From the Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724
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ABSTRACT |
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The human band 4.1-related protein-tyrosine
phosphatase PTPH1 was introduced into NIH3T3 cells under the control of
a tetracycline-repressible promoter. Ectopic expression of wild type
PTPH1 dramatically inhibited cell growth, whereas a catalytically
impaired mutant showed no effect. To identify the direct target of
PTPH1 in the cell, we generated a substrate-trapping mutant, in which
an invariant aspartate residue was changed to alanine (D811A in PTPH1).
The PTPH1-D811A mutant trapped primarily a 97-kDa
tyrosine-phosphorylated protein, which was determined to be VCP (also
named p97 or yeast CDC48), from various cell lysates in
vitro. However, when expressed in mammalian cells, the D811A
mutant was observed to contain high levels of phosphotyrosine and did
not trap substrates. Mutation of tyrosine 676 to phenylalanine (Y676F)
in the PTPH1-D811A mutant led to a marked reduction in phosphotyrosine
content. Furthermore, this double mutant specifically trapped VCP
in vivo and recognized the C-terminal tyrosines of VCP,
whose phosphorylation is important for cell cycle progression in yeast.
Like wild type PTPH1, this double mutant also inhibited cell
proliferation. Moreover, induction of wild type PTPH1 resulted in
specific dephosphorylation of VCP without changing the overall
phosphotyrosine profile of the cells. VCP has been implicated in
control of a variety of membrane functions, including membrane fusions,
and is a regulator of the cell cycle. Our results suggest that PTPH1
may exert its effects on cell growth through dephosphorylation of VCP,
thus implicating tyrosine phosphorylation as an important regulator of
VCP function.
Reversible protein tyrosine phosphorylation, coordinated by the
action of protein-tyrosine kinases and protein-tyrosine phosphatases (PTPs),1 is a key mechanism
in regulating many cellular activities. The diversity and complexity of
PTPs are beginning to rival that of protein-tyrosine kinases and it is
now clear that PTPs are equally important in delivering both positive
and negative signals for proper function of cellular machinery.
Approximately 80 PTPs have been identified, with ~300 members
estimated from genome sequencing efforts (1). However, information
about the function and regulation of most PTPs in cellular contexts is limited.
PTPH1 and PTPMEG are the prototypes of a growing family of PTPs
characterized by N-terminal segments containing sequence motifs with
homology to band 4.1 domains and PDZ domains (2-4). Band 4.1 domains
are responsible for targeting proteins to the cytoskeleton-membrane interface (5). PTPH1 was found to be mostly associated with membrane
structures in the cells and this association appears to be mediated by
the N-terminal portion of
PTPH1.2 PDZ domains have been
implicated in mediating protein-protein interactions, recognizing
C-terminal valine residues, or binding to other PDZ domains in their
targets (6). The PDZ domains of PTPBAS (also known as FAP-1), a member
of the PTPH1 family, were found to interact with the cell surface
receptor FAS and, as a result, it has been suggested to be involved in
FAS-mediated apoptosis (7). We have shown that the N-terminal segment
of PTPH1 exerts an inhibitory effect on its enzymatic activity in vitro (8). In addition, we have found that 20-50% of PTPH1 is
complexed with the 14-3-3 adaptor protein in cells, in a manner that is
dependent on the phosphorylation of PTPH1 and is regulated by mitogenic
signals (4). However, the biological function and physiological
substrate(s) of PTPH1 remain to be elucidated.
