Alternative Splicing of the Human CDC25B Tyrosine Phosphatase

Alternative splicing of the human CDC25B tyrosine phosphatase. Possible implications for growth control?

VeÂ ronique Baldin1, Christophe Cans1, Giulio Superti-Furga2 and Bernard Ducommun1

1Institut de Pharmacologie et de Biologie Structurale du CNRS UniversiteÂ Paul Sabatier, 205 route de Narbonne, 31077 Toulouse cedex France; 2EMBL, Meyerhofstraûe 1, D69012 Heidelberg, Germany

CDC25B2, a protein tyrosine phosphatase closely related CDK ATP-binding site (Gould and Nurse, 1989; to the putative CDC25B oncogene, was identi®ed in a Morgan, 1995). Tyrosine phosphorylation is ensured Burkitt lymphoma cDNA library. CDC25B2 diers from by the wee1 tyrosine kinase or related kinases that were CDC25B by a 14 residue insertion and a 41 residue ®rst identi®ed in yeast (Booher et al., 1993; Russell and deletion, which are both located in the amino-terminal Nurse, 1987) then in mammals (Igarashi et al., 1991; region of the protein, upstream of the catalytic domain. Watanabe et al., 1995). In ®ssion yeast, the CDC25 Examination of the genomic sequence revealed that tyrosine phosphatase catalyses the activating depho- CDC25B1 (formerly B) and CDC25B2 are splice sphorylation of the CDC2 protein kinase on tyrosine variants of the same gene. A third variant, CDC25B3, 15 and acts as an inducer of mitosis (Millar and that carries both the 14 and the 41 residue sequences was Russell, 1992; Russell and Nurse, 1986). In mammals, also identi®ed in the same cDNA library. All three three homologues of CDC25 called CDC25A, variants were detected in a panel of human primary CDC25B and CDC25C have been identi®ed so far. culture and cell lines, although at dierent levels. In All three tyrosine phosphatases are able to complement primary ®broblasts and in HeLa cells the CDC25B the premitotic arrest of a ®ssion yeast strain carrying a expression is cell cycle regulated, reaching a maximum thermosensitive mutation of the cdc25 gene (Galak- in G2-phase. In vitro, CDC25B1 phosphatase is slightly tionov and Beach, 1991; Nagata et al., 1991; Sadhu et more active than CDC25B2 and B3. However, episomal al., 1990). overexpression of the three CDC25B variants in ®ssion In ®ssion yeast, the activity of CDC25 is regulated yeast reveals that in vivo, CDC25B2 is largely more both at the mRNA and protein levels (Ducommun et active than either B1 or B3 (B24B34B1) both to al., 1990; Kovelman and Russell, 1996; Moreno et al., complement a thermosensitive S pombe CDC25 activity 1990). In HeLa cells, expression of CDC25B has been and to act as a mitotic inducer. Alternative splicing of reported to be low through out the cell cycle with a

CDC25B may therefore contribute to the control of cell sharp increase in G2, CDC25C is predominantly proliferation. expressed in G2 (Nagata et al., 1991; Sadhu et al., 1990) and CDC25A is abundant both at the mRNA

Keywords: cdc25B; cell cycle; protein phosphatase; and protein levels in late G1 (Jinno et al., 1994). splicing Phosphorylation of CDC25C by cdc2-cyclin B was shown and proposed to be part of the self-amplifica- tion mechanism of MPF at mitosis (Homann et al., 1993). Similarly, cdk2/cyclinE dependent phosphoryla- Introduction tion of CDC25A was demonstrated, indicating that a similar feed-back loop might regulate the progression The eukaryotic cell cycle is controlled by a family of in S-phase (Homan et al., 1994). Site directed cyclin-dependent kinases (CDKs) that regulate its key mutagenesis of the Xenopus CDC25C homologue was transitions. Their activities are regulated at dierent performed to identify the cdc2 phosphorylation sites. levels (Morgan, 1995). Firstly, as the name implies, This study pointed out ®ve serine and threonine association with a cyclin regulatory subunit is a general residues, elimination of which blocked the initiation feature of all members of the CDK family (Draetta, of M-phase (Izumi and Maller, 1993). These residues 1993). For instance, a complex formed by association are all located in the amino terminal part of the of CDC2 and cyclin B plays an essential role at the G2/ phosphatase and are mostly conserved in human M transition. It has recently been shown that the CDC25B and C. activity of CDK/cyclin complexes is also regulated by CDC25A and B have been shown to be associated speci®c inhibitors called cyclin-dependent kinase with the Raf1 protein kinase and with members of the inhibitors (CKI) that appear to play essential roles in 14-3-3 protein family. It has been suggested that these the transduction of extra- or intra-cellular signals such interactions might link the signal transduction machin- as DNA damage (Elledge and Harper, 1994) to the cell ery to cell cycle activation (Conklin et al., 1995; cycle machinery. Galaktionov et al., 1995). Another major regulatory mechanism is the phos- An important issue is whether the abnormal phorylation/dephosphorylation state of critical threo- regulation of positive cell cycle regulators such as nine and tyrosine residues that are located within the CDC25 tyrosine phosphatases might be involved in oncogenic processes. Several observations favour such Correspondence: B Ducommun a possibility. Overexpression of CDC25B has been Received 30 July 1996; revised 24 January 1997; accepted 27 January reported in two human cancer cell lines: a bladder 1997 carcinoma (T24) and SV40-transformed ®broblasts Splice variants of the CDC25B phosphatase VBaldinet al 2486 (GM637) (Nagata et al., 1991). Transformation of indicated in Figures 1 and 2a. First, a 42 bp insertion human primary ®broblasts with SV40 also caused an was found at nucleotide position 199 of the open important increase in CDC25B mRNA (Nagata et al., reading frame (Figure 1). This insertion encodes the 14 1991). More recently, in situ hybridisation studies amino-acid peptide SETPKSQVGTLLFR which con- performed on breast tumours have shown that tains a putative CDK phosphorylation site (underlined CDC25B was overexpressed in 32% of the neoplastic T, boxed in Figure 2b) that is conserved in CDC25 tissue samples (Galaktionov et al., 1995). In addition, sequences from a number of dierent organisms (Izumi CDC25B expression strongly correlated with micro- and Maller, 1993). Second, a 123 bp deletion of a vessel density, a negative prognostic feature, and was fragment spanning from nucleotide positions 418 to clearly associated with a high distant recurrence rate 541 in the CDC25B1 open reading frame led to the loss (Galaktionov et al., 1995). Finally, in rodent cells, of a 41 amino-acid peptide that also carries a potential human CDC25A and CDC25B, but not CDC25C were proline-directed phosphorylation site (Figure 2b) that is shown to cooperate with a mutant of Ras or with loss again conserved in a variety of CDC25 sequences. In of Rb1 in oncogenic transformation (Galaktionov et addition to these two major dierences, we found that al., 1995). in CDC25B2, the 3' untranslated region was shorter Here, we report the identi®cation of three splicing and totally diverged from CDC25B1 at nucleotide variants of the human CDC25B gene. Characterisation position 1978 where a stretch of polyA is located. An of the properties of these three proteins indicate that additional point dierence (T to A) was found in the alternative splicing is an important element in the upstream sequences at position 1962. The calculated regulation of their phosphatase activity in vivo. This molecular weight of CDC25B2 is 60 717 kDa (539 process might therefore play an important role in the residues), a value close to that of CDC25B1 control of cell proliferation. (63 402 kDa, 566 residues).

