Elimination of cdc2 phosphorylation sites in the cdc25 phosphatase blocks initiation of M-phase.

The cdc25 phosphatase is a mitotic inducer that activates p34cdc2 at the G2/M transition by dephosphorylation of Tyr15 in p34cdc2. cdc25 itself is also regulated through periodic changes in its phosphorylation state. To elucidate the mechanism for induction of mitosis, phosphorylation of cdc25 has been investigated using recombinant proteins. cdc25 is phosphorylated by both cyclin A/p34cdc2 and cyclin B/p34cdc2 at similar sets of multiple sites in vitro. This phosphorylation retards its electrophoretical mobility and activates its ability to increase cyclin B/p34cdc2 kinase activity three- to fourfold in vitro, as found for endogenous Xenopus cdc25 in M-phase extracts. The threonine and serine residues followed by proline that are conserved between Xenopus and human cdc25 have been mutated. Both the triple mutation of Thr48, Thr67, and Thr138 and the quintuple mutation of these three threonine residues plus Ser205 and Ser285, almost completely abolish the shift in electrophoretic mobility of cdc25 after incubation with M-phase extracts or phosphorylation by p34cdc2. These mutations inhibit the activation of cdc25 by phosphorylation with p34cdc2 by 70 and 90%, respectively. At physiological concentrations these mutants cannot activate cyclin B/p34cdc2 in cdc25-immunodepleted oocyte extracts, suggesting that a positive feed-back loop between cdc2 and cdc25 is necessary for the full activation of cyclin B/p34cdc2 that induces abrupt entry into mitosis in vivo.     

The G2/M checkpoint phosphatase cdc25C is located within centrosomes

cdc25C is a phosphatase which regulates the activity of the mitosis promoting factor cyclin B/cdk1 by dephosphorylation, thus triggering G(2)/M transition. The activity and the sub-cellular localisation of cdc25C are regulated by phosphorylation. It is well accepted that cdc25C has to enter the nucleus to activate the cyclin B/cdk1 complex at G(2)/M transition. Here, we will show that cdc25C is located in the cytoplasm at defined dense structures, which according to immunofluorescence analysis, electron microscopy as well as biochemical subfractionation, are proven to be the centrosomes. Since cyclin B and cdk1 are also located at the centrosomes, this subfraction of cdc25C might participate in the control of the onset of mitosis suggesting a further role for cdc25C at the centrosomes.     

The mitotic phosphatase cdc25C at the Golgi apparatus

cdc25C is a phosphatase which regulates the activity of the mitosis promoting factor cyclin B/cdk1 by dephosphorylation, thus triggering G(2)/M transition. The activity of cdc25C is regulated by phosphorylation which by itself is implicated in regulating the subcellular localization. It is well accepted that cdc25C has to enter the nucleus to activate the cyclin B/cdk1 complex at G(2)/M transition. Here, we will show that cdc25C is located in the cytoplasm at defined dense structures which by immunofluorescence analysis as well as by biochemical subfractionation turned out to be the Golgi apparatus. It will be further shown that cdc25C at the Golgi fraction is an active phosphatase suggesting an additional and new role of cdc25C at the Golgi apparatus.     

The NIMA protein kinase is hyperphosphorylated and activated downstream of p34cdc2/cyclin B: coordination of two mitosis promoting kinases.

Initiation of mitosis in Aspergillus nidulans requires activation of two protein kinases, p34cdc2/cyclin B and NIMA. Forced expression of NIMA, even when p34cdc2 was inactivated, promoted chromatin condensation. NIMA may therefore directly cause mitotic chromosome condensation. However, the mitosis-promoting function of NIMA is normally under control of p34cdc2/cyclin B as the active G2 form of NIMA is hyperphosphorylated and further activated by p34cdc2/cyclin B when cells initiate mitosis. To see the p34cdc2/cyclin B dependent activation of NIMA, okadaic acid had to be added to isolation buffers to prevent dephosphorylation of NIMA during isolation. Hyperphosphorylated NIMA contained the MPM-2 epitope and, in vitro, phosphorylation of NIMA by p34cdc2/cyclin B generated the MPM-2 epitope, suggesting that NIMA is phosphorylated directly by p34cdc2/cyclin B during mitotic initiation. These two kinases, which are both essential for mitotic initiation, are therefore independently activated as protein kinases during G2. Then, to initiate mitosis, we suggest that each activates the other's mitosis-promoting functions. This ensures that cells coordinately activate p34cdc2/cyclin B and NIMA to initiate mitosis only upon completion of all interphase events. Finally, we show that NIMA is regulated through the cell cycle like cyclin B, as it accumulates during G2 and is degraded only when cells traverse mitosis.     

