Proc. Natl. Acad. Sci. USAVol. 93, pp. 8278-8283, August 1996Biochemistry

p34cdc2 kinase activity is maintained upon activation of thereplication checkpoint in Schizosaccharomycespombe

(cyclin-dependent kinase/DNA synthesis/Radl/cell cycle/checkpoint control)

KAREN E. KNUDSEN*, ERIK S. KNUDSENt, JEAN Y. J. WANGt, AND SURESH SUBRAMANI*t*Department of Biology, University of California at San Diego, La Jolla, CA 92093-0322; and tCenter for Molecular Genetics, University of California at SanDiego, La Jolla, CA 92093-0347

Communicated by S. J. Singer, University of California at San Diego, La Jolla, CA, April 30, 1996 (received for review February 22, 1996)

ABSTRACT All eukaryotes use feedback controls to orderand coordinate cell cycle events. In Schizosaccharomycespombe, several classes of checkpoint genes serve to ensure thatDNA replication is complete and free of error before the onsetof mitosis. Wild-type cells normally arrest upon inhibition ofDNA synthesis or in response to DNA damage, although theexact mechanisms controlling this arrest are unclear. Geneticevidence in fission yeast suggests that the dependence ofmitosis upon completion of DNA replication is linked to theregulation of the p34cdc2 cyclin-dependent kinase. It has beenhypothesized that inhibition of DNA synthesis triggers down-regulation of p34cdc2 kinase activity, although this has neverbeen shown biochemically. We analyzed the activity of p34cdc2in wild-type and checkpoint-defective cells treated with a DNAsynthesis inhibitor. Using standard in vitro assays we demon-strate that p34cdc2 kinase activity is maintained in wild-typecells arrested at the replication checkpoint. We also used anovel in vivo assay for p34cdc2 kinase activity, in which weexpressed a fragment of the human retinoblastoma tumorsuppressor protein in fission yeast. Phosphorylation of thisfragment of the human retinoblastoma tumor suppressorprotein is dependent on p34cdc2 kinase activity, and thisactivity is also maintained in cells arrested at the replicationcheckpoint. These data suggest that the mechanism for cell-cycle arrest in response to incomplete DNA synthesis is notdependent on the attenuation of p34cdc2 activity.

To maintain genomic stability, all cells must ensure that DNAis without error and that its replication is complete before theonset of cell division. Over the past several years, studies ineukaryotes have uncovered the existence of an elaboratescheme of checkpoint controls, whose main purpose is tomaintain precise ordering of cell-cycle events (1). Loss ofcheckpoint control can lead to chromosomal rearrangements,amplifications, and loss of genetic material. Such genomicinstability is often seen in cancerous cells and is tightlyassociated with uncontrolled growth (2).

Schizosaccharomyces pombe has been a popular model or-ganism for investigations related to checkpoint control (3, 4).Families of checkpoint genes that are involved in the DNAdamage checkpoint, the DNA replication checkpoint, or bothhave been cloned (3). The radl gene product is required forboth checkpoints in fission yeast, as null mutations of radlimpart sensitivity to gamma radiation and to DNA synthesisinhibitors (5, 6). Radl-defective cells are unable to senseand/or relay signals in response to such genotoxic insults, andproceed into mitosis with either DNA damage or 1N DNAcontent (5, 6). The precise function of the Radl protein isunknown, and the mechanism by which it communicates withcell-cycle machinery is unclear.

