Loss of RCC1, a nuclear DNA-binding protein ... - Europe PMC

The EMBO Journal vol.10 no.6 pp.1555- 1564, 1991

Loss of RCC1, a nuclear DNA-binding protein, uncouples the completion of DNA replication from the activation of cdc2 protein kinase and mitosis Hideo Nishitani, Motoaki Ohtsubo, Katsumi Yamashita, Hiroshi lida, Jonathon Pines2, Hideyo Yasudo3, Yosaburo Shibata1, Tony Hunter2 and Takeharu Nishimoto4 Department of Molecular Biology, Graduate School of Medical Science and 'Department of Anatomy, School of Medicine, Kyushu University, Maidashi, Fukuoka, 812 Japan; 2Molecular Biology and Virology Laboratory, The Salk Institute for Biological Studies, PO Box 85800, San Diego, CA 92186, USA and 3Department of Pharnaceutical Science, Kanazawa University, Kanazawa 920, Japan 4Corresponding author Communicated by T.Hunt

The temperature-sensitive mutant cell line tsBN2, was derived from the BHK21 cell line and has a point mutation in the RCCI gene. In tsBN2 cells, the RCC1 protein disappeared after a shift to the non-permissive temperature at any time in the cell cycle. From S phase onwards, once RCC1 function was lost at the nonpermissive temperature, p34cdc2 was dephosphorylated and M-phase specific histone Hi kinase was activated. However, in G, phase, shifting to the non-permissive temperature did not activate p34cdc2 histone Hi kinase. The activation of p34cdc2 histone Hi kinase required protein synthesis in addition to the presence of a complex between p34cdc2 and cyclin B. Upon the loss of RCC1 in S phase of tsBN2 cells and the consequent p34cdc2 histone Hi kinase activation, a normal mitotic cycle is induced, including the formation of a mitotic spindle and subsequent reformation of the interphase-microtubule network. Exit from mitosis was accompanied by the disappearance of cyclin B, and a decrease in p34cdc2 histone Hi kinase activity. The kinetics of p34cdc2 histone Hi kinase activation correlated well with the appearance of premature mitotic cells and was not affected by the presence of a DNA synthesis inhibitor. Thus the normal inhibition of p34cdc2 activation by incompletely replicated DNA is abrogated by the loss of RCC1. Key words: cell cycle/PCC/RCC1/tsBN2/uncoupling

Introduction In the cell cycle, DNA oscillates between a condensed and decondensed state. After mitosis chromosomes are decondensed and express mRNAs required to initiate the cell cycle in GI phase (Prescott, 1976). In S phase, DNA is gradually condensed (Rao et al., 1976), and at the end of S phase, it is further condensed to enter mitosis. A major element involved in the entry into mitosis is M-phase promoting factor (MPF) which consists of two proteins; p34cdc2, a 34 kd protein-serine/threonine kinase homologous to the gene product of the cdc2 + gene in Oxford University Press

Saccharomyces pombe, and cyclin B, a 50-60 kd protein first identified in marine invertebrates (Evans et al., 1983; Simanis and Nurse, 1986; Arion et al., 1988; Dunphy et al., 1988; Lohka et al., 1988; Gautier et al., 1990). The activity of MPF is regulated by the phosphorylation state of p34cdc2 and the level of cyclin B through the cell cycle. In G1 phase there is very little cyclin B, p34cdc2 is unphosphorylated and no MPF activity is detectable (Lee et al., 1988; Morla et al., 1989). At the end of GI phase, phosphorylated p34cdc2 appears. The histone HI kinase activity of p34cdc2 increases sharply at the end of G2 phase (Meijer et al., 1989; Pines and Hunter, 1989; Dasso and Newport, 1990). Concomitant with the activation of p34cdc2 histone HI kinase, p34cdc2 is dephosphorylated (Dunphy and Newport, 1989; Gould and Nurse, 1989). During the transition from metaphase to anaphase, cyclin B is degraded and the activity of p34cdc2 histone Hi kinase decreases (reviewed in Nurse, 1990). Before the initiation of mitosis, genomic DNA must be completely replicated for chromosome separation to occur successfully. To ensure this, cells possess a regulatory mechanism that prevents the initiation of mitosis until the completion of DNA replication (Hartwell and Weinert, 1989). This regulatory mechanism can be overcome by drugs or mutations, such that chromosome condensation is induced before the completion of DNA synthesis. For example, caffeine induces premature chromosome condensation (PCC) in BHK21 cells and in the Xenopus egg cell-free system when DNA replication is blocked with hydroxyurea or aphidicolin (Schlegel and Pardee, 1986; Dasso and Newport, 1990). A similar uncoupling of mitosis from DNA replication has been reported for genetically defined mutants such as the bimE7 mutant in Aspergillus nidulans (Osmani et al., 1988), some wee mutants of fission yeast (Enoch and Nurse, 1990) and in tsBN2 cells (Nishimoto, 1988). In all except tsBN2 cells, mitosis is initiated at the restrictive temperature only when DNA replication is prevented. In contrast, in tsBN2 cells PCC can occur in the absence of a DNA synthesis inhibitor (Nishimoto et al., 1981). The tsBN2 cell line was isolated as a DNA- mutant from nitrosoguanidine-treated BHK21 cells by FUdR selection (Nishimoto and Basilico, 1978; Nishimoto et al., 1978 and 1981). Concomitant with PCC, mitosis-specific phenomena, such as the appearance of MPM-2 antigens and the phosphorylation of histones HI and H3 are induced in this cell line at the restrictive temperature, 39.5 'C (Ajiro et al., 1983; Yamashita et al., 1985). The wild-type RCCI gene was cloned by complemention of the tsBN2 mutation (Kai et al., 1986; Ohtsubo et al., 1987), and proved to be mutated in tsBN2 cells (Uchida et al., 1990). The RCCJ gene encodes a 45 kd protein, which has DNA binding activity and is located in the nucleus (Ohtsubo et al., 1989). In this paper we show that in S phase a 'normal' mitotic 1555

