©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Cloning and Characterization of a Saccharomyces cerevisiae Gene Encoding the Low Molecular Weight Protein-tyrosine Phosphatase (*)

(Received for publication, April 19, 1995)

Kirill Ostanin Christine Pokalsky Shuishu Wang Robert L. Van Etten (§)

From the Department of Chemistry, Purdue University, West Lafayette, Indiana 47907-1393

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The low molecular weight protein-tyrosine phosphatase (low M(r) PTPase) is an 18-kDa cytoplasmic enzyme of unknown function that has been previously found in several vertebrates. Using an oligonucleotide probe derived from the active site sequence of the mammalian low M(r) PTPases, a Saccharomyces cerevisiae gene that encodes a homolog of this enzyme was cloned by low stringency hybridization. This gene, LTP1, together with a neighboring gene, TKL1, is shown to be located on the right arm of chromosome XVI. The deduced amino acid sequence of its 161-amino acid residue product shows a 39% average identity with that of the mammalian enzymes. The yeast Ltp1 protein was expressed in Escherichia coli, purified to homogeneity, and shown to possess PTPase activity. The recombinant Ltp1 efficiently hydrolyzes phosphotyrosine and a phosphotyrosine-containing peptide, Tyr-fyn, but it shows low activity toward phosphoserine and phosphothreonine. The catalytic activity of Ltp1 toward a number of substrates was approximately 30-fold lower than the corresponding values measured for the bovine low M(r) PTPase. However, the yeast enzyme was markedly activated by adenine and some purine nucleosides and nucleotides, including cAMP and cGMP. In the case of adenine, the activity of Ltp1 was increased by approximately 30-fold. The high degree of evolutionary conservation of the low M(r) PTPases implies a significant role for this enzyme. However, neither the disruption of the LTP1 gene nor an approximately 10-fold overexpression of its product in S. cerevisiae caused any apparent phenotypic changes under the conditions tested. No proteins related to Ltp1 could be detected in extracts of the ltp1 null mutant, either by immunoblotting or by gel-filtration analysis accompanied by extended kinetic assays, consistent with the conclusion that LTP1 is the only low M(r) PTPase-encoding gene in S. cerevisiae.


INTRODUCTION

The important role of protein-tyrosine phosphatases (PTPases, EC 3.1.3.48) (^1)in the control of cell proliferation and differentiation is well documented in recent reviews (1, 2) . The regulation of these processes involves cascades of reversible protein phosphorylation events occurring on tyrosyl and/or seryl/threonyl residues that are induced by diverse extra- or intracellular signals. PTPases take part in such signaling cascades either by directly providing a signal via the dephosphorylation of one or more downstream target proteins (3, 4, 5) or by counteracting the activities of signal-transducing protein-tyrosine kinases.

The broad family of protein-tyrosine phosphatases includes a wide variety of both transmembrane and intracellular enzymes(6, 7) . Despite a significant diversity of primary sequences, the vast majority of PTPases contain one or two copies of a conserved catalytic domain of approximately 240 residues(1) . A signature motif, (I/V)HCXAGXXR(S/T)G, including catalytically essential cysteine and arginine residues, is located in its C-terminal region. At least two subfamilies, VH1-like and cdc25 dual specificity protein phosphatases, have been characterized whose sequence homology with other PTPases is restricted to approximately 30 residues surrounding the active site motif(1) . In addition, a nuclear protein-tyrosine phosphatase PRL-1 was recently identified that lacks any homology beyond the active site motif to the previously identified PTPases(8) . These findings indicate the existence of distantly related classes of PTPases, which may possibly be products of convergent evolution.

The low molecular weight protein-tyrosine phosphatase (low M(r) PTPase) represents a striking example of structural diversity among PTPases. This is a 157-amino acid residue cytoplasmic protein that has been isolated from a variety of mammalian tissues(9, 10, 11, 12, 13, 14, 15) . The cDNAs encoding the bovine and human low M(r) PTPases as well as the corresponding human gene have been cloned(16, 17, 18) . (^2)At least in some organisms, this enzyme is represented by two isoenzymes generated by alternative mRNA splicing(17, 18, 19) . Two mammalian isoenzymes share the conserved N- and C-terminal sequences, separated by a variable region of 34 residues(17, 18, 19, 20, 21, 22) . Phosphatases showing similar molecular masses and some common enzymatic properties are reportedly present in lower vertebrates, although no sequence information is available for them(23, 24, 25) . Initially identified as acid phosphatases, the low M(r) PTPases have since been shown to exhibit catalytic activity in vitro toward phosphotyrosyl, but not phosphoseryl or phosphothreonyl, proteins and peptides(11, 14, 26, 27) . Although these enzymes do not possess an overall sequence similarity to other families of PTPases, they do share a common active site motif CXXXXXR, which is a part of PTPase signature motif. This sequence includes a cysteine residue responsible for the transient formation of a phosphocysteinyl covalent intermediate in the course of enzymatic reaction and an arginine involved in the binding of a phospho group of substrate(16, 28, 29) . Recent crystallographic studies have demonstrated the near identity of the three-dimensional structures of the phosphate-binding loops of bovine low M(r) PTPase (BPTP)(30, 31) , human placenta PTP1B(32) , and Yersinia PTPase(33) . Thus, the low molecular weight PTPases represent a novel class of protein-tyrosine phosphatases.

