Dual-specificity phosphatases (dsPTPase) (
)hydrolyze
phosphoserine/threonine/tyrosine-containing substrates in vitro and exhibit a substrate preference in vitro and in
vivo for diphosphorylated (Thr(P)/Tyr(P)) mitogen-activated
protein kinase (MAPK) homologues (reviewed in (1) ). Most
dsPTPases are localized to the
nucleus(2, 3, 4) , and it has been suggested
that they are responsible for the nuclear dephosphorylation and
inactivation of MAPKs seen in vivo(5) . All dsPTPases
contain the sequence, HCXXGXXR(S/T), which has been
shown to correspond to the active site of PTPases(6) . The
essential cysteine forms a thiophosphate intermediate during
dsPTPase-catalyzed dephosphorylation(7, 8) . Several
investigators have shown that substitution of this Cys, by Ser, in the
dsPTPases, abolishes hydrolytic activity in vitro(7, 9, 10, 11, 12) and
function in vivo(10, 13, 14) .
Interestingly, transient expression of the Cys to Ser mutant prolongs
MAPK activation in vivo(12) , suggesting that it may
compete with native phosphatases for binding to phosphorylated MAPK.
Moreover, the mutant dsPTPase co-immunoprecipitates with phosphorylated
MAPK(10) , indicating that the catalytic cysteine is not
essential for substrate binding. This conclusion is also supported by
PTPase crystallographic studies showing that Cys to Ser mutants bind
sulfate (15) and tyrosine-phosphorylated peptide (16) in a manner identical with native enzymes.
This paper
describes the identification and isolation of a novel
phosphoserine/threonine/tyrosine-binding protein (STYX) that is related
in amino acid sequence to dsPTPases, but contains a naturally occurring
Gly residue in place of the active site, catalytic Cys. Residues which
have been shown to be important in binding of phosphorylated substrates
by dsPTPases are present in STYX. (
)Expression of
recombinant STYX showed that it had no phosphatase activity; however, a
single mutation of Gly
to Cys (STYX-G120C) confers
phosphatase activity to the recombinant mutant protein. The STYX-G120C
mutant had binding constants and kinetic parameters similar to
dsPTPases. We believe this is the first example of a naturally
occurring ``dominant negative''
phosphoserine/threonine/tyrosine-binding protein, structurally related
to dsPTPases.
EXPERIMENTAL PROCEDURES
PCR Subcloning and Northern Blot Analysis
Mouse
testis poly(A
) RNA was extracted from adult tissue
using TriZOL
Reagent (Life Technologies, Inc.), followed
by PolyATtract (Promega) purification according to manufacturer's
instructions. First strand cDNA synthesis was carried out with
oligo(dT) and random primers using the cDNA Cycle Kit (Invitrogen), and
DNA amplification was performed by polymerase chain reaction (PCR) with
primers to mouse testis expressed sequence tag F220A (GenBank
accession number L26737). Fifty pmol each of 5`-primer
(5`-GGATATTGCTGATAATCCAGTTGAAAAC-3`) and 3`-primer
(5`-GATATACCTGCCTTTGCTTCAGAGG-3`) were used with Taq polymerase (Boehringer Mannheim) for 35 cycles of 94 °C, 2
min; 50 °C, 2 min; 72 °C, 3 min. The single PCR product of 314
bp was ligated into pCR
II plasmid (Invitrogen), and
sequenced by dideoxy chain termination using Sequenase II polymerase
(U. S. Biochemical Corp.). The 314-bp insert of the pCR
II
subclone (TA314) was used as a template for probe synthesis by PCR as
above, using [
-
P]dATP in the reaction.
P-Labeled PCR product was used to probe an adult mouse
multiple tissue poly(A
) Northern blot (Clontech) at
2
10
cpm/ml, under manufacturer's
conditions. Final washing was performed at 50 °C in 0.1
SSC
(1
SSC is 150 mM NaCl, 15 mM Na
citrate, pH 7.0), 0.1% SDS for 40 min, and
hybridizing bands were visualized by autoradiography at -80
°C with intensifying screens. Washing at 65 °C for 60 min had
no effect on either the tissue distribution or number of bands seen.