It has been hypothesized that radl and the family of check-point genes to which it belongs are responsible for sending asignal to the key mitotic cyclin-dependent kinase (cdk) infission yeast, p34cdc2 (3, 7), whose activation is required forentry into mitosis in eukaryotic cells (8, 9). This activation iscontrolled via several processes, including interaction withcyclin B, activation by phosphorylation on Thr-167, and de-phosphorylation on Tyr-15 (8, 9). Conversely, the activation ofp34cdc2 iS inhibited by phosphorylation of Tyr-15, and geneticdata in fission yeast suggest that inhibition of mitosis inresponse to incomplete DNA synthesis acts through phospho-rylation of this residue (3, 10). This proposal was the result ofseveral observations: (i) Overexpression of p8Ocdc25, the acti-vating phosphatase that removes inhibitory phosphates onTyr-15, leads to hydroxyurea (HU) sensitivity (10); (ii) Exog-enous expression of a mutant form of p34cdc2 in which Tyr-15is changed to phenylalanine advances cdc2-deficient cells intopremature mitosis (11); and (iii) cdc2-3w, a mutant no longercorrectly regulated by Tyr-15 phosphorylation, is partiallysensitive to HU, although it is several orders of magnitude lesssensitive than Radl-defective cells (10). Data supporting thistheory are not completely consistent, however, as mutation ofplO7weel, the kinase that inactivates p34cdc2 through Tyr-15phosphorylation, does not lead to HU sensitivity (10).

Since the inhibition of p34cdc2 activity upon activation of thereplication checkpoint had not been tested biochemically, weanalyzed the kinase activity of p34cdc2 in wild-type cells andcompared it to that of Radl-defective cells, which lack thischeckpoint. We demonstrate that, in fact, p34cdc2 kinaseactivity is maintained throughout the replication checkpoint,as measured via standard in vitro assays as well as a novel in vivoassay for p34cdc2 kinase activity in S. pombe.

MATERIALS AND METHODSCell Culture and Drug Treatment. Strains used were wild-

type 972 (h-), K2 (h- ura4-D18 leul-32), K25 (h- his3 leul-32ura4-D18 rad1::ura4+), K27(h+ cdc25-22 ade6-M216 leul -32ura4-D18), and K57 (h- cdc2-33 ura4-D18 leul-32 ade6-M216). S. pombe strains were grown at 30°C except wheretemperature-sensitivity of the mutant was a factor. For thesestrains, the permissive and the nonpermissive temperatureswere 25°C and 36°C, respectively. Cells were cultured in yeastextract with supplements for nonselective conditions andEdinburgh minimal medium lacking the appropriate aminoacid for selective conditions (12). Nitrogen starvation of cellswas carried out for 16 hr at 25°C in Edinburgh minimalmedium lacking nitrogen sources. Transformations were per-formed using the lithium acetate procedure (13).

Abbreviations: cdk, cyclin-dependent kinase; HU, hydroxyurea; RB,human retinoblastoma tumor suppressor protein; LP, large pocket ofRB.ITo whom reprint requests should be addressed at: Department ofBiology, Room 4314 Bonner Hall, University of California at SanDiego, La Jolla, CA, 92093-0322. email: ssubramani@ucsd.edu.

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement" inaccordance with 18 U.S.C. §1734 solely to indicate this fact.

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Treatment of cells with HU (Calbiochem) was with a finalconcentration of 20 mM for the times indicated. Treatment ofcells with benomyl was carried out as described (14).

Septation Analysis. Cells were fixed at specific timepointswith iodine stain, as described (5). Cells were then viewed withphase-contrast microscopy for septum formation, and at leasttwo fields of 200 cells were tallied for each data point. Theproportion of cells with intact septa were used to determinethe percentage of cells that had undergone mitosis.

Plasmids. Plasmid pCMV-LP (15) contains the codingregion for amino acids 379-928 of the human retinoblastomatumor suppressor protein (RB). The coding region for this"large pocket" of RB (LP) was excised using BamHI andcloned into the BamHI site of the S. pombe expression vectorpART1 (16), placing expression of LP under the control of theconstitutive adh promoter. The resulting plasmid, pKL22, wasused to transform strains K2 and K57 to leucine prototrophy.

Immunofluorescence. Cells were fixed at indicated timeswith methanol and processed for immunofluorescence as perMoreno et al. (12). The antibody used to detect p34cdc2 was amouse monoclonal aPSTAIRE, and rabbit antibody KBD 56was used to monitor p56cdcl3. Both were generous gifts fromKathleen Gould (Vanderbilt University). Secondary antibod-ies were fluorescein isothiocyanate-conjugated goat anti-mouse and goat anti-rabbit antibodies, respectively (JacksonImmunoResearch).

