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.     

Cell cycle regulation of the p34cdc2 inhibitory kinases.

In cells of higher eukaryotic organisms the activity of the p34cdc2/cyclin B complex is inhibited by phosphorylation of p34cdc2 at two sites within its amino-terminus (threonine 14 and tyrosine 15). In this study, the cell cycle regulation of the kinases responsible for phosphorylating p34cdc2 on Thr14 and Tyr15 was examined in extracts prepared from both HeLa cells and Xenopus eggs. Both Thr14- and Tyr15- specific kinase activities were regulated in a cell cycle-dependent manner. The kinase activities were high throughout interphase and diminished coincident with entry of cells into mitosis. In HeLa cells delayed in G2 by the DNA-binding dye Hoechst 33342, Thr14- and Tyr15-specific kinase activities remained high, suggesting that a decrease in Thr14- and Tyr15- kinase activities may be required for entry of cells into mitosis. Similar cell cycle regulation was observed for the Thr14/Tyr15 kinase(s) in Xenopus egg extracts. These results indicate that activation of CDC2 and entry of cells into mitosis is not triggered solely by activation of the Cdc25 phosphatase but by the balance between Thr14/Tyr15 kinase and phosphatase activities. Finally, we have detected two activities capable of phosphorylating p34cdc2 on Thr14 and/or Tyr15 in interphase extracts prepared from Xenopus eggs. An activity capable of phosphorylating Tyr15 remained soluble after ultracentrifugation of interphase extracts whereas a second activity capable of phosphorylating both Thr14 and Tyr15 pelleted. The pelleted fraction contained activities that were detergent extractable and that phosphorylated p34cdc2 on both Thr14 and Tyr15. The Thr14- and Tyr15-specific kinase activities co-purified through three successive chromatographic steps indicating the presence of a dual-specificity protein kinase capable of acting on p34cdc2.     

Fission yeast p13 blocks mitotic activation and tyrosine dephosphorylation of the Xenopus cdc2 protein kinase

It has been demonstrated that the Xenopus homolog of the fission yeast cdc2 protein is a component of M phase promoting factor (MPF). We show that the Xenopus cdc2 protein is phosphorylated on tyrosine in vivo, and that this tyrosine phosphorylation varies markedly with the stage of the cell cycle. Tyrosine phosphorylation is high during interphase (in Xenopus oocytes and activated eggs) but absent during M phase (in unfertilized eggs). In vitro activation of pre-MPF from Xenopus oocytes results in tyrosine dephosphorylation of the cdc2 protein and switching-on of its kinase activity. The product of the fission yeast suc1 gene (p13), which inhibits the entry into mitosis in Xenopus extracts, completely blocks tyrosine dephosphorylation and kinase activation. However, p13 has no effect on the activated form of the cdc2 kinase. These findings suggest that p13 controls the activation of the cdc2 kinase, and that tyrosine dephosphorylation is an important step in this process.     

A cdc2-related kinase oscillates in the cell cycle independently of cyclins G2/M and cdc2.

The Eg1 gene in Xenopus laevis is related in sequence to the cdc2+ gene. We show here that the Eg1 gene product (cdk2) possesses histone H1 protein kinase activity and binds to PSTAIR antibodies as well as to Sepharose beads linked to the 13-kDa product of the suc 1 gene (p13suc1). Eg1 protein kinase is active only in an Mr approximately 200,000 complex with other proteins but is not associated with any of the three known Xenopus mitotic cyclins or with any newly synthesized protein in egg extracts that exhibit cell cycle oscillations in vitro. The protein kinase activity of Eg1 oscillates in the mitotic cell cycle, being high in M-phase and low in interphase. Hyperactivation of cdc2 kinase by the addition of cyclin A has no effect on the activity or oscillatory behavior of Eg1. Inhibition of cdc2 kinase activation by emetine or RNase treatment of oscillating extracts does not inhibit the activation of Eg1 but does block deactivation normally seen during exit from mitosis. These results indicate that Eg1 is regulated by a cell cycle clock independently of cyclin and cdc2 kinase.     

