p25rum1 promotes proteolysis of the mitotic B-cyclin p56cdc13 during G1 of the fission yeast cell cycle
1997; Springer Nature; Volume: 16; Issue: 15 Linguagem: Inglês
10.1093/emboj/16.15.4657
ISSN1460-2075
Autores Tópico(s)Genomics and Chromatin Dynamics
ResumoArticle1 August 1997free access p25rum1 promotes proteolysis of the mitotic B-cyclin p56cdc13 during G1 of the fission yeast cell cycle Jaime Correa-Bordes Jaime Correa-Bordes Cell Cycle Laboratory, Imperial Cancer Research Fund,44 Lincoln's Inn Fields, London, WC2A 3PX UK Search for more papers by this author Marie-Pierre Gulli Marie-Pierre Gulli Cell Cycle Laboratory, Imperial Cancer Research Fund,44 Lincoln's Inn Fields, London, WC2A 3PX UK Search for more papers by this author Paul Nurse Corresponding Author Paul Nurse Cell Cycle Laboratory, Imperial Cancer Research Fund,44 Lincoln's Inn Fields, London, WC2A 3PX UK Search for more papers by this author Jaime Correa-Bordes Jaime Correa-Bordes Cell Cycle Laboratory, Imperial Cancer Research Fund,44 Lincoln's Inn Fields, London, WC2A 3PX UK Search for more papers by this author Marie-Pierre Gulli Marie-Pierre Gulli Cell Cycle Laboratory, Imperial Cancer Research Fund,44 Lincoln's Inn Fields, London, WC2A 3PX UK Search for more papers by this author Paul Nurse Corresponding Author Paul Nurse Cell Cycle Laboratory, Imperial Cancer Research Fund,44 Lincoln's Inn Fields, London, WC2A 3PX UK Search for more papers by this author Author Information Jaime Correa-Bordes1,2, Marie-Pierre Gulli1,3 and Paul Nurse 1 1Cell Cycle Laboratory, Imperial Cancer Research Fund,44 Lincoln's Inn Fields, London, WC2A 3PX UK 2Departamento de Microbiologia, Facultad Ciencias, Avda Elvas S/N, 06071 Badajoz, Spain 3ISREC, Chemin des Boveresses 155, 1066 Epalinges/VD, Switzerland *Corresponding author. E-mail: [email protected] The EMBO Journal (1997)16:4657-4664https://doi.org/10.1093/emboj/16.15.4657 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions Figures & Info The fission yeast Schizosaccharomyces pombe CDK inhibitor p25rum1 plays a major role in regulating cell cycle progression during G1. Here we show that p25rum1 associates with the CDK p34cdc2/p56cdc13 during Gl in normally cycling cells and is required for the rapid proteolysis of p56cdc13. In vitro binding data indicate that p25rum1 has specificity for the B-cyclin p56cdc13 component of the CDK and can bind the cyclin even in the absence of the cyclin destruction box. At the G1–S-phase transition, p25rum1 levels decrease and p56cdc13 levels increase. We also show that on release from a G1 block, the rapid disappearance of p25rum1 requires the activity of the CDK p34cdc2/cig1p and that this same CDK phosphorylates p25rum1 in vitro.We propose that the binding of p25rum1 to p56cdc13 promotes cyclin proteolysis during G1, with p25rum1 possibly acting as an adaptor protein, promoting transfer of p56cdc13 to the proteolytic machinery. At the G1–S-phase transition, p25rum1 becomes targeted for proteolysis by a mechanism which may involve p34cdc2/cig1p phosphorylation. As a consequence, at this point in the cell cycle p56cdc13 proteolysis is inhibited, leading to a rise of p56cdc13 levels in preparation for mitosis. Introduction Cyclin-dependent kinases (CDKs) are important for controlling the major transitions during the cell cycle, including the onset of S-phase and mitosis. Their activities are tightly regulated by a combination of different mechanisms including transcription, phosphorylation, proteolysis and association with CDK inhibitors (CKIs) (Nasmyth, 1993; Nigg, 1995; Sherr and Roberts, 1995). These mechanisms ensure that CDK activities are kept low during those phases of the cell cycle when they are not required and only increase when they are needed to bring about cell cycle progression. An important example is the control of CDK activities during the G1 phase. This is best understood in the budding yeast where the controls appear to involve transcriptional regulation, specific cyclin proteolysis, phosphorylation and action of a CKI. Two types of cyclin are required for cell cycle progression in budding yeast, the CLNs acting during G1 and the CLBs acting during G1–S and then again at mitosis (Nasmyth, 1993). There are transcriptional controls