Results

Identi®cation of a new B-type CDC25 phosphatase In order to identify new human cdc2 regulatory proteins, a genetic screen was performed in the ®ssion yeast Schizosaccharomyces pombe. A strain carrying the cdc25-22 thermosensitive allele was transformed with a human cDNA library made from a Burkitt lymphoma cell line (see Materials and methods). Transcription was under the control of the strong constitutive S pombe ADH promoter. At non permissive temperature (35.58C) 20 clones were obtained that complemented the de®cient ®ssion yeast CDC25 activity and allowed growth and formation of colonies. Plasmid mapping and subsequent sequence analyses indicated that these 20 plasmids fell into three groups. Nine clones encoded the already identi®ed CDC25A tyrosine phosphatase (Galaktionov and Beach, 1991) and diered only by their 5'-length. Most of the clones lacked sequences encoding the amino terminal part of CDC25A, a domain that is only weakly conserved between dierent members of the CDC25 family and which is not necessary for catalytic activity (Xu and Burke, 1996). Four clones have not yet been characterised. The seven remaining clones were highly homologous to the CDC25B tyrosine phosphatase and encoded full length as well as 5'-truncated version of that cDNA. However, careful mapping and full DNA sequencing of one of the longest clones revealed a number of striking dierences. Because of its high homology to CDC25B, this clone will be subsequently called CDC25B2 and we suggest the name CDC25B1 be given to the ®rst B-type CDC25 clone identi®ed (Galaktionov and Beach, 1991; Nagata et al., 1991).

Sequence dierences between CDC25B1 and Figure 1 The nucleotide sequence of CDC25B2 cDNA. Below the nucleotide sequence (2002 bp) is the translation in standard CDC25B2 single letter amino acid code (539 amino acids). The initiating methionine is underlined. The 42 bp insertion is in bold and the Complete nucleotide and deduced amino-acid se- position of the 123 bp is marked with an open triangle (!). quences for CDC25B2 are shown in Figure 1. Terminating codon is marked with an asterisk (Accession number Sequence dierences between CDC25B1 and B2 are is Z68092) Splice variants of the CDC25B phosphatase VBaldinet al 2487 Genomic sequence analysis reveals that CDC25B1 and CDC25B2 are splicing variants a Examination of the nucleotide sequences surrounding the 42 bp insertion found in CDC25B2 revealed typical features of eukaryotic splicing sites (Horowitz and Krainer, 1994; Mount, 1982). As shown in Figure 3a, this additional sequence is preceded and followed by the CAG motif which is the classical consensus for the 5' half of a mammalian splicing donor site (Horowitz and Krainer, 1994). It is therefore very likely that alternative splicing of CDC25B pre-mRNA is responsible for the occurrence of the two CDC25B isoforms. The 42 bp insertion found in CDC25B2 is probably encoded by a short exon that is skipped during maturation of CDC25B mRNA. Using human genomic DNA as template and speci®c primers on both sides of the 42 bp fragment, we were unable to amplify by polymerase chain reaction any detectable fragment encompassing the CDC25B2-speci®c insertion. A likely explanation is that the intronic sequences that surround this small exon largely surpasses the polymerase chain extension capabilities. Nucleotide analysis of the 5' and 3' regions surrounding the 123 bp sequence that is deleted in CDC25B2 suggested the existence of acceptor/donor splice sequences (Horowitz and Krainer, 1994). We therefore used polymerase chain reaction to selectively amplify the CDC25B gene from human genomic DNA (see Methods for the sequence of the primers no. 7 and no. 8). The ampli®cation of a 186 bp cDNA region including the sequence that is deleted in CDC25B2 produced a 546 bp fragment when human genomic DNA was used as template. This genomic DNA fragment was fully sequenced (Figure 3b and accession b number X96436) and reveals the presence of two introns surrounding the CDC25B2 speci®c 123 bp exon. The length of these introns are 245 bp and 105 bp respectively and their junction with the exon sequences are in agreement with eukaryotic splice sites (Horowitz and Krainer, 1994; Mount, 1982). These results allow us to conclude that CDC25B1 and CDC25B2 are splice variants of the same gene (Figure 3c).

Evidence for a third CDC25B variant in the Burkitt lymphoma library Figure 2 Protein sequence comparison between CDC25B2 and others CDC25. (a) Alignment of CDC25B1 (top line) and Oligonucleotides that are speci®c for the 123 bp and CDC25B2 (bottom line) protein sequences showing the presence 42 bp insertion found in CDC25B1 and CDC25B2 of a 14 residue insertion and a 41 residue deletion within CDC25B2. (b) Alignment of several members of the CDC25 respectively were designed in order to amplify tyrosine phosphatase family in the region were the CDC25B2 selectively these two cDNA using polymerase chain insertion (top alignment) and deletion (bottom alignment) are reaction. Figure 4a shows a schematic view of the two occurring. Sequences were ®rst aligned using the pileup program CDC25B sequences with the position of the six (UWGCG package) and additional corrections were subsequently oligonucleotides that were used in the following made by eye with insertion of gaps to obtain the best alignment. The invariant residues are displayed in the consensus sequence experiment (see Material and methods for the (cons) with capital letters, and residues that are highly conserved complete sequences of the primers). These oligos were are displayed in small letters. The following sequences (in the ®rst tested with CDC25A, CDC25B1, and CDC25B2 range indicated with bracketes on the right side of the ®gure) used plasmids (Figure 4b). Oligonucleotide no. 2 together in this study are: H sapiens CDC25B (accession number M81934 (Galaktionov and Beach, 1991), H sapiens CDC25B2 (this study, with no. 4 were speci®c for the 42 bp insertion found accession number Z68092), H sapiens CDC25C (accession number in CDC25B2, as this pair of primers ampli®ed a M34065) (Sadhu et al., 1990), M musculus CDC25A (accession 1519 bp fragment from CDC25B2 cDNA but from number L16926) (Nargi and Woodford-Thomas, 1994), M auratus neither CDC25B1 nor CDC25A (Figure 4b, left panel). (hamster) CDC25 (accession number D10878), (Seki et al., Similarly, PCR with oligonucleotide no. 3 together unpublished), X laevis CDC25c (accession number M94264) (Kumagai and Dunphy, 1992), X laevis CDC25bb (accession with no. 4 was speci®c for the insertion present in number M96858) (Izumi et al., 1992), S scrofa (pig) (accession CDC25B1 but not in CDC25B2, since a 1328 bp number X78317) (Cui et al., unpublished) Splice variants of the CDC25B phosphatase VBaldinet al 2488

