(Received for publication, April 19, 1995)
From the
The low molecular weight protein-tyrosine phosphatase (low M
The important role of protein-tyrosine phosphatases (PTPases,
EC 3.1.3.48) ( 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 Despite
the fact that the structure and catalytic mechanism of the low M When this study was initiated, no low M
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.
Figure 1:
Southern blot hybridization analysis of S. cerevisiae genomic DNA. A 5`
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
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.
All of
the catalytically important amino acid residues identified by
site-directed mutagenesis in the bovine low M
Figure 3:
LTP1 mRNA analysis.
Poly(A
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 Although the low M
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) .
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).
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
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 (
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 For some time, our knowledge about low M
Figure 8:
Sequence
alignment of Ltp1 with the low M
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 The cloning of
the low M 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 During
the course of this study, we learned that a S. pombe homolog
of the mammalian genes encoding the low M
The
nucleotide sequence(s) reported in this paper has been submitted to the
GenBank®/EMBL Data Bank with accession number(s)
U11057[GenBank].
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
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
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
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
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
PTPase-encoding gene in S. cerevisiae.
)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.
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
PTPases as well as the corresponding human gene have been
cloned(16, 17, 18) . (
)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
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
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.
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
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.
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.
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 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
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
PO
, 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).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--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
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.
Cloning of a S. cerevisiae Gene Encoding a Low
M
Low stringency hybridization was
employed to search for a S. cerevisiae gene encoding a homolog
of the mammalian low MPTPase
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
(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
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
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
PTPase. The remainder of the clones gave
hybridization signals that were hardly distinguishable from the
background.
P-labeled
degenerate oligonucleotide derived from the active site sequence of
mammalian low M
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.
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.
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 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
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
PTPases, the adjacent region on its amino side has the features
of a tyrosine phosphorylation site only in the yeast enzyme.
PTPase(28, 29, 46, 47) ,
namely Cys
(responsible for the formation of the
phosphoenzyme intermediate), Arg
(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.
) 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
In order to confirm that the LTP1 gene encodes a low MPTPase
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
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
PTPases from bovine heart and
human placenta(14, 16, 17, 49) .
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.
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
-naphthyl
phosphate was substantially better as compared with its
-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 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. (
)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
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.
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.
Disruption and Overexpression of the LTP1 Gene
As
part of a study of the physiological role of the low M 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
-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.
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
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
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.
ltp1)
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.
(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).
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
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) .
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
PTPase (28, 29, 30, 46, 47) are in boldface.
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
PTPases
will be of particular interest, because they may reveal the basis of
the activation phenomena exhibited by these enzymes.
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
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
PTPase activity which could be detected in the wild type strain,
and it resulted in the disappearance of the low M
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.
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
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.
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,(
)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
proteintyrosine
phosphatase family.
PTPase, low molecular weight protein-tyrosine phosphatase; BPTP,
bovine low M
PTPase; HCPTP-A and HCPTP-B, A and B
isoenzymes of the low M
PTPase from human
placenta; pNPP, p-nitrophenyl phosphate; PDGF,
platelet-derived growth factor; bp, base pair(s); kb, kilobase(s); GST,
glutathione S-transferase.
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.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.