for both classes of cyclin gene which operate during the cell cycle and ensure that the cyclin proteins can only be made at the appropriate times (Amon et al., 1993). In addition to these controls there are specific proteolytic mechanisms. For example, during late G1, Cln2p first forms a complex with the E2 ubiquitin-conjugating enzyme Cdc34p, and then becomes ubiquitinated before degradation by the proteosome (Willems et al., 1996). Phosphorylation of Cln2p by the p34CDC28 CDK is thought to activate proteolysis by promoting the association of Cln2p with Cdc34p (Lanker et al., 1996; Willems et al., 1996). The CLB cyclins are targeted for degradation at the end of mitosis by the anaphase-promoting complex (APC) and this proteolysis persists throughout G1 (Amon et al., 1994; Irniger et al., 1995). The CKI p40SIC1 inhibits the CLB-dependent CDKs in G1 and is thought to be targeted for ubiquitination by a process involving Cdc34p and CLN-dependent phosphorylation (Schwob et al., 1994; Schneider et al., 1996). The removal of p40SIC1 by the CLN protein kinases allows an increase in CLB protein kinase to occur, leading to passage through G1. Thus, a combination of regulatory mechanisms control the appearance and disappearance of cyclins and their associated CDKs at the appropriate times during the cell cycle, resulting in an orderly progression through G1 into S-phase. In the fission yeast these controls are less well understood. Early G1 cells have low levels of mitotic p34cdc2/p56cdc13 protein kinase activity and of p56cdc13 (Hayles et al., 1994; Correa-Bordes and Nurse, 1995). These low levels are not brought about by regulating transcription of the cdc2 and cdc13 genes, because these transcripts can be detected in G1 cells (J.Hayles, unpublished results). The lack of a transcriptional control suggests that translational or proteolytic mechanisms may be more important in regulating p34cdc12 protein kinase activity in fission yeast G1 cells. Of relevance to these mechanisms is the rum1 gene which encodes the CKI p25rum1 (Moreno and Nurse, 1994; Correa-Bordes and Nurse, 1995; Martin-Castellanos et al., 1996). This CKI specifically inhibits the p34cdc2/p56cdc13 mitotic kinase and is present only in G1 cells when it forms a complex with p34cdc2/p56cdc13. In the absence of the rum1 gene, fission yeast cells blocked in G1 are unable to keep p34cdc2/p56cdc13 protein kinase levels low and, as a consequence, they can initiate mitosis without having undergone S-phase. These results indicate that p25rum1 plays a pivotal role in regulating the p34cdc2/p56cdc13 protein kinase during G1. Given these observations we have investigated the role of p25rum1 in G1 regulation further. In the present study, we show that the low levels of p56cdc13 during G1 are brought about by p25rum1 promoting rapid proteolysis of p56cdc13. p25rum1 associates in vitro with the p56cdc13 cyclin moiety, suggesting that p25rum1 may act as an adaptor protein which targets p56cdc13 for degradation. p25rum1 disappears upon onset of S-phase by a mechanism which may involve p34cdc2/cig1p phosphorylation, and as a consequence p56cdc13 levels rise in preparation for mitosis. This process allows an orderly progression through G1 of the fission yeast cell cycle. Results p25rum1 maintains high p56cdc13 turnover We have previously shown that the level of the B-cyclin p56cdc13 is much reduced in early G1 cells of fission yeast (Hayles et al., 1994; Correa-Bordes and Nurse, 1995). To investigate whether p25rum1 influences p56cdc13 levels during G1, we have monitored the effects of p25rum1 on the rates of p56cdc13 translation and turnover. The rate of translation of p56cdc13 was measured in normally cycling cells and in G1-arrested cells, using the strains cdc10-129 and cdc10-129 rum1Δ growing at 25°C, and 3.5 h after shift to 36°C. Cells were pulse-labelled with [35S]methionine for 30 min and immunoprecipitations performed using anti- p56cdc13 antibodies [lanes marked (+) in Figure 1;] and control pre-immune antibodies [lanes marked (−) in Figure 1]. Comparison of the (+) and (−) lanes showed that the anti-p56cdc13 antibodies specifically immunoprecipitated a protein of the correct molecular weight for p56cdc13. The rate of translation of p56cdc13 at 