it to act as an oncogene during tumorigenesis.

Amino Acids C-Terminal to the 14-3-3 Binding Motif in CDC25B Affect the Efficiency of 14-3-3 Binding

The phospho-site adapter protein 14-3-3 binds to target proteins at amino acid sequences matching the consensus motif Arg-X-X-Ser/Thr-X-Pro, where the serine or threonine residue is phosphorylated and X is any amino acid. The dual-specificity phosphatase CDC25B, which is involved in cell cycle regulation, contains five 14-3-3 binding motifs, but 14-3-3 preferentially binds to the motif at Ser309 in CDC25B1 (or Ser323 in CDC25B3). In the present study, we demonstrate that amino acid residues C-terminal to the 14-3-3 binding motif strongly affect the efficiency of 14-3-3 binding. Alanine substitutions at residues downstream of the Ser309 motif dramatically reduced 14-3-3 binding, although phosphorylation of Ser309 was unaffected. We also observed that binding of endogenous 14-3-3 to mutant CDC25B occurred less efficiently than to the wild type. Mutants to which 14-3-3 cannot bind efficiently tend to be located in the nucleus, although not as specifically as the alanine substitution mutant of Ser309. These results indicate that amino acid sequences C-terminal to the consensus binding site have an important role in the efficient binding of 14-3-3 to at least CDC25B, which may partly explain why some consensus sequences are inactive as 14-3-3 binding sites.

Synthesis and Biological Evaluation of Caulibugulones A—E.

AbstractFor Abstract see ChemInform Abstract in Full Text.

Binding of 14-3-3beta but not 14-3-3sigma controls the cytoplasmic localization of CDC25B: binding site preferences of 14-3-3 subtypes and the subcellular localization of CDC25B.

The dual specificity phosphatase CDC25B positively controls the G2-M transition by activating CDK1/cyclin B. The binding of 14-3-3 to CDC25B has been shown to regulate the subcellular redistribution of CDC25B from the nucleus to the cytoplasm and may be correlated with the G2 checkpoint. We used a FLAG-tagged version of CDC25B to study the differences among the binding sites for the 14-3-3 subtypes, 14-3-3beta, 14-3-3epsilon and 14-3-3sigma, and the relationship between subtype binding and the subcellular localization of CDC25B. All three subtypes were found to bind to CDC25B. Site-directed mutagenesis studies revealed that 14-3-3beta bound exclusively near serine-309 of CDC25B1, which is within a potential consensus motif for 14-3-3 binding. By contrast, 14-3-3sigma bound preferentially to a site around serine-216, and the presence of serine-137 and -309 enhanced the binding. In addition to these binding-site differences, we found that the binding of 14-3-3beta drove CDC25B to the cytoplasm and that mutation of serine-309 to alanine completely abolished the cytoplasmic localization of CDC25B. However, co-expression of 14-3-3sigma and CDC25B did not affect the subcellular localization of CDC25B. Furthermore, serine-309 of CDC25B was sufficient to produce its cytoplasmic distribution with co-expression of 14-3-3beta, even when other putative 14-3-3 binding sites were mutated. 14-3-3epsilon resembled 14-3-3beta with regard to its binding to CDC25B and the control of CDC25B subcellular localization. The results of the present study indicate that two 14-3-3 subtypes can control the subcellular localization of CDC25B by binding to a specific site and that 14-3-3sigma has effects on CDC25B other than the control of its subcellular localization.

Sulfonylated aminothiazoles as new small molecule inhibitors of protein phosphatases

Based on a previously identified lead structure, SC-ααδ9, we have developed a versatile new chemical scaffold that can be readily modified to generate libraries of both Tyr and dual specificity phosphatase inhibitors with reduced molecular weight and lipophilicity. The most potent analogue identified to date, aminothiazole 8z, inhibits the dual specificity phosphatase Cdc25B with a Ki of 4.6 ± 0.4 μM and a Hill coefficient of 2.

A dual-specificity phosphatase Cdc25B is an unstable protein and triggers p34(cdc2)/cyclin B activation in hamster BHK21 cells arrested with hydroxyurea.

By incubating at 30 degrees C in the presence of an energy source, p34(cdc2)/cyclin B was activated in the extract prepared from a temperature-sensitive mutant, tsBN2, which prematurely enters mitosis at 40 degrees C, the nonpermissive temperature (Nishimoto, T. , E. Eilen, and C. Basilico. 1978. Cell. 15:475-483), and wild-type cells of the hamster BHK21 cell line arrested in S phase, without protein synthesis. Such an in vitro activation of p34(cdc2)/cyclin B, however, did not occur in the extract prepared from cells pretreated with protein synthesis inhibitor cycloheximide, although this extract still retained the ability to inhibit p34(cdc2)/cyclin B activation. When tsBN2 cells arrested in S phase were incubated at 40 degrees C in the presence of cycloheximide, Cdc25B, but not Cdc25A and C, among a family of dual-specificity phosphatases, Cdc25, was lost coincidentally with the lack of the activation of p34(cdc2)/cyclin B. Consistently, the immunodepletion of Cdc25B from the extract inhibited the activation of p34(cdc2)/cyclin B. Cdc25B was found to be unstable (half-life < 30 min). Cdc25B, but not Cdc25C, immunoprecipitated from the extract directly activated the p34(cdc2)/cyclin B of cycloheximide-treated cells as well as that of nontreated cells, although Cdc25C immunoprecipitated from the extract of mitotic cells activated the p34(cdc2)/cyclin B within the extract of cycloheximide-treated cells. Our data suggest that Cdc25B made an initial activation of p34(cdc2)/cyclin B, which initiates mitosis through the activation of Cdc25C.