Phosphorylation and activation of human cdc25-C by cdc2--cyclin B and its involvement in the self-amplification of MPF at mitosis.

We have investigated the mechanisms responsible for the sudden activation of the cdc2-cyclin B protein kinase before mitosis. It has been found previously that cdc25 is the tyrosine phosphatase responsible for dephosphorylating and activating cdc2-cyclin B. In Xenopus eggs and early embryos a cdc25 homologue undergoes periodic phosphorylation and activation. Here we show that the catalytic activity of human cdc25-C phosphatase is also activated directly by phosphorylation in mitotic cells. Phosphorylation of cdc25-C in mitotic HeLa extracts or by cdc2-cyclin B increases its catalytic activity. cdc25-C is not a substrate of the cyclin A-associated kinases. cdc25-C is able to activate cdc2-cyclin B1 in Xenopus egg extracts and to induce Xenopus oocyte maturation, but only after stable thiophosphorylation. This demonstrates that phosphorylation of cdc25-C is required for the activation of cdc2-cyclin B and entry into M-phase. Together, these studies offer a plausible explanation for the rapid activation of cdc2-cyclin B at the onset of mitosis and the self-amplification of MPF observed in vivo.     

Activation of the phosphatase activity of human cdc25A by a cdk2-cyclin E dependent phosphorylation at the G1/S transition.

Progression through the cell cycle is monitored at two major points: during the G1/S and the G2/M transitions. In most cells, the G2/M transition is regulated by the timing of p34cdc2 dephosphorylation which results in the activation of the kinase activity of the cdc2-cyclin B complex. The timing of p34cdc2 dephosphorylation is determined by the balance between the activity of the kinase that phosphorylates p34cdc2 (wee1 in human cells) and the opposing phosphatase (cdc25C). Both enzymes are regulated and it has been shown that cdc25C is phosphorylated and activated by the cdc2-cyclin B complex. This creates a positive feed-back loop providing a switch used to control the onset of mitosis. Here, we show that another member of the human cdc25 family, cdc25A, undergoes phosphorylation during S phase, resulting in an increase of its phosphatase activity. The phosphorylation of cdc25A is dependent on the activity of the cdc2-cyclin E kinase. Microinjection of anti-cdc25A antibodies into G1 cells blocks entry into S phase. These results indicate that the cdc25A phosphatase is required to enter S phase in human cells and suggest that this enzyme is part of an auto-amplification loop analogous to that described at the G2/M transition. We discuss the nature of the in vivo substrate of the cdc25A phosphatase in S phase and the possible implications for the regulation of S phase entry.     

cdc25+ encodes a protein phosphatase that dephosphorylates p34cdc2.

To determine how the human cdc25 gene product acts to regulate p34cdc2 at the G2 to M transition, we have overproduced the full-length protein (cdc25Hs) as well as several deletion mutants in bacteria as glutathione-S-transferase fusion proteins. The wild-type cdc25Hs gene product was synthesized as an 80-kDa fusion protein (p80GST-cdc25) and was judged to be functional by several criteria: recombinant p80GST-cdc25 induced meiotic maturation of Xenopus oocytes in the presence of cycloheximide; p80GST-cdc25 activated histone H1 kinase activity upon addition to extracts prepared from Xenopus oocytes; p80GST-cdc25 activated p34cdc2/cyclin B complexes (prematuration promoting factor) in immune complex kinase assays performed in vitro; p80GST-cdc25 stimulated the tyrosine dephosphorylation of p34cdc2/cyclin complexes isolated from Xenopus oocyte extracts as well as from overproducing insect cells; and p80GST-cdc25 hydrolyzed p-nitrophenylphosphate. In addition, deletion analysis defined a functional domain residing within the carboxy-terminus of the cdc25Hs protein. Taken together, these results suggest that the cdc25Hs protein is itself a phosphatase and that it may function directly in the tyrosine dephosphorylation and activation of p34cdc2 at the G2 to M transition.     