Despite the fact that the structure and catalytic mechanism of the low M(r) PTPase has been well studied, little is known about the physiological role of this protein, beyond a report that overexpression of the bovine liver enzyme in murine fibroblasts caused a reduction in the level of autophosphorylation of the PDGF receptor, thereby inhibiting a mitogenic response to PDGF(34) . To better understand the role of the low M(r) PTPase in the cell, we focused our efforts on the search for a homologous gene in a genetically tractable organism, the budding yeast Saccharomyces cerevisiae.

When this study was initiated, no low M(r) PTPase gene or corresponding enzyme activity from the organisms other than vertebrates had been described. Using low stringency hybridization we have isolated a S. cerevisiae gene, designated LTP1, whose product shows 39% average sequence identity to the mammalian enzymes. After the present sequence had been deposited in GenBank® (accession number U11057), a report appeared which described a homologous gene (stp1) from the evolutionarily divergent fission yeast Schizosaccharomyces pombe(35) . Although the stp1-encoded protein was not directly demonstrated to be an active phosphatase, it was cloned as a multicopy suppressor of a mutation in the cdc25 gene that encodes a dual specificity phosphatase involved in mitotic control(35) . In addition to the cloning and chromosomal assignment of the LTP1 gene, we report here the heterologous expression and purification of the Ltp1 protein, characterize its phosphatase activity toward a number of substrates, including a phosphotyrosine-containing peptide, and describe the marked activation of the enzyme by purines. Finally, an ltp1 null mutation is constructed and tested for phenotypic consequences.


EXPERIMENTAL PROCEDURES

Yeast Strains and Plasmids

The S. cerevisiae strains used in the present study are listed in Table 1. Yeast culture growth, sporulation, and tetrad analysis were carried out using standard protocols(36) . Yeast transformation was performed by the lithium acetate method(37) . The S. cerevisiae cDNA library in the vector ACT was kindly provided by Dr. S. Elledge (Baylor College of Medicine, Houston, TX), the Saccharomyces cerevisiae genomic library in the plasmid YCp50 was a gift of Dr. S. Liebman (University of Illinois at Chicago, Chicago, IL), whereas the plasmid pBTM116 was obtained from Dr. S. Fields (State University of New York at Stony Brook, Stony Brook, NY). The - and cosmid-clone grid filters representing the S. cerevisiae genome were kindly provided by Dr. M. Goebl (Indiana University Medical School, Indianapolis, IN).



Screening of the Yeast Libraries and DNA Sequencing

A degenerate 44-mer oligonucleotide, 5`-TT(A/G)TT(T/C)GT(T/C)TG(T/C)TT(A/G)GG(T/C)AA(T/C)AT(T/C)TG(T/C)AG(A/G)TC(T/C/A)CC(A/T)AT(C/T)GC(C/T)GA-3`, was used as a probe to isolate the yeast low M(r) PTPase cDNA. This oligonucleotide was derived from the sequence of residues 9-23, i.e. LFVCLGNICRSPIAE, which are identical in all known mammalian low M(r) PTPases. Escherichia coli strain LE392 was transfected with a S. cerevisiae cDNA library generated in the ACT vector, followed by plaque screening with 5`-end P-labeled probe according to standard protocols (38) . Duplicate nitrocellulose membrane plaque lifts were washed with 2 SSPE (0.2 M NaH(2)PO(4), pH 7.4, 3 M NaCl, 20 mM EDTA), 0.5% SDS during 1 h at 55 °C and subjected to prehybridization and hybridization for 4 and 12 h, respectively, at 50 °C in 5 SSPE, containing 2.5 Denhardt's solution, 0.1% SDS, and 0.1 mg/ml denatured salmon sperm DNA. Following hybridization, the membranes were subsequently washed twice for 30 min with 2 SSPE containing 0.1% SDS at room temperature, the same solution at 50 °C for 3 min, and finally with 0.2 SSPE, 0.1% SDS at room temperature for 2 h. The dried membranes were exposed to x-ray film for 48 h with an intensifying screen. The positive phage clones were converted to the plasmid form (39) and subjected to sequence analysis. DNA sequencing was performed by the chain termination method of Sanger using Sequenase, Version 2 (U. S. Biochemical Corp.). Isolated LTP1 cDNA was labeled by a random priming method (U. S. Biochemical Corp.) and used as a probe to screen a S. cerevisiae genomic library in the plasmid YCp50. The library was amplified in E. coli strain HB101. The hybridization of colony lifts onto nitrocellulose was performed in 5 SSPE, 2.5 Denhardt's solution, 0.1% SDS, 0.1 mg/ml denatured salmon sperm DNA at 60 °C for 16 h. The filters were washed with 2 SSPE, 0.1% SDS at the same temperature for 30 min prior to autoradiography.

Plasmid Construction

An E. coli expression vector providing for the synthesis of Ltp1 under control of the T7 polymerase promoter was constructed based on the plasmid pT7-7(40) . The coding sequence of the LTP1 gene was amplified by a polymerase chain reaction using the plasmid YCp50(LTP1)-5 and two 26-mer primers, 5`-AACACGCATATGACAATTGAAAAACC-3` and 5`-TTTTACTCGATGTAGACCGCACCATC-3`. As a result, an NdeI restriction site including the initiation codon of LTP1 was generated. Following treatment with NdeI and NheI, the resulting 487-bp fragment was subcloned into the NdeI, XbaI-digested plasmid pUC18. The resulting plasmid was digested with NdeI and BamHI, and a fragment carrying the coding sequence of LTP1 was isolated and subsequently ligated with the plasmid pT7-7 that had been treated with the same restriction enzymes. This procedure gave rise to the expression vector pT7-7(LTP1).