Figure 3 CDC25B1 and CDC25B2 are splice variants of the same gene. (a) Putative intron/exon organisation of the sequence surrounding the 42 bp CDC25B2 speci®c insertion. The length of the two introns has not been determined. (b) Intron/exon organisation of the region of CDC25B encompassing the 123 bp deletion found in CDC25B2. A 546 bp genomic fragment was ampli®ed by polymerase chain reaction using speci®c oligonucleotides (see Materials and methods) and human genomic DNA as template. Intron sequences of 245 and 105 bp respectively are found on both sides of the 123 bp CDC25B1 speci®c sequence (accession number X96436). (c) Schematic representation of the predicted CDC25B intron-exon organisation and the three CDC25B splicing variants. Introns 1 and 2 are of indetermined size. Introns 3 and 4 are 245 bp and 105 bp respectively. Exon 5 is not shown at the same scale as the rest of the sequence

fragment was ampli®ed from CDC25B1 cDNA but not fragment ampli®ed with pair no. 2 ± no. 4 was sub from CDC25B2 and CDC25A (Figure 4b, right panel). cloned and fully sequenced it was found to encode Oligonucleotides no. 2 and no. 3 in combination alternatively spliced form of a CDC25B cDNA that with no. 4 were subsequently used to check whether carries both the CDC25B1- and CDC25B2-speci®c CDC25B splice variants were present in the Burkitt insertions. This third variant encodes a 64 946 kDa lymphoma cDNA library from which CDC25B2 was protein (580 residues) and we suggest that it be obtained (Figure 4c). A 1328 bp fragment that designated CDC25B3. corresponds to a cDNA containing the CDC25B1- Only one 675 bp fragment was ampli®ed from the speci®c insertion was ampli®ed from the Burkitt library Burkitt cDNA library when primers no. 5 and no. 6 (Bk lane) using the no. 3 ± no. 4 pair of oligos (Figure (located on both sides of the CDC25B catalytic 4c). domain) were used (data not shown). This fragment As expected, a fragment migrating at the same size is identical in length to the ampli®cation product as CDC25B2 was also detected in the library and obtained with a CDC25B2 plasmid. It is therefore ampli®ed with the no. 2 ± no. 4 pair of oligos but, in likely that no other splicing variants are present in our addition, a very abundant and slightly larger fragment cDNA library for this part of CDC25B. was also ampli®ed. These two PCR products were further separated (Figure 4d) then the top band was mRNA expression of CDC25B variants in dierent cell puri®ed and subjected to further ampli®cation with types oligonucleotides no. 3 and no. 4. A fragment that migrated at the expected size for CDC25B1 was The relative expression of the CDC25B splicing detected in the reampli®cation product with this variants was examined in human primary ®broblasts combination of primers. This observation indicated (IMR-90) and in primary retinal pigment epithelial that a cDNA containing both the 42 bp and 123 bp cells (RPE) as well as in spontaneously immortalised insertions was present in the library. When the PCR and in transformed cells (Figure 5). RT ± PCR with a Splice variants of the CDC25B phosphatase VBaldinet al 2489 a a

b b B1 B2 B3 12345678 910 2-4 3-4 2,322 2,027

Figure 5 mRNA expression of CDC25B variants in dierent cell types. (a) A schematic view of the CDC25B sequence with the relative position of the two primers (no. 9 and no. 10) that were B2 B1 A B2 B1 A used to amplify CDC25B1, B2 and B3 in RT ± PCR studies. The sequence of the primers are provided in the Materials and methods section. (b) The three ®rst lanes are the ampli®cation products of a PCR reaction on puri®ed CDC25B1, B2 and B3 c d 2-4 3-4 2-4 3-4 cDNA using the primers no. 9 and no. 10. Molecular weight markers are from top to bottom: 738, 615, 492, 369 bp. RT ± PCR was performed as described in the Materials and methods section on RNA that were prepared from IMR90 human diploid ®broblasts (lane 1), PYC endometrial spontaneously immortalised cells (lane 2), MCF-7 human breast adenocarcinoma cells Bk B2 Bk B1 (lane 3), human keratinocytes (lane 4), MDAH-041 Li ± Fraumeni ®broblasts (lane 5), IGROV1 human ovarian carcinoma cells Figure 4 Detection of three CDC25B variants using polymerase (lane 6), NIH-OVCAR3 human ovarian carcinoma cells (lane 7), chain reaction. (a) Linear representation of CDC25B and HeLa cells (lane 8), RPE retinal pigment epithelial cells primary CDC25B2 cDNA indicating the relative position of the culture (lane 9), SARCCR2 human epithelioid sarcoma cell line oligonucleotides used in this study. (b) Plasmids cDNA encoding (lane 10) CDC25B1 `B1', CDC25B2 `B2' and CDC25A `A' were used as templates for ampli®cation with the indicated pairs of oligonucleotides. Sequences of the oligos and ampli®cation conditions are provided in the Materials and methods section. (c) A Burkitt after nocodazole treatment, CDC25B1 and B2 mRNA lymphoma cDNA library (Bk) and both CDC25B1 and B2 level was also more abundant. These results suggest plasmid cDNA were used as templates for ampli®cation with the that expression of these three variants is increased indicated pairs of oligos. (d) After ampli®cation of Burkitt during the G -phase of the cell cycle. lymphoma cDNA library with the no. 2 ± no. 4 pair of primers, 2 the top band (white arrow) was puri®ed and re-ampli®ed using We next investigated the cell cycle regulation of the no. 3 ± no. 4 primers CDC25B3 mRNA expression in human IMR90 ®broblasts. In these cells, the CDC25B3 mRNA was found to be present in exponentially growing cells but pair of primers no. 9 and no. 10, (see Materials and was virtually absent from cells harvested at con¯uence methods for the sequences) that selectively amplify all (Figure 6b). We examined the variation of expression three variants was used in this study. Figure 5a shows of this variant upon serum deprivation and re-addition a schematic view of the CDC25B sequence indicating (Baldin et al., 1993). The synchrony of the cell the relative position of the two primers. As shown in population entering S-phase was monitored in a Figure 5b, only the CDC25B3 variant was detected in parallel culture by BrdU incorporation (Figure 6c). human diploid IMR90 ®broblasts and in NIH- The expression of CDC25B3 mRNA was considerably OVCAR3 cells (lanes 1 and 7). In contrast, the three reduced in 72 h serum starved cells. However, after variants were present, although at dierent levels in serum re-addition (20%), a large increase in the level of most of the cell lines that were examined. However, in CDC25B3 mRNA was detected. The pattern of IGROV1 and in RPE cells, the CDC25B2 mRNA was accumulation of CDC25B3 mRNA showed a max- barely detectable. imum after 28 h when most of the cells had already

undergone S-phase and remained high in G2.In primary ®broblasts, the mRNA expression of Cell cycle regulation of CDC25B mRNA expression CDC25B therefore also appears to be cell cycle We ®rst investigated the expression of CDC25B regulated. mRNA variants in HeLa cells that were drug-arrested at dierent stages of the cell cycle. As shown in Figure Characterisation of the CDC25B variants phosphatase 6a, lovastatine treated cells were mostly arrested in the activities in vitro G1-phase. Hydroxyurea treated cells were arrested at