Protein Extraction. Approximately 50 ml of cells wereharvested and washed once in cold harvest buffer (1 mMNaN3/150mM NaCl/50mM NaF/10mM EDTA, pH 8.0). Fornative protein extraction, pellets were resuspended in 20 Al ofIPCDC2 buffer (10 mM Tris HCl, pH 7.5/5 mM EDTA, pH8.0/130 mM NaCl/1% Triton X-100/50 mM NaF/25 mMNaPPi/1 mM phenylmethylsulfonyl fluoride/i mMNa3VO4/10 ,tg of leupeptin per ml/15 mMp-nitrophenylphos-phate/60 mM f3-glycerophosphate/20 ,ug of aprotinin per ml)(17). For denatured extracts, pellets were resuspended in 20 ,tlof RIPA buffer (0.15 mM NaCl/0.05 mM Tris HCl, pH8.0/1% Nonidet P-40/0.5% sodium deoxycholate/0.1% SDS)supplemented with protease and phosphatase inhibitors. Pel-lets were subjected to glass bead lysis for 5 min at 4°C. Proteinswere recovered in 1 ml of the appropriate buffer and clarifiedat 13000 x g (4°C for 15 min).Kinase Assays and Immunoblots. To isolate p34cdc2 com-

plexes, 1.5 jig of pl3sucl-Sepharose (gift from John Newport,University of California at San Diego) was added to 100 ,ug ofnative extracts and incubated at 4°C with rotation for 2 hr.Beads were washed eight times (washes one and two withIPCDC2 buffer plus 500 mM NaCl; washes three and four withIPCDC2 plus 200 mM NaCl; washes five and six with IPCDC2;and washes seven and eight with kinase buffer) (17). Kinasebuffer consisted of 25 mM Tris HCl (pH 7.5), 10 mM MgCl2,and 1 mM DTT. After the last wash, the beads were resus-pended in 35 ,ul of kinase buffer. Kinase reactions began uponaddition of 150 ,M ATP, 1.5 jig of histone Hi, and 40 ,uCi of[-y-32P]ATP. Kinase reactions were carried out at room tem-perature for 10 min with shaking and stopped by addition of3x SDS/PAGE loading buffer. Aliquots were boiled andimmediately loaded onto a SDS/12% polyacrylamide gel.After electrophoresis and transfer to Immobilon polyvinyli-dene difluoride (PVDF; Millipore), incorporation of['y-32P]ATP into histone Hi was detected by autoradiography.Where indicated, kinase activity was quantitated using anIS1000 Digital Imaging System (Alpha Innotech, San Leandro,CA). Following autoradiography, the blots were probed forp34Cdc2 protein levels using the aPSTAIRE antibody. Goatanti-mouse horseradish peroxidase (Bio-Rad) at 1:2000 wasused for antibody visualization via enhanced chemilumines-cence (ECL; Amersham).For detection of the LP, denatured extracts of the strains

harboring pKL22 were used. After clarification, 100 ,ug of each

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FIG. 1. p34cdc2 histone Hi kinase activity remains high in HU-blocked cells. (A) Septation indices of wild-type (972) and Radl-defective cells (K25) released from nitrogen starvation at time 0(Upper) and treated with HU for the indicated times (Lower). (B)p34Cdc2-associated kinase activities for cells described in A. Cellsreleased from Gl arrest at time 0 (lanes 1 and 6) were treated with HUafter 0.5 hr (lanes 2 and 7). Aliquots from HU-treated cells wereanalyzed for p34cdc2 kinase activity at 2, 4, and 5.5 hr from release(lanes 3-5 and 8-10).

lysate was loaded into SDS/10% polyacrylamide gel. Follow-ing electrophoresis and transfer to nitrocellulose, blots wereprobed with either 851 (18) or G99-549 (PharMingen) anti-body at 1:1000. G99-549 recognizes only the unphospho-rylated forms of LP, whereas 851 recognizes all forms of LP.Protein A (for 851) or goat anti-mouse (for G99-549) conju-gated horseradish peroxidase (Bio-Rad) at 1:2000 was used forantibody detection.