Subclone TA228 was produced by PCR amplification of mouse testis cDNA
described above, with 5`-primer (5`-TTGTCCACCAACTTCAGCTCTGGCTGTC-3`)
and 3`-primer (5`-AGCTAAGGACCTTTCTATTTGG-3`) for 35 cycles of 94
°C, 2 min; 47 °C, 2 min; 72 °C, 3 min, followed by ligation
and sequencing of the 228-bp product as above.
Library Screening
Approximately 10
subclones were screened from both a Lambda Max1 mouse testis 5`-stretch
cDNA library (Clontech) and Uni-Zap
XR mouse diaphragm
cDNA library (Stratagene) per manufacturer's instructions.
Subclone TA314 was used for probe synthesis by PCR as described above.
Final washing of nitrocellulose filters was performed at 65 °C in
0.2
SSC, 0.1% SDS for 60 min. Positive subclones were
identified by autoradiography and plaque-purified. Phagemids were
excised and rescued from purified phage according to the manufacturer.
Stratagene library phagemid, ST9, contained an insert of 1150 bp which
overlapped the sequence of all the other phagemid clones isolated. All
rescued phagemids were sequenced on both strands as above.
In Vitro Transcription/Translation
Phagemid ST9
was used as template for in vitro transcription/translation
using [
S]Met and the TNT
Coupled
Reticulocyte Lysate System (Promega), according to the manufacturer.
Protein products were resolved by SDS-PAGE and identified by
autoradiography without enhancement.
Overexpression and Purification of GST Fusion
Proteins
Coding sequence contained within the 886-bp fragment of
an NcoI/XhoI partial digest of ST9 was ligated into
the NcoI/XhoI sites of bacterial expression plasmid,
pGEX-KG(18) , to produce pGEX-Native (NA) plasmid. Mutation of
Gly
to Cys (G120C) was accomplished by PCR amplification
with pGEX-specific 5`-primer (kindly provided by Dr. G. Zhou) and
mutated ST9 3`-primer (5`-ACTTCTAGAGATACCTGCATTRCAATGGAC-3`) for 35
cycles of 94 °C, 2 min; 45 °C, 1.5 min; 72 °C, 1.5 min. The
383-bp XbaI fragment of pGEX-NA was replaced with the
corresponding XbaI fragment of the PCR reaction to produce
pGEX-G120C. The identities of both pGEX constructs were confirmed by
sequencing and differed only in the two nucleotides that convert
Gly
codon (GGG) to cysteine (TGC). The pGEX-NA and -G120C
plasmids were used separately to transform competent BL21/DE3 bacteria
to produce GST-NA and GST-G120C fusion proteins by established
procedures(18) . GST-fusion proteins were separated from
bacterial proteins by incubation with glutathione-agarose beads,
washing, and elution off the beads in the presence of glutathione. The
purity of eluted proteins was confirmed by SDS-PAGE and Coomassie Blue
staining.
Kinetic Assays
Three different quantitative
enzymatic assays were performed on GST-G120C, exactly as described for
human dsPTPase, VHR(8) . All assays were performed at 30
°C, in a three-component buffer consisting of 0.05 M Tris,
0.05 M Bis-Tris, and 0.1 M acetate. Hydrolysis of
PTPase substrate, p-nitrophenyl phosphate (pNPP), was
followed as an increase in absorbance at 405 nm of the reaction
product, p-nitrophenol. Initial rates at various initial
substrate concentrations were fitted directly to the Michaelis-Menten
equation using the nonlinear least squares program, Kinetasyst for the Macintosh (IntelliKinetics, State College, PA). A
continuous spectrophotometric assay was used to follow the Tyr(P)
dephosphorylation of Tyr(P)/Thr(P)-containing peptides as described
previously(8) . The complete time course of the reaction was
fitted to a modified integrated form of the Michaelis-Menten equation
using a nonlinear least squares algorithm to obtain kinetic parameters k
and K
. The third method
involved reverse phase HPLC separation and quantitation of the
substrates and products from the reaction of GST-GC with
MAP
kinase peptide DHTGFLpTEpYVATR(8) .
