Activity of novel Cdc25 inhibitors and preliminary evaluation of their potentiation of chemotherapeutic drugs in human breast cancer cells

Dual-specific phosphatases Cdc25 play a critical role in the cell cycle regulation by activating kinases of Cdk/cyclin complexes. Three Cdc25 isoforms (A, B and C) have been identified in mammalians. Cdc25A and B display oncogenic properties and are over-expressed in different tumors. Cdc25 phosphatases are therefore attractive targets for therapeutic strategies. Novel maleic anhydride derivatives bearing a fatty acid chain of variable size have been synthesized and tested for their Cdc25 inhibitory potential using an in vitro assay. We report biological activity of ineffective, moderate, and efficient inhibitors on breast cancer cells (MCF7) and its counterpart resistant to vincristine (Vcr-R). The most potent compounds induced Cdk2 inhibition and accumulation in G0/G1 phase of the cell cycle. Moreover, apoptosis was triggered within 48-h treatment, without oxidative burst and modulation of the Bax to Bcl-2 ratio. When used as pre-treatments, these derivatives were also able to potentiate adriamycin and cisplatin toxicity in both cell lines. Thus, maleic anhydride derivatives may mediate apoptosis through a cell cycle blockage via inhibition of Cdc25. This class of inhibitors may present potential interest in therapeutic strategies against cancer.     

Synthesis and biological evaluation of a targeted library of protein phosphatase inhibitors

Phosphorylation of serine, threonine, and tyrosine controls fundamental mammalian cell events and is achieved by kinases which, in turn, are in dynamic relationship with phosphatases. Few selective inhibitors of protein tyrosine and dual specificity phosphatases are readily available. Based on SAR studies of naturally occurring phosphatase inhibitors and following up on previously published research, we have designed a new pharmacophore model V and synthesized a new library of functional analogues of V. All synthetic steps were carried out and optimized employing combinatorial chemistry methods on Wang resin. All compounds were tested in vitro for their ability to inhibit recombinant human protein tyrosine (PTP1B) and dual-specificity (Cdc25B(2) and VHR) phosphatases. Three of the approximately 70 compounds in our library inhibited Cdc25B(2) by 50% at 375-490 microM. No compounds inhibited PTP1B, and only one blocked VHR. Cell-culture studies revealed no toxicity to human breast cancer cells with two of the phosphatase inhibitors.     

Halenaquinone and xestoquinone derivatives, inhibitors of Cdc25B phosphatase from a Xestospongia sp

Separation of an extract of a Xestospongia sp., guided by bioassay against Cdc25B, led to the isolation of nine compounds, halenaquinone (1), xestoquinone (2), adociaquinones A (3) and B (4), 3-ketoadociaquinones A (5) and B (6), tetrahydrohalenaquinones A (7) and B (8), and 13-O-methyl xestoquinol sulfate (9). The structures of the new natural products 6 and 9 were established on the basis of extensive one- and two-dimensional NMR studies. Compounds 1, 4, and 6 inhibited recombinant human Cdc25B in vitro with IC50 values of 0.7, 0.07, and 0.2 microM, respectively, and were 19- to 150-fold less active against two related protein phosphatases. Compound 4 blocked cell cycle progression through mitosis.     

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.     