Mitotic phosphorylation of the dynein light intermediate chain is mediated by cdc2 kinase

Cytoplasmic dynein, a large minus-end-directed microtubule motor, performs multiple functions during the cell cycle. In interphase, dynein moves membrane organelles, while in mitosis it moves chromosomes and helps to form the mitotic spindle. The cell-cycle regulation of dynein activity may be controlled, at least in part, by the phosphorylation of its light intermediate chains (DLIC), since a 10-fold increase in light intermediate chain phosphorylation correlates with a decrease in dynein-based membrane transport of similar magnitude in mitosis. In this study, we sought to identify the kinase responsible for this potentially important phosphorylation event. We show that bacterially-expressed chicken light intermediate chain (chDLIC) will undergo mitosis-specific phosphorylation when added to Xenopus egg extracts. Mutation of a conserved cdc2 kinase consensus site (Ser197) abolishes this phosphorylation event, and mass spectroscopy analysis confirms that the wild-type DLIC is stoichiometrically phosphorylated at this site when incubated with metaphase but not interphase extracts. We also show that purified cdc2 kinase phosphorylates purified DLICs at Ser197 in vitro and that Ser197 phosphorylation is dramatically reduced in metaphase extracts depleted of cdc2 kinase. These results indicate that cdc2 kinase directly phosphorylates dynein and thus may be an important regulator of dynein activity in the cell cycle.     

Cdc2 H1 kinase is negatively regulated by a type 2A phosphatase in the Xenopus early embryonic cell cycle: evidence from the effects of okadaic acid.

In Xenopus embryos, the cell cycle is abbreviated to a rapid alternation between interphase and mitosis. The onset of each M phase is induced by the periodic activation of the cdc2 kinase which is triggered by a threshold level of cyclins and apparently involves dephosphorylation of p34cdc2. We have prepared post-ribosomal supernatants from eggs sampled during interphase (interphase extracts) and just before the first mitosis of the early embryonic cell cycle (prophase extracts). In 'interphase extracts', the cdc2 kinase never activates spontaneously upon incubation at room temperature whereas in 'prophase extracts' it does. We show here that in 'interphase extracts', specific inhibition of type 2A phosphatase by okadaic acid induces cdc2 kinase activation. This requires a subthreshold level of cyclin and the presence of a particulate factor in the extract. Inhibition of type 1 phosphatases by inhibitor 1 and inhibitor 2 never results in cdc2 kinase activation. These results demonstrate that during the period of cyclin accumulation, cdc2 kinase activation is inhibited by a type 2A phosphatase. In 'prophase extracts', spontaneous activation of the cdc2 kinase is inhibited by beta-glycerophosphate and NaF, but not by okadaic acid, inhibitor 1 and inhibitor 2 or divalent cation chelation. This demonstrates that when enough cyclin has accumulated, cdc2 kinase activation involves a protein phosphatase which must be distinct from the type 1 and 2A phosphatases, and from the calcium-dependent (type 2B) and magnesium-dependent (type 2C) phosphatases.     

An okadaic acid-sensitive phosphatase negatively controls the cyclin degradation pathway in amphibian eggs.

Inhibition of okadaic acid-sensitive phosphatases released the cyclin degradation pathway from its inhibited state in extracts prepared from unfertilized Xenopus eggs arrested at the second meiotic metaphase. It also switched on cyclin protease activity in a permanent fashion in interphase extracts prepared from activated eggs. Even after cdc2 kinase inactivation, microinjection of okadaic acid-treated interphase extracts pushed G2-arrested recipient oocytes into the M phase, suggesting that the phosphatase inhibitor stabilizes the activity of an unidentified factor which shares in common with cdc2 kinase the maturation-promoting factor 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.     

