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(Received for publication, August 3, 1995; and in revised form, October 23,
1995) From the
Low M Low M
Low M Little is known
about the regulation of the activity of cytosolic PTPases. From some
research it appears that these enzymes can be subject to tyrosine
phosphorylation, and, for some of them, the regulatory meaning of such
modification has been demonstrated. PTP1C, containing two SH2 domains,
is phosphorylated in vivo in response to colony stimulating
factor-1 (CSF-1) (8) and insulin stimulation (9) and
following v-src transformation of fibroblasts(10) . It
is also tyrosine-phosphorylated by pp60 pp60 The oncogenic
form, pp60 In this study we demonstrate that
low M
To follow the auto-dephosphorylation of the enzyme,
1 µg of inactivated PTPase, which had been previously
v-Src-phosphorylated, was mixed with 0.05 µg of active recombinant
PTPase. The incubation was carried out in 10 mM acetate, pH
4.9, 1 mM EDTA, 1 mM dithiothreitol at room
temperature. The control reaction mixture did not include the active
PTPase. At various time intervals, aliquots of the reaction mixture
were sampled and boiled in 2 To subsequently determine the activity of the
phosphorylated enzyme, the kinase reaction included active low M
When the phosphopeptide
was the substrate, the reaction was stopped by the addition of
trichloroacetic acid (3% final concentration), and the mixture was
centrifuged at 18,000 rpm for 5 min. Aliquots of the supernatant were
used to determine the phosphate released by the PTPase activity with
the malachite green colorimetric assay(25) .
After transfection with pSVPTP, v-src transformed
NIH/3T3 cells displayed high overexpression levels of low M To investigate the possible effects of such
PTPase activity on the cellular phosphorylation balance, we obtained
whole cell lysates and analyzed them by SDS-polyacrylamide gel
electrophoresis, followed by immunoblotting with anti-Tyr(P) antibody (Fig. 1A). No significant differences between PTPase
overexpressing v-src transformed NIH/3T3 and control cells
were evidenced, except for a protein of approximately 18 kDa that
reacted significantly with anti-Tyr(P) antibodies in cells
overexpressing the low M
Figure 1:
Low M
Similar analysis conducted on PTPase-transfected NIH/3T3
cells, either normal or transformed by the deregulated tyrosine kinase
coded by v-erbB, did not show any tyrosine phosphorylation on
the overexpressed enzyme (data not shown). This observation and the
obvious evidence that Src-tyrosine kinase activity is the major cause
for Tyr phosphorylation in v-src transformed NIH/3T3 cell
line, led us to hypothesize the direct involvement of this kinase in
low M To verify this
hypothesis we immunoprecipitated the pp60
Figure 2:
Low M
The auto-dephosphorylation was
confirmed by incubating PAO-inactivated and previously phosphorylated
PTPase with the active enzyme. Fig. 3shows that the enzyme was
capable of dephosphorylating itself intermolecularly. Analysis of the
phosphorylated amino acids conducted on the in vitro phosphorylated enzyme revealed the significant presence of
phosphotyrosine, while neither phosphoserine nor phosphothreonine was
detected.