G1/S and were in S-phase by 4 h after drug removal. The CDC25B1-3 splicing variants mRNA encode

From nocodazole treated cells (mostly in G2/M), proteins that are 63 402 kDa, 60 717 kDa and mitosis-arrested cells were recovered in the media 64 946 kDa respectively. In vitro translated after plate shaking. The determination of cdc2 mRNA CDC25B1, B2 and B3 are all recognised by commer- expression was used as an internal control. The three cially available antibodies against CDC25B (Figure CDC25B variants mRNA were detected at every stage 7b). However the minor dierence in their apparent of the cell cycle. However, CDC25B3 (and B1 to a molecular weight and the presence of phosphorylated lower extend) was more abundant in nocodazole forms do not allow an accurate identi®cation of these treated cells than in G1- and S-phase. Furthermore, three proteins on Western-Blot of total cellular extracts in mitotic cells that were recovered by mitotic shake-o (data not shown). Splice variants of the CDC25B phosphatase VBaldinet al 2490

a c Exp Lov. HU HUr Noc Noc* B3 B1— B2 Cdc2

b Exp Conf 0h 8h 16h 20h 24h 28h 32h 615—

492—

Figure 6 Cell cycle regulation of CDC25B mRNA expression. (a) The expression of CDC25B variants was analysed by RT ± PCR (with primer no. 9 and no. 10 as in Figure 5) on HeLa cells arrested at various stages of the cell cycle. Cdc2 mRNA level was analysed as a control. Exponentially growing cells (Exp), cells treated during 24 h with 60 mM Lovastatine (Lov), cells treated during 20 h with 10 mM Hydroxyurea (HU) and rinsed and incubated with fresh media during 4 h (HUr), cells treated during 20 h with 250 ng/ml Nocodazole (Noc) and shaked-o to recover mitosis-arrested cells (Noc*). The cell cycle distribution was analysed by ¯ow cytometry after propidium iodine staining. (b) The mRNA expression of the CDC25B3 variant in human IMR90 ®broblasts was analysed by RT ± PCR on puri®ed RNA from cells that were exponentially growing (Exp), con¯uent (Conf), serum-starved (72 h (time 0 h), and restimulated with 20% serum (time 8 h, 16 h, 20 h, 24 h, 28 h and 32 h). (c) On a parallel culture of IMR90 ®broblasts, entry into S-phase was monitored by BrdU incorporation. The percentage of positive cells as judged by ¯uorescence microscopy is indicated

In order to characterise in vitro the phosphatase transformed in the ®ssion yeast strain SP587 carrying activity of the three CDC25B splicing variants, their the cdc25-22ts allele. In addition, two N-terminal cDNAs were cloned in a pGEX vector and fusion truncated CDC25B proteins that were identi®ed in proteins with glutathione S-transferase produced in E our initial screen were also examined. CDC25B-S2 and coli. Recombinant proteins were puri®ed by anity CDC25B-S6 encode truncated proteins that lack the chromatography on glutathione Sepharose, eluted with ®rst 274 and 405 amino-acids respectively (CDC25B1 free glutathione and dialysed. As shown in Figure 7c, numbering), S6 is therefore identical to the no. 67-2 the three CDC25B phosphatases were able to activate a clone identi®ed by Nagata and colleagues (Nagata et cdc2-cyclin B complex isolated from ®ssion yeast (see al., 1991) and encodes only the catalytic domain of Materials and methods) although B1 seemed to be CDC25B. The complementing activity of these slightly more ecient. Phosphatase activities of the constructs was tested after colony streaking and three variants were also measured according to transfer to non-permissive temperature (35.58C). As (Homann et al., 1993), with the modi®cation shown in Figure 8, while the empty vector was indeed described in the Materials and methods section, using unable to support growth, all CDC25B constructs pNPP as substrate. As shown in Figure 7d, for complemented the ®ssion yeast cdc25-22ts allele and concentrations of puri®ed phosphatase ranging from 1 allowed the formation of colonies. However, cells to 10 mg of protein, recombinant CDC25B1 was slightly containing CDC25B1 seemed to grow more slowly more active (1.5-fold) than CDC25B2 and CDC25B3. and thus this variant appears to be less eective in this assay than CDC25B2 and B3. In ®ssion yeast, overexpression of cdc25+ results in the appearance of Complementation of a thermosensitive allele of CDC25 the `wee' phenotype, that is cell division at reduced cell in ®ssion yeast length (Russell and Nurse, 1986). Accordingly, we Recently, it has been proposed that cytoplasmic measured the length of cells expressing the CDC25B accumulation of CDC25B is involved in the control variants in liquid culture at both 258C and at 35.58C of the initiation of mitosis in HeLa cells (Gabrielli et (Table 1). At permissive temperature, all constructs al., 1996). Both CDC25B1 (Nagata et al., 1991) and were able to induce mitosis at a smaller size than CDC25B2 (this work) have been isolated by virtue of control cells transformed with the empty vector. The their ability to complement the mitotic function of a eect was most striking in the case of CDC25B2, and thermosensitive allele of cdc25 in ®ssion yeast. To cells overexpressing CDC25B2 displayed an obvious compare the activity of the three splicing variants we reduction in cell size at mitosis (data not shown). At therefore investigated their ability to behave as inducer non permissive temperature, control cells were unable of mitosis and to complement a thermosensitive cdc25 to divide and rapidly displayed a terminal cdc7 allele. All three variants were sub-cloned under the phenotype. CDC25B2 and CDC25B-S6 (D405) trans- control of the constitutive ADH promoter and formed cells entered mitosis at a similar size at both Splice variants of the CDC25B phosphatase VBaldinet al 2491

a b

kDa

94 —

66 —

43 — B1 B2 B3

c d

Figure 7 In vitro characterisation of CDC25B protein phosphatases. (a) Schematic representation of the predicted CDC25B1, B2 and B3 variant proteins. (b) Immunodetection of in vitro translated CDC25B1 ± 3 splicing variants using antipeptide antibodies raised against the carboxy terminus of CDC25B. The apparent molecular weight of the three variants are larger than the calculated. (c) Activity of cdc2 protein kinase (in c.p.m., using histone H1 as substrate) after incubation or not with 3 mg of puri®ed GST, GST- CDC25B1, GST-CDC25B2 and GST-CDC25B3. Values are the average of duplicate determinations. (d) CDC25B1, B2 and B3 phosphatases activities. Increasing amount of CDC25B1 (&), B2 (^) and B3 (*) GST-fusion proteins as well as the GST alone (~) were incubated in the presence of 20 mM pNPP for 20 min at 308C (see Materials and methods). The reactions performed in triplicate were stopped and the absorbance at 410 nm were measured