With this method, the rate of enzyme-catalyzed hydrolysis at Thr(P) was
determined as above.
RESULTS AND DISCUSSION
Identification, PCR Subcloning, and Tissue
Distribution
We identified an expressed sequence tag isolated
from mouse testis (GenBank
accession number L26737) that
contained a partial open reading frame homologous to the active site of
dual-specificity protein-tyrosine phosphatases (dsPTPases)(1) .
Several amino acids critical for phosphatase activity were conserved (8) ; however, there appeared to be a glycine substitution for
the conserved cysteine within the active site of the putative
phosphatase (Fig. 1A). Site-directed mutagenesis of the
corresponding Cys, to Ser, has been shown previously to inactivate
several dsPTPases
catalytically(7, 9, 10, 11, 12, 13, 14) ,
while preserving substrate
binding(10, 15, 16) . Since it was possible
that the Gly codon reflected errors in the original data base entry (i.e. a sequencing or PCR error), we initially attempted to
confirm the sequence of the open reading frame. Primers were designed
to specifically amplify a 314-bp sequence encompassing the Gly
substitution and were used in PCR reactions with cDNA of
reverse-transcribed mouse testis poly(A
) RNA. As a
consequence of the Gly substitution (Val-His-Gly), a unique NcoI site was created (5`-GTC CAT GGG-3`) which would not be
found at the active site (Val-His-Cys) of PTPases (5`-GTN CAY TGY-3`).
PCR reactions yielded a single product of 314 bp which was completely
digested by NcoI (not shown), suggesting not only that the Gly
codon substitution existed in the poly(A
) RNA, but
that a Cys-containing codon (i.e. catalytically active
homologue) did not exist. The entire PCR reaction product was ligated
into vector, and the sequence of 30 subclones all contained the glycine
substitution (Fig. 1B). Similar results were obtained
using mouse genomic DNA, reverse-transcribed mouse testis total RNA,
and reverse-transcribed rat pancreas poly(A
) RNA as
templates for PCR (not shown), suggesting that the glycine codon did
not arise from post-transcriptional modification and that its
expression was not restricted to mouse. Hybridization of the 314-bp PCR
product to mouse poly(A
) RNA demonstrated bands at
4.6, 2.4, 1.5 and 1.2 kb, with highest abundance in skeletal muscle,
testis, and heart (Fig. 1C). Collectively, these
observations suggested that the Gly codon was present in the expressed
sequence tag, was also present in genomic DNA as well as
poly(A
) RNA, and that it was expressed in several
tissues of both mouse and rat.
Figure 1:
Identification, isolation, and
tissue distribution of the glycine codon substitution. A,
amino acid comparison between PTP active site consensus (PTP)
and putative partial open reading frame of expressed sequence tag F220A (EST, GenBank
L26737). The apparent glycine
substitution is highlighted. B, autoradiograph of
sequencing reaction for PCR product TA314, subclone 11. The nucleotide
and putative amino acid sequence is shown. An asterisk denotes
the glycine codon. C, autoradiograph of mouse multiple tissue
Northern probed with
P-labeled TA314 insert as described
under ``Experimental Procedures.'' The position of size
standards in kilobases is shown.
Isolation of cDNA Library Subclones
In an effort
to obtain a full-length subclone, cDNA libraries from mouse testis and
skeletal muscle were screened with the 314 bp PCR product described
above. Several overlapping subclones were rescued and sequenced, with
the longest clones from the two libraries (ST9 and CT20) schematically
represented in Fig. 2A. Additional subclones were
identified by PCR (TA228) and data base searches (EST2). All subclones
contained identical nucleotide sequence in their 5` ends (bases
1-759), including the Gly codon in the putative active site;
however, the 3` ends appeared to encode two distinct amino acid
sequences. One subset of clones (EST1, TA314, and TA228) contained
nucleotides 760-895 which code for the carboxyl terminus seen in
the original expressed sequenced tag. The remaining clones (CT20, EST2,
and ST9) lacked these 136 nucleotides and consequently encoded a
different carboxyl end. Interestingly, nucleotides 896-1273 were
identical for all the subclones, suggesting that the two forms were
alternatively spliced products of the same mRNA (entire composite
nucleotide sequence deposited as GenBank
U34973).