To construct an E. coli expression vector providing for the synthesis of the Ltp1 fused to the glutathione S-transferase (GST), a 0.7-kb HpaI-XhoI fragment from pL10 was subcloned into pGEX-KG (41) that has been treated with EcoRI, Klenow fragment, and XhoI. The resulting plasmid pGST-LTP1 contained the coding regions of glutathione S-transferase and the LTP1 genes separated by a sequence encoding a thrombin cleavage site, a glycine kinker, and including 9 bp of the 5`-noncoding region of LTP1.

A plasmid for the disruption of the LTP1 gene was constructed as follows. A 3.5-kb BglII-EcoRI fragment from the plasmid YCp50(LTP1) was subcloned into BamHI-EcoRI-digested pBR327 to generate pBR327(LTP1). A 1.7-kb BamHI fragment containing the S. cerevisiae HIS3 gene was subcloned into the same site of pUC18. The fragment of the same size was released by treatment of the resulting plasmid with SmaI and XbaI and ligated with HpaI, NheI-digested pBR327(LTP1). As a result, a fragment of the LTP1 gene, including 9 bp of the 5`-noncoding region, the entire coding sequence, and 1 bp of 3`-noncoding sequence, was replaced by the HIS3 gene. The 3.8-kb PvuII-EcoRI fragment carrying the disrupted ltp1::HIS3 gene was purified and used for yeast transformation.

A vector for the overexpression of the LTP1 gene in S. cerevisiae was constructed based on the plasmid pBTM116 which carried the lexA gene inserted between the promoter and transcriptional terminator of the ADH1 gene. To replace lexA by LTP1, this plasmid was subjected to partial digestion with HindIII followed by blunt-ending with Klenow fragment, digestion with BamHI, and ligation with a HpaI-BglII fragment of the LTP1 cDNA. As a result, a plasmid pALA1 was generated.

Heterologous Expression and Purification of the Recombinant Ltp1

The GST-Ltp1 fusion protein was expressed in E. coli strain HB101 transformed with the plasmid pGST-LTP1 and purified using affinity chromatography on a glutathione-agarose column (41) . The Ltp1 protein was released by on-column thrombin cleavage. The nonfusion Ltp1 was expressed as follows. An overnight culture of E. coli strain BL21(DE3) bearing the plasmid pT7-7(LTP1) was diluted 100-fold with 2 liters of LB medium supplemented with ampicillin (50 mg/liter) and grown at 37 °C. When the culture reached the optical density of 2.0 at 600 nm, isopropyl-beta-D-thiogalactopyranoside was added up to a final concentration of 0.2 mM, and the culture was grown for an additional 4 h. The recombinant protein was purified by chromatography on SP-Sephadex C-50 column followed by gel-filtration on Sephadex G-50 using a procedure developed for the recombinant bovine low M(r) PTPase(16) .

Immunoblotting

Immunodetection of Ltp1 in the yeast cell extracts was performed as follows. The cells (total optical density at 600 nm of 5.0) were boiled in 200 µl of SDS-polyacrylamide gel electrophoresis loading buffer for 10 min and cell debris was removed by centrifugation at 12,000 g for 5 min. The samples (25-50 µl) were electrophoresized in SDS-12.5% polyacrylamide gel, and the proteins were electrotransferred onto a nitrocellulose membrane at 50 V for 6 h. The membrane was blocked in phosphate-buffered saline (10 mM sodium phosphate, pH 7.2, 150 mM sodium chloride), containing 1% bovine serum albumin and 0.1% Tween 20, incubated with 5000-fold diluted Ltp1 rabbit antiserum and then with 20,000-fold diluted goat anti-rabbit immunoglobulins conjugated with horseradish peroxidase (Pierce) in the blocking solution. Each incubation was performed at room temperature for 1 h, followed by washing with the same solution without bovine serum albumin. The immunoreactive bands were visualized using an ECL chemiluminescence detection kit (Amersham Corp.).

Enzyme Activity Assay

The enzymatic assays were performed in 100 mM sodium acetate, pH 5.0, 1 mM EDTA at the ionic strength of 0.15 M which was adjusted using sodium chloride. The rate of substrate dephosphorylation was established by measuring the release of inorganic phosphate using a Malachite Green assay procedure (42) or the liberation of p-nitrophenol that was determined from the absorbance at 405 nm. A phosphotyrosine-containing peptide, Tyr-fyn (Ac-FTATEPQY(P)QPGENL-COOH), derived from the autophosphorylation site of the protein-tyrosine kinase fyn was a gift from Dr. Terry Higgins (Sterling Drug Co., Inc.).

Fluorescence Spectral Measurements

The effect of adenine on the tryptophan fluorescence spectra of the Ltp1 protein was studied at room temperature using a Hitachi F-2000 fluorescence spectrophotometer. The protein was present at a final concentration of 0.12 mg/ml in 100 mM sodium acetate, pH 5.0, 1 mM EDTA, and adenine concentrations ranged from 0 to 10 mM. The ionic strength of the solution was maintained at 0.15 M using sodium chloride. The sample, contained in a NSGT-52 dual path length cell (1 cm 4 mm), was excited at a wavelength of 300 nm with a bandwidth of 10 nm. Emission was observed at a wavelength range from 310 to 400 nm with a bandwidth of 10 nm. K values were determined using the tryptophan fluorescence intensity changes observed at 345 nm(43) . Emission spectra were corrected using NIST SRM 1931 standards.


