(Received for publication, October 18, 1994)
From the
An expression and purification method was developed to obtain
the recombinant human dual-specific protein tyrosine phosphatase
(PTPase) VHR in quantities suitable for both kinetic studies and
crystallization. Physical characterization of the homogeneous
recombinant protein verified the mass to be 20,500 ± 100 by
matrix-assisted laser desorption mass spectrometry, confirmed the
anticipated NH-terminal amino acid sequence and
demonstrated that the protein exists as a monomer. Conditions were
developed to obtain crystals which were suitable for x-ray structure
determination. Using synthetic diphosphorylated peptides corresponding
to MAP
(mitogen-activated protein) kinase
(DHTGFLpTEpYVATR), an assay was devised which permitted the
determination of the rate constants for dephosphorylation of the
diphosphorylated peptide on threonine and tyrosine residues. The
diphosphorylated peptides are preferred over the singly phosphorylated
on tyrosine by 3-8-fold. The apparent second-order rate constant k
/K
for
dephosphorlyation of phosphotyrosine on DHTGFLpTEpYVATR was 32,000 M
s
while
dephosphorylation of phosphothreonine was 14 M
s
(pH 6). The reaction
of DHTGFLpTEpYVATR with VHR is ordered, with rapid dephosphorylation on
tyrosine occurring first followed by slow dephosphorylation on
threonine. Similar results were obtained with F(NLe)(NLe)pTPpYVVTR, a
peptide corresponding to a MAP kinase-like protein
(JNK1
) which is involved in the stress response
signaling pathway.
A class of protein tyrosine phosphatases (PTPases), ()referred to as ``dual-specific PTPases'', is
emerging as important regulators of cell cycle control and mitogenic
signal transduction. The first dual-specific PTPase identified
corresponded to the H1 open reading frame in Vaccinia virus.
This phosphatase was called VH1 for Vaccinia open reading
frame H1 and was capable of hydrolyzing phosphate monoesters
from both phosphotyrosine and phosphoserine containing peptides (Guan et al., 1991), distinguishing this catalyst from the
tyrosine-specific PTPases. The VH1-like phosphatases and the PTPases
share the active site sequence motif HCxxGxxR, but
show limited sequence identity beyond this region. Following the
discovery of VH1, it was shown that p80
(a protein
necessary for the passage through mitosis) shared sequence identity to
VH1. Several laboratories demonstrated that p80
had
intrinsic tyrosine phosphatase activity and that it would
dephosphorylate the cyclin-dependent protein kinase p34
on Thr-14 and Tyr-15. The dephosphorylation then led to the
activation of p34
and subsequent entry into mitosis
(Dunphy and Kumagai, 1991; Galaktionov and Beach 1991; Gautier et
al., 1991; Kumagai and Dunphy, 1991; Millar et al., 1991;
Strausfeld et al., 1991).
Ishibashi et al.,(1992) identified a dual-specific PTPase, VHR (for VH1-Related) using an expression cloning strategy (Fig. 1). The human fibroblast cDNA clone encoded a protein of 185 amino acids. The bacterially expressed glutathione S-transferase/VHR fusion protein was shown to be a protein phosphatase with dual specificity based upon its ability to hydrolyze phosphoserine from casein as well as phosphotyrosine from a number of tyrosine-phosphorylated growth factor receptors (Ishibashi et al., 1992).
Figure 1: A schematic diagram of the protein structure of the dual-specific protein tyrosine phosphatases.
Two laboratories have independently identified an identical dual-specific phosphatase (Cdi1 or KAP) (Gyuris et al., 1993; Hannon et al., 1994). Both laboratories demonstrated that Cdi1 (KAP) interacts with the cyclin-dependent kinases CDK2 and cdc2, suggesting that these PTPases may be directly involved in cell cycle control. It is important to note that activation of these cyclin-dependent kinases appears to be dependent upon the phosphorylation state of adjacent tyrosine and threonine residues. The list of dual-specific phosphatases (Table 1) includes the mammalian proteins PAC-1 and MKP-1 (Fig. 1). These phosphatases have been shown to dephosphorylate and inactivate threonine- and tyrosine-phosphorylated MAP (mitogen-activated protein) kinase in vivo (Ward et al., 1994; Sun et al., 1993). MAP kinase must be phosphorylated on adjacent tyrosine 185 and threonine 183 residues for activation (Her et al., 1993). Both MKP-1 and PAC-1 appear to be immediate-early gene products. Dual-specific phosphatases are also present in yeast with recent reports suggesting involvement of MSG5 in the mating pathway (Doi et al., 1994) and of YVH1 in nitrogen regulation (Guan et al., 1992), (Table 1, Fig. 1). Based upon the number of recently reported sequences of dual-specific phosphatases, it would appear that these proteins constitute a large family of catalysts.