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cycle, starting from the activation of p34cdc2 histone HI kinase, is induced in tsBN2 cells by a temperature shift without the addition of DNA synthesis inhibitors, and this correlates with the disappearance of the RCC 1 protein. The activation of p34cdc2 histone HI kinase by the loss of RCC1 function appears to require one or more newly synthesized protein(s) in addition to cyclin B and p34cdc2.

Results

Fig. 1. Appearance of 'rounded up' cells. Cultures of tsBN2 cells synchronized at the GI/S boundary were incubated at 39.5'C. Every 2 h, cells were fixed with 3% paraformaldehyde and then stained with Hoechst 33258. (A) Phase contrast photographs. (B) Hoechst staining of the same field as A. The number on the left side indicates the time (h) after temperature shift to 39.50C.

Transient induction of mitotic events in tsBN2 cells by temperature shift When cultures of tsBN2 cells, synchronized at the GI/S boundary with hydroxyurea, were incubated at the restrictive temperature, 39.5°C, 'rounded up' cells with condensed chromosomes appeared after a 2 h incubation (Figure 1). After 4 h these cells began to disappear, and cells with micronuclei and flat morphology appeared. These results suggested that tsBN2 cells had entered and exited from M phase at the restrictive temperature. Consistent with this interpretation, the 'rounded up' cells which had condensed chromosomes also appeared to have a normal mitotic spindle when stained with anti-f-tubulin antibodies (Figure 2B). Mitotic spindles extended from both sides of the cell, which suggested that the centrosomes duplicated while DNA replication was blocked, and that they divided properly at the onset of mitosis (McIntosh and Koonce, 1989). After a 6 h incubation, interphase microtubule arrays reformed and cells contained micronuclei with a nuclear membrane, nuclear pores and nuclear lamina (Figure 2C -I). In these cells, some diffuse lamin staining was still observed with

Fig. 2. Mitotic spindle formation and reformation of the nuclear membrane. In cultures of the tsBN2 cells shown in Figure 1, mitotically 'rounded up' cells (A and B) or flattened cells with micronuclei (C-H) were fixed and stained with Hoechst 33258 (A, D and G), with anti-B-tubulin (B and E), or anti-lamin (H) antibodies. Phase contrast photographs were also taken (C and F). (A, B), (C, D, E) and (F, G, H) show the fields respectively. Reformed micronuclei were investigated by electron microscopy (I). A to H are at the same magnification. The bar in H and I represents 20 /m and 1 zm, respectively. same

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the anti-lamin antibody (Figure 2H). Furthermore, antigens recognized by the mitosis-specific monoclonal antibody MPM-2 (Davis et al., 1983), which were observed in cells with condensed chromosomes, disappeared in cells with micronuclei (Figure 7h). Taken together, these results suggest that after 6 h at 39.5°C many cells have apparently completed the prematurely-induced mitosis, and that therefore the tsBN2 mutation is not a mitosis-arrest mutant, unlike bimE7 in Aspergillus (Osmani et al., 1988). RCC1 protein of tsBN2 cells disappears at high temperature TsBN2 cells have a single mis-sense mutation in the RCCI gene, which can be complemented by the wild-type RCCI

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Fig. 3. Disappearance of RCC1 in tsBN2 cells upon temperature shift. (A) tsBN2 cells exponentially growing on coverslips were incubated at 33.5°C (left) and at 39.5°C (right) for 3 h, fixed and stained with the anti-Xenopus RCC1 antibody and rhodamine conjugated anti-rabbit IgG. (B) total protein extracted from tsBN2 and BHK21 cells incubated at 39.5'C for indicated time was electrophoresed and analyzed by immunoblotting with the anti-Xenopus RCC1 antibody using an alkaline phosphatase linked second antibody for detection. Only the position of RCC1 is shown. (C) Cultures of tsBN2 and BHK21 cells were labeled for 3 h with [35S]methionine at 33.5°C and then shifted to 39.5°C in fresh medium. As a control, one of the cultures of tsBN2 cells was kept at 33.5°C. RCC1 was immunoprecipitated with the anti-Xenopus RCC1 antibody and analyzed on a 10% SDS-polyacrylamide gel. M; 14C-labeled marker proteins. Lanes 1 -3; precipitates from BHK21 cells collected at 0, 1 and 3 h after temperature shift. Lanes 4-6; from tsBN2 cells collected at 0, 1 and 3 h after temperature shift. Lane 7; from tsBN2 cells kept at 33.5°C for 3 h. The upper arrow indicates 46 kd and the lower one indicates 45 kd RCC1 (B, C). Molecular weights are given in kd.