Binding and inhibition of Cdc25 phosphatases by vitamin K analogues

A synthetic K vitamin analogue, 2-(2-mercaptothenol)-3-methyl-1,4-naphthoquinone or Cpd 5, was previously found to be a potent inhibitor of cell growth [Nishikawa et al., (1995) J. Biol. Chem. 270, 28304-28310]. The mechanisms of cell growth were hypothesized to include the inactivation of cellular protein tyrosine phosphatases, especially the Cdc25 family [Tamura et al. (2000) Cancer Res. 60, 1317-1325]. In this study, we synthesized PD 49, a new biotin containing Cpd 5 derivative, to search for evidence of direct interaction of these arylating analogues with Cdc25A, Cdc25B, and Cdc25C phosphatases. PD 49 was shown to directly bind to GST-Cdc25A, GST-Cdc25B, their catalytic fragments, and GST-Cdc25C. The binding could be competed with excess glutathione or Cpd 5, and a cysteine-to-serine mutation of the catalytic cysteine abolished binding. This was consistent with an involvement in binding of cysteine in the catalytic domain. This interaction between PD 49 and Cdc25 also occurred in lysates of treated cells. PD 49 also bound to protein phosphatases other than Cdc25. We found that the new analogue also inhibited Hep3B human hepatoma cell growth. This growth inhibition involved ERK1/2 phosphorylation and was inhibited by a MEK antagonist. The results demonstrate a direct interaction and binding between this growth-inhibiting K vitamin derivative with both purified as well as with cellular Cdc25A, Cdc25B, and Cdc25C.     

Ectopic Expression of Cdc25A Accelerates the G1/S Transition and Leads to Premature Activation of Cyclin E- and Cyclin A-Dependent Kinases

Human Cdc25 phosphatases play important roles in cell cycle regulation by removing inhibitory phosphates from tyrosine and threonine residues of cyclin-dependent kinases. Three human Cdc25 isoforms, A, B, and C, have been discovered. Cdc25B and Cdc25C play crucial roles at the G(2)/M transition. In the present study, we have investigated the function of human Cdc25A phosphatase. Cell lines that express human Cdc25A in an inducible manner have been generated. Ectopic expression of Cdc25A accelerates the G(1)/S-phase transition, indicating that Cdc25A controls an event(s) that is rate limiting for entry into S phase. Furthermore, we carried out a detailed analysis of the expression and activation of human Cdc25A. Activation of endogenous Cdc25A occurs during late G(1) phase and increases in S and G(2) phases. We further demonstrate that Cdc25A is activated at the same time as cyclin E- and cyclin A-dependent kinases. In vitro, Cdc25A dephosphorylates and activates the cyclin-Cdk complexes that are active during G(1). Overexpression of Cdc25A in the inducible system, however, leads to a premature activation of both cyclin E-Cdk2 and cyclin A-Cdk2 complexes, while no effect of cyclin D-dependent kinases is observed. Furthermore, Cdc25A overexpression induces a tyrosine dephosphorylation of Cdk2. These results suggest that Cdc25A is an important regulator of the G(1)/S-phase transition and that cyclin E- and cyclin A-dependent kinases act as direct targets.     

Cdc25B and Cdc25C differ markedly in their properties as initiators of mitosis.

We have used time-lapse fluorescence microscopy to study the properties of the Cdc25B and Cdc25C phosphatases that have both been implicated as initiators of mitosis in human cells. To differentiate between the functions of the two proteins, we have microinjected expression constructs encoding Cdc25B or Cdc25C or their GFP-chimeras into synchronized tissue culture cells. This assay allows us to express the proteins at defined points in the cell cycle. We have followed the microinjected cells by time-lapse microscopy, in the presence or absence of DNA synthesis inhibitors, and assayed whether they enter mitosis prematurely or at the correct time. We find that overexpressing Cdc25B alone rapidly causes S phase and G2 phase cells to enter mitosis, whether or not DNA replication is complete, whereas overexpressing Cdc25C does not cause premature mitosis. Overexpressing Cdc25C together with cyclin B1 does shorten the G2 phase and can override the unreplicated DNA checkpoint, but much less efficiently than overexpressing Cdc25B. These results suggest that Cdc25B and Cdc25C do not respond identically to the same cell cycle checkpoints. This difference may be related to the differential localization of the proteins; Cdc25C is nuclear throughout interphase, whereas Cdc25B is nuclear in the G1 phase and cytoplasmic in the S and G2 phases. We have found that the change in subcellular localization of Cdc25B is due to nuclear export and that this is dependent on cyclin B1. Our data suggest that although both Cdc25B and Cdc25C can promote mitosis, they are likely to have distinct roles in the controlling the initiation of mitosis.     