Figure 3:
Low M
Other PTPases are known to be substrates of tyrosine
kinases, and, in some cases, the regulatory significance of the
subsequent modification has been demonstrated: PTP1D-Syp is
tyrosine-phosphorylated in response to the stimulation of receptors by
various growth factors (11) as well as in cells transformed by
the tyrosine kinase pp120 In order to assess if tyrosine
phosphorylation of low M
Figure 4:
Activity enhancement of phosphorylated low M
We
had previously observed that parental NIH/3T3 cells overexpressing low M The overexpression of low M The preceding evidence points
to a selective activity of this PTPase on receptor tyrosine kinases. In
order to ascertain a possible effect of this enzyme on the mitogenic
signal cascade downstream of growth factor receptors, we decided to
transfect the enzyme in v-src transformed NIH/3T3 cells. It is
well known that pp60 PTPase-transfected v-src-NIH/3T3 show levels of
overexpression much higher then those obtained when normal NIH/3T3 were
transfected; moreover, the enzyme is activated when it is
tyrosine-phosphorylated by pp60
Figure 5:
Growth kinetics of parental and low M
PTPase tyrosine phosphorylation could in part
explain how a cytosolic enzyme would act selectively upon membrane
substrates. In fact, this enzyme has neither sequential nor structural
features that can mediate its association with the membrane: it does
not have SH2 domains like SHP or SH-PTP1 or myristoylation sites like a Dictyostelium PTPase, nor does it have cytoskeletal
protein-like domains as does a PTPase found in HeLa cells(5) . If pp60
Volume 271,
Number 3,
Issue of January 19, 1996 pp. 1278-1281
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Phosphorylates and Activates Low Molecular Weight
Phosphotyrosine-protein Phosphatase (*)
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
phosphotyrosine-protein phosphatase
belongs to the non-receptor cytosolic phosphotyrosine-protein
phosphatase subfamily. It has been demonstrated that this enzyme
dephosphorylates receptor tyrosine kinases, namely the epidermal growth
factor receptor in vitro and the platelet-derived growth
factor receptor in vivo. phosphotyrosine-protein phosphatase is constitutively
tyrosine-phosphorylated in NIH/3T3 cells transformed by
pp60. The same tyrosine kinase,
previously immunoprecipitated, phosphorylates this enzyme in vitro as well. Phosphorylation is enhanced using phosphatase inhibitors
and phenylarsine oxide-inactivated phosphatase, consistently with the
existence of an auto-dephosphorylation process. Intermolecular
dephosphorylation is demonstrated adding the active enzyme in a
solution containing the inactivated and previously phosphorylated one.
This tyrosine phosphorylation correlates with an increase in catalytic
activity. Our results provide evidence of a physiological mechanism of
low M
phosphotyrosine-protein phosphatase activity
regulation.
phosphotyrosine-protein phosphatase
(PTPase) (
)is an enzyme that possesses the characteristic
PTPase CXXXXXR motif in the active site(1) . The
cysteine residue present in this sequence is absolutely necessary for
the activity of the enzyme, as it forms a phosphointermediate during
the reaction mechanism(1, 2) . The phosphate-binding
loop shows a striking structural similarity to the same loop of the
cytosolic phosphotyrosine-protein phosphatase PTP1B. All these
features, together with the overall recently described 
/
structure(3) , identify low M PTPase as
belonging to the PTPase family. The enzyme was originally localized in
the cytosol(4) , and it can be considered a non-receptor
cytosolic PTPase. Its structure does not display any features that can
mediate its stable or temporary association with the membrane as occurs
in the case of a PTPase containing Src-homology 2 (SH2) domains (SHP,
SH-PTP1), myristoylation sites (a PTPase from Dictyostelium yeast), or cytoskeletal protein-like domains (a PTPase found in
HeLa cells)(5) . Nevertheless, its ability to act upon membrane
substrates has been demonstrated: it dephosphorylates the EGF-r in
vitro(6) and particularly the PDGF-r in
vivo(7) . When the enzyme is overexpressed in the NIH/3T3
cell line, the response to PDGF is inhibited dramatically, and the
receptor appears to be phosphorylated to a much lesser degree. This
evidence suggests that the PDGF-activated receptor can be
down-regulated by low M PTPase. This was further
confirmed by experiments performed in NIH/3T3 cells transfected with
the negative dominant form of the enzyme, obtained by substituting the
Cys-12 residue with Ser, showing the co-immunoprecipitation of PDGF-r
with low M PTPase. (
)in vitro, with a resulting increase in its
activity. Another PTPase that is highly homologous to the preceding,
named PTP1D or Syp, is tyrosine-phosphorylated in response to EGF or
PDGF stimulation (11) and in cells transformed by the tyrosine
kinase pp120
(12) . In this case, it
has been hypothesized that the modification may mediate the enzyme
association with the SH2 domains of other tyrosine-phosphorylated
proteins, which may become substrates of the PTPase activity.