gene (Uchida et al., 1990). Based on the nature of this mutation, Uchida et al. (1990) suggested that the mutant RCC1 protein will have a profoundly different structure at the restrictive temperature. Therefore the behavior of RCC 1 was investigated in tsBN2 cells at 39.5'C, using an antibody prepared against Xenopus RCC 1, which recognizes RCC 1 in frog, hamster and human cells (Nishitani et al., 1990). When exponentially growing tsBN2 cells were shifted to 39.5°C, RCC 1 became undetectable by immunofluorescence (Figure 3A, right) and by immunoblotting analysis (Figure 3B). Both the 45 and 46 kd forms of RCC 1 in tsBN2 cells became undetectable after 1 h incubation at 39.5°C, whereas in the wild-type BHK21 cell line, the 45 and 46 kd bands were unchanged after 8 h incubation at 39.5°C. Based on the electrophoretic patterns of total cellular protein on SDS -PAGE, the overall protein composition of tsBN2 cells did not change significantly during incubation at 39.5°C for 8 h (data not shown). To confirm the disappearance of BN2-RCC 1, exponentially growing cultures of tsBN2 and BHK21 cells were labeled with [35S]methionine for 3 h at 33.5°C, then incubated in fresh medium at both 33.5°C and 39.5°C, and RCC 1 was analyzed by immunoprecipitation with anti-RCC 1 antibody (Figure 3C). The amount of RCC 1 did not change in BHK21 cells during a 3 h incubation at 39.50C. In contrast, in tsBN2 cells the amount of both the 45 and 46 kd forms of RCC1 was significantly reduced after 1 h

Fig. 4. Correlation between the loss of RCC 1 and activation of at the p34cdc2 histone HI kinase. Cultures of tsBN2 cells synchronized G1/S boundary with hydroxyurea were divided into 2 series and incubated in the presence of hydroxyurea (2.5 mM) at 39.5°C, either without (A), or with cycloheximide (10 Ag/ml) (B). At the indicated time, cells were lysed and subjected to the following analysis. The content of RCC1 was analyzed by immunoblotting with the antiXenopus RCC1 and [125I]protein A [p45(RCC1)]. The mobility of cdc2 protein was investigated with immunoblot using the antibody to the PSTAIR peptide of p34cdc2 and an alkaline phosphatase conjugated second antibody [p34(cdc2)]. Histone HI kinase activity of the immunoprecipitated p34cdc2 protein was assayed as described in Materials and methods [histone HI].

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activity of p34cdc2 and the mobility of p34Cdc2 on SDS-PAGE. After 1 h incubation at 39.5°C, more than 80% of RCC1, as estimated by densitometric scanning, had disappeared and the histone HI kinase activity of p34cdc2 had increased (Figure 4A). Consistent with previous reports, the increase in histone HI kinase activity was accompanied by a decrease in the amount of the upper, phosphorylated band of p34cdc2 and the appearance of the lower, dephosphorylated form (Dunphy and Newport, 1989; Gautier et al., 1989; Yamashita et al.,1990). Three hours

after the shift to the non-permissive temperature the histone HI kinase activity of p34Cdc2 was maximal and almost all

the p34cdc2 was dephosphorylated. The same kinetics of histone HI kinase activation was also observed in immunoprecipitates with anti-cyclin B antiserum

(data not shown).

Fig. 5. Presence of the p34Cdc2/ cyclin B complex in tsBN2 cells synchronized at the GI/S boundary. (A) Immunoblotting with the anticyclin B antibody. Cultures of tsBN2 cells were synchronized in ileumedium at GI and then at the GI/S boundary by incubating at 33.5°C in the presence of hydroxyurea. Cell lysates were prepared at the GI phase; after isoleucine deprivation (lane 1) and at the GI/S boundary; after hydroxyure treatment (lane 2) and then, every hour after the temperature shift for 6 h (lanes 3-8). These lysates were electrophoresed and immunoblotted with anti cyclin B antiserum. Only bands corresponding to cyclin B are shown.(B) Immunoprecipitation analysis of cyclin B and p34cdc2. Cultures of tsBN2 cells labeled with [35S]methionine for the last 3 h during synchronization at 33.5°C at the G1/S boundary were lysed. Lysates were immunoprecipitated either with the antibody to human cyclin B (lane 1) or with that to the Cterminus of human p34cdc2 (lane 2). Precipitates were electrophoresed on 10% SDS-polyacrylamide gel. The radioactivity was detected by fluorography. The positions of cyclin B and p34cdc are marked, as cyclin B and p34 respectively. Molecular weights are given in kd.