25°C in cycling cells which were predominantly in G2 was compared with that in cells arrested in G1 by shifting to 36°C (Figure 1, compare lanes 2 and 4). After correcting for the rate of cell labelling using the unspecific upper band shown in Figure 1 as the control, the rate of incorporation into p56cdc13 was found to be slightly reduced at 36°C to 80% of the value at 25°C. This result indicates that the rate of p56cdc13 translation is approximately similar in G1 and G2 cells, consistent with the fact that cdc13 transcript levels are constant throughout the cell cycle (J.Hayles, unpublished results). The rates of p56cdc13 translation were also similar in cycling and G1-arrested cells, whether the rum1 gene was present (Figure 1, lanes 2 and 4) or absent (Figure 1, lanes 6 and 8). After correcting for cell labelling as before, the rate of incorporation into p56cdc13 was slightly increased at 36°C to 140% of the value at 25°C. Therefore, despite the fact that p56cdc13 is at a much higher level in G2 cells compared with G1 cells, there is no significant difference in the rate of synthesis of the protein between the two stages of the cell cycle. Figure 1.The rate of p56cdc13 translation in G1 is not affected by rum1. Cells of cdc10-129 and cdc10-129 rum1Δ strains were pulse-labelled with [35S]methionine for 55 min at 25°C or 30 min after 3.5 h at 36°C. Immunoprecipitates using anti-p56cdc13 antibodies [lanes marked (+)] and control pre-immune antibodies [lanes marked (−)] were analysed by SDS–PAGE followed by fluorography. Download figure Download PowerPoint This conclusion suggests that the differences in p56cdc13 levels observed in the presence and absence of rum1 are due to changes in p56cdc13 turnover. To test this directly, we attempted to perform the appropriate pulse–chase experiments but were unable to perform the chase using normal physiological growth conditions. As an alternative,we estimatedp56cdc13 turnover by following p56cdc13 levels after switching off cdc13transcription. cdc13 was expressed using the medium-level nmt repressible promoter of pREP41 stably integrated into the cdc10-129 cdc13Δ and cdc10-129 cdc13Δ rum1Δ strains. The cells of both strains were arrested largely in G1 by shifting to 36°C for 2.5 h, the nmt promoter switched off by adding thiamine, and the levels of p56cdc13 monitored by Western blot analysis. It can be seen from Figure 2 that p56cdc13 is proteolysed at a higher rate in G1 cells when p25rum1 is present; p56cdc13 has a half-life of ∼10 min in the presence of rum1 and 30 min in the absence of rum1. No difference in the kinetics of p56cdc13 proteolysis was observed after switching off cdc13 transcription at 25°C, where exponentially growing cells are mainly in G2 (Figure 2A). The increase in half-life of p56cdc13 from 10 min to 30 min that occurs when rum1 is deleted explains why the rate of incorporation into p56cdc13 changes to 140% of control in the absence of rum1 compared with 80% of control in the presence of rum1. If the rate of p56cdc13 translation in both situations were approximately similar, then decreased turnover in the absence of rum1 would result in a higher level of detected incorporation compared with cells containing rum1. Figure 2.p25rum1 is required for rapid proteolysis of p56cdc13 in G1. (A) The cdc13+ gene controlled by the nmt1 promoter was expressed in acdc10-129 cdc13Δ strain in the presence or absence of rum1. The cells of both strains were arrested in G1 at 36°C for 2.5 h, and the nmt promoter switched off by adding thiamine. As a control the same experiment was performed at 25°C. (B) Abundance of the p56cdc13 in the G1 extracts was determined by densitometry after immunoblotting with anti-p56cdc13 antibodies. Download figure Download PowerPoint The increase in p56cdc13 stability in rum1Δ cells could be explained by p25rum1 being specifically required for rapid proteolysis of the mitotic cyclin in G1, or could be due to a rise in mitotic kinase activity caused by the absence of p25rum1 resulting in cells moving from G1 to some state later in the cell cycle. To investigate this further, we have monitored the levels of p42cut2 in G1 in the presence or absence of rum1. p42cut2 is degraded in anaphase and remains unstable during G1 (Funabiki et al., 1996a), and it has been reported that cells arrested using a cdc10ts mutant have reduced levels of p56cdc13 and p42cut2 (Funabiki et al., 1996b). Therefore, p42cut2 turn-over acts as a marker for a cell being in G1. We found that p56cdc13 was undetectable and p42cut2 levels were reduced 2- to 3-fold in a cdc10-129 mutant (Figure 3, lane 2). When rum1 was deleted, a similar drop in p42cut2 levels was still observed but in contrast p56cdc13 remained at a high level (Figure 3, lane 4). Extracts from exponentially growing cut2HA+ cells were used to confirm that the anti-cut2 antibodies were detecting p42cut2 (Figure 3, lane 5). This result shows that G1 down-regulation of p42cut2 still occurs in the double mutant cdc10-129 rum1Δ at the restrictive temperature, indicating that p25rum1 is required specifically for p56cdc13 proteolysis in G1, but is not required for p42cut2 proteolysis. This suggests that the effects of p25rum1 on p56cdc13 proteolysis are likely to be specific and are not due to cells moving from G1 to a later state in the cell cycle. In the absence of p25rum1, proteolysis still occurs but the rate is reduced to a level which is insufficient to counteract the continuing translation of p56cdc13. As a consequence, the level of p56cdc13 remains high in G1 cells lacking p25rum1. Figure 3.p25rum1 is not required for down-regulation of p42cut2 in G1. cdc10-129 and cdc10-129 rum1Δ strains were grown in minimal medium at 25°C and then shifted to 36°C for 4 h. Samples were taken to make total extracts as described. Proteins (25 mg) from these extracts were Western blotted and probed with anti-p56cdc13 and anti-p42cut2 antibodies. Total extracts of exponentially growing cut2HA+ cells were used as a control for anti-p42cut2 antibodies. Download figure Download PowerPoint p25rum1 binds p56cdc13 in vitro The above results indicate that p25rum1 promotes p56cdc13 turnover in G1-arrested cells. Our earlier work showed that p25rum1 associates directly with p56cdc13 in cells arrested in G1 (Correa-Bordes and Nurse, 1995). To investigate this process of association further, we have examined the ability of p25rum1 to form complexes with p56cdc13 in vitro. We first tested whether p25rum1 could bind either p56cdc13 alone or p56cdc13 complexed with p34cdc2. Both p34cdc2 and p56cdc13 were translated in vitro using [35S]methionine and the Promega-TNT system. They were then tested separately and after mixing to determine whether they could form complexes with p25rum1. This was carried out by adding purified bacterially produced p25rum1 to the in vitro translation mixes for 30 min, followed by immunoprecipitation with anti-p25rum1 antibodies. In Figure 4, the labelled proteins produced by in vitro translation are shown by the lanes marked 'Input', while the immunoprecipitations with anti-p25rum1 and control antibodies are shown respectively as (+) and (−). It can be seen that p25rum1 could form specific complexes with p56cdc13 and with p34cdc2/p56cdc13 complexes but not with p34cdc2 alone. We next tested whether the destruction box which is required for B-cyclin proteolysis in mitosis was required for p25rum1 binding. The cdc13Δ90 mutant lacks this destruction box, but the mutant protein could still form an in vitro complex with p25rum1 (Figure 4). Finally, we tested if p25rum1 could form in vitro complexes with the B-cyclins encoded by cig1 and cig2 or with complexes of these cyclins with p34cdc2. No specific p25rum1 association was detected with these cyclins, either alone or in association with p34cdc2. Figure 4.p25rum1 has in vitro binding specificity for p56cdc13. In vitro-translated [35S]methionine-labelled products of the indicated cyclins and p34cdc2 were incubated with (lanes +) or without (lanes −) p25rum1. Immunocomplexes obtained using anti-p25rum1 antibodies were separated by SDS–PAGE followed by fluorography. Download figure Download PowerPoint We conclude that p25rum1 can become stably associated either with free p56cdc13 or with p56cdc13 complexed with p34cdc2 and that this association does not require the cyclin destruction box. In addition, the association appears to be specific to the B-cyclin p56cdc13 because there is no detectable association with other B-cyclins encoded