RESULTS

Cloning of a S. cerevisiae Gene Encoding a Low M(r)PTPase

Low stringency hybridization was employed to search for a S. cerevisiae gene encoding a homolog of the mammalian low M(r) PTPases. A degenerate 44-mer oligonucleotide based on an active site sequence LFVCLGNICRSPIAE, which is identical in all of the known sequences of mammalian enzymes (16, 17, 21, 22) and includes three catalytically important residues, namely, Cys, Cys, and Arg^18(28, 29) , was used as a probe to screen the S. cerevisiae cDNA library. This oligonucleotide hybridized with a single fragment of XbaI-digested yeast total chromosome DNA under optimized conditions (Fig. 1). Screening of the S. cerevisiae cDNA library performed under the same conditions allowed us to isolate twelve putative positive clones among approximately 100,000 plaques tested. However, Southern hybridization of purified plasmid DNA indicated that only one of them, designated pL10, produced a signal comparable with that of the cDNA encoding the low M(r) PTPase from bovine heart. A 719-bp cDNA fragment purified from clone pL10 was found to contain an open reading frame encoding a protein sequence that was 39% identical to the mammalian low M(r) PTPases. Two other isolated clones exhibited approximately 5-10 times weaker signals on Southern blots, but their nucleotide sequences showed no relation to the low M(r) PTPase. The remainder of the clones gave hybridization signals that were hardly distinguishable from the background.


Figure 1: Southern blot hybridization analysis of S. cerevisiae genomic DNA. A 5` P-labeled degenerate oligonucleotide derived from the active site sequence of mammalian low M(r) PTPases (see ``Experimental Procedures'') was used as a probe. Yeast genomic DNA was digested with XbaI, subjected to electrophoresis in 0.7% agarose gel, and transferred to nitrocellulose. Hybridization was performed under the same conditions as described under ``Experimental Procedures'' for cDNA library screening.



Screening of a S. cerevisiae genomic library using the isolated cDNA from clone pL10 as a probe made possible the isolation of 19 positive clones. All of them contained a 120-bp HpaI-XbaI fragment that was also present in pL10 cDNA, and they were all related to each other as indicated by the presence of other common restriction sites. One of the identified clones, YCp50(LTP1)-5, carrying a 5.4-kb insert of yeast genomic DNA, was further characterized by partial sequencing. The nucleotide sequence of a 1329-bp fragment was shown to comprise an open reading frame which was identical to that found in the previously isolated cDNA (Fig. 2). Based on the high homology of the deduced protein sequence to that of mammalian low M(r) PTPases, the isolated gene was designated LTP1 (low molecular weight protein tyrosine phosphatase). In addition, we found that the sequence overlapped at the 5` terminus with a previously published sequence of the gene TKL1 encoding transketolase(44) . A common fragment that encompassed 261 bp of the TKL1 coding sequence and 120 bp of its 3`-noncoding region was found, with no differences between the present results and the previously published sequence data. Thus, the coding sequence of LTP1 is located downstream from the TKL1 gene and is separated from the stop codon of the latter by a 377-bp fragment (Fig. 2). Unfortunately, the chromosome localization of the TKL1 gene has not been described previously. To map the LTP1 gene, a set of - and cosmid-clone grid filters representing 99% of S. cerevisiae genome (45) was probed with a 3.8-kb PvuII-EcoRI fragment containing this gene and 3`-region of the TKL1 (Fig. 2A). This probe hybridized specifically with clone number 1712, leading to to the conclusion that the LTP1 and TKL1 genes are located on the right arm of the chromosome XVI of S. cerevisiae.


Figure 2: Restriction map (A) and nucleotide and deduced amino acid sequence of the LTP1 gene of S. cerevisiae (B). A, restriction map of the 5.4-kb fragment isolated from S. cerevisiae genomic library as a part of the YCp50(LTP1)-5 clone is shown. The region that was sequenced is indicated by a dashed line. The boxes represent the coding sequences of the LTP1 and TKL1 genes. B, the nucleotide sequence reported here begins at the stop codon of the TKL1 gene. A putative TATA-box consensus motif is in boldface.



Sequence Analysis

The resulting LTP1 gene of S. cerevisiae was predicted to encode a 161-amino acid residue protein with a calculated molecular mass of 18,675 Da (Fig. 2B). A search for similar protein sequences in the NCBI data base using the program BLAST did not reveal any identical sequences. The primary structure of the Ltp1 protein was in fact most similar to that of the mammalian low M(r) PTPases. The yeast enzyme is three or four residues longer than the latter enzymes, depending on whether its N-terminal methionine is removed by posttranslational cleavage or not. It is known that the mammalian low M(r) PTPases do not contain methionine at the N terminus but instead contain an N-acetylated alanine, derived from the second predicted residue(27, 28) . It is noteworthy that the percentages of identity between the S. cerevisiae PTPase and two different groups of mammalian isoenzymes are nearly the same. For example, the A and B isoenzymes from human placenta (17) show 39 and 40% sequence identity to the yeast enzyme, respectively. Among a number of specific protein sites and signature motifs recognized by the program PROSITE in the software package PC-GENE, only a putative phosphorylation site on Tyr may be of any possible significance. Although this tyrosine residue is conserved in all of the known low M(r) PTPases, the adjacent region on its amino side has the features of a tyrosine phosphorylation site only in the yeast enzyme.

All of the catalytically important amino acid residues identified by site-directed mutagenesis in the bovine low M(r) PTPase(28, 29, 46, 47) , namely Cys (responsible for the formation of the phosphoenzyme intermediate), Arg^18 (involved in the binding of phospho group of substrate), Asp (which is a proton donor for substrate leaving group), as well as Cys and His, have obvious homologs in the sequence of yeast enzyme. In addition, other residues which according to the crystal structure of the bovine enzyme may contribute to the enzyme activity and specificity(30) , such as Ser, Glu, Asp, Ser, and Asp, are also perfectly conserved.