In vitro microtubule-nucleating activity of spindle pole bodies in fission yeast Schizosaccharomyces pombe: cell cycle-dependent activation in xenopus cell-free extracts

The spindle pole body (SPB) is the equivalent of the centrosome in fission yeast. In vivo it nucleates microtubules (MTs) during mitosis, but, unlike animal centrosomes, does not act as a microtubule organizing center (MTOC) during interphase. We have studied the MT-nucleating activity of SPBs in vitro and have found that SPBs in permeabilized cells retain in vivo characteristics. SPBs in cells permeabilized during mitosis can nucleate MTs, and are recognized by two antibodies: anti-gamma-tubulin and MPM-2 which recognizes phosphoepitopes. SPBs in cells permeabilized during interphase cannot nucleate MTs and are only recognized by anti-gamma-tubulin. Interphase SPBs which cannot nucleate can be converted to a nucleation competent state by incubation in cytostatic factor (CSF)-arrested Xenopus egg extracts. After incubation, they are recognized by MPM-2, and can nucleate MTs. The conversion does not occur in Xenopus interphase extract, but occurs in Xenopus interphase extract driven into mitosis by preincubation with exogenous cyclin B. The conversion is ATP dependent and inhibited by protein kinase inhibitors and alkaline phosphatase. Purified, active, cdc2 kinase/cyclin B complex in itself is not effective for activation of MT nucleation, although some interphase SPBs are now stained with MPM-2. These results suggest that the ability of SPBs in vitro to nucleate MTs after exposure to CSF-arrested extracts is activated through a downstream pathway which is regulated by cdc2 kinase.     

Protein kinase A regulates caspase-9 activation by Apaf-1 downstream of cytochrome c

The cyclic AMP signal transduction pathway modulates apoptosis in diverse cell types, although the mechanism is poorly understood. A critical component of the intrinsic apoptotic pathway is caspase-9, which is activated by Apaf-1 in the apoptosome, a large complex assembled in response to release of cytochrome c from mitochondria. Caspase-9 cleaves and activates effector caspases, predominantly caspase-3, resulting in the demise of the cell. Here we identified a distinct mechanism by which cyclic AMP regulates this apoptotic pathway through activation of protein kinase A. We show that protein kinase A inhibits activation of caspase-9 and caspase-3 downstream of cytochrome c in Xenopus egg extracts and in a human cell-free system. Protein kinase A directly phosphorylates human caspase-9 at serines 99, 183, and 195. However, mutational analysis demonstrated that phosphorylation at these sites is not required for the inhibitory effect of protein kinase A on caspase-9 activation. Importantly, protein kinase A inhibits cytochrome c-dependent recruitment of procaspase-9 to Apaf-1 but not activation of caspase-9 by a constitutively activated form of Apaf-1. These data indicate that extracellular signals that elevate cyclic AMP and activate protein kinase A may suppress apoptosis by inhibiting apoptosome formation downstream of cytochrome c release from mitochondria.     

Src kinase induces calcium release in Xenopus egg extracts via PLCgamma and IP3-dependent mechanism

Mobilization of intracellular calcium is an indispensable step of fertilization-induced egg activation. Recently, this process has been shown to require the sequential activation of Src family tyrosine kinases, phospholipase Cgamma (PLCgamma), and inositol-1,4,5-trisphosphate (IP3)-dependent receptor of endoplasmic reticulum. In the present study, we made an attempt to recapitulate the early events of egg activation by stimulating Src kinase activity in the cell-free extracts of Xenopus eggs. We found that enhanced Src kinase activity can initiate calcium response of low magnitude in cytostatic factor (CSF)-arrested mitotic extracts without releasing them into interphase. The addition of catalytically active recombinant Src kinase, as well as the activation of endogenous Xenopus Src family kinase by hydrogen peroxide (H2O2), increased total tyrosine phosphorylation, tyrosine phosphorylation of PLCgamma, and IP3 production in the extracts. The treatment with the Src family kinase-specific inhibitor, PP1, or PLC inhibitor, U73122, or IP3 receptor antagonist, heparin, prevented calcium release in the extracts. We conclude, therefore, that possible mechanism of Src/H2O2 action in the extracts might involve tyrosine phosphorylation and activation of PLCgamma, accompanied by the increase in IP3 content and subsequent calcium release from IP3-regulated calcium stores. These results also suggest that monitoring calcium signals induced in the Xenopus egg extracts by various components of signaling pathways may provide a particularly useful approach to investigating their role in the signal transduction.     