Regulation of Cdc25C by ERK-MAP kinases during the G2/M transition

Induction of G(2)/M phase transition in mitotic and meiotic cell cycles requires activation by phosphorylation of the protein phosphatase Cdc25. Although Cdc2/cyclin B and polo-like kinase (PLK) can phosphorylate and activate Cdc25 in vitro, phosphorylation by these two kinases is insufficient to account for Cdc25 activation during M phase induction. Here we demonstrate that p42 MAP kinase (MAPK), the Xenopus ortholog of ERK2, is a major Cdc25 phosphorylating kinase in extracts of M phase-arrested Xenopus eggs. In Xenopus oocytes, p42 MAPK interacts with hypophosphorylated Cdc25 before meiotic induction. During meiotic induction, p42 MAPK phosphorylates Cdc25 at T48, T138, and S205, increasing Cdc25's phosphatase activity. In a mammalian cell line, ERK1/2 interacts with Cdc25C in interphase and phosphorylates Cdc25C at T48 in mitosis. Inhibition of ERK activation partially inhibits T48 phosphorylation, Cdc25C activation, and mitotic induction. These findings demonstrate that ERK-MAP kinases are directly involved in activating Cdc25 during the G(2)/M transition.     

Dephosphorylation of cdc25-C by a type-2A protein phosphatase: specific regulation during the cell cycle in Xenopus egg extracts.

We have examined the roles of type-1 (PP-1) and type-2A (PP-2A) protein-serine/threonine phosphatases in the mechanism of activation of p34cdc2/cyclin B protein kinase in Xenopus egg extracts. p34cdc2/cyclin B is prematurely activated in the extracts by inhibition of PP-2A by okadaic acid but not by specific inhibition of PP-1 by inhibitor-2. Activation of the kinase can be blocked by addition of the purified catalytic subunit of PP-2A at a twofold excess over the activity in the extract. The catalytic subunit of PP-1 can also block kinase activation, but very high levels of activity are required. Activation of p34cdc2/cyclin B protein kinase requires dephosphorylation of p34cdc2 on Tyr15. This reaction is catalysed by cdc25-C phosphatase that is itself activated by phosphorylation. We show that, in interphase extracts, inhibition of PP-2A by okadaic acid completely blocks cdc25-C dephosphorylation, whereas inhibition of PP-1 by specific inhibitors has no effect. This indicates that a type-2A protein phosphatase negatively regulates p34cdc2/cyclin B protein kinase activation primarily by maintaining cdc25-C phosphatase in a dephosphorylated, low activity state. In extracts containing active p34cdc2/cyclin B protein kinase, dephosphorylation of cdc25-C is inhibited, whereas the activity of PP-2A (and PP-1) towards other substrates is unaffected. We propose that this specific inhibition of cdc25-C dephosphorylation is part of a positive feedback loop that also involves direct phosphorylation and activation of cdc25-C by p34cdc2/cyclin B. Dephosphorylation of cdc25-C is also inhibited when cyclin A-dependent protein kinase is active, and this may explain the potentiation of p34cdc2/cyclin B protein kinase activation by cyclin A. In extracts supplemented with nuclei, the block on p34cdc2/cyclin B activation by unreplicated DNA is abolished when PP-2A is inhibited or when stably phosphorylated cdc25-C is added, but not when PP-1 is specifically inhibited. This suggests that unreplicated DNA inhibits p34cdc2/cyclin B activation by maintaining cdc25-C in a low activity, dephosphorylated state, probably by keeping the activity of a type-2A protein phosphatase towards cdc25-C at a high level.     

Regulation of the cdc25 protein during the cell cycle in Xenopus extracts.

The cdc25 protein is a highly specific tyrosine phosphatase that triggers mitosis by dephosphorylating the cdc2 protein kinase. Using Xenopus extracts, we have found that the cdc25 protein is active at a low level throughout interphase. Near the onset of mitosis, the cdc25 protein undergoes a marked elevation in phosphatase activity that coincides with an extensive phosphorylation of the protein in its N-terminal region. In vitro dephosphorylation of this hyperphosphorylated form of cdc25 reduces its phosphatase activity back to the interphase level. Moreover, treatment of interphase Xenopus extracts with okadaic acid, a phosphatase inhibitor that accelerates the entry into mitosis, elicits both the premature hyperphosphorylation of cdc25 and the stimulation of its cdc2-specific tyrosine phosphatase activity. These experiments demonstrate the existence of a cdc25 regulatory system consisting of both a stimulatory kinase that phosphorylates a putative regulatory domain of the cdc25 protein and an inhibitory serine/threonine phosphatase that counteracts this kinase activity.     

Periodic changes in phosphorylation of the Xenopus cdc25 phosphatase regulate its activity.