In the few cases that have been investigated, these phosphatases appear to display a marked preference for protein kinases which can be phosphorylated on tyrosine and threonine in close proximity (Table 1). Despite the growing number of dual-specific PTPases identified, little is known about the biochemical mechanisms employed by this family of enzymes. The current investigation begins to explore the catalytic properties of the dual-specific phosphatases. Our strategy was to select a prototype of this class of phosphatases, express and purify large quantities of the recombinant enzyme, characterize the protein biochemically and crystallize the enzyme for x-ray structure determination. Since human VHR contains the catalytic domain which is common to all members of this class (Fig. 1), this enzyme was an ideal choice for analysis. Here, we describe the overexpression, purification, and biochemical and kinetic characterization of VHR.
Size exclusion chromatography was performed on a Pharmacia fast protein liquid chromatography system using a Superose 12 column and a mobile phase buffer consisting of 50 mM Tris, 150 mM NaCl, 1 mM EDTA (pH 7.4), flowing at a rate of 0.25 ml/min.
The general approach to phosphopeptide synthesis used in this study,
global phosphorylation, involved post-synthetic phosphorylation while
the peptide was still attached to the solid-phase support with all of
its protecting groups intact except those on selected hydroxyl groups
(Kitas et al., 1991; Andrews et al., 1991). The
hydroxyamino acids were introduced during synthesis as the FMOC-amino
acids with no side chain protection. Tyr, Thr, and Ser residues which
were not to be phosphorylated contained t-butyl ether
protecting groups. The NH-terminal amino acid was
incorporated as the t-Boc-protected amino acid. The resin was
dried for 24 h on a vacuum system at room temperature before swelling
in anhydrous DMF. Reaction with a 20-fold molar excess of the
phosphoramidite and 50-fold molar excess of tetrazole for 1-2 h
was similar to previous reports (Kitas et al., 1991; Andrews et al., 1991). The resin was washed thoroughly with dry DMF
and then subjected to oxidation with 0.5 Mt-butyl
peroxide in DMF for times ranging from 0.5 to 4 h at room temperature.
The phosphorylated peptide resin was then washed thoroughly with DMF,
methylene chloride, dried, and subjected to cleavage and deprotection
in 90% trifluoroacetic acid and 5% thioanisole, 3% ethanedithiol, 2%
anisole. The cleaved peptide was then precipitated by addition of 15
volumes cold diethylether.
One of the synthetic peptides (DHTGFLpTEpYVATR) exhibited only low levels of phosphorylation. The use of alternative solvents or longer reaction times did not significantly improve the extent of phosphorylation. This was attributed to steric effects of the amino-terminal segment since shorter forms of this peptide were quite efficiently phosphorylated. The approach used was to synthesize the peptide by normal procedures except that the synthesis was stopped after 8 residues, the FMOC protecting group left on the Leu residue, and the peptide resin subjected to the normal phosphorylation protocol. After the oxidation step, the peptide was thoroughly washed and placed back on the synthesizer, and the last 5 amino acid residues coupled. A significant amount of dephosphorylation occurred at the Thr residue during the piperidine deprotection step in the subsequent cycles. This was reduced by shortening the piperidine deprotection step in each cycle and incorporating a t-Boc-protected amino acid in the amino-terminal position.