RESULTSp34cdc2 Histone Hi Kinase Activity Is Not Affected upon

Activation ofReplication Checkpoint. Wild-type cells normally

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Proc. Natl. Acad. Sci. USA 93 (1996)

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FIG. 2. Comparison of histone Hi kinase activity at different cell-cycle stages. (A) Septation indices of wild-type and Radl-defective cellsreleased from nitrogen starvation at time 0 (Top), following HU addition (Middle), or benomyl addition (Bottom). HU was added to the cells 30min after release from nitrogen stravation, and benomyl was added 270 min postrelease. (B) p34Cdc2-associated kinase activities from experimentdescribed inA (lanes 1-10). Kinase activity of K27 strain (temperature-sensitive for p80cdc25) (lanes 11 and 12). (C) Quantitation of kinase activitiesat different cell-cycle stages. Activities during the benomyl block were similar in 972 and K25 cells and were arbitrarily set at 100% for each strain.

arrest in the presence of the DNA synthesis inhibitor HU,whereas Radl-defective strains proceed into a lethal, prema-ture mitosis (5, 6). Wild-type (972) and Radl-defective cells(K25) from midlogarithmic phase cultures were harvested,washed, and synchronized by nitrogen starvation for 16 hr (19).At time 0, the Gl population of cells was released fromnitrogen starvation into medium rich with yeast extract andsupplements. After 30 min, HU was added to half of eachculture. Aliquots were taken to monitor cell division for thenext 6 hr. The percentage of cells with visible septa (indicating

completion of mitosis) were counted for each timepoint (Fig.1A). Untreated cells entered the cell cycle in a synchronousfashion (Fig. 1A, Upper). As expected, 972 cells treated withHU failed to enter mitosis throughout the course of treatment,as seen by a septation index close to zero. Septation inHU-treated K25 cells increased, as these cells proceeded intomitosis unchecked (Fig. 1A, Lower). Previous studies haveshown that cell death correlates directly with this lack of arrest(5, 6). After addition of HU, cells were harvested at theintervals indicated and assayed for p34cdc2-associated histone

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Proc. Natl. Acad. Sci. USA 93 (1996) 8281

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a-cdc2 a-cdcl 3FIG. 3. p34cdc2 and p56cdcl3 localize to the nucleus in HU-arrested

cells. Wild-type cells blocked in HU for 5 hr were analyzed by indirectimmunofluorescence for the subcellular localization of p34cdc2 andp%6cdcl3

Hi kinase activity and p34cdc2 protein content (Fig. 1B). Therewas little p34Cdc2 kinase activity or p34cdc2 protein present inthe nitrogen-starved, Gl-arrested 972 and K25 cells (Fig. iB,lanes 1 and 6) (19, 20). Upon entry into the cell cycle (Fig. 1B,lanes 2 and 7), both protein content and histone Hi kinaseactivity increased in 972 and K25 cells. As expected, p34cdc2kinase activity remained high throughout treatment of the K25strain with HU, as these cells continued into mitosis (Fig. 1B,lanes 8-10). However, kinase activity also remained highthroughout the HU-induced arrest in wild-type cells (Fig. 1B,lanes 3-5). This same phenomenon was observed in asynchro-nous populations of wild-type cells blocked with HU (data notshown). These results suggest that arrest in response to inhi-bition of DNA synthesis is not dependent on down-regulationof p34cdc2 kinase activity.To more accurately assess the degree of kinase activity seen