incubation at 39.5°C and was almost undetectable 3 h later (Figure 3C lane 6). At 33.5°C, no reduction in the 45 and 46 kd forms of RCC1 was observed in tsBN2 cells after a 3 h incubation (Figure 3C, lane 7). Thus the disappearance of BN2-RCCI is due to its instability at 39.5°C. When tsBN2 cells were incubated at 33.5°C after the disappearance of RCC1 at 39.5'C, RCC1 did not reappear in the presence of cycloheximide (data not shown), suggesting that it is degraded at 39.5°C. Disappearance of RCC1 correlates with activation of p34cdc2 Hi kinase and PCC The above results suggested that the loss of RCC1 caused the premature induction of mitosis. One of the key elements in the initiation of mitosis is the activation of p34cdc2 (Nurse, 1990). Therefore we investigated the relationship between the disappearance of BN2-RCC1 and the activation of p34cdc2 Cultures of tsBN2 cells were blocked in G1 by isoleucine starvation, released and then arrested at the G1/S boundary with hydroxyurea. Cells were shifted to 39.5°C in the presence of hydroxyurea, and at 1 h intervals cells were lysed to determine the amount of RCC 1, the histone HI kinase 1558

A new protein(s), other than cyclin is required to activate the cdc2/H1 kinase Previously we reported that protein synthesis was required for PCC induction in tsBN2 cells following a temperature shift (Nishimoto et al., 1981). Therefore we investigated whether protein synthesis is required for the activation of p34cdc2 histone HI kinase (using the same synchronization regime as for Figure 4A). At 39.5'C, RCCl disappeared at the same rate in the presence or absence of cycloheximide (compare Figure 4A and B), but with cycloheximide the activity of cdc2/HI kinase never increased (Figure 4B). In addition, there was no increase in the lower, dephosphorylated form of p34cdc2 during a 4 h incubation with cycloheximide at 39.5°C. Thus, protein synthesis is required for the activation of histone HI kinase prior to the dephosphorylation of p34cdc2. The synthesis of cyclins is essential for entry into mitosis (Nurse, 1990). Therefore we used an anti-human cyclin B antibody (Pines and Hunter, 1989), and an antibody to p34cdc2 to determine the amount of cyclin B present, and its association with p34cdc2, in tsBN2 cells arrested at the GI/S boundary with hydroxyurea after isoleucine deprivation (as

above).

The anti-cyclin B antibody co-immunoprecipitated the phosphorylated forms of p34cdc2 with hamster cyclin B, as previously reported for human cells (Pines and Hunter, 1989) (Figure SB, lane 1), and the anti-cdc2 antibody coimmunoprecipitated cyclin B (Figure SB, lane 2). Thus, cyclin B is already present and complexed with p34cdc2 in tsBN2 cells synchronized at the GI/S boundary, as previously reported (Yamashita et al., 1990). The amount of cyclin B did not significantly change during the increase in p34cdc2 histone Hl kinase activity (Figure SA), and

became undetectable concomitant with the inactivation of

p34cdc2 histone H 1 kinase as previously reported (Pines and Hunter, 1989). These data suggest that the synthesis of a protein(s) other than cyclin B is required for the activation of p34cdc2 upon the loss of RCC1 function.

Uncoupling p34cdc2 activation from the completion

of DNA replication The inhibition of DNA synthesis is essential for caffeine to induce PCC (Schlegel and Pardee, 1986) and the caffeineinduced PCC index is increased by extended treatment with hydroxyurea to synchronize cells at the G1/S boundary (R.Schlegel, personal communication). This indicates that


Fig. 6. Activation of cdc2/H1 kinase by temperature shift in cultures of tsBN2 cells travelling through S phase after release from synchronization in GI phase. (A) Cultures of tsBN2 cells were synchronized at GI in ileumedium and then allowed to grow in fresh normal medium. At 12 h (time 0) after release from synchronization, cultures were divided into four series (1, 2, 3 and 4). Two of them (3 and 4) were shifted to 39.5'C and others were incubated at 33.5°C. Each series of cultures at both temperatures (1 and 3) was given aphidicolin at the time 0. As a control, cultures of BHK21 cells (5 and 6) were similarly treated and incubated at 39.5°C with (5) or without (6) aphidicolin. Every hour, cells were lysed and the activity of p34cdc2 histone HI kinase was analyzed as shown in Figure 5 . The content of cgclin B and the mobility of p34cdc2 was analyzed b immunoblot analysis using anti-cyclin B or anti-cdc2 PSTAIR antibody and detected with [l 5I]protein A. The positions of cyclin B and p34cdc are shown as cyclin B and p34, respectively. The activity of cdc2/Hl kinase shown as HI was estimated by autoradiography of labeled histone HI. (B) Flow cytometric analysis of distribution of DNA content in tsBN2 cells traversing S phase either at 33.5'C, or at 39.5°C (shown in panel A-2 and 4). Cultures of tsBN2 cells synchronized at GI with ileumedium (a) were given fresh normal medium and allowed to grow for 12 h at 33.5°C (b) and then half of cultures were shifted to 39.5°C and incubated for 3 or 6 h either at 33.5°C (c, 3 h; d, 6 h) or at 39.5°C (e, 3 h; f, 6 h). At the indicated time, cells were prepared for the flow cytometric analysis. Total cell number was adjusted to 10 000. The second peak in (a) corresponds to the GI peak of tetraploid cells which frequently appears in cultures of tsBN2 cells (Nishimoto et al., 1978). Since cells with more than 4C were out of scale, total cell number in panels c-f was < 10 000.