Three different types of quantitative enzymatic assays were employed
in these studies. A three-component buffer consisting of 0.05 M Tris, 0.05 M Bis-Tris, and 0.1 M acetate was
used in all the kinetic analyses. This buffer maintains constant ionic
strength over its useful pH range (Ellis and Morrison, 1982). Since
preliminary studies (data not shown) indicated that enzymatic activity
was sensitive to ionic strength, maintaining constant ionic strength
was critical. This buffer exhibited no inhibitory effects on enzymatic
activity. All assays were performed at 30 °C. Using pNPP as a
substrate, the phosphatase reaction was followed as an increase in
absorbance at 405 nm of the product para-nitrophenolate (Zhang
and Van Etten, 1991). Initial rates were determined from the change in
absorbance upon addition of 1 N NaOH. Rates were determined
over the linear region of the reaction using the molar extinction
coefficient of 18,000 M cm
for the product para-nitrophenolate. Inital rates at
various initial substrate concentrations were then fit directly to the
Michaelis-Menten using the nonlinear least-squares program Kinetasyst for the Macintosh (IntelliKinetics, State College,
PA).
Figure 2: SDS-polyacrylamide gel electrophoresis of the purification steps for VHR. Lane 1, polyethyleneimine (0.5%) precipitation, supernatant; lane 2, 35-65% ammonium sulfate precipitation; lane 3, S-Sepharose cation-exchange column; lane 4, Sephadex G-75 gel filtration column. Amount of protein in lanes 3 and 4 are 6 and 10 µg, respectively.
To establish the oligomeric state of the enzyme, VHR was subjected to size exclusion chromatography as outlined under ``Materials and Methods'' (Fig. 3). The purified enzyme was injected onto the column at a concentration of 10 mg/ml. The elution time corresponded to a protein with an apparent molecular weight of 20,600, which was in good agreement with the mass of the monomer predicted by amino acid sequence composition (20,500 for 185 amino acids).
Figure 3: Size exclusion chromatography of VHR. Enzyme was injected at a concentration of 10 mg/ml and eluted in 50 mM Tris, 1 mM EDTA, 150 mM NaCl (pH 7.4) using a Superose 12 gel filtration column as described under ``Materials and Methods.'' Molecular size standards were bovine serum albumin (66,000), egg albumin (44,000), carbonic anhydrase (29,000), and cytochrome (12,400).
To establish the integrity of the purified enzyme,
VHR was subjected to mass spectral, amino acid, and amino-terminal
sequence analysis. The first six cycles of amino-terminal sequencing
established that the protein begins with Ser-Gly-Ser-Phe-Glu-Leu. This
is identical to the predicted sequence except that the initiator Met
was removed. Amino acid analysis and the amino acid content predicted
by the cDNA sequence were in excellent agreement (data not shown).
Matrix-assisted laser desorption mass spectrometry analysis yielded an
apparent mass of 20,500 ± 100, in good agreement with the
predicted molecular weight. Consistent with these results, the purified
enzyme contains 184 amino acids with a molecular weight of 20,400.
Based upon amino acid analysis and UV absorbance measurements of
purified VHR, the molar extinction coefficient at 280 nm was 11,500 M cm
. All subsequent
concentrations of VHR were determined by using this molar extinction
coefficient.
Figure 4:
Crystal of VHR. Crystal was grown by
repeated macroseeding as described under ``Results.'' This
typical crystal (0.38 0.2
0.1 mm
)
diffracted to 2.3 Å resolution.
To explore the dual specificity and the substrate specificity
of VHR, the kinetic parameters k, K
, and k
/K
were determined for a variety of synthetic peptides including
diphosphorylated MAP
(DHTGFLpTEpYVATR) kinase
and JNK1
kinase (F(Nle)(Nle)pTPpYVVTR), (Table 3). Phosphotyrosine dephosphorylation can be followed
continuously using the spectrophotometric methods (Fig. 5)
described under ``Materials and Methods.'' The k
/K
parameter is the
apparent second-order rate constant for the reaction between free
substrate and free enzyme and involves both the steps in binding of substrate and catalysis. The k
/K
value is therefore an
excellent indicator of the quality of a particular substrate. On the
other hand, the kinetic parameter k
involves
only the steps in catalysis and product release. The diphosphorylated
peptides corresponding to MAP and JNK kinases (Table 3) yielded
the highest k
/K
values of
all substrates, 32,300 and 26,600 M
s
, respectively. For comparison, the
Neu
(DAEEpYLVPQQG) peptide (Zhang et
al., 1993), monophosphorylated on tyrosine, yielded a k
/K
value of 4080 M
s
that was only
slightly higher than pNPP (3240 M
s
). For VHR, the tyrosine-monophosphorylated
MAP kinase peptide DHTGFLTEpYVATR yielded a k
/K
value of 11,200 M
s
.