25˚C 35.5˚C Figure 8 In vivo activity of the CDC25B splicing variants. Schizosaccharomyces pombe cells (strain SP587), carrying a temperature sensitive mutation of CDC25 (CDC25-22) were transformed with vector alone or plasmid encoding CDC25B1, B2, B3 and the N- terminal truncated forms S2 (D248) and S6 (D405) under the control of the constitutive ADH promoter. Colonies were streaked on selective media plates and were incubated at either 258C (left panel) or 35.58C (right panel) for 6 days temperatures, whilst CDC25B3 and especially signi®cantly less active. A truncated form of CDC25B CDC25B1 were generally longer and poorly comple- that encodes only the catalytic domain is also an mented the S pombe cdc25 defect. Together these eective CDC25 phosphatase. The in vivo function of results indicate that CDC25B2 is an eective tyrosine the regulatory N-terminal domain of the CDC25B phosphotase, however, CDC25B3 and CDC25B1 are phosphatase appears therefore to be modulated by the Splice variants of the CDC25B phosphatase VBaldinet al 2492 Table 1 Expression of human CDC25B splicing variants in a fission Kakizuka et al. (1992) is a CDC25B3 cDNA yeast Schizosaccharomyces pombe strain carrying a temperature- according to our nomenclature. sensitive allele of cdc25 Splicing variants of other cell cycle regulatory Average cell length at division (mm) +s.d. proteins have also been recently reported. Two cDNA 258C 35.58Ca `PISLRE', CDK-related proteins that are probable Controlb 16.7+2.3 cdcc splice variants of the same gene have been cloned CDC25B1 15.2+2.3 25.5+5.6 CDC25B2 13.2+2.8 16.8+3.9 (Brambilla and Draetta, 1994; Grana et al., 1994) and CDC25B3 15.0+2.1 21.3+7.2 a cyclin E splicing variant that lacks 49 amino-acids CDC25B D405 15.2+1.4 17.3+5.1 within the cyclin box and is therefore unable to bind Cell lengths of 50 cells with a complete septum were measured cdk2 has been found in several cell lines (Sewing et al., microscopically. aThe cells were grown for 6 h at non-permissive 1994). Alternate splicing resulting in the production of temperature before measurement. bControl cells are transformed with c a novel cyclin D1 transcript has also been identi®ed the empty vector. Cell cycle arrest. and appears to be modulated by A/G polymorphism located at the splice donor region of exon 4 (Betticher et al., 1995). However, this polymorphism is not speci®c for tumour cells, although it might be presence or the absence of the two short 14 and 41 associated with higher risk of non-small cell lung amino-acids domains that are encoded by the two cancer relapse. alternatively spliced exons that we have identi®ed. We have demonstrated here that alternative splicing play a role in the regulation of the CDC25B expression. What therefore are the mechanisms that Discussion regulate the alternative splicing of CDC25B (Green, 1991), what are the splicing factors involved (Horowitz In this article we report the identi®cation and and Krainer, 1994) and are they speci®cally dedicated characterisation of three CDC25B tyrosine phospha- to CDC25B pre-mRNA? These are some of the tase splicing variants. Functional complementation of a questions that remain to be investigated. CDC25 thermosensitive allele in ®ssion yeast led to the The list of cyclin-dependent kinases that have been identi®cation of a cDNA closely related to the identi®ed so far is probably far from complete (Pines, CDC25B sequence reported by Galaktionov and 1995). Therefore, the number of CDK-cyclin complexes Beach (1991) and Nagata et al. (1991). Following that we are currently considering, with respect to their genomic sequence analysis and ampli®cations with regulation through the activity of CDC25 phospha- various speci®c primers we reached the conclusion tases, is certainly largely underestimated. In our screen that both cDNAs were splicing variants of the same for suppressors of CDC25 thermosensitive mutation in CDC25B gene which we now designate CDC25B1 (the ®ssion yeast we have been unable to identify CDC25 original isolate) and CDC25B2 (this study). A third genes other than the already cloned CDC25A and variant that carries both domains that were detected in CDC25B. We did not retrieve a CDC25C cDNA, but CDC25B1 and CDC25B2 was also identi®ed by PCR this result is not surprising since it has been reported in the Burkitt lymphoma cDNA library, by RT ± PCR that CDC25C does not fully rescue a cdc25-22 allele at in human ®broblasts and in other cell lines. We strict non-permissive temperature (Jinno et al., 1994). designate this variant CDC25B3. A careful database Similarly, we did not retrieve CDC25B1 in our initial search in the EST tags sub-section revealed that a short screen and this is also likely to be due to its poor CDC25 cDNA fragment containing both the 42 bp ability to complement a cdc25-22ts allele at 35.58C. One insertion (CDC25B2 speci®c) and the 123 bp insertion should therefore consider the possibility that other (CDC25B1 speci®c) had previously been sequenced human CDC25-related genes might exist but, as for from a 73 day post-natal infant whole brain cDNA CDC25C, they are not as potent as CDC25A and B in library (Soares 1NIB). This sequence was deposited by the complementation of a ®ssion yeast mitotic CDC25 the IMAGE consortium (accession number H14343). defect. Other cloning strategies should therefore We performed a PCR ampli®cation using speci®c de®nitely be used to sort this question. primers and a partial sequencing of that EST cDNA Whether or not other CDC25 genes exist, alternative (data not shown) and found that it is identical to the splicing might be a major regulatory mechanism by CDC25B3 splicing variant that we have identi®ed. which to generate a larger family of CDC25 proteins. Our results indicate that at least three CDC25B These might share common features in their catalytic variants are derived from the alternative splicing of a and structurally important domains but be divergent in single CDC25 pre-mRNA. The putative intron-exon domains that are speci®cally involved in other speci®c structure of CDC25B in the region coding these functions such as the regulation of catalytic activity or splicing events is schematised in Figure 3c. Introns 1 substrate