LTP1 mRNA Analysis

A single poly(A) RNA species hybridizing with LTP1 cDNA was detected by Northern blot hybridization (Fig. 3), and its estimated size of 0.75 kb is in good agreement with the length of the isolated cDNA (719 bp). Although systematic studies were not conducted on this point, the LTP1 mRNA appears to be a relatively low abundance messenger. Based on the results of hybridization experiments using total yeast RNA (data not shown), the level of the LTP1-encoded transcript is at least 1 order of magnitude lower than that of HIS3 mRNA, which is of average abundance. Such a low abundance for the LTP1 mRNA is consistent with the relatively low codon bias index value calculated for LTP1, which is 0.11. The codon bias index, which reflects the preferred codon usage in yeast, has been shown to be well correlated with the level of the corresponding mRNA(48) . Values of the index comparable with that observed for the LTP1 gene are usually characteristic of poorly expressed genes.


Figure 3: LTP1 mRNA analysis. Poly(A) RNA from strains OK17-2D (wild type) and OK17-2C (ltp1 null mutant) was purified, electrophoresed in a formaldehyde, 1.5% agarose gel, transferred to nitrocellulose, and probed with LTP1 cDNA following published protocols(36) . A total of 25 µg of poly(A) RNA from each preparation was loaded on the gel. A 0.16-1.77-kb RNA ladder (Life Technologies, Inc.) was used as a size marker.



Expression and Properties of the Recombinant Yeast Low M(r)PTPase

In order to confirm that the LTP1 gene encodes a low M(r) PTPase, it was initially expressed in E. coli using a glutathione S-transferase fusion expression vector. In the resulting expression vector, the coding region of the LTP1 gene was joined to an upstream sequence encoding a thrombin-cleavage site by a sequence coding for the peptide PGISGGGGGINTK. Consequently, the recombinant Ltp1 was expected to be 13 amino acids longer and to carry the latter peptide at the N terminus. Approximately 10 mg of homogenous recombinant protein (apparent molecular mass of 18 kDa) was purified to homogeneity from 2 liters of E. coli culture. It hydrolyzed p-nitrophenyl phosphate (pNPP) and exhibited highest activity at 5.0. This is also the pH optimum for mammalian low M(r) PTPases when hydrolyzing this acidic, artificial substrate. However, the specific activity of the yeast enzyme (3.2 units/mg) was 10-30-fold lower than the corresponding values for native and recombinant low M(r) PTPases from bovine heart and human placenta(14, 16, 17, 49) .

To determine whether the activity of the recombinant enzyme was lowered due to the presence of 13 additional residues at the N terminus, we employed a pT7-7 vector-based E. coli expression system providing for the synthesis of nonfusion proteins. In this case, almost 50 mg of nearly homogenous Ltp1 could be isolated from 2 liters of the culture medium by a two-step purification procedure. The N-terminal sequence of the recombinant protein was determined to be Thr-Ile-Glu-Lys-Pro-Lys, which is identical with that predicted from the nucleotide sequence with the exception that no N-terminal methionine was present. The kinetic parameters of the nonfusion enzyme toward pNPP were effectively identical to those measured for homogeneous Ltp1 carrying the 13 extra N-terminal residues (k values were 1.24 and 1.15 s and K values were 17 and 19 µM, respectively). This purified nonfusion recombinant protein was used for further subsequent studies.

Although the low M(r) PTPase from budding yeast has a somewhat lower phosphatase activity, its substrate specificity was similar to that of its mammalian counterparts (Table 2). Like the bovine and human enzymes, the Ltp1 protein showed little or no activity toward phosphoserine and phosphothreonine, but was capable of hydrolyzing phosphotyrosine, as well as a representative phosphotyrosine-containing peptide, Tyr-fyn, at nearly the same efficiency as pNPP. In addition, as in the case of the mammalian enzymes(15, 17) , the substrate beta-naphthyl phosphate was substantially better as compared with its alpha-isomer. Significantly, because the k and the K values were each reduced by approximately the same degree for the yeast enzyme as compared with the corresponding values for the bovine enzyme, the k/K ratios of the two enzymes were effectively identical for most substrates. For example, for p-nitrophenyl phosphate, the k/K values were 72,900 and 90,000 M s for the yeast and bovine enzymes, respectively.



Activation of the Ltp1 Phosphatase by Purines

A moderate activation of the mammalian and avian low M(r) PTPases by purine nitrogen bases, nucleosides, and nucleotides has been described previously(50, 51, 52) . Consistent with this, we found that the rate of hydrolysis of pNPP catalyzed by the recombinant bovine enzyme was increased by approximately 3-fold in the presence of 10 mM adenine. (^3)However, the yeast enzyme exhibited a much higher degree of activation: at 25 °C and pH 5.0, the rate of hydrolysis of pNPP was increased 31-fold by the presence of 5 mM adenine. A number of other purine compounds, including adenine and guanine nucleosides and nucleotides, were also tested as potential modulators of yeast low M(r) PTPase under the same conditions. All of them were shown to activate the enzyme, although the observed effects were smaller than that caused by adenine. Among them, cGMP and cAMP were moderately strong activators, causing a 13- and 7-fold elevation of specific activity at concentration of 5 mM at 25 °C.