The cdc25 tyrosine phosphatase is known to activate cdc2 kinase in the G2/M transition by dephosphorylation of tyrosine 15. To determine how entry into M-phase in eukaryotic cells is controlled, we have investigated the regulation of the cdc25 protein in Xenopus eggs and oocytes. Two closely related Xenopus cdc25 genes have been cloned and sequenced and specific antibodies generated. The cdc25 phosphatase activity oscillates in both meiotic and mitotic cell cycles, being low in interphase and high in M-phase. Increased activity of cdc25 at M-phase is accompanied by increased phosphorylation that retards electrophoretic mobility in gels from 76 to 92 kDa. Treatment of cdc25 with either phosphatase 1 or phosphatase 2A removes phosphate from cdc25, reverses the mobility shift, and decreases its ability to activate cdc2 kinase. Furthermore, the addition of okadaic acid to egg extracts arrested in S-phase by aphidicolin causes phosphorylation and activation of the cdc25 protein before cyclin B/cdc2 kinase activation. These results demonstrate that the activity of the cdc25 phosphatase at the G2/M transition is directly regulated through changes in its phosphorylation state.     

Parallel activation of the NIMA and p34cdc2 cell cycle-regulated protein kinases is required to initiate mitosis in A. nidulans

We show that in Aspergillus nidulans, p34cdc2 tyrosine dephosphorylation accompanies activation of p34cdc2 as an H1 kinase at mitosis. However, the nimA5 mutation arrests cells in G2 with p34cdc2 tyrosine dephosphorylated and fully active as an H1 kinase. Activation of NIMA is therefore not required for p34cdc2 activation. Furthermore, mutation of nimT, which encodes a protein with 50% similarity to fission yeast cdc25, causes a G2 arrest and prevents tyrosine dephosphorylation of p34cdc2 but does not prevent full activation of the NIMA protein kinase. Mitotic activation of p34cdc2 by tyrosine dephosphorylation is therefore not required for activation of NIMA. These data suggest that activation of either the p34cdc2 protein kinase or the NIMA protein kinase alone is not sufficient to initiate mitosis. Parallel activation of both cell cycle-regulated protein kinases is required to trigger mitosis.     

Multiple splicing variants of cdc25B regulate G2/M progression.

Progression through G2 phase into mitosis is regulated by the activation of the mitotic cyclin/cdk complexes, which are in turn activated cdc25B and cdc25C phosphatases. Here we report that alternate splicing produces at least five variants of cdc25B, although only cdc25B2 and cdc25B3 are detectable as proteins. Analysis of these two variants shows that cdc25B2 is expressed at lower levels relative to cdc25B3 in all cell lines tested, and the expression of both increased markedly during G2 and mitosis. Overexpression of the catalytically inactive version of either cdc25B variant produced a G2 arrest implicating both in regulating G2/M progression.     

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.     

Filamin A stimulates cdc25C function and promotes entry into mitosis

The activity of the dual specificity phosphatase cdc25C is required for mitotic progression though the mechanisms by which cdc25C is activated prior to mitosis in human cells remain unclear. The data presented herein show that the actin binding protein Filamin A forms a complex with cdc25C in vivo and binds preferentially to the mitotic form of cdc25C. Co-expression of Filamin A with cdc25C results in an increase in PCC induced by cdc25C, while knocking down Filamin A expression reduces the levels of PCC induced by cdc25C overexpression. Further, only a Filamin A fragment that forms a complex with both cdc25C and cyclin B1 and retains the dimerization domain can stimulate the ability of cdc25C to induce PCC. These results suggest that Filamin A provides a platform for the assembly of the cyclin B1-cdk1- cdc25C complex resulting in cdk1 activation and mitotic progression.     

Distinct Pools of cdc25C Are Phosphorylated on Specific TP Sites and Differentially Localized in Human Mitotic Cells

Background

The dual specificity phosphatase cdc25C was the first human cdc25 family member found to be essential in the activation of cdk1/cyclin B1 that takes place at the entry into mitosis. Human cdc25C is phosphorylated on Proline-dependent SP and TP sites when it becomes active at mitosis and the prevalent model is that this phosphorylation/activation of cdc25C would be part of an amplification loop with cdk1/cyclin B1.

Methodology/Principal Findings

Using highly specific antibodies directed against cdc25C phospho-epitopes, pT67 and pT130, we show here that these two phospho-forms of cdc25C represent distinct pools with differential localization during human mitosis. Phosphorylation on T67 occurs from prophase and the cdc25C-pT67 phospho-isoform closely localizes with condensed chromosomes throughout mitosis. The phospho-T130 form of cdc25C arises in late G2 and associates predominantly with centrosomes from prophase to anaphase B where it colocalizes with Plk1. As shown by immunoprecipitation of each isoform, these two phospho-forms are not simultaneously phosphorylated on the other mitotic TP sites or associated with one another. Phospho-T67 cdc25C co-precipitates with MPM2-reactive proteins while pT130-cdc25C is associated with Plk1. Interaction and colocalization of phosphoT130-cdc25C with Plk1 demonstrate in living cells, that the sequence around pT130 acts as a true Polo Box Domain (PBD) binding site as previously identified from in vitro peptide screening studies. Overexpression of non-phosphorylatable alanine mutant forms for each isoform, but not wild type cdc25C, strongly impairs mitotic progression showing the functional requirement for each site-specific phosphorylation of cdc25C at mitosis.

