INTRODUCTION

The Saccharomyces cerevisiae CDC14 gene was phenotypically defined by Pringle and Hartwell (1) as a temperature-sensitive mutant strain carrying the cdc14-1^ts allele. Like other temperature-sensitive CDC mutant strains, this strain grows normally at permissive temperatures (30 °C) but enters cell cycle arrest at non-permissive temperatures (37 °C). The cdc14-1^ts arrest occurs in late mitosis and is characterized by large-budded uninucleate cells with duplicated spindle plaques and extended spindles (1). Analysis of the CDC14 point of action suggests that CDC14 acts in late nuclear division, perhaps playing a role in preparation for DNA replication during the subsequent cell cycle (2). Recent evidence further supports a role for CDC14p in the assembly of origin of replication complexes (3). The CDC14 gene was isolated by complementation of the cdc14-1^ts arrest phenotype (4). The open reading frame was shown to specify a protein possessing a putative protein-tyrosine phosphatase (PTPase)¹ domain, with a unique PTPase active site signature motif, VHCKAGLGRTG (5).

Recently, a human gene product sharing sequence similarity with the yeast CDC14p was identified as a candidate tumor suppressor gene (6-8). This gene alternately named PTEN (phosphatase and tension homologue deleted on chromosome 10) or MMAC1 (mutated in multiple advanced cancers) is similar to the CDC14 gene throughout the phosphatase domain and shares a Ser/Thr-rich C-terminal domain with CDC14p. Deletion, frameshift, and point mutations at the PTEN/MMAC1 locus have been detected in several primary tumors and tumor cell lines, and these mutations would be expected to affect the phosphatase activity and/or substrate binding of the PTEN/MMAC1 protein (6-8).

This report describes the isolation of two human cDNAs encoding proteins with sequence identity to the yeast CDC14 gene product and demonstrates that these transcripts can functionally complement the yeast cdc14-1^ts arrest. Another distinct, but closely related, cDNA isolated in our screen was shown to encode the PTEN/MMAC1 protein. This gene does not complement the cdc14-1^ts arrest at 37 °C. While the human and yeast CDC14 proteins appear to form a family of putative phosphatases, the PTEN/MMAC1 protein is most similar to an open reading frame in yeast known as YNL128W. We demonstrate that both human CDC14p and PTEN/MMAC1 proteins have phosphatase activities similar to other dual specific phosphatases. The human CDC14p and PTEN/MMAC1 proteins are also localized to different subcellular compartments.

MATERIALS AND METHODS

S. cerevisiae CDC14p sequence (GenBank^TM accession number D55715) was used in BLAST searches of GenBank^TM non-redundant and dbEST data bases (9). Two human expressed sequence tag (EST) clones were found to encode either an exact match to the CDC14p PTPase active-site sequence, VHCKAGLGRTG (GenBank^TM accession number N92068, IMAGE Consortium clone 293403), or the closely related sequence, IHCKAGKGRTG (GenBank^TM accession number N48030, IMAGE clone 272092).

The insert for clone 293403 was radioactively labeled and used to screen both an oligo(dT)-primed human fetal spleen cDNA library and a random + oligo(dT)-primed human heart cDNA library (both from Stratagene) according to the manufacturer's instructions. Purified library clones, and all subsequent subclone constructs, were completely characterized by dideoxynucleotide sequencing of both strands. The longest subclones from each library shared 957 bp of overlapping sequence and comprised a full-length cDNA of 4151 bp, designated hCDC14A (GenBank^TM accession number AF000367). One heart library subclone contained a full-length 2630-bp insert comprising a unique hCdc14A-related cDNA, designated hCDC14B (GenBank^TM accession number AF023158). The complete coding sequence of hCDC14A (bases 400-2140) was amplified by polymerase chain reaction (PCR), using 5 primer (5-CGGGGTACCAAAAAAATGAAAGATCGGTTATATTTTG-3) and 3 primer (5-CACTCTCCACTCGAGTTAGTAATGAACATATTCAGACT-3), and inserted into the KpnI/XhoI sites of yeast expression vector pYES2 (Invitrogen) to form construct pLWL-A. A truncated hCdc14A coding sequence (nucleotides 400-1492) was amplified by PCR, using the 5 primer above and 3 primer (5-CACAGCGGCCGCCTAGGGAAAACTTACCTCTCC-3), and inserted into the KpnI/NotI sites of pYES2 to form construct pLWL-AS.