Moreover, examination of nucleotides 760-895 revealed a strong
similarity to the consensus sequences of intron borders (see (19) for review), except for substitution of cytosine for
guanine at the +1 position of the putative 5` donor site (Fig. 2B). Substitution for guanine at this position
has been shown to attenuate intron splicing efficiency (19) and
is implicated in the retention of introns in mRNA from genes underlying
disease states(20) . Thus, the alternative splicing of this
region could give rise to proteins with different carboxyl ends (Fig. 2A, Spliced and Unspliced),
while preserving the glycine codon within the putative active site.
Attempts to isolate the 136-bp ``intron'' from commercial
cDNA libraries were unsuccessful; however, semiquantitative reverse
transcription-PCR with poly(A
) mouse testis RNA showed
a relative abundance of spliced to unspliced product of approximately
100:1, respectively (not shown).
Figure 2:
Schematic representation of subclones and
identification of alternative splice variant. A, schematic
representations of nucleotide sequences are aligned and numbered in
relation to Stratagene library subclone ST9. Subclones are labeled as
described under ``Experimental Procedures,'' with
GenBank
expressed sequence tags L26737 and L26718 shown as EST1 and EST2, respectively. Horizontal bars represent putative open reading frames. The broken line spanning 760-895 denotes a gap in nucleotide sequence.
Nucleotides 896-972 are identical for all the subclones shown and
represent either 3`-untranslated sequence or coding sequence for the
unspliced and spliced forms, respectively. Asterisks denote
in-frame stop codons. The number of adenosines in putative
3`-polyadenylation sites (A) are listed. Striped boxes designate putative carboxyl-terminal ends of spliced and unspliced
forms. The relative position of the glycine substitution coding
sequence is shown (VHG). B, consensus nucleotide sequences of
intron borders and branch point (19) are shown in comparison to
subclone TA228. Nucleotide numbering for TA228 corresponds to A. Non-consensus sequences are condensed (slashes)
for clarity. Nucleotide identities are represented by vertical
lines. Putative 5` and 3` splice sites are shown (> and <,
respectively). Asterisks denote highly conserved positions of
the consensus sequences. The cytosine for guanine substitution at the
putative 5` donor site is highlighted.
Cell-free Expression and Amino Acid Similarity to
VHR
Since the size of the ST9 subclone (1.16 kb) approximately
equaled the smallest poly(A
) RNA species seen by
Northern analysis (Fig. 1C), we examined the nature of
the cell-free translation products expressed in a rabbit reticulocyte
system. Protein products of
25 and 23 kDa (Fig. 3A) agreed very well with the predicted sizes
(25.4 and 22.6 kDa) of proteins utilizing methionine codons at
nucleotide positions 256-258 and 328-330 (GenBank
U34973), respectively. Since both methionines reside in the same
open reading frame, the upstream Met
was
designated residue +1 of an open reading frame coding for 223
amino acids (Fig. 3B). There remains the possibility
that the ST9 sequence represents a partial open reading frame contained
within longer mRNAs (Fig. 1C); however, additional
support for Met
as a translational start site
comes from surrounding nucleotide similarity (5`-GGG ACC ATG G-3`) with
the consensus sequence of vertebrate start sites (5`-GCC RCC ATG
R-3`)(17) . Met
also correlates with
the known start site of human dsPTPase, VHR (9) (Fig. 3B), which operationally defines the
smallest PTPase domain.
Figure 3:
Cell-free transcription/translation of
STYX cDNA and amino acid alignment with dsPTPase, VHR. A,
autoradiograph of [
S]Met-labeled proteins from
cell-free in vitro transcription/translation of Stratagene
subclone ST9 (Fig. 2A) resolved by SDS-PAGE. The
position of molecular mass standards in kDa is shown at right.
An arrowhead denotes the
25-kDa protein corresponding to
the open reading frame translation below. B, amino acid
comparison of ST9-Met
open reading frame (STYX)
with dsPTPase, VHR(9) . The alternative carboxyl-terminal end
contained in TA228 (Fig. 2A) is shown (STNS). Similar
amino acids between STYX, STNS, and VHR are highlighted. Asterisks denote in-frame stop codons. An arrowhead marks the glycine substitution shown in Fig. 1A.