in HU-treated cells, kinase activity was compared with that ofcells blocked in mitosis. Both 972 and K25 cells arrestedtransiently in response to benomyl in metaphase, at which timep34Cdc2 kinase activity is considered to be maximal (14, 21).Cells were blocked in Gl via nitrogen starvation (time 0) andreleased synchronously into the cell cycle (Fig. 2A, Top). After30 min, HU was added to a fraction of the cells, and thetreatment continued for an additional 330 min. Throughoutthe HU treatment, 972 cells arrested, whereas K25 cells did not(Fig. 2A, Middle). Benomyl was added to cycling cells 270 minafter release and the block continued for 90 min. Both 972 andK25 cells transiently arrested in response to this drug (Fig. 2A,Bottom). Aliquots were taken from nitrogen-starved cells(time 0), HU-treated cells (time 180), benomyl-blocked cells(time 360), or untreated cells from the analogous time points.Again we observed that kinase activity remained high in bothwild-type (S phase-blocked) (Fig. 2B, lane 3) and Radl-defective (cycling) cells (Fig. 2B, lane 7); these levels wereunchanged relative to those observed in the absence of HU(Fig. 2B, compare lanes 2 and 3 and lanes 7 and 8). Aspredicted, kinase activity appeared highest in the metaphase-blocked cells (Fig. 2B, lanes 5 and 10). Specific kinase activitywas quantitated from two sets of experiments (Fig. 2C).Activity at the benomyl block was considered to be 100% (peakpossible kinase activity). Again kinase activity in HU-arrestedcells was comparable to that of cells continuing to advancethrough the cell cycle.To ensure that our in vitro kinase assay conditions accurately

reflected the state of p34CdC2 activity in vivo, we used a strain(K27) harboring a temperature-sensitive mutation of p8ocdc25.p80cdc25 is the phosphatase that activates p34CdC2 (22). At thepermissive temperature, p34cdc2 kinase activity was high (Fig.2B, lane 11), whereas 3 hr after the shift to the restrictivetemperature, there was no detectable p34cdc2-associated kinaseactivity (Fig. 2B, lane 12). Identical results were seen using

-p aG99-549

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pLPLP __

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1 2FIG. 4. LP phosphorylation is dependent on p34cdc2 kinase activity

in S. pombe. (A) Immunoblot for the LP in wild-type strain K2 carryingthe parent plasmid (pART1) (lane 1) or the LP-expressing plasmidpKL22 (lane 2). a851 antibodies that detect all forms of LP (Upper)or aG99-549 antibodies, which detect only the unphosphorylated form(Lower), were used. pLP, phosphorylated form of LP; LP, unphos-phorylated form of LP. (B) Phosphorylation pattern of LP in the K57(temperature-sensitive for cdc2) strain. Phosphorylation of LP at 25°C(lane 1) and 36°C (lane 2) were monitored by immunoblotting withantibodies a851 (Upper) and aG99-549 (Lower).

strain K57 (cdc2 temperature-sensitive strain) (data notshown). In addition, we used antisera against either p34Cdc2 orp56cdc13 to isolate complexes released synchronously into thecell cycle in the presence or absence ofHU. Histone Hi kinaseactivity seen in these experiments was indistinguishable fromthat seen upon isolation of p34cdc2 using pl3sucl-Sepharose(data not shown).

p34cdc2 and p56cf1t3 Remain in the Nucleus in the Presenceof HU. Immunofluorescence was used to determine whetherp34CdC2 and its cyclin partner (p56cdcl3) were localized to thenucleus, as would be expected for active p34cdc2/p56cdcl3complexes. In nitrogen-starved 972, no signals were detectedfor p34cdc2 or p56cdcl3 (data not shown). In HU-arrested 972cells, however, both p34cdc2 and p56cdcl3 localized to thenucleus in all of the cells examined (Fig. 3). Similar results wereobserved for K25 cells (data not shown).

p34cdc2 Kinase Activity is Unchanged in Vivo in the PresenceofHU. Recent research in higher eukaryotes has identified cdk

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1 2 3FIG. 5. LP phosphorylation in HU-blocked cells. Phosphorylation

of LP in wild-type strains arrested in Gl (lane 1), released for 4 hr in

the absence (lane 2) or presence (lane 3) of HU.