Adenine was found to quench the tryptophan fluorescence of the yeast enzyme, and the decrease in fluorescence showed a dependence on adenine concentration that followed a typical saturation curve (data not shown). This curve showed a good correlation with the adenine concentration dependence of enzyme activity that was determined using both pNPP and phosphotyrosine as substrates (Fig. 4). The dissociation constant of the enzyme-adenine complex, as determined from the fluorescence quenching data (see ``Experimental Procedures'') was 3.1 mM. This value is effectively identical to the corresponding value for the BPTP (43) .^3 Therefore, the larger activation of the yeast enzyme as compared with the bovine one cannot be explained by a higher affinity of adenine. Instead, it must be due to structural differences between these enzymes.


Figure 4: Activation of Ltp1 by adenine and cGMP. Enzyme activity was assayed using 10 mM pNPP (filled triangles) or 10 mM phosphotyrosine (open triangles) in the presence of different adenine concentrations at 37 °C or using 10 mM pNPP in the presence of cGMP at 25 °C (open circles).



Disruption and Overexpression of the LTP1 Gene

As part of a study of the physiological role of the low M(r) PTPase in yeast S. cerevisiae, we explored the phenotypic effects of an ltp1 null mutation. A knockout of LTP1 was performed as shown in Fig. 5A. The LTP1 gene was disrupted in vitro by replacing its entire coding region with a fragment carrying the yeast HIS3 gene as a selective marker. In order to generate the heterozygous disruptant strain OK17, a diploid strain OK15 was transformed with a linear fragment carrying the disrupted gene. A chromosomal disruption of the LTP1 wild type allele was verified by Southern hybridization (Fig. 5B). The resulting strain OK17 was subjected to sporulation followed by tetrad dissection and the analysis of spore viability at 30 °C on YEPD plates. Most of the analyzed tetrads yielded four viable spores, and the segregation of markers (HIS3 and MAT) was 2:2. A proper segregation of the LTP1 wild type and mutant alleles in one tetrad was confirmed by Southern hybridization (data not shown). The wild type and disruptant mutant segregants of both mating types were selected for further analysis. No effects of the ltp1 mutation on the rate of growth in rich media containing glucose, galactose, sucrose, or glycerol as a source of carbon or in the minimal SD medium at 22, 30, and 37 °C were observed. Both wild type and mutant strains were found to grow at approximately the same rate in a high osmolarity medium containing 1 M sorbitol or in the presence of 2 mM sodium vanadate (an inhibitor of PTPases), 250 mM 3-aminotriazole, 1 M NaCl, or 100 mM LiCl. The mating efficiency and level of alpha-factor secretion were also examined, but no effects were apparent. Finally, the homozygous diploid ltp1 mutant strain was obtained, and its efficiency of sporulation was shown to be similar to that of the wild type strain. Thus, disruption of LTP1 did not cause any phenotypic consequences under the conditions that were tested.


Figure 5: Disruption of LTP1. A, a 496-bp HpaI-NheI fragment containing 9 bp of the 5`-noncoding region, the entire coding sequence, and 1 bp of the 3`-noncoding region of the LTP1 gene was replaced by a 1.7-kb SmaI-XbaI fragment carrying the S. cerevisiae HIS3 gene. A 3.8-kb PvuII-EcoRI fragment containing ltp1::HIS3 was introduced into a diploid S. cerevisiae strain OK15 to generate a heterozygous disruptant strain OK17. The coding region of LTP1 is shown by an open box. A filled box indicates the fragment containing the S. cerevisiae HIS3 gene. E, EcoRI; H, HpaI; Nh, NheI; Pv, PvuII; Sm, SmaI; X, XbaI. B, Southern hybridization analysis of PvuII, EcoRI-digested chromosomal DNA from the strains OK15 (lane 1) and OK17 (lane 2). A 2.6-kb PvuII-EcoRI fragment carrying the wild type LTP1 was used as a probe. The same analysis of the haploid segregants obtained from a single tetrad confirmed the disruption of LTP1 (data not shown).



The failure to find any visible phenotypes of the ltp1 null mutant may result from the presence of more than one functionally redundant isoenzymes of the low M(r) PTPase in S. cerevisiae. However, no protein species that were immunologically related to the Ltp1 protein were detected when extracts of the mutant strain were immunoblotted using antisera to the recombinant yeast enzyme (Fig. 6). Furthermore, a S. cerevisiae phosphatase was detected that eluted from a Sephadex G-150 column at the same volume as the low M(r) bovine PTPase (Fig. 7A; data for the bovine enzyme are not shown). The apparent molecular mass of each enzyme estimated by gel-filtration was 15 kDa. Further corroborating that this phosphatase was a product of the LTP1, the former enzyme was shown to be activated more than 20-fold in the presence of 5 mM adenine. A knockout of the LTP1 gene completely abolished the corresponding peak of activity (Fig. 7B). These data strongly indicate that the low M(r) PTPase is represented by a single LTP1-encoded species in budding yeast. The experiments described above enabled us to estimate that the level of expression of Ltp1 in the budding yeast strain studied here was approximately 0.01% of the total protein. Thus, consistent with the observation of a relatively low level of LTP1 mRNA, this enzyme is a rather poorly expressed protein.


Figure 6: Immunoblot analysis of extracts of ltp1 null mutant (lane 1) and wild type strain (lane 2). Immunodetection using antisera to the Ltp1 was performed as described under ``Experimental Procedures.''