Regulation of Cdc2/cyclin B activation in Xenopus egg extracts via inhibitory phosphorylation of Cdc25C phosphatase by Ca(2+)/calmodulin-dependent protein [corrected] kinase II

Activation of Cdc2/cyclin B kinase and entry into mitosis requires dephosphorylation of inhibitory sites on Cdc2 by Cdc25 phosphatase. In vertebrates, Cdc25C is inhibited by phosphorylation at a single site targeted by the checkpoint kinases Chk1 and Cds1/Chk2 in response to DNA damage or replication arrest. In Xenopus early embryos, the inhibitory site on Cdc25C (S287) is also phosphorylated by a distinct protein kinase that may determine the intrinsic timing of the cell cycle. We show that S287-kinase activity is repressed in extracts of unfertilized Xenopus eggs arrested in M phase but is rapidly stimulated upon release into interphase by addition of Ca2+, which mimics fertilization. S287-kinase activity is not dependent on cyclin B degradation or inactivation of Cdc2/cyclin B kinase, indicating a direct mechanism of activation by Ca2+. Indeed, inhibitor studies identify the predominant S287-kinase as Ca2+/calmodulin-dependent protein kinase II (CaMKII). CaMKII phosphorylates Cdc25C efficiently on S287 in vitro and, like Chk1, is inhibited by 7-hydroxystaurosporine (UCN-01) and debromohymenialdisine, compounds that abrogate G2 arrest in somatic cells. CaMKII delays Cdc2/cyclin B activation via phosphorylation of Cdc25C at S287 in egg extracts, indicating that this pathway regulates the timing of mitosis during the early embryonic cell cycle.     

The Chk1 protein kinase and the Cdc25C regulatory pathways are targets of the anticancer agent UCN-01

A checkpoint operating in the G(2) phase of the cell cycle prevents entry into mitosis in the presence of DNA damage. UCN-01, a protein kinase inhibitor currently undergoing clinical trials for cancer treatment, abrogates G(2) checkpoint function and sensitizes p53-defective cancer cells to DNA-damaging agents. In most species, the G(2) checkpoint prevents the Cdc25 phosphatase from removing inhibitory phosphate groups from the mitosis-promoting kinase Cdc2. This is accomplished by maintaining Cdc25 in a phosphorylated form that binds 14-3-3 proteins. The checkpoint kinases, Chk1 and Cds1, are proposed to regulate the interactions between human Cdc25C and 14-3-3 proteins by phosphorylating Cdc25C on serine 216. 14-3-3 proteins, in turn, function to keep Cdc25C out of the nucleus. Here we report that UCN-01 caused loss of both serine 216 phosphorylation and 14-3-3 binding to Cdc25C in DNA-damaged cells. In addition, UCN-01 potently inhibited the ability of Chk1 to phosphorylate Cdc25C in vitro. In contrast, Cds1 was refractory to inhibition by UCN-01 in vitro, and Cds1 was still phosphorylated in irradiated cells treated with UCN-01. Thus, neither Cds1 nor kinases upstream of Cds1, such as ataxia telangiectasia-mutated, are targets of UCN-01 action in vivo. Taken together our results identify the Chk1 kinase and the Cdc25C pathway as potential targets of G(2) checkpoint abrogation by UCN-01.     

3,3��-Diindolylmethane inhibits breast cancer cell growth via miR-21-mediated Cdc25A degradation

3,3'-Diindolylmethane (DIM) is a potential cancer preventive phytochemical derived from Brassica vegetables. The effects of DIM on cell-cycle regulation in both estrogen-dependent MCF-7 and estrogen receptor negative p53 mutant MDA-MB-468 human breast cancer cells were assessed in this study. DIM inhibited the breast cancer cell growth in vitro and in vivo, and caused cell-cycle arrest by down-regulating protein levels of cell-cycle related kinases CDK1, CDK2, CDK4, and CDK6, as well as Cyclin B1 and Cdc25A. Meanwhile, it was revealed that Ser(124) phosphorylation of Cdc25A is primarily responsible for the DIM-induced Cdc25A degradation. Furthermore, treatment of MCF-7 cells with DIM increased miR-21 expression and down-regulated Cdc25A, resulting in an inhibition of breast cancer cell proliferation. These observations collectively suggest that by differentially modulating cellular signaling pathways DIM is able to arrest the cell-cycle progression of human breast cancer cells.     