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.     

Coupling of mitosis to the completion of S phase in Xenopus occurs via modulation of the tyrosine kinase that phosphorylates p34cdc2.

In cell-free extracts derived from Xenopus eggs which oscillate between S phase and mitosis, incompletely replicated DNA blocks the activation of p34cdc2-cyclin by maintaining p34cdc2 in a tyrosine-phosphorylated form. We used a recombinant cyclin fusion protein to generate a substrate to measure the ability of the tyrosine kinase(s) to phosphorylate and inactivate p34cdc2 in the absence of tyrosine phosphatase activity. p34cdc2 tyrosine phosphorylation is highly regulated during the cell cycle, being elevated in S phase and attenuated in mitosis. The elevation in p34cdc2 tyrosine phosphorylation rate occurs in response to the presence of incompletely replicated DNA. Moreover, okadaic acid and caffeine, which uncouple the dependence of mitosis on the completion of S phase, increase unphosphorylated p34cdc2 by attenuating tyrosine kinase function. These data indicate that the control system, which monitors the state of DNA replication, modulates the function of the tyrosine kinase by a phosphorylation/dephosphorylation mechanism, ensuring that mitosis occurs only when S phase is complete.     

The Xenopus cdc2 protein is a component of MPF, a cytoplasmic regulator of mitosis

In Xenopus, a cytoplasmic agent known as MPF induces entry into mitosis. In fission yeast, genetic studies have shown that the cdc2 kinase regulates mitotic initiation. The 13 kd product of the suc1 gene interacts with the cdc2 kinase in yeast cells. We show that the yeast suc1 gene product (p13) is a potent inhibitor of MPF in cell-free extracts from Xenopus eggs. p13 appears to exert its antagonistic effect by binding directly to MPF. MPF activity is quantitatively depleted by chromatography on a p13 affinity column. Concomitantly, the Xenopus counterpart of the yeast cdc2 protein is adsorbed to the column. A 42 kd protein also binds specifically to the p13 affinity matrix. These findings suggest that the Xenopus cdc2 protein and the 42 kd protein are components of MPF.     

The c-mos proto-oncogene protein kinase turns on and maintains the activity of MAP kinase, but not MPF, in cell-free extracts of Xenopus oocytes and eggs.

During studies of the activation and inactivation of the cyclin B-p34cdc2 protein kinase (MPF) in cell-free extracts of Xenopus oocytes and eggs, we found that a bacterially expressed fusion protein between the Escherichia coli maltose-binding protein and the Xenopus c-mos protein kinase (malE-mos) activated a 42 kDa MAP kinase. The activation of MAP kinase on addition of malE-mos was consistent, whereas the activation of MPF was variable and failed to occur in some oocyte extracts in which cyclin A or okadaic acid activated both MPF and MAP kinase. In cases when MPF activation was transient, MAP kinase activity declined after MPF activity was lost, and MAP kinase, but not MPF, could be maintained at a high level by the presence of malE-mos. When intact oocytes were treated with progesterone, however, the activation of MPF and MAP kinase occurred simultaneously, in contrast to the behaviour of extracts. These observations suggest that one role of c-mos may be to maintain high MAP kinase activity in meiosis. They also imply that the activation of MPF and MAP kinase in vivo are synchronous events that normally rely on an agent that has still to be identified.     

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.     

cdc25 is one of the MPM-2 antigens involved in the activation of maturation-promoting factor.