recognition. In this report we have shown and 2 are probably very large since we have not been that CDC25B exon skipping is responsible for the able to amplify them with PCR. On the contrary, presence or absence of two domains that are partially introns 3 and 4 are only 245 bp and 105 bp conserved in the human CDC25 gene family. These respectively. At this stage, one cannot exclude the exons are shown to be important for the in vivo possibility that the organisation of this gene is more regulation of CDC25B activity. We have clearly complex, although we have not yet detected other size demonstrated using the ®ssion yeast system that variants that may re¯ect the existence of additional CDC25B2 is more ecient than CDC25B3 and splice sites. The two alternatively spliced exons are CDC25B1, both as an inducer of mitosis and to probably conserved in other vertebrates since the rescue a thermosensitive allele of the CDC25 S pombe mouse CDC25B clone (M2-cdc25) reported by phosphatase. Our results suggest that the 14 amino- Splice variants of the CDC25B phosphatase VBaldinet al 2493 acid domain that is present in CDC25B2 had an necessary to re-investigate with speci®c probes some of activatory eect on the activity in vivo. In contrast, the the ®ndings previously reported. 41 amino-acid domain that is speci®c for CDC25B1, The overexpression of CDC25B phosphatase in appears to play an inhibitory role. In agreement with human breast cancer has recently been reported (see this model, the CDC25B3 phosphatase that carries introduction). That study was performed using a probe both the 14 and the 41 residue insertions displays a that could not distinguish between the three variants intermediate activity. It has been shown recently in that we have identi®ed. It is now also essential to HeLa cells that the CDC25B phosphatase is involved reconsider these results by investigating the speci®c in the control of the initiation of mitosis (Gabrielli et expression of the three variants. Similarly, the al., 1996). Since in ®ssion yeast CDC25 is only required demonstration of a potentially oncogenic role of at the G2/M transition our results obtained in this CDC25B in association with Ras emphasises the model organism are likely to re¯ect the real properties importance of our ®ndings. Strikingly, CDC25B3 is of the three human CDC25B variants. the only variant that is expressed in primary ®broblasts It has been suggested by Xu and Burke (1996) that whereas the three forms are present in immortalised the amino-terminus of CDC25B is not required for the ®broblasts. It is therefore crucial to determine whether interaction of the phosphatase with the cyclin A-cdk2 the transforming activity of CDC25B is common to all complex, and has a negative regulatory eect on the the three members of the CDC25B family or is speci®c CDC25 phosphatase activity (Xu and Burke, 1996). to one of the variants. The CDC25B gene was mapped We have also observed that a truncated version of by FISH to chromosome 20p13 (Demetrick and CDC25B that carries only the catalytic domain, is an Beach, 1993; Lane et al., 1993), an area which is not ecient tyrosine phosphatase that complements a known to present karyotypic alteration in tumours. cdc25-22ts allele in ®ssion yeast. However, our results However, mutations of splice sites contributes sig- indicate that the regulatory function of the N-terminal ni®cantly to human genetic disease by aecting pre- domain of CDC25B is more subtle and that the splice mRNA splicing. The study of the contribution of variant forms of CDC25B have dierent activities in CDC25B splicing in carcinogenesis is therefore an vivo. important issue. As already demonstrated for CDC25A and CDC25C, CDC25B phosphatases are phosphorylated upon incubation with cellular extracts (data not Materials and methods shown), but the identity of the kinase that is involved in that process as well as the consequences of this cDNA library construction phosphorylation on the two alternatively spliced exons The cDNA library used in this study is described in remain unclear. Alternatively, it is possible that the two (Superti-Furga et al., 1996). Brie¯y, that library was made domains encoded by the spliced exons are involved in from a Burkitt lymphoma B cell line called BJA-B. The protein ± protein interaction and may for instance complexity was 16106 original clones with a 1.5 kb associate regulatory partners that modify the catalytic average insert size. The S pombe vector pADH-X used activities of these phosphatases. Alternative splicing carries the ura4+ gene and the ARS1 from Schizosacchar- might also be an important mechanism for the omyces pombe. The cDNA was cloned under the control of regulation of stability, or for the intracellular localisa- the constitutive SpombeADH promoter. tion of speci®c CDC25B sub-types, or for the ®ne tuning of the substrate speci®city of that class of Yeast strains, transformation and screening enzymes toward the multiple CDK-cyclin complexes The S pombe strain used in this study was SP587 (h+, during the major transitions of the cell cycle. Further CDC25-22, leu1-32, ura4, ade6.210), a gift of D Beach. investigations are now needed to examine these Cells were grown in minimal supplemented media (BIO 101 dierent hypotheses. Inc). Transformation of the cells with the cDNA library On the basis of both expression studies and micro- was performed by electroporation using the Biorad Gene- injection experiments, it was thought that CDC25B pulser as described (Prentice, 1992). Transformants were plated on selective media and transferred at non permissive and C act as mitotic regulators in late G2 while CDC25A is likely to function earlier in the cell cycle temperature (35.58C) after an overnight incubation at and be involved in the regulation of the cyclin 258C. After 10 days, plasmids recovered from growing dependent kinases that regulate G /S transition (Jinno colonies were re-transformed to ensure the speci®city of 1 complementation, then mapped and sequenced. A total of et al., 1994; Nagata et al., 1991). It has been shown by 20 clones that conferred the complementing activity were Gabrielli and co-workers that in HeLa cells, CDC25B selected from approximately 