inhibitors that act to modify cdk activity in response to external

cues (1,23). We wished to establish an in vivo assay inS. pombe

for p34cdc2 kinase activity to eliminate the possibility that an

inhibitor of p34cdc2 was displaced by the in vitro assay. It has

been shown in Saccharomyces cerevisiae that human RB can be

exogenously expressed, and its phosphorylation is strictly

dependent on the activity of p34cdc28 (24). We cloned the DNA

encoding the LP region of RB under the adh promoter in a S.

pombe expression vector. LP contains 10 of the 16 cdk-

consensus phosphorylation sites present in full-length RB (25).

As was the case in Saccharomyces cerevisiae, no overt effect on

cell growth or morphology was seen as a result of LP expres-

sion in S. pombe (data not shown). LP could be specifically

detected in strains transformed with pKL22 (Fig. 4A, lane 2),

whereas cells harboring only the parental plasmid (pART1)

exhibited no reactivity in immunoblots (Fig. 4A, lane 1). The

protein migrated as a series of bands that could be visualized

when probed with an antibody that detects all forms of RB

(Fig. 4A, Upper), consistent with the behavior of this protein

in mammalian cells (25). Using aG99-549 antibody, which

detects only unphosphorylated LP, only a single band was

detected (LP) (Fig. 4A, Lower). These results show that the

mobility shift of LP seen using the a851 antibody (Fig. 4A,

Upper) is indeed due to phosphorylation of LP.

To derhonstrate that the phosphorylation of LP was cata-

lyzed by p34cdc2, we used a temperature-sensitive

expressing LP. After shift of strain K57 (containing the cdc2-33

mutation) to the restrictive temperature for 5 hr, LP was

present only in its unphosphorylated form (Fig. 4B, lanes 1

versus 2). This demonstrates that as in budding yeast, LP

phosphorylation in vivo is dependent on p34`cc2 kinase activity.

Wild-type cells expressing LP were then used to monitor in

vivo p34cdc2 kinase activity in response to HU. K2 cells

expressing LP were synchronized and released, followed by

treatment of a fraction of the cells with HU. Cells blocked in

Gi contained only the unphosphorylated form of LP (Fig. 5,

lane 1). This is expected, as p34cdc2 kinase activity monitored

in vitro was low in nitrogen-starved cells (Fig. 1, 2). Cells

harvested 5 hr after release into nitrogen-rich media contained

LP in all states of phosphorylation, similar to that seen in

asynchronously-growing cells (Fig. 5, lane 2). In extracts from

cells that were treated with HU 30 min after release and

harvested 4.5 hr later, septation indices remained close to zero

throughout the block (data not shown). The phosphorylation

state of LP at the HU block was comparable to that of

untreated cells (Fig. 5, lanes 2 and 3). This was also seen in

asynchronous wild-type cells expressing LP and blocked at the

replicationcheckpoint (data not shown). These data confirm

the in vitro results, which demonstrate that p34cdc2 kinase

activity remains high in HU-blocked cells.

DISCUSSION

We have shown that p34cdc2 kinase activity remains elevated

and unchanged in cells arrested at the replication checkpoint

when compared with cells in which the replication checkpointhas not been activated. Standard histone Hi kinase assays(Figs. 1 and 2) demonstrated that p34cdc2 kinase activity inwild-type cells is indistinguishable from that of checkpoint-defective cells when treated with a DNA synthesis inhibitor.We also used a novel in vivo assay for monitoring p34cdc2 kinaseactivity in S. pombe. As has been previously shown for Sac-charomyces cerevisiae, human RB protein (or fragmentsthereof) is phosphorylated in S. pombe, and this phosphory-lation is dependent on the activity of p34cdc2 (Fig. 4). Weobserved that LP is indeed phosphorylated in wild-type cellsblocked at the replication checkpoint (Fig. 5). This resultsupports our in vitro observations and leads us to conclude thatarrest in response to inhibition of DNA synthesis in S. pombeis not simply a function of down-regulating p34cdc2 kinaseactivity. We propose that arrest in response to blocked DNAreplication is more complicated than was originally suggestedby the genetic data.