We have named the sequence defined by the
ST9-Met
reading frame, STYX, for
phosphoserine/threonine/tyrosine interaction protein. The putative
alternative carboxyl terminus (Unspliced, Fig. 2A) is referred to as STNS, for alternatively
spliced intron of STYX. Amino acid comparison of STYX with VHR revealed
extensive sequence similarity (46.4%), including the conservation of
VHR residues Asp
and Arg
, previously shown
to be important for substrate binding (8) .
Bacterial Expression, Conversion, and Kinetics
The
STYX coding sequence shown in Fig. 3B was ligated into
pGEX-KG vector (18) and expressed as a GST-fusion protein.
Purified GST-STYX fusion protein failed to show any hydrolytic activity
toward the PTPase substrate, para-nitrophenyl phosphate (pNPP) (Fig. 4A). Since STYX contained all the
structural elements thought to be important for dsPTPase
activity(8) , except the active-site Cys, we thought that
replacing the naturally occurring Gly with Cys might restore catalytic
activity. Mutation of Gly
to Cys (STYX-G120C) conferred
phosphatase activity to the fusion protein, as demonstrated by the
hydrolysis of pNPP (Fig. 4A), with a k
of 4.6 s
and a K
of 9.4 mM. Interestingly, preliminary
studies showed that native STYX can inhibit the pNPP
hydrolytic activity of the Cys-containing mutant. (
)The
dual-specific nature of the STYX-G120C mutant was demonstrated through
its dephosphorylation of both Tyr(P) and Thr(P) of diphosphorylated MAP
kinase peptide (Fig. 4B). Removal of GST via thrombin
cleavage had no effect on the activity of either native STYX or the
G120C mutant (not shown). Surprisingly, the kinetics of hydrolysis for
STYX-G120C mutant were comparable to native dsPTPase, VHR(8) ,
including inhibition by vanadate, (
)suggesting that the
Gly-containing STYX possessed all the structural components necessary
for phosphorylated substrate binding similar to dsPTPases. We realize
that the artificial substrates, pNPP and diphosphorylated
peptides, only serve to indicate the potential function of STYX. The
search for an in vivo phosphoprotein(s) which selectively bind
to STYX is currently underway.
Figure 4:
Hydrolytic activity of native and mutant
GST-STYX fusion proteins. A, hydrolysis of pNPP
measured by spectrophotometric absorbance of product, para-nitrophenol, at 405 nm. Assays were performed as
described under ``Experimental Procedures'' with native STYX (diamonds) and STYX-G120C (circles) fusion proteins
at 480 nM for 1 min at 30 °C, pH 6.0. Data points are
representative of three experiments. B, HPLC elution profile
of products of MAPK peptide hydrolysis by STYX-G120C fusion protein.
Aliquots of the reaction were taken at the times indicated, after
incubation of G120C mutant (13.1 µM final concentration)
with 1 mM diphosphorylated peptide (DHTGFLpTEpYVATR) at 30
°C, pH 6.0. The same volume of each sample was loaded on a C18
column and resolved by reverse phase HPLC as described under
``Experimental Procedures.'' Peaks of absorbance at 220 nm
are labeled with the phosphorylation state of the peptide as determined
by standards(8) . Elution profiles are offset for
clarity.
We believe that STYX is the first
example of a naturally occurring binding domain that is structurally
similar to dsPTPases. Recent enzymatic (8) and crystallographic
studies
have implicated aspartic acid and arginine
residues, as well as the amide backbone of the conserved catalytic
loop, in coordinating and binding substrate phosphoryl groups by
dsPTPases. Since STYX contains these structural components of the
binding pocket, it may share a degree of overlap in substrate
preference with dsPTPases. If this is the case, the function of STYX
may be to bind phosphorylated dsPTPase substrates and thereby protect
them from serine/threonine phosphatases. The existence of a
``protective factor'' has been proposed recently for MAPK
signaling(5) , and STYX would be predicted to have properties
consistent with this function. The biological properties of STYX are
currently under investigation.