MPM-2 antigens, a discrete set of phosphoproteins that contain similar phosphoepitopes recognized by the monoclonal antibody MPM-2, are phosphorylated during M-phase induction. Our previous studies suggested that certain MPM-2 antigens are involved in the appearance of maturation-promoting factor (MPF) activity. Because the central mitotic regulator cdc2 kinase has been shown to exhibit MPF activity, we explored the possibility that certain MPM-2 antigens are regulators of cdc2 kinase. We found that MPM-2 binding of its antigens would inhibit the autoamplification of cdc2 kinase in Xenopus oocytes and interfere with cyclin-activation of cdc2 kinase in Xenopus interphase egg extract. Immunodepletion of MPM-2 antigens from cyclin-induced M-phase egg extract caused the inactivation of cdc2 kinase, which was accompanied by an inhibitory phosphorylation of p34cdc2 on Thr 14 and Tyr 15, indicating that at least one MPM-2 antigen is a positive regulator of p34cdc2 dephosphorylation. We then showed that cdc25 from M-phase arrested egg extract is an MPM-2 antigen. These results suggest that phosphorylation of the epitope recognized by MPM-2 may be a crucial event in the activation of cdc25 and that the kinase(s) that phosphorylates this MPM-2 epitope may be an important regulator of cdc2 kinase activation.     

Phosphorylation by cdc2-CyclinB1 kinase releases cytoplasmic dynein from membranes

Movement of various cargoes toward microtubule minus ends is driven by the microtubule motor cytoplasmic dynein (CD). Many cargoes are motile only during certain cell cycle phases, suggesting that CD function may be under cell cycle control. Phosphorylation of the CD light intermediate chain (DLIC) has been suggested to play a crucial role in modulating CD function during the Xenopus embryonic cell cycle, where CD-driven organelle movement is active in interphase but greatly reduced in metaphase. This down-regulation correlates with hyperphosphorylation of DLIC and release of CD from the membrane. Here we investigate the role of the key mitotic kinase, cdc2-cyclinB1, in this process. We show that DLIC within the native Xenopus CD complex is an excellent substrate for purified Xenopus cdc2-glutathione S-transferase (GST) cyclinB1 (cdc2-GSTcyclinB1) kinase. Mass spectrometry of native DLIC revealed that a conserved cdc2 site (Ser-197) previously implicated in the metaphase modulation of CD remains phosphorylated in interphase and so is unlikely to be the key regulatory site. We also demonstrate that incubating interphase membranes with cdc2-GSTcyclinB1 kinase results in substantial release of CD from the membrane. These data suggest that phosphorylation of DLIC by cdc2 kinase leads directly to the loss of membrane-associated CD and an inhibition of organelle movement.     

Cdc25 regulates the phosphorylation and activity of the Xenopus cdk2 protein kinase complex.

The Xenopus cdk2 gene encodes a 32-kDa protein kinase with sequence similarity to the 34-kDa product of the cdc2 gene. Previous studies have shown that the kinase activity of the protein product of the cdk2 gene oscillates in the Xenopus embryonic cell cycle with a high in M-phase and a low in interphase. In the present study cdk2 was found not to be associated with any newly synthesized proteins during the cell cycle, but the enzyme did undergo periodic changes in phosphorylation. Upon exit from metaphase, cdk2 became increasingly phosphorylated on both tyrosine and serine residues, and labeling on these residues increased progressively until entry into mitosis, when tyrosine residues were markedly dephosphorylated. Phosphopeptide mapping of cdk2 demonstrated the major sites of phosphorylation were in a phosphopeptide with a pI of 3.7 that contained both phosphoserine and phosphotyrosine. This phosphopeptide accumulated in egg extracts blocked in S-phase with aphidicolin and was not evident in cdc2 immunoprecipitated under the same conditions. Under the same conditions cdc2 was phosphorylated primarily on a phosphopeptide containing both phosphothreonine and phosphotyrosine residues, most likely threonine 14 and tyrosine 15. Affinity-purified human GST-cdc25 was able to dephosphorylate and activate cdk2 isolated from interphase cells. Phosphopeptide mapping demonstrated that the phosphate was specifically removed from the same phosphopeptide identified as the major in vivo site of phosphorylation. These results demonstrate that cdk2 is regulated in the cell cycle by phosphorylation and dephosphorylation on both serine and tyrosine residues. Moreover, the increased phosphorylation of cdk2 in aphidicolin-blocked extracts and the ability of cdc25 to mediate cdk2 dephosphorylation in vitro suggest the possibility that cdk2 is part of the mechanism ensuring mitosis is not initiated until completion of DNA replication. It also implies cdc25 may have other functions in addition to the regulation of cdc2 kinase activity.