Genetic evidence linking down-regulation of p34cdc2 kinaseactivity to the replication checkpoint could have been mis-leading due to the nature of the experiments performed;overexpression of p8ocdc25 or mutation of p34cdc2-Tyr15 mayhave overridden the effects of whatever normal brakes the celluses to prevent mitosis in the absence of DNA replication. Inconcluding this, we are left to speculate about how the cell isable to arrest despite high p34cdc2 activity. Although we haveshown that p34cdc2 kinase activity remains elevated in HU-arrested cells, it is possible that p34cdc2 is prevented access tokey substrates that it must phosphorylate for mitotic progres-sion. We have shown that p34cdc2 is generally in its properlocation for activation of mitotic substrates (Fig. 3), but thiscertainly does not exclude the possibility of subnuclear seques-tration of active p34cdc2. A similar possibility could be thatalthough p34cdc2 is active is our assays, it may be inactiveagainst at least one key mitotic substrate in vivo. Specificity forsubstrate is generally controlled via interaction with cyclins(23). p34cdc2 is known to interact with the p56cdcl3 B-type cyclinin S. pombe, and while this manuscript was in preparation,reports emerged describing p34cdc2 in active complexes withthe cig2 and cigl cyclins (26, 27). These reports suggest thatpS6cdcl3 drives mitosis, but can also serve as a Gl cyclin in theabsence of cigl and cig2, suggesting redundant functions forcyclins in S. pombe and indicating that different cyclins mayinteract with an overlapping set of substrates. It is importantto note that when p34cdc2 complexes were captured usingantibodies against p56cdc13 or p34cdc2, the level of histone Hikinase activity was not changed upon activation of the repli-cation checkpoint (see Results). Lastly, it is possible that whileactivep34cdc2 may have complete access to the requiredsubstrates, perhaps this is insufficient to drive a cell into mitosis.

Research in other organisms has failed to uncover a com-mon mechanism for arrest at the replication checkpoint. InXenopus laevis, it has been postulated that an unstable orinsoluble inhibitor of p34cdc2 may down-regulate p34cdc2 kinaseactivity in response to replication inhibition (28, 29). Thisarrest is known to be unrelated to inhibitory phosphorylationon Tyr-15 or Thr-14 (28, 29). Based on our experiments, it isunliklely that there exists an analogous inhibitor in S. pombe,as we were able to detect p34cdc2 kinase activity both in vitroand in vivo. In Saccharomyces cerevisiae, it has been shown thatp34cdc28 kinase activity remains high during the HU block,despite phosphorylation on Tyr-19 (analogous to Tyr-15 infission yeast) (30, 31). There is some evidence that activatedp34cdc28/cyclin complexes shift to a lower molecular weight inresponse to HU-arrest, indicating that an activator or targetingmolecule required for mitotic progression is lost from p34cdc28complexes (32). It is possible that a situation such as this maybe used in S. pombe and should be investigated. In mammaliancells, the presence of numerous cdks and cyclins complicatesthe assessment of cdk activities. It has been shown that

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Proc. Natl. Acad. Sci. USA 93 (1996) 8283

HU-blocked HeLa cells still exhibit high Cyclin A/Cdk2activity, which is required in cycling cells for progressionthrough S phase (33). However, expression of p34Cdc2 mutants,which are nonphosphorylatable on inhibitory sites, is dominantin these cells as well, leading to premature mitotic events (34).

In summary, we conclude that while p34Cdc2 may be involvedin the coupling of S phase to mitosis, cell-cycle arrest at thischeckpoint is not dependent on the down-regulation of p34cdc2kinase activity.

This work was supported by Grant GM31253 to S.S. E.S.K. issupported by National Institutes of Health Grant CA58320 to J.Y.J.W.KIE.K. is supported by National Institutes of Health Training GrantCA09345.

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