is a widely expressed tyrosine kinase
that can associate with membranes thanks to an amino-terminal acylation
site. This kinase is implicated in various cell functions, including
cell to cell contact, neural differentiation, and cell
proliferation(13) , being known to be located downstream of the
PDGF receptor in the mitogenic signal cascade that proceeds from this
growth factor stimulation(14) . It is known to phosphorylate a
number of substrates, among which we can cite GTPase activating protein
of Ras (GAP), pp125 focal adhesion kinase (FAK), pp120, and other
components of the cytoskeleton(15, 16) . Furthermore,
it can autophosphorylate on Tyr-416 (13) .
, is permanently active
since it lacks the carboxyl-terminal region comprising Tyr-527 (17) . In fact, the phosphorylation in
pp60
of this residue determines its
intramolecular binding with the SH2 domain, resulting in the
inaccessibility of the active site with the subsequent inhibition of
the kinase activity(18) .
PTPase is phosphorylated by Src-kinase in vitro and in NIH/3T3 cells transformed by v-src.
This in vitro phosphorylation causes an in-crease in the
enzyme activity and could be a way to regulate it in vivo as
well.
Cell Culture and Transfection
v-src transformed NIH/3T3 murine fibroblasts were obtained from the
Laboratory of Cellular and Molecular Biology of the NCI, National
Institutes of Health and have been described(19, 20) .
The cells were routinely cultured in a humidified atmosphere of 5%
CO at 37 °C in Dulbecco's modified Eagle's
medium (Sigma D7777) supplemented with 10% fetal calf serum (complete
medium). The cells were co-transfected with pSVPTP plasmid, containing
a synthetic DNA sequence coding for low M PTPase (21) under the control of the SV40 early region promoter, and
with pK0neo, expressing neomycin (G418) resistance, using the calcium
phosphate precipitation method as described previously(22) .
Stable G418-resistant clones were selected by supplementing the culture
medium with 400 µg/ml antibiotic. The clones were periodically
cultured in the same selective medium to maintain a stable expression.Evaluation of PTPase Overexpression
Northern blot
analysis of total RNA was performed under standard conditions,
according to Ramponi et al.(22) , using the P-labeled PTPase synthetic gene as a probe. The low M
PTPase content was evaluated by a noncompetitive
immunoenzymatic assay as described previously(23) . The
activity of the overexpressed enzyme was checked in both parental and
low M PTPase transfected cell lysates using p-nitrophenyl phosphate (pNPP) as a substrate. The
overexpressed activity was totally inhibited by vanadate.Electrophoresis and Immunoblotting
The cells were
lysed in 66 mM Tris-HCl, pH 6.8, containing 2% SDS, 10 mM EDTA, 10% glycerol, and bromphenol blue, and were boiled for 10
min. After a 10-min centrifugation at 13,000 rpm, the total protein
content was determined by the bicinchoninic acid solution (BCA) protein
reagent assay; equal protein amounts from whole cell extracts were
subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) on a 12%
gel and then electrotransferred to a nitrocellulose membrane
(Sartorius). After blocking with 1% bovine serum albumin (BSA) in
Tris-buffered saline (TBS) (25 mM Tris, pH 7.4, 0.8% NaCl,
0.2% KCl) containing 0.1% Tween 20 for 60 min, the membrane was
incubated with RC20 anti-phosphotyrosine antibody conjugated to
horseradish peroxidase (Transduction Laboratories) for an additional 60
min. After an extensive washing with TBS-T (0.5% Tween 20 in TBS), the
immunoreactive proteins were visualized by enhanced chemiluminescence
(ECL) (Amersham) detection following the manufacturer's
instructions. After incubation for 30 min at 50 °C in stripping
buffer (62.5 mM Tris-HCl, pH 6.7, 100 mM 2-mercaptoethanol, 2% SDS) and extensive washing, the membranes
were reprobed with polyclonal anti-low M PTPase
horseradish peroxidase-conjugated antibodies (produced and conjugated
in our department as described previously(23) ), following the
same procedure.pp60
v-src transformed NIH/3T3 cells were grown to confluence on a 100-mm
dish, washed twice with phosphate-buffered saline, and then collected
in 500 µl of IPA buffer (10 mM Tris-HCl, pH 7.45, 0.15 M NaCl, 0.1% SDS, 1% Triton X-100, 1% sodium deoxycholate, 5
mM EGTA, 1 mM phenylmethylsulfonyl fluoride, 1 mM sodium vanadate). After 10 min on ice, the cell lysate was
centrifuged at 12,000 rpm for 10 min, and 10 µl of mouse
anti-pp60 Immunoprecipitation
monoclonal antibody (Chemicon
International Inc. MAB4030, ascites fluid) were added to the
supernatant that was maintained on ice for 1 h. 25 µl of Protein
A-Sepharose (Sigma), previously incubated for 1 h on ice with 5 µl
of rabbit antibody to mouse IgG, were mixed with the above lysate.