Figure 7: Separation of the phosphatases from wild type strain (A) and ltp1 null mutant (B) by gel-filtration on Sephadex G-150 column. The S. cerevisiae strains OK17-2D (wild type) and OK17-2C (Deltaltp1) were grown in 250 ml of YEPD up to the late exponential phase. Cells were resuspended in 10 ml of 0.1 M Tris-HCl, pH 8.0, containing 150 mM NaCl, 1 mM dithiothreitol, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 5 µg/ml leupeptin, 5 µg/ml pepstatin A, 0.1% Nonidet P-40, and lysed by vortexing with glass beads (450-600 µm, Sigma) for 3 min. Cell debris was removed by centrifugation at 25,000 g for 30 min, and the supernatant was concentrated to 2 ml using the ultrafiltration membrane PM10 (Amicon). A concentrated sample was loaded on the Sephadex G-150 column (3.5 100 cm) equilibrated with 10 mM Tris-HCl, pH 8.0, 1 mM EDTA, and the proteins were eluted at a flow rate of 18 ml/h. The fraction volume was 6 ml. To assay phosphatase activity, aliquots of the protein fractions were diluted 10-fold (open circles) or 2-fold (open triangles) with 100 mM sodium acetate, pH 5.0, 1 mM EDTA, 10 mM PNPP, or 10-fold with the same buffer containing 10 mM adenine (open squares) and incubated at 37 °C for 60 min. The reaction was stopped by adding 1 M NaOH (0.5 volume) and followed by measuring the absorbance at 405 nm.



To study the effect of LTP1 overexpression, a multicopy plasmid pALA1 carrying this gene under the control of the strong ADH1 promoter was introduced into the wild type strain PJ51-3A. The resulting expression level of Ltp1 was elevated by approximately 10-fold compared with that in the control strain, as estimated by immunoblotting. The same phenotypes as for the case of the knockout mutant were examined for the Ltp1-overexpressing strain, but again no changes were apparent. In addition, the effects of LTP1 knockout and overexpression in the background of a mih1 mutation were explored. The MIH1 gene of S. cerevisiae is a homolog of S. pombe cdc25(53) . Activation of cyclin B-associated Cdc2 protein kinase via its dephosphorylation on a single tyrosine residue by Cdc25 in S. pombe represents a key event in the promotion of the onset of mitosis(54, 55) . In contrast to the S. pombe cdc25 null mutation, which is lethal(56) , the inactivation of S. cerevisiae MIH1 has little effect on cell division, apparently due to a poor level of the phosphorylation of Cdc28, which is a homolog of Cdc2 of S. pombe. However, the MIH1 gene becomes essential when the stoichiometry of Cdc28 phosphorylation is increased by overexpressing the S. pombe Wee1 kinase in budding yeast(53) . We found that the growth defect of the mih1 mutant that was expressing Wee1 kinase under the control of the galactose-inducible GAL1 promoter was not suppressed by the moderate 10-fold overexpression of LTP1. Therefore, Ltp1 phosphatase does not appear to be able to contribute to the activation of Cdc28 under normal conditions. The absence of apparent effects of a knockout of LTP1 in the background of the mih1 null mutation was also demonstrated by tetrad analysis of a diploid strain obtained by the crossing of strains OK17-2C and PR24 (Table 1).


DISCUSSION

For some time, our knowledge about low M(r) PTPases has been restricted to the enzymes from vertebrate tissues. Using low stringency hybridization, we have now isolated a S. cerevisiae LTP1 gene whose product reveals 39% average sequence identity to the mammalian enzymes (Fig. 8). Consistent with the moderately high degree of homology, the Ltp1 protein expressed in E. coli exhibited phosphatase activity and its substrate specificity was generally similar to those of its mammalian counterparts(14, 15, 17, 49) . In particular, although it shows very low activity toward phosphoserine and phosphothreonine, this enzyme efficiently hydrolyzes phosphotyrosine and a phosphotyrosine-containing peptide, Tyr-fyn. The yeast enzyme exhibited a lower specific activity than mammalian low M(r) PTPases. However, because the k and K values of most substrates were each reduced by approximately 30-fold for the yeast enzyme compared with the corresponding values for the bovine enzyme, the result is that the values of the k/K ratio were surprisingly similar(49) . In terms of kinetic equations describing the postulated catalytic mechanism, such a difference may be expected to result from a decrease in the rate of hydrolysis of the phosphoenzyme intermediate that occurs during the course of the enzymatic reaction (47) .


Figure 8: Sequence alignment of Ltp1 with the low M(r) PTPases from S. pombe (Stp1(35) ), bovine heart (BHPTP(16) ), and human placenta (HCPTP-A and HCPTP-B isoenzymes (HCPTP-A and HCPTP-B)(17) ). Residues that are identical (asterisk) or similar (&cjs1219;) in all of the sequences are indicated. The residues corresponding to those that have been shown to be catalytically important in the bovine low M(r) PTPase (28, 29, 30, 46, 47) are in boldface.



Although the Ltp1 phosphatase exhibited relatively low activity in the absence of effectors, it was considerably activated by a number of purine compounds. The strongest effect was observed in the presence of adenine, which caused a 31-fold elevation of the Ltp1 activity at 25 °C. Some modulation of the activity of the mammalian low M(r) PTPases by purine derivatives has been described earlier(50, 51, 52) , but the observed effects were much smaller. The values of the dissociation constants of adenine-enzyme complexes estimated for the yeast and bovine enzyme were nearly identical. Therefore, the larger degree of activation of Ltp1 cannot be explained by the higher affinity of the yeast enzyme for the effector. The biological significance of the activation effect is not immediately apparent, considering that it is observed at moderately high, presumably nonphysiological concentrations of purines. It is possible to hypothesize that the activity of Ltp1 may be regulated in vivo by other compounds that exhibit tighter binding to the enzyme. One intriguing possibility is that the enzyme is activated by its protein substrate, and thus the presence of an activating determinant in the substrate structure serves to enhance or determine the substrate specificity. Comparative studies of the structure and mechanism of the yeast and mammalian low M(r) PTPases will be of particular interest, because they may reveal the basis of the activation phenomena exhibited by these enzymes.