Figure 5:
VHR- catalyzed hydrolysis of
phosphotyrosine on JNK1 kinase. Initial
concentration of F(NLe)(NLe)pTPpYVVTR was 1.8 mM, and enzyme
concentration was 5 µM. Conditions: 30 °C and pH 6. Solid line is a fit of the data to , and the circles represent 200 data points.
Because the
continuous spectrophotometric assay for phosphotyrosine-specific
hydrolysis was invisible to the hydrolysis at phosphothreonine, we
developed an HPLC-based assay that would allow us to monitor all
possible reactions within a single experiment. This method can provide
information on the order of hydrolysis at each site as well as the
relative rates of hydrolysis. The apparent second-order rate constant k/K
for dephosphorylation
of phosphothreonine on the diphosphorylated DHTGFLpTEpYVATR
(MAP
kinase) and F(NLe)(NLe)pTPpYVVTR
(JNK1
kinase) peptides was determined by
utilizing HPLC to separate and quantitate all four possible
phosphorylation states, -TxY-, -pTxY-,
-TxpY-, and -pTxpY-, as described under
``Materials and Methods.'' All peaks in the HPLC elution
profiles were identified by a combination of mass spectral analysis and
by comparison with the retention times of the authentic peptides. Fig. 6shows the elution profile at different reaction times when
VHR was reacted with diphosphorylated MAP
kinase peptide DHTGFLpTEpYVATR. Within the first 6 min, the
DHTGFLpTEpYVATR peptide was rapidly (32,000 M
s
) and completely dephosphorylated on
tyrosine, thus generating phosphothreonine peptide DHTGFLpTEYVATR which
was then slowly hydrolyzed by VHR, yielding completely dephosphorylated
peptide DHTGFLTEYVATR. No detectable amount of monophosphorylated
phosphotyrosine peptide was observed in these experiments with either
MAP
kinase or JNK1
kinase. The amount of unlabeled peptide was calculated by amino
acid analysis of the peak and was correlated with peak area to
determine the concentration of peptide at each time point.
Alternatively, the amount of peptide formed was determined by
correlating the peak area to the amount of starting peptide. Fig. 7shows the data from the time course of reaction for
hydrolysis at phosphothreonine with diphosphorylated
MAP
kinase peptide (DHTGFLpTEpYVATR) and the
fit to the integrated Michaelis-Menten . The fit yielded a k
/K
value of 13.5 ±
0.3 M
s
(pH 6) which was
about 200-fold lower than the rate obtained with pNPP and about
2000-fold slower than phosphotyrosine hydrolysis on the same
diphosphorylated peptide. Because the phosphothreonine peptide
substrate DHTGFLpTEYVATR was not present at saturating levels (i.e.K
1 mM), the parameter k
could not be determined with good accuracy. At
pH 7, the k
/K
value
decreased to 3.5 ± 0.3 M
s
, consistent with the decrease in values seen
with both the phosphotyrosine peptides and pNPP as substrates. These
results suggest that the pH optimum is close to pH 6, with activity
decreasing at higher pH. Using the same HPLC-based analysis, a similar k
/K
value of 16.8 ±
0.3 M
s
for
phosphothreonine hydrolysis of diphosphorylated JNK1
kinase peptide (F(NLe)(NLe)pTPpYVVTR) was determined.
Figure 6:
HPLC elution profile of the products of
diphosphorylated MAP kinase peptide
(DHTGFLpTEpYVATR)-catalyzed hydrolysis by VHR. Results represent
reactions done at 30 °C and pH 6.0. Initial peptide concentration
was 300 µM, and the VHR concentration was 1.65
µM. Details are given under ``Materials and
Methods.''
Figure 7:
Phosphothreonine hydrolysis of
diphosporylated MAP kinase peptide
(DHTGFLpTEpYVATR) catalyzed by VHR. Data points represent the
quantitation of the results shown in Fig. 6as described under
``Materials and Methods.'' Solid line is a fit of
the data to .