some factor(s) needed for MPF activation, such as cyclin B, accumulates during hydroxyurea treatment. We have previously shown that the inhibition of DNA synthesis is not required to induce PCC in tsBN2 cells after a temperature shift (Nishimoto et al., 1978 and 1981), and we have now confirmed that the activation of p34cdc2 histone HI kinase

does not require the inhibition of DNA synthesis in tsBN2 cells. To avoid any ambiguity due to the accumulation of MPF

activator(s) during hydroxyurea synchronization, we synchronized tsBN2 cells in GI by isoleucine deprivation. After release from this block, PCC can first be induced at 1559

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DNA ont Fig. 7. Correlated flow cytometric analysis of DNA content and MPM-2 staining of tsBN2 cells showing PCC. Cultures of tsBN2 cells synchronized at the GI/S boundary with hydroxyurea (a) were incubated at 39.5°C, for 3 h in the presence of hydroxyurea (b, e), or allowed to grow in fresh medium either at 33.5°C for 9 h in the presence of 0.4 ug/ml of nocodazole (c, f), or at 39.5°C for 3 h (d, g). Cells were doubly stained for MPM-2 antibody reactivity and DNA content and analyzed with flow cytometry. (a) to (d) show dot blot analysis of MPM-2 staining. Distribution of DNA content of cells in panels b, c and d are shown in panels e, f and g. 2c and 4c indicate the G0 and G2 DNA content. The immunofluorescence micrograph of tsBN2 cells showing PCC and MPM-2 antigen staining was shown in (h). Cells were doubly stained with MPM-2 and Hoechst 33258.

the beginning of S phase (Nishimoto et al., 1981). Thus a series of tsBN2 cultures were shifted to 39.5°C for 12 h after release from the GI block, when cells were entering S phase (Figure 6B and Figure8). BHK21 cells were similarly treated as a control. At the same time (12 h after release from synchronization), a series of cultures was given aphidicolin, another inhibitor of DNA synthesis. In the normal cell cycle, blocking DNA synthesis prevents activation of p34cdc2 histone HI kinase. However, inhibiting DNA synthesis with aphidicolin did not prevent the activation of p34cdc2 histone HI kinase in tsBN2 cells at 39.5°C (Figure 6A-3). No increase in p34cdc2 histone HI kinase 1560

activity was seen in aphidicolin-treated tsBN2 cells at 33.50C (Figure 6A-1) or in BHK21 cells at 39.5°C (Figure 6A-5), although both cyclin B and the phosphorylated form of p34cdc2 accumulated. The p34cdc2 histone HI kinase of tsBN2 cells was activated with the same kinetics, regardless of the presence or absence of aphidicolin at 39.5°C (compare Figure 6A-3 with 6A-4), and in the absence of aphidicolin DNA replication proceeded normally for 3 h after the temperature shift, as verified by flow cytometry (Figure 6B). After a 1 h incubation at 39.50C, p34cdc2 histone HI kinase activity clearly increased, and was maximal 2 h later. One hour before the peak of p34cdc2 histone HI kinase activity,


4 h after the temperature shift and before the reduction of p34cdc2 histone H 1 kinase activity (Figure 6A-3 and 6A-4), indicating that cells had entered anaphase (Minshull et al., 1989). The number of 'rounded up' cells increased 3 h after temperature shift and then decreased with the same kinetics as the decline in p34cdc2 histone HI kinase activity (data not shown). Thus a 'normal' cycle of p34cdc2 histone HI kinase activation and inactivation was induced by the loss of RCC1 function in S phase tsBN2 cells, with or without the addition of a DNA synthesis inhibitor.

Fig. 8. Effect of temperature shift on p34 dc2 histone H I kinase in the phase of tsBN2 cells. Cultures of tsBN2 cells synchronized at GI by ileu- deprivation were allowed to grow in normal medium as described in Figure 7. (A) DNA synthesis and activity of p34cdc2 histone HI kinase through the cell cycle after release from a GI phase block: DNA synthesis and the activity of p34cd2 histone HI kinase were followed at 33.5°C as described in Materials and methods. The dotted and solid lines represent the incorporation of [3H]thymidine and the activity of HI kinase, respectively. (B) Change in the molecular mass of p34cdc2 through the cell cycle after release from a GI phase block: At the indicated times after release from the ileublock, cells were lysed and the mobility of p34cdc2 protein was determined by immunoblotting analysis using the anti-cdc2 PSTAIR antibody and an alkaline phosphatase conjugated second antibody. (C) Activation of p34cdc2 histone H1 kinase by temperature shift: At the indicated time after release from the ileublock, one dish of cells was shifted to 39.5°C and the activity of p34cdc2 histone HI kinase was measured 3 h later as described in Materials and methods. (D) Absence of the slower migrating p34cdc2 after temperature shift to 39.5°C in G, phase: At 4 h after release from the ileublock, a series of cultures was shifted to 39.5°C and incubated with (+) or without (-) vanadate. The molecular mass of p34CdC2 was analyzed 6. 8 and 10 h later.corresponding to 10, 12 and 14 h after release from the ileublock, respectively. Control cells were incubated at 33.5°C. with or without addition of vanadate. Lanes a-c: incubated at 33.50C without vanadate for 4, 10, or 14 h. Lanes d and e; incubated at 33.5°C for 6 or 10 h with vanadate. Lanes f-k; incubated at 39.5°C without (f-h) or with (i-k) vanadate, respectively. The time (h) after release from the ileublock is shown.

