Oncogene (1997) 14, 2485 ± 2495 1997 Stockton Press All rights reserved 0950 ± 9232/97 $12.00 Alternative splicing of the human CDC25B tyrosine phosphatase. Possible implications for growth control? Ve ronique Baldin1, Christophe Cans1, Giulio Superti-Furga2 and Bernard Ducommun1 1Institut de Pharmacologie et de Biologie Structurale du CNRS Universite 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).