We have described a simple and convenient purification strategy for obtaining large amounts of the native human dual-specific PTPase VHR. Because results obtained from kinetic and biochemical analyses can be altered by engineered proteins, VHR was overexpressed as the native enzyme coded for by its cDNA sequence and not as a fusion protein nor as a histidine-tagged protein. This has enabled us to crystallize the enzyme for structure determination, physically characterize the enzyme and investigate its substrate specificity. Other than demonstrating the general phosphatase activity of this growing class of PTPases, there has been no report on the details of this reaction, the relative rates of dephosphorylation on the putative Thr-183 and Tyr-185 of MAP kinase and the substrate requirements of this important class of enzymes.
After VHR was purified to
homogeneity, numerous physical methods were performed to ensure the
integrity of the enzyme. Results from amino acid analysis, MALDI mass
spectral analysis, and size-exclusion chromatography are consistent
with the predicted mass of 20.4 kDa. With NH-terminal amino
acid sequencing revealing that the initiator Met was cleaved, the
protein contains 184 amino acids.
Once the integrity of the enzyme
was confirmed, the substrate specificity was determined. VHR will
effectively dephosphorylate purified recombinant MAP kinase; however,
because phosphorylation of MAP kinase by MAP kinase kinase was never
stoichiometric on both tyrosine 185 and threonine 183, an alternative
assay was developed to unambiguously determine the rates of
dephosphorylation. Using stoichiometrically diphosphorylated
MAP kinase (DHTGFLpTEpYVATR) and
JNK1
kinase (F(Nle)(Nle)pTPpYVVTR) peptides in
the continuous spectrophotometric assay and the HPLC method described
here, the rates of the VHR-catalyzed hydrolysis at each site were
determined. The diphosphorylated peptides are clearly the best
substrates in terms of phosphotyrosine hydrolysis, with K
values between 100 and 200 µM and k
/K
values of 30,000 M
s
. In comparison,
peptides monophosphorylated on tyrosine have K
values above 1 mM. When the diphosphorylated peptides
are reacted with VHR, no monophosphotyrosine peptide was ever detected,
indicating that the dephosphorylation is ordered. The phosphotyrosine
residue is rapidly hydrolyzed first at a rate of 30,000 M
s
followed by the slow
(15 M
s
) rate of
hydrolysis at the threonine position (Fig. 8).
Figure 8:
The ordered dephosphorylation of
diphosphorylated MAP kinase peptide
(DHTGFLpTEpYVATR) and JNK1
kinase
F(NLe)(NLe)pTPpYVVTR by VHR. The numbers refer to the relative
rates of dephosphorylation at tyrosine (2000) and
threonine(1) .
Both the
dual-specific PTPases and the PTPases contain an essential cysteine
residue that is thought to be the nucleophile which attacks the
phosphorus atom, forming a thiol-phosphate intermediate along the
catalytic pathway. We have previously established the importance of
Cys-124 in the VHR-catalyzed reaction toward both phosphotyrosine and
phosphoserine/threonine containing peptides (Zhou et al.,
1994). When this cysteine is replace with serine, the enzyme is
functionally inactive toward both types of substrates, suggesting a
similar mechanism and the same active site with both types of
substrates. The proposed intermediate was trapped and characterized
when VHR was mixed with
[P]phosphotyrosine-labeled Raytide. However,
attempts to trap the intermediate with
phosphoserine/threonine-containing peptides failed. This apparent
inconsistency can be explained with the results observed in the current
study. The amount of intermediate formed in the reaction is directly
proportional to the ratio of the net rate of breakdown to the net rate
of formation of the intermediate. Given that the k
/K
value for
phosphothreonine peptide hydrolysis is about three orders of magnitude
slower than hydrolysis of phosphotyrosine containing peptide, the
amount of intermediate would also be expected to drop by the same
magnitude. The k
/K
parameter involves the steps of binding through the release of
the first product, the alcohol. Thus, this rate constant measures the
net rate of formation of the intermediate. Since a common
thiol-phosphate intermediate would be formed with both phosphothreonine
and phosphotyrosine substrates, the rate of breakdown of the
intermediate is expected to be identical. As a result, the levels of
trapped intermediate with phosphoserine/threonine substrates would be
negligible and therefore undetectable by the described trapping method.