by cig1 and cig2, at least in vitro. These results suggest that p25rum1 specifically associates either with free p56cdc13 or with p56cdc13/p34cdc2 complexes present in G1 cells and that these associations may play a role in targeting p56cdc13 for proteolysis during this phase of the cell cycle. p25rum1 association with p56cdc13/p34cdc2 during the cell cycle If p25rum1 targets p56cdc13 for proteolysis when cells are arrested in G1, then p25rum1 association with p56cdc13 or with p56cdc13/p34cdc2 complexes might be expected to vary during the normal cell cycle, peaking in G1. We attempted to test this possibility by immunoprecipitations using anti-p25rum1 antibodies followed by Western blotting, but the limited amounts of material available from selection synchronous cultures prevented this approach from being successful. As an alternative, we monitored whether p25rum1 was associated with p56cdc13/p34cdc2 protein kinase activity, because enzymatic assays can be more sensitive than Western blotting. In a first experiment, anti-p25rum1 antibodies were used to immunoprecipitate from extracts prepared from asynchronously growing wild-type, rum1Δ and the temperature-sensitive cdc2-33 strains. An H1-histone kinase activity was detected in the extracts from the wild-type cells but not from the rum1Δ cells grown at 25°C (Figure 5, lanes 3 and 4). This activity entirely disappeared when assayed at 40°C in the extract prepared from the cdc2-33 strain, but was still present in the wild-type extract at 40°C (Figure 5, lanes 5 and 6). Because the p34cdc2 H1-histone kinase activity is temperature-sensitive in this strain—as shown by assays performed with immunoprecipitates prepared using anti-p34cdc2 antibodies (Figure 5, lanes 7–10)—we conclude that the H1-histone kinase activity associated with p25rum1 is due to p34cdc2. This protein kinase activity was shown to be derived mostly from p56cdc13/p34cdc2 complexes, by exploiting the fact that these complexes are very sensitive to the addition of bacterially produced p25rum1 to the in vitro protein kinase assays, while complexes of the cig2 cyclin are only partially sensitive and complexes of the cig1 cyclin are completely insensitive to p25rum1 (Correa-Bordes and Nurse, 1995). Addition of p25rum1 to the anti-p25rum1 immunoprecipitates completely inhibited the H1-histone kinase activity, establishing that p56cdc13 is the major B-cyclin involved in the active complexes (Figure 5, lanes 1 and 2). These results indicate that p25rum1 immunoprecipitates contain active p56cdc13/p34cdc2 protein kinase activity. This activity can be inhibited by the addition of further p25rum1 in vitro, behaviour similar to that observed with p21cip1 immunoprecipitates from mammalian cells which contain CDK protein kinase activity inhibited by the addition of further p21cip1 (Zhang et al., 1994). Possibly the complexes between p25rum1 and p56cdc13/p34cdc2 formed in the immunoprecipitates become dissociated under the conditions of the in vitro protein kinase assay, leading to restoration of enzymatic activity. Figure 5.p25rum1-associated kinase is p34cdc2 dependent. p25rum1 (lanes 1–6) and p34cdc2 (lanes 7–10) were immunoprecipitated from extracts of the indicated strains grown at 25°C and assayed for H1-histone kinase activity at either 25 or 40°C. p25rum1 immunoprecipitates from wild-type cells were assayed for H1-histone kinase activity in the absence (lane 1) or presence of 2 nM p25rum1 (lane 2). Download figure Download PowerPoint Having established that p25rum1 immunoprecipitates contain p56cdc13/p34cdc2 protein kinase activity, extracts were prepared from different time points of a synchronous culture and immunoprecipitations performed using the anti-p25rum1 antibodies (Figure 6). These antibodies detected a peak in H1-histone kinase activity 20 min before the peak of septation, timing which corresponded to the period of the cell cycle when cells were traversing through G1. These data are consistent with p25rum1 becoming associated with p56cdc13/p34cdc2 protein kinase activity during the G1 phase of the cell cycle, and we propose that this association is an early step in the process which leads to p56cdc13 proteolysis. As a consequence, the p56cdc13/p34cdc2 protein kinase activity