The insert for clone 272092 was radioactively labeled and used to probe an oligo(dT)-primed human fetal brain cDNA library (Stratagene) as above to obtain a subclone containing a novel 1940-bp insert. Subsequent sequence analysis revealed that this insert encoded the recently described PTEN/MMAC1 gene product (6, 7). The coding sequence of PTEN/MMAC1 was amplified by PCR as above, using 5 primer (5-AAAGGCAGAATTCGGAAAATGACAGCCATCATCAAAGAG-3) and 3 primer (5-GATGTACTCGAGTTAGACTTTTGTAATTTGTGTATG-3), and inserted into the EcoRI/XhoI sites of pYES2 to form pLWL-PTEN.

A human multiple tissue Northern blot (CLONTECH) was probed separately with random-primed radioactively labeled fragments of hCDC14-A coding sequence (nucleotides 795-1492), 3 UTR (nucleotides 2126-4151), and the 3 UTR of hCDC14-B (nucleotides 2013-2630) according to the manufacturer's instructions.

Human 293 and HeLa cell lines were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 100 µg/ml streptomycin. The coding sequences of hCDC14A, hCDC14AS, and CDC14B were amplified by PCR and subcloned as above, in-frame with the 3 end of the enhanced green fluorescent protein (EGFP) sequence of the pEGFP C-1 vector (CLONTECH). Prior to transfection, 1 × 10⁵ cells were plated onto coverslips in 6-well dishes and allowed to recover for 24 h. Cells were transfected with 2 µg of DNA/well using LipofectAMINE^TM (Life Technologies, Inc.) according to the manufacturer. The transfected cells were grown for an additional 48 h prior to slide mounting and analysis with a Zeiss Axioskop fluorescence microscope.

The coding sequences of hCDC14A and PTEN/MMAC1 were PCR subcloned as above into a modified version of the bacterial expression vector, pT7-7, containing a lac operator and an N-terminal His₆-tag (gift of James Clemens). Protein expression and purification was carried out as described (10), and kinetic analysis was performed according to Gottlin et al. (11).

S. cerevisiae strain YC27D (Mata, cdc14-1^ts) and plasmids containing the wild-type S. cerevisiae CDC14 gene and the corresponding inactivation variant, pJWC100 and pJWC102, respectively, were gifts of Dr. Grunstein (4). S. cerevisiae transformation was carried out as described (12). Strain YC27D was transformed with plasmids pJWC100, pJWC102, pYVH1, pYES2, pLWL-A, pLWL-AS, and pLWL-PTEN. Transformants were selected on SC uracil plates at room temperature. Single colonies from each transformation were transferred to SC uracil, +galactose plates and incubated at 30 °C for either 5 or 15 h. Plates were then transferred to 37 °C and incubated for 2-5 days before growth was assessed.

RESULTS AND DISCUSSION

Using the corrected S. cerevisiae CDC14 protein sequence (12), we performed a BLAST search in GenBank^TM non-redundant and dbEST data bases. Two human expressed sequence tag clones were found to encode peptides that shared sequence identity with S. cerevisiae CDC14p. Clone 293403 (IMAGE Consortium) encoded an exact match to the yeast CDC14 PTPase active site sequence, VHCKAGLGRTG, while clone 272092 (IMAGE Consortium) encoded a closely related sequence, IHCKAGKGRTG. Using clone 293403, we screened human cDNA libraries from both heart and fetal spleen and isolated several partial length subclones. Sequence analysis showed that two of the clones contained 957 bp of overlapping sequence, which together comprise a 4151-bp cDNA that we designated hCDC14A (entire composite nucleotide sequence deposited as GenBank^TM AF000367). hCDC14A encodes a single open reading frame of 580 amino acids (Fig. 1A), with a predicted Kozak initiator methionine codon (5-GAG TTC ATG A-3) (13) at nucleotide 400, preceded by several in-frame stop codons. The hCDC14A protein is 64% identical to the yeast CDC14p protein and contains a putative PTPase domain located between amino acids 60 and 330. Conservation of numerous Ser/Thr residues between human and yeast proteins in the C terminus raises the possibility that phosphorylation may play an important role in the regulation of these proteins. To determine the role of the C-terminal tail, we designed constructs encoding a truncated hCDC14A protein, containing amino acids 1-365 followed by a four amino acid extension (Val-Ser-Phe-Pro) and stop codon. The truncated protein, designated hCDC14AS, contains the entire hCDC14A PTPase but lacks all of the putative Ser/Thr phosphorylation sites found in the C-terminal tails of both hCDC14A and yeast CDC14p.