After 1 h of incubation in a shaker at 4 °C, the beads were
collected by centrifugation and washed four times with 1 ml of IPA
buffer.
Expression and Purification of Recombinant Low M
The enzyme used in the in vitro phosphorylation experiments was obtained as a fusion protein with
glutathione S-transferase(24) . The fusion protein was
cleaved by thrombin, and PTPase was then purified. The enzyme was fully
active. PTPaseLow M
The
recombinant enzyme was incubated for 30 min on ice in TBS containing 1
mg/ml BSA and 100 µM PAO to achieve its total
inactivation. Unreacted PAO was removed by dialysis. PTPase InactivationIn Vitro Low M
To evaluate the extent of PTPase
phosphorylation, the kinase reaction mixture included PAO-inactivated
recombinant low M PTPase Phosphorylation and
Dephosphorylation PTPase (1 µg), kinase buffer
(25 mM Hepes pH 7.5, 10 mM MgCl, 10
mM MnCl, 0.1% Nonidet P-40, 100 mM NaCl,
10% glycerol), BSA (1.5 µg), 100 mM sodium vanadate, 0.2
mM ATP, 22.5 µCi of [-
P]ATP,
and Protein A-Sepharose beads (5 µl) bound to
pp60
, in a total reaction volume of 15
µl. The control reaction mixture did not include low M
PTPase. The reaction was carried out at 30
°C. Samples were withdrawn at 30 and 120 min, mixed with 2 
Laemmli sample buffer, boiled, and run on a 12% SDS-PAGE. The gel was
then dried and exposed to a film (Kodak x-ray) to obtain the
autoradiography. Laemmli sample buffer. After
SDS-PAGE, the PTPase phosphorylation extent was evaluated by
autoradiography. PTPase, kinase buffer containing 60 mM 2-mercaptoethanol, BSA, -thioATP (Boehringer Mannheim), and
Protein A-Sepharose beads bound to pp60
in
the same quantities as in the phosphorylation test. The control
reaction mixture did not include
-thioATP. The incubation was
carried out at 30 °C. Supernatant aliquots were assayed at various
time intervals for their PTPase activity.
PTPase Activity Assay
The activity of the enzyme
was determined at 37 °C in 0.1 M acetate, 1 mM EDTA, pH 5.5, using alternatively as substrates p-nitrophenyl phosphate (4 mM) or the peptide
corresponding to the sequence 767-776 of the PDGF receptor (0.5
mM), containing a phosphotyrosine.Determination of Growth Kinetics
Control and low M PTPase overexpressing v-src-NIH/3T3
cells were seeded at low density (10,000 cells/cm) on a
multiwell plate in complete medium. Cell number was determined every 24
h for the following 3 days using a Bürker chamber.
Four wells were counted every day.Phosphoamino Acid Analysis
An aliquot of the in vitro phosphorylated low M PTPase was
sampled from the supernatant of the reaction mixture before Laemmli
buffer addition and was partially digested with 6 M HCl at 110
°C for 2 h. The phosphoamino acid separation was performed in high
pressure liquid chromatography on a DEAE-column equilibrated with 10
mM potassium phosphate, pH 3.0, 12.5% methanol. After a
post-column derivatization with o-phthalaldehyde, the amino
acids were detected through their fluorescence emission and collected.