is kept at a low level during this phase of the cell cycle, preventing inappropriate entry into S-phase or mitosis. Figure 6.Oscillation of p25rum1-associated p34cdc2 kinase during the cell cycle. Small wild-type cells in early G2 were selected by elutriation and reinoculated in minimal medium at 32°C, and samples taken for determination of septation index and H1-histone kinase activity present in p25rum1 immunoprecipitates. Download figure Download PowerPoint As cells exit G1 and proceed through S-phase, p56cdc13/p34cdc2 protein kinase levels begin to rise. This rise is associated with a drop in p25rum1 levels, as shown in Figure 7B where temperature-sensitive cdc10-V50 cells have been released from a G1 block. Cells were arrested in G1 by incubation for 4 h at 36°C and were then shifted down to 25°C. Within 60 min of shift-down, cells had entered S-phase, and by 90 min S-phase was largely complete (Figure 7A). Western blotting showed that p25rum1 had disappeared from cells by 60 min and by this time p56cdc13 had also begun to accumulate (Figure 7B). This result suggests that the disappearance of p25rum1 at the end of G1 is important in allowing p56cdc13 levels to accumulate, and thus the reduction of p25rum1 levels at the end of G1 may be an important regulatory mechanism of cell cycle progression. Figure 7.The drop of p25rum1 levels at the onset of S-phase depends on cig1p/p34cdc2 activity. (A) FACS analysis of G1 block and release experiments of indicated strains. Cells were arrested in G1 by incubation for 4 h at 36°C and shifted down to 25°C. (B) Western blots from total extracts of the experiments described above were probed with anti-p25rum1, anti-cig2p and anti-p56cdc13 antibodies. Download figure Download PowerPoint cig1p-associated p34cdc2 kinase phosphorylates p25rum1 Phosphorylation has been implicated in targeting a number of proteins for proteolysis, and p25rum1 has several consensus phosphorylation sites for the p34cdc2 protein kinase (Moreno and Nurse, 1994). Therefore we decided to investigate whether p34cdc2 phosphorylation of p25rum1 might have a role in reducing the level of p25rum1 as cells exit G1. Firstly, we tested whether p25rum1 was an in vitro substrate for the p34cdc2 protein kinase. Immunoprecipitates from a wild-type extract were prepared using antibodies against p34cdc2 and the three B-cyclins encoded by cdc13, cig1 and cig2 (Hagan et al., 1988; Booher et al., 1989; Moreno et al., 1989; Bueno et al., 1991; Obara-Ishihara and Okayama, 1994). p25rum1 kinase activity was detected with both the anti-p34cdc2 and anti-cig1p antibodies, but not with the other two (Figure 8). This result indicates that p25rum1 can be phosphorylatedin vitro by the p34cdc2 protein kinase activity associated with the cig1-encoded B-cyclin, but not with the other two B-cyclins. Figure 8.cig1p/p34cdc2 complex phosphorylates p25rum1 in vitro. p34cdc2 associated with different cyclins was immunoprecipitated from wild-type extracts and assayed for protein kinase activity using p25rum1 as a substrate. Download figure Download PowerPoint We next investigated whether the cig1 gene had any in vivo effects on p25rum1 levels. These experiments were carried out in a cdc10-V50 strain in which cig1 had been deleted, together with control cells in which cig2 was deleted. In cig1Δ cells, p25rum1 was still present at high levels 90 min after release from the cdc10-V50-imposed G1 block, 60 min longer than controls (Figure 7B), indicating that cig1 is required for the rapid turnover of p25rum1 that occurs when cells are released from a G1 block. Therefore the direct phosphorylation of p25rum1 by the cig1p-associated p34cdc2 protein kinase may play a role in regulating p25rum1 turnover on progression from G1 to S-phase. The cig1Δ cells were also delayed from entering S-phase on release from the cdc10-V50 block by ∼30–45 min compared with cdc10-V50 control (Figure 7A). In contrast, cdc10-V50 cig2Δ showed no significant differences in either p25rum1 or S-phase entry compared with cdc10-V50 (Figure 7A and B). Two further points can be made from these experiments. Firstly, the rapidity with which p56cdc13 appears after release from the G1 block suggests that inactivation of the p56cdc13 proteolytic machinery, possibly