4.76105 transformants, which is present in interphase but strongly increases in is about 50% of the total complexity of the library. prophase and is involved in the regulation of prophase Complementation assays were performed after transforma- microtubules nucleation (Gabrielli et al., 1996). tion of the SP587 strain with the pADH-X vector alone or Accordingly, we have shown here that the expression containing CDC25B splicing variant inserts. The clones S2 of CDC25B3 (and to a minor extend B1 and B2) are and S6 that encode N-terminal truncated (D274 and D405) forms of CDC25B were also used in that study. These partial cell cycle regulated with a maximum in G2-phase. As shown in Figure 7, the sizes of the three CDC25B clones were recovered from our initial screening. Colonies variants are very similar (566, 539 and 580 residues for were streaked in duplicate on selective media and grown at 258C or 35.58C. To determine cell size at mitosis, cells were B1, B2 and B3 respectively), and most of the studies grown in liquid at permissive temperature and transferred at have been performed using antibodies that are unable 35.58C for 6 h. A minimum of 50 cells with a complete to distinguish between the three splicing variants. In septum were measured microscopically and the average+s.d. light of our results and depending on the expression of was calculated. Cytological observations were performed as these three proteins in dierent cell types, it might be described (Moreno et al., 1991). Splice variants of the CDC25B phosphatase VBaldinet al 2494 Polymerase chain reaction Sequence analyses Primers sequences used in this study to selectively amplify Sequence editing, comparisons and alignments were CDC25B or CDC25B2 were: performed using the Best®t and Gap software from the Primer no. 1: 5'-TCCAGCCAGCCTTCTGC-3' UWGCG package. Primer no. 2: 5'-AGAGTCAGGTAGGGACC-3' Primer no. 3: 5'-CGTGCTTCGGAACATCAC-3' Expression and puri®cation of recombinant human CDC25B Primer no. 4: 5'-CATGGACAGCACCTGAG-3' Primer no. 5: 5'-GGGGATACTGAGGAAGATG-3' Human CDC25B1, B2 and B3 were cloned in the pGEX-2T Primer no. 6: 5'-ACAGTGGAAAATGAGGATG-3' vector (Pharmacia) and expressed in Escherichia coli (TG1) Primer no. 7: 5'-GGCTTCCATGGCATCTTG-3' as fusion with glutathione S-transferase. After isopropyl b- Primer no. 8: 5'-GTTTGCCATCAGACGCTTCC-3' D-thiogalactoside (IPTG) induction (0.4 mM ®nal concentration) for 4 h at 258C, the three fusion proteins were Primers no. 1 to no. 6 were used to amplify CDC25B1, B2 recovered as described (Smith and Johnson, 1988) by and B3 from the Burkitt lymphoma library (100 ng) and from anity chromatography on glutathione-Sepharose beads plasmid (10 ng). Primers no. 7 and no. 8 were used to (Pharmacia) and eluted by 10 mM glutathione in 50 mM amplify the genomic DNA that includes the sequence Tris-HCl, pH 8.0, 50 mM NaCl and 1 mM DTT. Eluted deleted in CDC25B2. proteins were extensively dialysed overnight against the PCR products were obtained after 40 cycles (948C, same buer in the absence of glutathione. Contamination 1.5 min; 608C, 1.5 min; 728C, 1.5 min) followed by 10 min of these preparations with the co-puri®ed 70 kDa dnaK E extension at 728C using a temperature cycler (Biometra). The coli gene product (Yu-Sherman and Golberg, 1992) was reactions were performed in 50 ml PCR buer containing 1 quanti®ed by scanning densitometry and taken in account unit of Taq polymerase (Extrapol I, Eurobio), 100 mM each to calculate the actual CDC25B protein concentration. dNTP and 200 nM each primers. The reaction products were separated on 1 or 2% agarose gel (Eurobio). Phosphatase assays Phosphatase assays using cdc2 as substrate were performed as following: a ®ssion yeast strain carrying the cdc25-22 RT ± PCR thermosensitive allele was transformed with the pREP1- Total RNA from 106 cells was prepared by using the Trizol cdc2HA vector encoding for the Spombecdc2 kinase reagent (Gibco BRL) following the manufacturer's instruc- tagged with the HA epitope under the control of the tions. RNA (1.5 mg) was ®rst incubated with random thiamine repressible promoter (Basi et al., 1993). Exponen- hexamer (10 mg/ml), heated 5 min at 958C and cooled tially growing cells were shifted during 4.5 h at 35.58Cto 5 min on ice before addition of 100 Units of Maloney block their cell cycle at G2/M and the cells were harvested. murine leukaemia virus reverse transcriptase (Promega), Cell pellets were resuspended in three volumes of buer A 2U/ml RNAse inhibitor and 250 mM dNTP. A control (50 mM Tris HCl pH 7.5, 250 mM NaCl, 5 mM EDTA, without addition of reverse transcriptase was always 0.1% triton X100, 1 mM dithiothreitol (DTT) supplemen- performed to ensure that ampli®cation did not proceed ted with proteases inhibitors: 5 mg/ml leupeptin, 2 mg/ml from residual genomic DNA. PCR was subsequently aprotinin, 20 mg/ml soybean trypsin inhibitor, 10 mg/ml performed as described above using 10 ml of the reverse tosyl lysine chloromethyl ketone (TLCK), 20 mg/ml tosyl transcription product per reaction. phenylalanine chloromethyl ketone (TPCK), 17 mg/ml The primers used to amplify simultaneously the three phenyl-methyl-sulfonyl ¯uoride (PMSF), 1 mg/ml pepsta- CDC25B splicing variants were the following: tin and phosphatases inhibitors: 50 mM NaF, 1 mM Na3VO4, 100 mM b-glycerophosphate). The cells were Primer no. 9: 5'-GCTTCCTCGCCGGTCACCAC-3' lysed by vortexing in the presence of glass beads and the Primer no. 10:5'-CCTGCGGCTGGCCCACTC-3' soluble proteins were recovered after three centrifugations (14 000 r.p.m., 10 min at 48C). The Cdc2-HA/cyclin B The primers used to amplify CDC2 were the following: complexes were immunoprecipitated from 250 mgof Forward primer: 5'-GGATCTACCATACCATTGACT-3' proteins using monoclonal anti-HA antibodies for 1 h at Reverse primer: 5'-CAAGCCATTTTCATCCAAGTT-3' 48C. After addition of rabbit polyclonal anti-mouse IgG (for 1 h at 48C) and centrifugation (5 min, 14 000 r.p.m. at 48C), the supernatants were incubated with 10 mlof Cell culture protein-A-Sepharose for 1 h at 48C. The immunoprecipitates were then washed twice in buer A without Human diploid lung ®broblasts (IMR-90), HeLa cells, phosphatases inhibitors and once in a phosphatase buer:

human spontaneously immortalised keratinocytes, and 50 mM Tris HCl pH 7.5, 10 mM MgCl2 and 1 mM DTT MDAH-041 Li ± Fraumeni ®broblasts (Agarwal et al., (Parker and Piwnica-Worms, 1992) then incubated for 1995), were cultured in Dulbecco's modi®ed Eagle medium 15 min at 308C in a ®nal volume of 80 ml with phosphatase (DMEM) supplemented with 10% heat inactivated foetal buer containing recombinant fusion protein GST- calf serum (FCS), 2 mM glutamin, penicillin (100 U/ml) CDC25B1, GST-CDC25B2, GST-CDC25B3 or GST. The and streptomycin (100 mg/ml) in a humidi®ed atmosphere immunoprecipitates were washed three times with 1 ml of

containing 5% CO2 at 378C. Human breast adenocarcino- buer A without phosphatases inhibitors, once with kinase

ma cells (cell line MCF-7, (Mazars et al., 1995)) were buer (50 mM Tris HCl pH 7.5, 10 mM MgCl2,1mMDTT, grown at 378C in RPMI 1640 medium supplemented with 100 mM ATP). The kinase activity on H1 histone was 5% FCS, 2 mM glutamin, insulin (1 mM) and gentamycin assayed by 10 min incubation at 308C, in a ®nal volume of (25 mg/ml). Human spontaneously immortalised endome- 25 ml kinase buer containing 0.5 mg/ml histone H1 and trium epithelial cells (PYC) and epithelioid sarcoma cell 5 mCi of [g32P]ATP. After SDS ± PAGE electrophoresis, the line (SARCCR2) (generous gifts of Dr S Jozan, Centre H1 histone proteins were excised from the gel and counted Claudius Regaud, Toulouse, France), IGROV1 human in Packard 16500 TR counter (Cerenkov eect). ovarian carcinoma cells (a gift of Dr J BeÂ nard, Villejuif) For phosphatase assays with para nitro-phenyl phospate and NIH-OVCAR3 human ovarian carcinoma cells as substrate, the bacterially-expressed GST-CDC25B1, B2 or (ATCC HTB161) were cultured in the same condition as B3 proteins were incubated in a ®nal volume of 200 ml with MCF7 cells. 20 mM para nitro-phenyl phosphate (Boehringer), 0.1% b- Splice variants of the CDC25B phosphatase VBaldinet al 2495 mercaptoethanol, 1 mM EDTA, 50 mM Tris-HCl pH 8.2 for Acknowledgements 20 min at 308C. The reaction was stopped through the We gratefully acknowledge N Chaudury for technical addition of 800 ml 0.2 M NaOH and the absorbance at assistance, A Valette, D Beach, J BeÂ nard JM, Darbon S, 410 nm was measured. Jozan N, Lamb A, Nagata and P Russell for the gift of cell lines, Spombestrains and plasmids, C Jessus and X Cayla for technical advice and N Lamb and J Hyams for advice In vitro translation, electrophoresis and Western-blot and comments on the manuscript. This work was The CDC25B1-B3 open reading frame were cloned in the supported by l'Institut National de la santeÂ et de la pET14b vector (Novagen) and the protein were in vitro Recherche MeÂ dicale (CRE93053), l'Association pour la translated in rabbit reticulocyte lysate using the TNT Recherche sur le Cancer, la Ligue Nationale Contre le transcription and translation kit (Promega) in the presence Cancer and la Ligue Contre le Cancer comiteÂ de la of unlabelled aminoacids. After electrophoresis on 10% Dordogne and comiteÂ de la Haute-Garonne. acrylamide SDS ± PAGE and transfer to nitro-cellulose, the CDC25B variants were detected by Western blot using rabbit polyclonal IgG raised against the carboxy terminus of mouse CDC25B (Santa Cruz, cat no. sc-326).

References

AgarwalML,AgarwalA,TaylorWRandStarkGR.(1995). Jinno S, Suto K, Nagata A, Igashari M, Kanaoka Y, Nojima Proc. Natl. Acad. Sci. USA, 92, 8493 ± 8497. H and Okayama H. (1994). EMBO J., 13, 1549 ± 1556. Baldin V, Lukas J, Marcote MJ, Pagano M and Draetta G. Kakizuka A, Sebastian B, Borgmeyer U, Hermans-Borg- (1993). Genes & Dev., 7, 812 ± 821. meyer I, Bolado J, Hunter T, Hoekstra MF and Evans Basi G, Schmid E and Maundrell K. (1993). Gene., 123, 131 ± RM. (1992). Genes & Dev., 6, 578 ± 590. 136. Kovelman R and Russell P. (1996). Mol. Cell. Biol., 16, 86 ± Betticher DC, Thatcher N, Altermatt HJ, Hoban P, Ryder 93. WD and Heighway J. (1995). Oncogene, 11, 1005 ± 1011. Kumagai A and Dunphy W. (1992). Cell, 70, 139 ± 151. Booher RN, Deshaies RJ and Kirschner MW. (1993). EMBO Lane SA, Baker E, Sutherland GR, Tonks I, Hayward N and J., 12, 3417 ± 3426. Ellem K. (1993). Genomics, 15, 693 ± 694. Brambilla R and Draetta G. (1994). Oncogene, 9, 3037 ± Mazars P, Barboule N, Baldin V, Vidal S, Ducommun B and 3041. Valette A. (1995). FEBS Lett., 362, 295 ± 300. Conklin DS, Galaktionov K and Beach D. (1995). Proc. Millar J and Russell P. (1992). Cell, 68, 407 ± 410. Natl. Acad. Sci. USA, 92, 7892 ± 7896. Moreno S, Klar A and Nurse P. (1991). In Gutrie C and Fink Demetrick DJ and Beach DH. (1993). Genomics, 18, 144 ± G (eds.), Guide to yeast genetics and molecular biology. 147. Academic Press: New York, Vol. 194, pp. 795 ± 826. Draetta G. (1993). TCB, 3, 287 ± 289. Moreno S, Nurse P and Russell P. (1990). Nature, 344, 549 ± Ducommun B, Draetta G, Young P and Beach D. (1990). 552. Biochem. Biophys. Res. Commun., 167, 301 ± 309. Morgan DO. (1995). Nature, 374, 131 ± 134. Elledge SJ and Harper JW. (1994). Cur. Op. Cell Biol., 6, Mount SE. (1982). Nuc. Acids Res., 10, 459 ± 472. 847 ± 852. Nagata A, Igarashi M, Jinno S, Suto K and Okayama H. Gabrielli BG, De Souza CPC, Tonks ID, Clarck JM, (1991). The new Biologist, 3, 959 ± 968. Hatward NK and Ellem KAO. (1996). J. Cell. Science, Nargi JL and Woodford-Thomas TA. (1994). Immunoge- 109, 1081 ± 1093. netics, 39, 99 ± 108. Galaktionov K and Beach D. (1991). Cell, 67, 1181 ± 1194. Parker LL and Piwnica-Worms H. (1992). Science, 257, Galaktionov K, Jessus C and Beach D. (1995). Genes & Dev., 1955 ± 1957. 9, 1046 ± 1058. Pines J. (1995). Biochem J., 308, 697 ± 711. Gould KL and Nurse P. (1989). Nature, 342, 39 ± 45. Prentice H. (1992). Nucleic Acids Research, 20, 621. GranaX,ClaudioPP,DeLucaA,SangNandGiordanoA. Russell P and Nurse P. (1986). Cell, 45, 145 ± 53. (1994). Oncogene, 9, 2097 ± 2103. Russell P and Nurse P. (1987). Cell, 49, 559 ± 567. Green MR. (1991). Annu.Rev.CellBiol.,1991, 559 ± 599. Sadhu K, Reed SI, Richardson H and Russell P. (1990). Homan I, Draetta I and Karsenti G. (1994). EMBO J., 13, Proc.Natl.Acad.Sci.USA,87, 5139 ± 5143. 4302 ± 4310. Sewing A, Ronicke V, Burger C, Funk M and Muller R. Homan I, Clarke P, Marcote MJ, Karsenti E and Draetta (1994). J. Cell Science, 107, 581 ± 588. G. (1993). EMBO J., 12, 53 ± 63. Smith D and Johnson K. (1988). Gene, 67, 31 ± 40. Horowitz DS and Krainer AR. (1994). TIG, 10, 100 ± 106. Superti-Furga G. Jonsson K and Courtneidge SA. (1996). Igarashi M, Nagata A, Jinno S, Suto K and Okayama H. Nature Biotech., In press. (1991). Nature, 353, 80 ± 83. Watanabe N, Broome M and Hunter T. (1995). EMBO J., Izumi T and Maller JL. (1993). Mol. Cell. Biol., 4, 1337 ± 14, 1878 ± 1891. 1350. Xu X and Burke SP. (1996). J. Biol. Chem., 271, 5118 ± 5124. Izumi T, Walker DH and Maller JL. (1992). Mol. Biol. Cell, Yu-Sherman M and Golberg AL. (1992). EMBO J., 11, 71 ± 3, 927 ± 939. 79.