The P-labeled amino acids were quantified in a liquid
scintillation counter.
PTPase as demonstrated by its determination in a
noncompetitive immunoenzymatic assay and confirmed by Northern blot RNA
analysis. The overexpressed enzyme was active on pNPP, and this
activity was completely sensitive to vanadate inhibition, as all
PTPases are known to be. PTPase. The
nitrocellulose membrane was then stripped and incubated with
anti-PTPase horseradish peroxidase-conjugated antibodies (Fig. 1B). The ECL exposition revealed that the
tyrosine-phosphorylated 18-kDa protein in PTPase-overexpressing cells
was specifically recognized by anti-low M PTPase
antibodies.
PTPase overexpression and tyrosine phosphorylation in
v-src-NIH/3T3. Whole cell lysates were run on a 12% SDS-PAGE. Lanes 1 and 3, control v-src-NIH/3T3; lanes 2 and 4, PTPase overexpressing
v-src-NIH/3T3. A, immunoblotting with monoclonal RC20
anti-phosphotyrosine-horseradish peroxidase antibody. B,
immunoblotting with polyclonal anti-low M PTPase-horseradish peroxidase antibody. Immunoreactive proteins
were visualized by ECL.
PTPase phosphorylation. from v-src-NIH/3T3 and used the immobilized enzyme in a
kinase assay including low M
PTPase and
[-
P]ATP. In these conditions, the enzyme
was poorly phosphorylated. The phosphorylation level increased notably
if the PTPase was inactivated previously with PAO and vanadate was
added to the kinase mixture, indicating a probable
auto-dephosphorylation when the enzyme was active. The phosphorylation
was evidenced by analysis of the reaction mixture on SDS-PAGE and
autoradiography (Fig. 2).
PTPase in
vitro phosphorylation. PAO-inactivated low M PTPase was added to a kinase reaction mixture including
[-
P]ATP, vanadate, BSA, and
immunoprecipitated pp60
. Incubation
was performed at 30 °C. At 30 min (A) and 120 min (B), aliquots were withdrawn, boiled in 2
Laemmli
sample buffer, and run on a 12% SDS-PAGE. The control reaction mixture
did not include the PTPase and was stopped at 120 min (C). The
gel was dried and exposed to a Kodak x-ray
film.
PTPase auto-dephosphorylation. Inactivated and
previously phosphorylated low M PTPase was mixed
with active PTPase and allowed to incubate at room temperature. At zero
time (A) and 60 min (B), aliquots were withdrawn and
boiled in 2 Laemmli sample buffer. The control reaction mixture (C) did not include the active PTPase and was stopped at 60
min. After a 12% SDS-PAGE, autoradiography was
performed.
(12) . In the
former case, the authors report a slight activation of the enzyme,
while in the latter case the authors did not find any variation in the
enzyme activity, since it rapidly auto-dephosphorylates; in observing
the co-immunoprecipitation of this PTPase with other
tyrosine-phosphorylated proteins containing SH2 domains, they
hypothesize that it may associate with them after its phosphorylation
and catalyze their dephosphorylation. Another PTPase, named PTP1C, is
phosphorylated in vivo in response to CSF-1 (8) and by
the activated insulin receptor both in vivo and in
vitro(9) ; evidence also suggests it is phosphorylated by
pp60
both in vivo and in
vitro(10) ; in any case, a 4-fold activation follows this
modification. The authors hypothesize a negative regulatory role of
activated PTP1C on insulin signaling.