Novel benzofuran inhibitors of human mitogen-activated protein kinase phosphatase-1

Protein tyrosine phosphatases have a central role in the maintenance of normal cellular functionality. For example, PTP1B has been implicated in insulin-resistance, obesity, and neoplasia. Mitogen-activated protein kinase phosphatase-1 (MKP-1 or DUSP1) dephosphorylates and inactivates mitogen-activated protein kinase (MAPK) substrates, such as p38, JNK, and Erk, and has been implicated in neoplasia. The lack of readily available selective small molecule inhibitors of MKP family members has severely limited interrogation of their biological role. Inspired by a previously identified inhibitor (NSC 357756) of MKP-3, we synthesized seven NSC 357756 congeners, which were evaluated for in vitro inhibition against several protein phosphatases. Remarkably, none displayed potent inhibition against MKP-3, including the desamino NSC 357756 analog NU-154. Interestingly, NU-154 inhibited human PTP1B in vitro with an IC(50) value of 24 +/- 1 microM and showed little inhibition against Cdc25B, MKP-1, and VHR phosphatases. NU-126 [2-((E)-2-(5-cyanobenzofuran-2-yl)vinyl)-1H-indole-6-carbonitrile] inhibited MKP-1 and VHR in vitro but was less active against human MKP-3, Cdc25B, and PTP1B. The inhibition of MKP-1 by NU-126 was independent of redox processes. The benzofuran substructure represents a new potential scaffold for further analog development and provides encouragement that more selective and potent inhibitors of MKP family members may be achievable.     

Normal cell cycle and checkpoint responses in mice and cells lacking Cdc25B and Cdc25C protein phosphatases

The Cdc25 family of protein phosphatases positively regulates cell division by activating cyclin-dependent protein kinases (CDKs). In humans and rodents, there are three Cdc25 family members--denoted Cdc25A, Cdc25B, and Cdc25C--that can be distinguished based on their subcellular compartmentalizations, their abundances and/or activities throughout the cell cycle, the CDKs that they target for activation, and whether they are overexpressed in human cancers. In addition, murine forms of Cdc25 exhibit distinct patterns of expression throughout development and in adult tissues. These properties suggest that individual Cdc25 family members contribute distinct biological functions in embryonic and adult cell cycles of mammals. Interestingly, mice with Cdc25C disrupted are healthy, and cells derived from these mice exhibit normal cell cycles and checkpoint responses. Cdc25B-/- mice are also generally normal (although females are sterile), and cells derived from Cdc25B-/- mice have normal cell cycles. Here we report that mice lacking both Cdc25B and Cdc25C are obtained at the expected Mendelian ratios, indicating that Cdc25B and Cdc25C are not required for mouse development or mitotic entry. Furthermore, cell cycles, DNA damage responses, and Cdc25A regulation are normal in cells lacking Cdc25B and Cdc25C. These findings indicate that Cdc25A, or possibly other phosphatases, is able to functionally compensate for the loss of Cdc25B and Cdc25C in mice.     

Androgens Upregulate Cdc25C Protein by Inhibiting Its Proteasomal and Lysosomal Degradation Pathways