GI

much of p34cdc2 shifted

on

SDS -PAGE from the slowest

to the fastest migrating, dephosphorylated form (Figure 6A). The activation of p34Wc2 histone H1 kinase was transient. After 6 h at 39.5 'C the p34cdc2 histone HI kinase activity of tsBN2 cells had decreased to the level of interphase cells, whereas it had reached a high level in BHK2 1 cells at 39.5 0C and in tsBN2 cells at 33.50C (Figure 6A). The amount of cyclin B decreased and became undetectable in tsBN2 cells

Uncoupling mitosis from the completion of DNA replication That PCC induction in tsBN2 cells does not require the inhibition of DNA synthesis was further confirmed by twoparameter flow cytometry analysis, using the monoclonal antibody MPM-2 and propidium iodide. Cells were synchronized at the G,/S boundary with hydroxyurea and released in the presence or absence of nocodazole, either at 33.5°C or at 39.5°C. In normal mitotic cells arrested with nocodazole at 33.5°C, MPM-2 antigens appeared at the position corresponding to G2 DNA content (4n) (Figure 7c). In tsBN2 cells incubated for 3 h at 39.5°C with hydroxyurea, MPM-2 antigens appeared in cells with G1 DNA content (2n) (Figure 7b), consistent with PCC induction (Figure 1). MPM-2 specifically stained cells showing PCC (Figure 7h). In tsBN2 cells incubated at 39.5°C without hydroxyurea, the DNA content increased to that of mid-S phase in 3 h, and these cells also contained MPM-2 antigens (Figure 7d,g). Thus mitosis is induced by a temperature shift in tsBN2 cells undergoing DNA replication, in contrast to caffeine-induced PCC.

p34cdc2 histone

H 1 kinase is not activated in G1 by loss of RCC 1 function We wished to determine whether the loss of RCC 1 function results in the activation of p34cdc2 histone H1 kinase at times other than S phase in the cell cycle, since tsBN2 cells will also arrest in GI upon temperature shift (Nishimoto et al., 1978 and 1981). We had thought that the GI arrest was caused by partial chromosome condensation which might inhibit the transcription of mRNAs required for progression through G1 phase (Nishimoto et al., 1981; Ajiro et al., 1983). However, according to recent theories on the activation of MPF (Hunt, 1989), p34cdc2 cannot be activated in GI since there is no cyclin B present (Lee et al., 1988; Morla et al., 1989; Pines and Hunter, 1989). Therefore we synchronized tsBN2 cells in GI by isoleucine deprivation and released them into fresh medium. In GI phase, only the fastest migrating, non-phosphorylated form of p34cdc2 was observed (Figure 8B). By 10 h after release from the GI block, DNA synthesis had begun (Figure 8A) and the two slower migrating, phosphorylated forms of p34cdc2 appeared (Figure 8B), consistent with previous reports (Lee et al., 1988; Morla et al., 1989). Up to this time p34cdc2 histone H1 kinase could not be activated by temperature shift (Figure 8C), even though RCC1 disappeared. The activity of p34cdc2 histone H 1 kinase induced by the temperature shift gradually increased through S phase (Figure 8C). consistent with the accumulation of cyclin B and the phosphorylated form of p34cdc2 (Figures 6 and 8). Thus a temperature shift in GI phase in tsBN2 cells does not activate p34cdc2 histone H1 kinase. 1 561

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Additionally, the phosphorylated forms of p34cdc2 did not appear in tsBN2 cells during a further 10 h incubation at 39.5°C following a temperature shift 4 h after release from isoleucine deprivation (Figure 8D), whereas they had appeared by this time at 33.5°C. Phosphorylation of p34cdc2 was not observed at 39.5°C even when vanadate was added to inhibit phosphotyrosine phosphatases (Tonks et al., 1988; Morla et al., 1989). Thus in the absence of RCC 1, cells in GI phase are unable to phosphorylate p34cdc2.