PTPase could modulate its
activity, we had to obtain a phosphorylated form of the enzyme in the
absence of any PTPase inhibitor. For this reason, the kinase reaction,
including immunoprecipitated pp60 and
PTPase, was carried out using
-thioATP in place of ATP. The
resulting thio-phosphorylated residues were relatively resistant to
phosphotyrosine phosphatase activity(26) . The activity of the
thio-phosphorylated enzyme was assayed at various times during the
kinase reaction using both pNPP and a tyrosine-phosphorylated peptide
of the PDGF receptor as substrates. We observed an activation that
proceeded along with the phosphorylation of the enzyme. As can be seen
in Fig. 4, the activity of the phosphorylated enzyme reaches
190% on pNPP and 140% on the phosphopeptide, given the activity of the
control mixture containing the unphosphorylated enzyme as 100%.
PTPase. The enzyme was incubated at 30 °C
with immunoprecipitated pp60 in the
presence of
-thioATP. At zero time, 30, 60, and 120 min, aliquots
were sampled from the reaction mixture and assayed for their PTPase
activity using alternatively as substrates pNPP or the peptide
corresponding to the sequence 767-776 of the PDGF-r, containing a
phosphotyrosine (P-peptide). The activity was evaluated with
respect to a control reaction mixture which did not include
-thioATP and whose activity was given as 100% at any time. Similar
results were obtained in three independent
experiments.
PTPase showed a reduced mitogenic potential, and
this effect was particularly striking when cells were stimulated with
PDGF-BB. We demonstrated that such an effect was associated with the
ability of the overexpressed PTPase to dephosphorylate the activated
PDGF-r, thus suppressing the mitogenic signal which departs from the
receptor after its binding with the specific growth factor(7) .
The co-immunoprecipitation of the PDGF-r with the dominant negative
mutant of the PTPase in cells overexpressing this form of the enzyme
confirms this result. PTPase in NIH/3T3 cells transformed by
v-erbB, coding for the truncated form of the EGF receptor, led
to an analogous reduction in growth rate and in the ability to form
colonies in soft agar(22) . In this case as well, we presumed a
direct activity of the PTPase on the autophosphorylated product of
v-erbB, this hypothesis being validated by the observed
activity of low M PTPase on the autophosphorylated
EGF-r in vitro(6) . is located downstream of the PDGF-r
in the mitogenic signal cascade(14) . The mitogenic signal that
proceeds from pp60
seems to be independent
of PDGF stimulation; in fact, if we stimulate PTPase overexpressing
v-src-NIH/3T3 fibroblasts with PDGF-BB, we do not observe any
phosphorylation of the specific receptor or any increase in the
mitogenic rate with respect to control (data not shown).
in
vitro. Nevertheless, no differences in the tyrosine
phosphorylation pattern can be observed, except for the phosphorylation
of the overexpressed Low M
PTPase. Furthermore,
when we evaluate the growth rate of PTPase-transfected
v-src-NIH/3T3, we do not find any significant difference as
compared to control v-src-fibroblasts (Fig. 5). This
result could be an indirect confirmation of the fact that, at least in
NIH/3T3 fibroblasts, the phosphorylated PDGF receptor is a target of
low M PTPase anti-proliferative action, since in
the absence of such a substrate we do not observe any depression of the
mitotic potential.
PTPase overexpressing v-src-NIH/3T3.
10,000 cells/cm were seeded on a multiwell plate in
complete medium. Four wells were counted every 24 h for the following 3
days. These data come from one out of three experiments that gave
qualitatively identical results.
is also capable of
tyrosine-phosphorylating the low M
PTPase; then we
can trace a hypothetical pathway in which the Src-kinase, recruited and
activated by the stimulated PDGF-r, phosphorylates the low M PTPase, thus allowing its association with some
protein containing an SH2 domain: this could produce a redistribution
of the enzyme, possibly favoring its interaction with the PDGF-r. Our in vitro evidence suggests that this modification would also
result in a significant increase in PTPase activity, and so a more
efficient dephosphorylation of the PDGF-r might be obtained. The low M PTPase could thus participate in a feedback
control mechanism of PDGF receptor activity.
))
We are grateful to Prof. M. Stefani (Dept. of
Biochemical Sciences, University of Firenze) for the generous gift of
P-peptide.
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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