Cdc25C is a cell cycle protein of the dual specificity phosphatase family essential for activating the cdk1/Cyclin B1 complex in cells entering into mitosis. Since altered cell cycle is a hallmark of human cancers, we investigated androgen regulation of Cdc25C protein in human prostate cancer (PCa) cells, including androgen-sensitive (AS) LNCaP C-33 cells and androgen-independent (AI) LNCaP C-81 as well as PC-3 cells. In the regular culture condition containing fetal bovine serum (FBS), Cdc25C protein levels were similar in these PCa cells. In a steroid-reduced condition, Cdc25C protein was greatly decreased in AS C-33 cells but not AI C-81 or PC-3 cells. In androgen-treated C-33 cells, the Cdc25C protein level was greatly elevated, following a dose- and a time-dependent manner, correlating with increased cell proliferation. This androgen effect was blocked by Casodex, an androgen receptor blocker. Nevertheless, epidermal growth factor (EGF), a growth stimulator of PCa cells, could only increase Cdc25C protein level by about 1.5-fold. Altered expression of Cdc25C in C-33 cells and PC-3 cells by cDNA and/or shRNA transfection is associated with the corresponding changes of cell growth and Cyclin B1 protein level. Actinomycin D and cycloheximide could only partially block androgen-induced Cdc25C protein level. Treatments with both proteasomal and lysosomal inhibitors resulted in elevated Cdc25C protein levels. Immunoprecipitation revealed that androgens reduced the ubiquitination of Cdc25C proteins. These results show for the first time that Cdc25C protein plays a role in regulating PCa cell growth, and androgen treatments, but not EGF, greatly increase Cdc25C protein levels in AS PCa cells, which is in part by decreasing its degradation. These results can lead to advanced PCa therapy via up-regulating the degradation pathways of Cdc25C protein.     

Redox regulation of Cdc25C

The Cdc25 family of dual specific phosphatases are critical components of cell cycle progression and checkpoint control. Certain stresses such as ultraviolet light stimulate the rapid and selective destruction of Cdc25A protein through a Chk1 protein kinase-dependent pathway. We demonstrate that in contrast to cellular stresses previously examined, hydrogen peroxide exposure affects Cdc25C but not Cdc25A levels. Pharmacological inhibition of Chk1 activity or a mutant of Cdc25C that lacks the Chk1 phosphorylation site still undergoes degradation in response to oxidants. We also demonstrate that in vitro hydrogen peroxide stimulates an intramolecular disulfide bond between the active site cysteine at position 377 and another invariant cysteine at position 330. The in vivo stability of Cdc25C is substantially reduced by the mutation of either of these two cysteine residues. In contrast, a double (C2) mutant of both cysteine 330 and cysteine 377 results in a protein that is more stable than wild type Cdc25C and is resistant to oxidative stress-induced degradation. In addition, the C2 mutant, which is unable to form an intramolecular disulfide bond, has reduced binding to 14-3-3 in vitro and in vivo. These results suggest that oxidative stress may induce cell cycle arrest in part through the degradation of Cdc25C.     

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.     

Discovery of small molecule human C5a receptor antagonists

A novel series of small molecule C5a antagonists is reported. In particular, in vitro metabolic studies and solution based combinatorial synthesis are demonstrated as useful tools for the rapid identification of antagonists with low in vitro clearance. Members of this series specifically inhibited the binding of ¹²⁵I-labeled C5a to human recombinant C5a receptor (C5aR). In functional cell assays these compounds displayed surmountable antagonism against C5a and did not demonstrate any detectable agonist activity.     

Identification of epidermal growth factor receptor as a target of Cdc25A protein phosphatase

Cdc25A, a dual-specificity protein phosphatase, plays a critical role in cell cycle progression. Although cyclin-dependent kinases are established substrates, Cdc25A may also affect other proteins. We have shown here that Cdc25A interacts with epidermal growth factor receptor (EGFR) both physically and functionally in Hep3B human hepatoma cells. Cdc25A inhibitor Cpd 5, a vitamin K analog, inhibited Cdc25A activity in the Cdc25A-EGFR immunocomplex and consequently caused prolonged EGFR tyrosine phosphorylation. Both purified GST-Cdc25A protein and endogenous Hep3B cellular Cdc25A dephosphorylated tyrosine-phosphorylated EGFR, and Cpd 5 antagonized the phosphatase activity of Cdc25A. A functional Cdc25A-EGFR interaction was seen in NR-6 fibroblasts expressing ectopic EGFR but not with a receptor lacking the C terminus or a mutated kinase domain. These data link the cell cycle control Cdc25A phosphatase to an EGFR-linked mitogenic signaling pathway specifically involving EGFR dephosphorylation.     