The cloning of the low M(r) PTPase gene from S. cerevisiae made it possible to initiate an exploration of the physiological role of this enzyme by the methods of yeast genetics. Despite the high evolutionary conservation of the low M(r) PTPase, which suggests that this enzyme may be involved in a fundamental cellular process, a strain bearing an ltp1 null mutation did not confer any visible phenotypes. Since this mutation caused the apparently complete disappearance of the low M(r) PTPase activity which could be detected in the wild type strain, and it resulted in the disappearance of the low M(r) PTPase band that was present on immunoblots, the existence of the additional structurally similar isoenzymes is unlikely. However, it is impossible to rule out the possibility that the activity of the Ltp1 in the cell may overlap with that of structurally unrelated (or distantly related) proteins.

Alternatively, the Ltp1 phosphatase might act cooperatively with other proteins. Such a hypothesis is well illustrated by the fact that a knockout of the S. cerevisiae gene PTP2, which encodes a PTPase, is not deleterious for the yeast cell by itself, but it does cause a synthetic growth defect when present together with a mutation in the PTC1 gene which encodes a homolog of mammalian serine/threonine phosphatase 2C(57) . The Ptp2 phosphatase was implicated in tyrosine dephosphorylation of a HOG1-encoded MAP kinase involved in an osmosensing signal transduction pathway in budding yeast(58) . Since the members of MAP kinase family require phosphorylation on both tyrosine and threonine residues to be activated, the function of Ptc1 protein was hypothesized to consist in threonine dephosphorylation of the Hog1 kinase. In this case, the absence of both Ptp2 and Ptc1 is required for the constitutive activation of Hog1 kinase that, in turn, may impair the vegetative growth(57, 58) . The question of whether the ltp1 null mutation may be lethal in the background of other mutations is currently being addressed by the use of a synthetic lethal screen.

Recently, an involvement of mammalian low M(r) PTPases in the control of cell proliferation was proposed based on the observations that overexpression of the bovine enzyme negatively affects the growth of the murine fibroblast cell cultures (59, 60) and reduces the tyrosine phosphorylation level of the PDGF receptor(34) . The latter finding provides no clue about the role of the phosphatase Ltp1, since receptor tyrosine kinases have not yet been discovered in budding yeast. Furthermore, in contrast to the effect of overexpression of the bovine enzyme, we did not observe any growth inhibition of the Ltp1-overproducing S. cerevisiae strain, although in both cases the expression levels were elevated to approximately the same degree when compared with the normal level. It is possible that the functions of the yeast and mammalian enzymes are different. Alternatively, it is reasonable to suggest that further corroboration is needed to show that the PDGF receptor may be the true physiological substrate of the mammalian low M(r) PTPase, since enzyme overexpression can result in nonspecific protein dephosphorylation that, in turn, may cause the growth inhibition. For example, the requirement of functional cdc25 in fission yeast can be bypassed by overexpression of human T-cell PTPase (61) and placental PTPase 1B(62) , which are normally irrelevant to cell cycle control. Exploring the effect of the gene knockout, rather than that of its overexpression, on the state of protein tyrosine phosphorylation appears to be more reliable way to the identification of the physiological substrates. Such a study utilizing an ltp1 mutant strain is currently being performed.

During the course of this study, we learned that a S. pombe homolog of the mammalian genes encoding the low M(r) PTPase, stp1, (Fig. 8) had been cloned as a multicopy suppressor of a cdc25 mutation(35) . Based on the observation that the disruption of stp1 did not affect cell size at division, that study concluded that the product of stp1 was not involved in mitotic control. Consistent with the present study, they also found that an stp1 null mutation did not cause any visible phenotypic changes. In this regard, it is worth noting that although the central role of protein tyrosine phosphorylation in a variety of cellular processes is well established, most of the S. cerevisiae PTPase genes identified to date, including MIH1(53) , PTP1(63) , PTP2(64, 65, 66) , PTP3,(^4)and MSG5(67) have been shown to be nonessential. Consequently, the investigations of their cellular roles required additional approaches. The present study should facilitate such extensions for the low M(r) proteintyrosine phosphatase family.


FOOTNOTES

*
This work was supported by the United States Department of Health and Human Services Research Grant GM 27003. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank®/EMBL Data Bank with accession number(s) U11057[GenBank].

§
To whom correspondence should be addressed. Fax: 317-494-0239.

^1
The abbreviations used are: low M(r) PTPase, low molecular weight protein-tyrosine phosphatase; BPTP, bovine low M(r) PTPase; HCPTP-A and HCPTP-B, A and B isoenzymes of the low M(r) PTPase from human placenta; pNPP, p-nitrophenyl phosphate; PDGF, platelet-derived growth factor; bp, base pair(s); kb, kilobase(s); GST, glutathione S-transferase.

^2
G. L. Bryson and R. L. Van Etten, unpublished data.

^3
C. Pokalsky, unpublished results.

^4
R. Deschenes, personal communication.


ACKNOWLEDGEMENTS

We thank Drs. Stephen Elledge, Susan Liebman, Paul Russell, Stanley Fields, Philip James, and Dmitry Gordenin for various S. cerevisiae libraries, strains, and plasmids. We are also grateful to Dr. Mark Goebl for the S. cerevisiae genomic clone-grid filters. We thank John Wilder for the antisera to Ltp1.


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