involving cig1, occurs very rapidly following activation of the cdc10 function. Secondly, deleting cig2 had no effect on the timing of S-phase onset in the cdc10-V50 G 1 block and release experiments. In these experiments, the cells are larger than normal and it is possible that in the enlarged cells formed in these circumstances, the cig2-associated p34cdc2 protein kinase is no longer limiting for G1 progression (Fisher and Nurse, 1996). Discussion The fission yeast B-cyclin p56cdc13/p34cdc2 protein kinase is able to bring about the onset of mitosis in G1 cells (Hayles et al., 1994) and therefore needs to be kept under tight control to avoid a catastrophic segregation of unreplicated chromosomes. We have previously shown that the CDK inhibitor p25rum1 is crucially involved in this control and that it acts as a potent inhibitor of p56cdc13/p34cdc2 protein kinase activity (Moreno and Nurse, 1994; Correa-Bordes and Nurse, 1995). In the present work we have demonstrated that p25rum1 is required for rapid proteolysis of p56cdc13 in G1 cells and that the regulation of p25rum1 during the cell cycle may involve the p34cdc2 protein kinase associated with the B-cyclin cig1p. Specifically, we have made the following observations: (i) in G1-arrested cells, p56cdc13 disappears rapidly and this disappearance requires rum1. Another protein proteolysed in G1, p42cut2 (Funabiki et al., 1996a), does not require rum1 for turnover. (ii) The rate of translation of p56cdc13 in G1 cells is the same in the presence or absence of the rum1 gene, but the rate of p56cdc13 proteolysis is increased in the presence of rum1. (iii) p25rum1 binds specifically in vitro either to p56cdc13 or to the p56cdc13/p34cdc2 complex, even when the cyclin destruction box is lacking. (iv) p25rum1 associates with the p56cdc13/p34cdc2 protein kinase during G1 of the normal cell cycle. (v) The cig1p/p34cdc2 protein kinase phosphorylates p25rum1 in vitro and, in the absence of cig1p, the disappearance of p25rum1 is delayed when cells are released from a G1 block. From these observations we conclude that an important role for p25rum1 in cell cycle regulation is to associate with p56cdc13 or p56cdc13/p34cdc2 complexes in G1 cells and to promote p56cdc13 proteolysis. We imagine that, as well as acting as a direct inhibitor of p56cdc13/p34cdc2 protein kinase activity, p25rum1 has a second role as an adaptor promoting transfer of p56cdc13 to the proteolytic machinery. Given that p56cdc13 translation is not reduced in G1 cells, the p25rum1-mediated promotion of p56cdc13 proteolysis must be a major mechanism preventing the accumulation of active p56cdc13/p34cdc2 protein kinase activity in G1 cells. This is of considerable importance because p34cdc2 is not inhibited by Y15 phosphorylation during G1 (Hayles and Nurse, 1995) and, as a consequence, p56cdc13/p34cdc2 protein kinase activity could appear in G1 cells and bring about an inappropriate entry into S-phase or a catastrophic entry into mitosis. Proteolysis of p56cdc13 can still occur in cells lacking rum1, and is clearly sufficient to allow exit from mitosis. However, the rate of proteolysis is insufficient to reduce the levels of p56cdc13 during the G1 phase of the cell cycle. p25rum1 may act as an adaptor, with the binding of p25rum1 to p56cdc13 enhancing the transfer of cyclin to the nuc2/cut9 proteosome machinery (Hirano et al., 1988; Samejima and Yanagida, 1994). Such a mechanism would share characteristics with other proteolytic processes where association with a specific adaptor protein targets a substrate protein for rapid turnover via the proteosome. For example, the E6 protein binds p53 and targets it for ubiquitination, and antizyme targets ornithine decarboxylase to the proteosome (Ciechanover, 1994). p25rum1 appears to have an effect on p56cdc13 proteolysis only in G1 cells and not during mitotic exit. Perhaps there are differences in the cyclin proteolytic mechanisms at these two different stages of the cell cycle with p25rum1 only having a significant targeting role during G1. We have shown that proteolysis of another protein p42cut2 during G1 is not affected by the absence of rum1, suggesting that the effect of p25rum1 on p56c
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