Conclusions/Significance

These results show for the first time that in human mitosis, distinct phospho-isoforms of cdc25C exist with different localizations and interacting partners, thus implying that the long-standing model of a cdc25C/cdk1 multi-site auto amplification loop is implausible.     

Purification and biochemical analysis of catalytically active human cdc25C dual specificity phosphatase

We describe a reliable and efficient method for the purification of catalytically active and mutant inactive full-length forms of the human dual specificity phosphatase cdc25C from bacteria. The protocol involves isolating insoluble cdc25C protein in inclusion bodies, solubilization in guanidine HCL, and renaturation through rapid dilution into low salt buffer. After binding renatured proteins to an ion exchange resin, cdc25C elutes in two peaks at 350 and 450 mM NaCl. Analysis by gel exclusion chromatography and enzymatic assays reveals the highest phosphatase activity is associated with the 350 mM NaCl with little or no activity present in the 450 mM peak. Furthermore, active cdc25C has a native molecular mass of 220 kDa consistent with a potential tetrameric complex of the 55-kDa cdc25C protein. Assaying phosphatase activity against artificial substrates pNPP and 3-OMFP reveals a 220 kDa form of the phosphatase is active in a non-phosphorylated state. The protein effectively activates cdk1/cyclin B prokinase complexes in vitro in the absence of cdk1 kinase activity in an orthovanadate sensitive manner but is inactivated by A-kinase phosphorylation. In vitro phosphorylation of purified cdc25C by cdk1/cyclin B1, cdk2/cyclin A2 and cdk2/cyclin E shows that distinct TP/SP mitotic phosphorylation sites on cdc25C are differentially phosphorylated by these 3 cdk/cyclin complexes associated with different levels of cdc25C activation. Finally, we show that endogenous native cdc25C from human cells is present in high molecular weight complexes with other proteins and resolves mostly above 200-kDa. These data show that untagged cdc25C can be purified with a simple protocol as an active dual specificity phosphatase with a native molecular mass consistent with a homo-tetrameric configuration.     

Specific activation of cdc25 tyrosine phosphatases by B-type cyclins: evidence for multiple roles of mitotic cyclins.

Two previously unidentified human cdc25 genes have been isolated, cdc25A and cdc25B. Both genes rescue a cdc25ts mutant of fission yeast. Microinjection of anti-cdc25A antibodies into HeLa cells causes their arrest in mitosis. cdc25A and cdc25B display endogenous tyrosine phosphatase activity that is stimulated several-fold, in the absence of cdc2, by stoichiometric addition of either cyclin B1 or B2 but not A or D1. Association between cdc25A and cyclin B1/cdc2 was detected in the HeLa cells. These findings indicate that B-type cyclins are multifunctional proteins that not only act as M phase regulatory subunits of the cdc2 protein kinase, but also activate the cdc25 tyrosine phosphatase, of which cdc2 is the physiological substrate. A region of amino acid similarity between cyclins and tyrosine PTPases has been detected. This region is absent in cdc25 phosphatases. The motif may represent an activating domain that has to be provided to cdc25 by intermolecular interaction with cyclin B.     

SAPK/JNK regulates cdc2/cyclin B kinase through phosphorylation and inhibition of cdc25c

Cells undergo M phase arrest in response to stresses like UV irradiation or DNA damage. Stress-activated protein kinase (SAPK, also known as c-Jun N-terminal kinase, JNK) is activated by such stress stimuli. We addressed the potential effects of SAPK activation on cell cycle regulatory proteins. Activation of SAPK strongly correlated with inhibition of cdc2/cyclin B kinase, an important regulator of G2/M phase. SAPK directly phosphorylated the cdc2 regulator, cdc25c, in vitro on serine 168 (S168). This residue was highly phosphorylated in vivo in response to stress stimuli. cdc25c phosphorylated on S168 in cells lacks phosphatase activity, and expression of a S168A mutant of cdc25c reversed the inhibition of cdc2/cyclin B kinase activity by cell stress. Antibodies directed against phosphorylated S168 detect increased phosphorylation of S168 after cell stress. We conclude that SAPK regulates cdc2/cyclin B kinase following stress events by a novel mechanism involving inhibitory phosphorylation of the cdc2-activating phosphatase cdc25c on S168.