Discussion In the cell there is a regulatory mechanism which ensures that the initiation of mitosis only occurs after the completion of DNA replication (Hartwell and Weinert, 1989). However, the loss of RCC 1 overrides this control mechanism. Upon temperature shift at any time in the cell cycle, RCC1 in tsBN2 cells becomes immunologically undetectable. There is a tight correlation between the loss of RCC 1 and the premature initiation of 'normal' mitotic events beginning with the activation of p34cdc2 histone HI kinase. The kinetics of activation of this kinase is not affected by the addition of an inhibitor of DNA synthesis. In the absence of such an inhibitor, DNA replication proceeds for at least 3 h after the temperature shift, while p34cdc2 histone HI kinase activity increases. Thus, in the absence of RCC1 function, the activation of p34cdc2 histone HI kinase is uncoupled from the completion of DNA replication. The phenotype of the tsBN2 cell line upon the loss of RCC 1 depends on the stage of the cell cycle, notably on the state of p34 dI2. In S phase, p34cdc2 is phosphorylated and complexed with cyclin B, and the loss of RCC1 function induces a mitotic cycle starting with the activation of p34cdc2 histone HI kinase. After prolonged incubation at the restrictive temperature, the nuclear membrane reforms and cells enter GI phase, but p34cdc2 phosphorylation does not occur. In GI phase, p34cdc2 is not yet phosphorylated nor complexed with cyclin B, and so the loss of RCC1 cannot induce the activation of p34cdc2 histone Hi kinase. Indeed, it is possible that RCC1 is required for the appearance of the phosphorylated form of p34cdc2 , and therefore the loss of RCC1 in GI phase would maintain the unphosphorylated state of p34Cdc2, such that p34cdc2 could not be activated. In this regard, we could postulate that RCC1 affects a kinase(s) which phosphorylates p34cdc2 and is needed to keep p34cdc2 inactive until the completion of DNA replication. In fission yeast, the cdc2+ gene regulates the cell cycle in both GI/S and G2/M transition (Nurse and Bissett, 1981), and similarly RCC 1 also seems to be required at both points in the mammalian cell cycle. In S and G2 phases, RCC 1 seems to play a role in the regulation of mitosis by preventing the activation of p34cdc2 histone HI kinase until DNA replication is complete. This activation requires the dephosphorylation of p34cdc2 complexed with cyclin B (Nurse, 1990) which may be carried out by an 'M-phase specific phosphatase' (Hunt, 1989). Our results indicate that the loss of RCC 1 function induces the synthesis of one or more proteins involved in the dephosphorylation of p34cdc2. Which protein is newly synthesized to activate the p34Cdc2 histone HI kinase? Since cyclin B is already present in tsBN2 cells synchronized at the G1/S boundary, it is not likely to be the protein whose synthesis is induced upon the loss of

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RCC 1. However, in the Xenopus in vitro system, cyclin is the only protein required to be newly synthesized for MPF activation (Murray and Kirschner, 1989) and its gradual accumulation induces an abrupt activation of p34cdc2 (Solomon et al., 1990). Thus, if the threshold of cyclin required to activate p34cdc2 is lowered in the absence of RCC 1, the slight increment in the amount of cyclin B during S phase will be enough to activate p34cdc2 histone HI kinase. In contrast to Xenopus oocytes, the overproduction of cyclin (p63cdc1 3) in fission yeast does not result in a wee phenotype, suggesting that the amount of cyclin is not ratelimiting for the activation of p34cdc2 (Booher and Beach, 1988; Hagan et al., 1988). To clarify the requirement for cyclin B, it is necessary to inject cyclin B into tsBN2 cells at the GI/S boundary in the presence of cycloheximide. Cyclin A might be the newly synthesized protein required to activate p34cdc2 histone HI kinase, and its mRNA induces GVBD in Xenopus oocytes (Swenson et al., 1986). However, cyclin A appears in advance of cyclin B in the HeLa cell cycle (Pines and Hunter, 1990), and therefore cyclin A is probably present in tsBN2 cells at the G1/S

boundary. The 'M-phase specific phosphatase' also seems to be present at S phase, since okadaic acid (a potent inhibitor of type 1 and type 2 phosphatase) activates p34cdc2 histone HI kinase and induces PCC without protein synthesis in both BHK21 and tsBN2 cells synchronized with hydroxyurea (Yamashita et al., 1990). Thus, the newly synthesized protein could be a phosphatase inhibitor and act in a similar manner to okadaic acid. In this regard it is interesting to note that the level of phosphatase inhibitor-2 oscillates in the cell cycle (Brautigan et al., 1990).

Another candidate protein is the cdc25 protein. Genetic analysis in yeast indicates that cdc25 in S.pombe activates p34cdc2 histone HI kinase by positively regulating the dephosphorylation of p34cdc2 (Gould and Nurse, 1989). In relation to the tsBN2 phenotype, it should be noted that cdc25 expression is increased in G2 phase of HeLa cells (Sadhu et al., 1990), and that the overproduction of cdc25 abolishes the dependence of cell division on the completion of DNA replication in S.pombe (Enoch and Nurse, 1990). Previously we thought that the GI arrest due to the loss of RCC1 was caused by partial chromosome condensation inhibiting the transcription of some gene(s) essential to cell cycle progression. However, since we could detect neither PCC nor the activation of p34cdc2 histone HI kinase in GI phase in the absence of RCC 1, the GI arrest may be due to another mechanism. When tsBN2 cells arrested in GI phase at the nonpermissive temperature were shifted down to the permissive temperature, the cells entered S phase after a delay corresponding to the length of time spent at the nonpermissive temperature (Nishimoto et al., 1978). Thus, the loss of RCC1 inhibits the accumulation of a factor(s) required to enter S phase which is synthesized and accumulates throughout G1. One possibility is that RCC 1 is required for the synthesis of a GI cyclin (Richardson et al., 1989) and for the activation of a GI cyclin/p34cdc2 complex which may be required for the initiation of S phase (D'Urso et al., 1990). The gradual increase in p34cdc2 histone HI kinase activity induced by temperature shift in late GI phase (Figure 8C), is similar to the ratio of cells entering S phase at high temperature after release from an