Identification of new Cdc25 dual specificity phosphatase inhibitors in a targeted small molecule array

Dual specificity protein phosphatases (DSPases) are key regulators of signal transduction, oncogenesis and the cell cycle. Few potent or specific inhibitors of DSPases, however, are readily available for these pharmacological targets. We have used a combinatorial/parallel synthetic approach to rigidify the variable core region and modify the side chains of 4-(benzyl-(2-[2,5-diphenyl-oxazole-4-carbonyl)-amino]-ethyl)-carbamoyl)- 2-decanoylamino butyric acid (or SC-alphaalphadelta9), which is the most active element in a previously described library of phosphatase inhibitors (Rice, R. L.; Rusnak, J. M.; Yokokawa, F.; Yokokawa, S.; Messner, D. J.; Boynton, A. L.; Wipf, P.; Lazo, J. S. Biochemistry 1997, 36, 15965). Several analogues were identified as effective inhibitors of the protein tyrosine phosphatase (PTPase) PTP1B and the DSPases VHR and Cdc25B2. Two compounds, FY3-alphaalpha09 and FY21-alphaalpha09, were partial competitive inhibitors of Cdc25B2 with Ki values of 7.6+/-0.5 and 1.6+/-0.2 microM, respectively. FY21-alphaalpha09 possessed only moderate activity against PTP1B. Consistent with its in vitro anti-phosphatase activity, FY21-alphaalpha09 inhibited growth in MDA-MB-231 and MCF-7 human breast cancer cell lines. FY21-alphaalpha09 also inhibited the G2/M transition in tsFT210 cells, consistent with Cdc25B inhibition. Several architectural requirements for DSPase inhibition were revealed through modification of the side chain moieties or variable core region of the pharmacophore, which resulted in decreased compound potency. The structure of FY21-alphaalpha09 provides a useful platform from which additional potent and more highly selective phosphatase inhibitors might be generated.     

Protein kinase A regulates resumption of meiosis by phosphorylation of Cdc25B in mammalian oocytes

In mammalian oocytes, meiosis arrests at prophase I. Meiotic resumption requires activation of Maturation-Promoting Factor (MPF), comprised of a catalytic Cyclin-dependent kinase-1 (Cdk1) and a regulatory subunit cyclin B, and results in germinal vesicle breakdown (GVBD). Cyclic AMP (cAMP)-mediated Protein Kinase A (PKA) activity sustains prophase arrest by inhibiting Cdk1. However, the link between PKA activity and MPF inhibition remains unclear. Cdc25 phosphatases can activate Cdks by removing inhibitory phosphates from Cdks. Thus one method for sustaining prophase arrest could be inhibition of the activity of the Cdc25 protein required for MPF activation. Indeed, studies in Xenopus identify Cdc25C as a target of PKA activity in meiosis. However, in mice, studies suggest that Cdc25B is the phosphatase essential for GVBD and, therefore, the likely target of PKA activity. To assess these questions, we targeted a potential PKA substrate, a highly conserved serine 321 residue of Cdc25B and evaluated the effect on oocyte maturation. A Cdc25B-Ser321Ala point mutant mRNA induces GVBD when injected into prophase-arrested oocytes more rapidly than wild type mRNA. Using fluorescently-tagged proteins we also determined that the mutant protein enters the nucleus more rapidly than its wildtype counterpart. These data suggest that phosphorylation of the Ser321 residue plays a key role in the negative regulation and localization of Cdc25B during prophase arrest. PKA also phosphorylates a wildtype Cdc25B protein but not a Ser321Ala mutant protein in vitro. Mutation of Ser321 in Cdc25B also affects its association with a sequestering protein, 14-3-3. Our studies suggest that Cdc25B is a direct target of PKA in prophase-arrested oocytes and that Cdc25B phosphorylation results in its inhibition and sequestration by the 14-3-3 protein.