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Characterization of additional clones obtained from the human heart cDNA library resulted in the isolation of another CDC14-related clone. This cDNA encodes a protein of 454 amino acids which we designated hCDC14B (Fig. 1A). hCDC14B is 85% identical to hCDC14A protein, suggesting that it is encoded by a distinct, but closely related gene. The extreme NH₂ terminus does not align well with either the yeast CDC14p or human CDC14A protein. Although the PTPase domain of hCDC14B is highly conserved among the other proteins, the C terminus is not rich in Ser/Thr residues as seen in the yeast CDC14p and hCDC14A proteins. Despite the lack of overall similarity, the C terminus of hCDC14B shares three regions of identity with the yeast CDC14p and hCDC14A protein, including the T(I/V)LR sequence which is followed by a stop codon in the hCDC14B protein (Fig. 1A).

Using clone 272092, we screened a human fetal brain cDNA library and obtained a subclone encoding a protein of 405 amino acids which was identical to the recently identified protein encoded by the putative tumor suppressor gene, PTEN/MMAC1 (6-8). Previous studies have noted the similarity between PTEN/MMAC1 protein with the yeast CDC14p and another yeast open reading frame YNL128W (GenBank^TM Z71404) (6, 7). Our analysis of the PTEN/MMAC1 protein suggested that it is more closely related to YNL128W as opposed to CDC14p (Fig. 1B). The yeast and human proteins share 45% identity which extends their entire length. Although YNL128W and PTEN/MMAC1 can be aligned with the CDC14p active site domain, using either primary sequence or secondary structure prediction,² both the N and C termini of PTEN/MMAC1 have only a limited number of amino acid sequence identities with the corresponding regions of yeast or human CDC14 gene products. Collectively, our results suggest that there are two related, but distinct, families of human and yeast phosphatases corresponding to CDC14 and YNL128W/PTEN/MMAC1, respectively.

Hybridization of hCDC14A to human poly(A⁺) RNA demonstrated bands of ~4.4 and 1.8 kb in all the tissues, with the strongest signals in kidney, heart, and skeletal muscle (Fig. 2A). Additional bands at ~7.0 and 2.8 kb were present in muscle, kidney, and heart. The unique 3 untranslated region of hCDC14A hybridized solely to the 4.5-kb RNA, indicating that it corresponds to the hCDC14A transcript (not shown). The other hybridizing bands may represent alternatively spliced hCDC14A transcripts or RNAs for closely related proteins. It is unlikely that these RNAs correspond to CDC14B since the 3 UTR fragment of this cDNA hybridized to an RNA of 6 kb (Fig. 2A).

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To determine the subcellular localization of hCDC14 proteins, we prepared fusion constructs expressing the enhanced green fluorescent protein (EGFP) in-frame with hCDC14 coding sequences. The localization of human CDC14 fusion proteins in 293 cells is shown in Fig. 2B. While the control EGFP protein was present throughout the entire cell, the EGFP-hCDC14A fusion protein was specifically localized to the nucleus. The EGFP-hCDC14AS protein was also found in the nucleus (Fig. 2B), demonstrating that the Ser/Thr-rich domain of hCDC14A does not play a role in the subcellular localization of the full-length protein. EGFP-hCDC14B protein also localized specifically in the nucleus. Nuclear localization of the fusion proteins was also seen in HeLa cells (not shown), indicating that these results were not cell type-specific. The nuclear localization of CDC14 proteins contrast with the reported cytoplasmic localization of PTEN/MMAC1 (8).