ileublock (Nishimoto et al., 1978), and may reflect the formation of a GI cyclin/p34cdc2 complex required to initiate S phase. The SRMI gene in S. cerevisiae is homologous to the RCCI gene, and so far two mutants in the SRMI gene have been isolated. These are srnl (Clark and Sprague, 1989) and prp2O (Aebi et al., 1990). The srml mutant was isolated as a suppressor mutation that restores mating to a or ae factor receptorless mutants, and arrests cells in GI after a temperature shift. The prp2O mutant was isolated as a mutant defective in mRNA processing, so it is possible that the loss of RCC1 arrests cells in GI by interrupting the synthesis of a protein needed to traverse G1 phase.

Materials and methods Synchronization of cells BHK21 and tsBN2 cells were grown in Dulbecco's modified Eagle's medium (DMEM) containing 10% calf or fetal bovine serum in a humidified atmosphere containing 10% CO2. Cells were synchronized at GI phase with isoleucine-free medium (ileu-), and at the GI/S boundary with 2.5 mM hydroxyurea as described (Nishimoto et al., 1981).

Immunofluorescence Cells grown on coverslips were rinsed with phosphate-buffered saline (PBS), fixed for 20 min with 3 % (w/v) paraformaldehyde in PBS, permeabilized for 15 min with 1% NP40 in PBS plus glycine, and then stained with the antibodies as described (Ohtsubo et al., 1989). For tubulin immunofluorescence, coverslips were rinsed with microtubulestabilizing buffer (0.1 M PIPES, pH 6.9, 1 mM EGTA, 1 mM MgCl2, 4 M glycerol) at 37°C and treated with the same buffer containing 0.5% Triton X-100 for 3 min at 37°C. Cells were then rinsed twice with the stabilizing buffer and fixed with methanol at 20°C for 10 min. Following rehydration with PBS for 5 min, cells were stained with a monoclonal anti3-tubulin antibody (1:500, Amersham) for 1 h at room temperature, and visualized with rhodamine-conjugated goat anti-mouse IgG (1:100). The coverslips were rinsed and mounted. DNA was stained with Hoechst 33258 (1 ig/rml) for 5 min.

Immunoblotting and immunoprecipitation The antibodies used were an anti-Xenopus RCC1 antiserum (Nishitani et al., 1990), anti-PSTAIR and anti-human p34cdc2 C-terminal peptide antisera (prepared for this experiment) and an anti-human cyclin B antiserum (Pines and Hunter, 1989). The procedure for immunoblotting was as described (Nishitani et al., 1990). The immunoprecipitation was performed as described (Yamashita et al., 1990). Flow cytometry Flow cytometry analysis of cellular DNA content was carried out on cells treated with 50% methanol and RNase A (200 yglmi), and stained with 50 ug/ml of propidium iodide. The 2-parameter flow cytometry analysis was performed as follows. Both floating and trypsinized cells were collected and fixed in 3 % (w/v) paraformaldehyde for 20 min, then in 50 % methanol for 30 min. After washing with PBS, cells were incubated sequentially with MPM-2 antibody (1:500) in PBS containing 10% horse serum at 4'C overnight, then with 10 Ag/ml biotinylated anti-mouse IgG (Vector) in PBS containing 10% horse serum for 6 h at 4°C, and then with 40 plg/mn fluorescein-labeled avidin DCS (Vector) in NaHCO3 buffered saline, pH 8.2 for 30 min at 4°C. The cells were washed with NaHCO3 buffer and suspended in a solution containing 50 Azg/ml propidium iodide, 0.1% sodium citrate and 200 Ag/ml RNaseA. Cell fluorescence was measured on FACScan (Becton Dickinson).

Histone HI kinase assay Histone HI kinase assays were performed on immunoprecipitates made with the anti-cdc2 C-terminal peptide antibody from 10 jig of lysate, as previously described (Yamashita et al., 1990).

Acknowledgments We thank Dr P.N.Rao (Texas, USA) and Dr L.Gerace (La Jolla, USA) for generous gifts of MPM-2 and anti-lamin antibodies, respectively. We thank Mariko Ohara for reading the manuscript. This work was supported

by Grants-in-Aid for Scientific Research and for Cancer Research from the Ministry of Education, Science and Culture and the Science and Technology Agency, and by the Mitsubishi Foundation.

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Received on January 2, 1991; revised on March 4, 1991

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