Human CDC14A and PTEN/MMAC1 have been described as putative phosphatases based upon their similarity to PTPase active site consensus sequences. We show that purified recombinant hCDC14A and PTEN/MMAC1 proteins are both phosphatases as measured by the hydrolysis of the artificial PTPase substrates, p-nitrophenyl phosphate (pNPP) and 3-O-methylfluorescein phosphate (OMFP) (Table I). The k_cat/K_m values of both hCDC14A and PTEN/MMAC1 show a 20-fold enhancement for OMFP over p-nitrophenyl phosphate, indicating that OMFP is the better substrate for both CDC14A and PTEN/MMAC1. Previous work has shown that OMFP is a preferred substrate for dual specific PTPases as opposed to protein-tyrosine phosphatases, which showed little or no substrate preference (11) (Table I). The k_cat values of hCDC14A and PTEN/MMAC1 are in the range of 10⁴ s¹, which is similar to those previously reported for dual specificity phosphatase, rVH6 (14). Although hCDC14A and PTEN/MMAC1 have relatively low activities toward these artificial substrates, a similar correlation of low activity using artificial substrates has also been seen with a distantly related phosphatase, p80^cdc25 (11).

Table I. Kinetic constants of dual specificity phosphatases using artificial substrates Dual specific PTPase Substrate Km kcat kcat/Km -Fold increasea µM s1 µM1·s1 rVH6b OMFP 130 0.014 110 138 pNPP 10,000 8.0  × 103 0.8 hCDC14A OMFP 62 7.5  × 104 12 20 pNPP 1300 7.5  × 104 0.6 PTEN/MMAC1 OMFP 110 2.4  × 104 2.1 21 pNPP 4500 4.4  × 104 0.1 a Calculated as (kcat/Km)OMFP/(kcat/Km)pNPP. pNPP, p-nitrophenyl phosphate. b Obtained from Gottlin et al. (11).

We set out to determine whether human CDC14 proteins were functionally equivalent to their putative homologues in yeast, by complementation in mutant yeast strains. To this end, we disrupted the YNL128W gene by homologous recombination in yeast, but could find no detectable phenotypic abnormalities associated with this alteration (not shown). However, S. cerevisiae strain YC27D is a haploid strain carrying the cdc14-1^ts allele. YC27D grows normally at 30 °C, but enters a cell cycle arrest at 37 °C. The complementation of this arrest by extragenic wild-type CDC14 was the basis for the original isolation of the yeast gene (4). Sequence similarity between the yeast and human CDC14 proteins suggests that the hCDC14A proteins may be capable of providing CDC14 activity to a yeast CDC14 mutant strain. To test this idea, we placed hCDC14A, hCDC14AS, and PTEN/MMAC1 open reading frames under the control of the pGAL1 promoter in the pYES2 yeast expression plasmids. Yeast wild type CDC14 under its own promoter (pJWC100), a His cassette-disrupted variant of this plasmid (pJWC102), and the empty pYES2 vector were used as controls. As an additional control for specificity, we also expressed the known dual-specific PTPase YVH1 in pYES2 (15). Upon transformation into strain YC27D, plasmids expressing hCDC14A and hCDC14AS specifically rescue the cdc14-1^ts cell cycle arrest (Fig. 3A). As expected, the arrest was also rescued in the presence of pJWC100, but not pJWC102 or pYES2 alone. Neither PTEN/MMAC1 nor YVH1 were able to complement the cdc14-1^ts arrest under these conditions. Collectively, these results suggest that hCDC14A is the functional homologue to the yeast CDC14p. It is likely that the C-terminal Ser/Thr-rich domain does not play a role in this aspect of CDC14 function, since the C-terminal truncated construct, hCDC14AS, efficiently complements the mutant strain. We observed that PTEN/MMAC1 was partially capable of complementing the cdc14-1^ts arrest if the transformed strain is first allowed to grow at permissive temperature for an extended time (15 h) prior to shifting to 37 °C (Fig. 3B). These results reinforce the idea that CDC14 and PTEN/MMAC1 are two closely related, but distinct, families of proteins.

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We have established that both human CDC14A and PTEN/MMAC1 are protein-tyrosine phosphatases. Both proteins have relatively low, but easily characterizable, k_cat and K_m constants obtained using two artificial substrates (Table I). The structural and functional equivalence of the yeast and human CDC14 PTPases also suggests that the human proteins may play an essential role in controlling mammalian cell cycle events. Although the natural substrates of yeast CDC14p have not been characterized, their homologues (3) are logically the best candidates for testing CDC14 function in humans. Likewise, due to the sequence similarity between PTEN/MMAC1 and YNL128W, the yeast protein may also serve as a model for PTEN function in humans.