To understand the function of PTPH1, it is critical to identify its
physiological substrates. PTP catalytic domains consist of ~240
residues containing a number of invariant amino acids, including those
in the PTP signature motif (9). Of particular interest are the
invariant cysteine residue, which functions in nucleophilic attack upon
the phosphate group of the substrate, and the invariant aspartate,
which acts both as a general acid to facilitate the protonation of the
tyrosyl leaving group and as a general base to facilitate hydrolysis of
the cysteinyl-phosphate intermediate (10). Mutation of the aspartate
residue to alanine dramatically slows catalysis, with minimal effect on
substrate affinity, thus stabilizing the enzyme-substrate complex (11). This observation led us to develop a "substrate trapping" approach for identifying physiological substrates of various PTPs (11-13). In
this approach, substrates are isolated as proteins that bind specifically to the Asp We expressed the substrate-trapping mutant of PTPH1 as a GST fusion
protein, GST-PTPH1-D811A, and found that it interacted primarily with a
97-kDa protein from various cell lysates in vitro. Peptide
sequencing revealed that the 97-kDa protein was the
valosin-containing protein (VCP),
an ATPase belonging to the AAA (ATPase
associated with different cellular activities)
family (14). However, upon expression in mammalian cells, we observed a
high phosphotyrosine content in the PTPH1-D811A mutant which appeared
to curtail its trapping ability in a cellular context. By mutating
tyrosine 676, which defines one side of the catalytic cleft, to
phenylalanine in PTPH1-D811A, we created a mutant in which
phosphotyrosine content was reduced and which displayed the ability to
trap VCP specifically in mammalian cells with concomitant inhibition of
cell proliferation. Importantly, the induction of wild type PTPH1 in
stable cell lines also inhibited cell proliferation and led to specific
dephosphorylation of VCP, without changes in the overall level and
pattern of tyrosine-phosphorylated proteins. These results suggest that
PTPH1 may exert its effect on cell growth through dephosphorylation of
VCP.
Antibodies--
Monoclonal antibodies against phosphotyrosine
were either developed in our laboratory (G98) (13) or purchased from
Upstate Biotechnology (4G10) and Transduction Labs (PY20). Polyclonal antibodies against FAK (SC-558) and cyclin D1 (SC-753) were from Santa
Cruz Biotechnology. Agarose-coupled anti-phosphotyrosine antibody PT66
was from Sigma. Ascites containing monoclonal antibodies against
epitopes HA (12CA5) and Myc (9E10) were produced in our laboratory.
Polyclonal antiserum CS531 against VCP was raised in rabbit using a
peptide (GGSVYTEDNDDDLYG), corresponding to the last 15 residues of
murine VCP, conjugated to keyhole limpet hemocyanin (Pierce). All
monoclonal antibodies against PTPH1 (mAbZ1, Z2, Z56, Z63, Z64) were
made using recombinant PTPH1, purified from Sf9 insect cells, as
antigen (4).
Lysates, Immunoprecipitation, and Immunoblotting--
After
washing twice with phosphate-buffered saline, cells were lysed in one
of the following three buffers: (i) Nonidet P-40 buffer, which consists
of 10 mM sodium phosphate, pH 7.0, 150 mM NaCl,
2 mM EDTA, 1% Nonidet P-40, 50 mM sodium
fluoride, 1 mM Na3VO4, and 1 × protease inhibitor mixture (5 µg/ml leupeptin, 5 µg/ml
aprotinin, 1 mM benzamidine, 1 mM
phenylmethylsulfonyl fluoride); (ii) RIPA buffer, which is Nonidet P-40
buffer supplemented with 1% sodium deoxycholate and 0.1% SDS; or
(iii) hypotonic buffer, which contains 20 mM HEPES, pH 7.5, 200 mM sucrose, 20 mM sodium fluoride, 1 mM EDTA, 1 mM dithiothreitol, 1 mM
Na3VO4, 50 mM sodium fluoride, and
1 × protease inhibitor mixture. Cell lysates were clarified by
centrifugation for 10 min at 10,000 × g.
Immunoprecipitations were performed with various antibodies bound to
protein A-Sepharose CL-4B (Pharmacia) as described previously (4).
Immunoprecipitates, or cell lysates, were solubilized with SDS sample
buffer, resolved by SDS-PAGE on 8% gels and transferred onto
Imobilon-P membranes (Millipore). Immunoblot analysis was performed in
a buffer containing 5% nonfat dry milk, 150 mM NaCl,
0.05% Tween 20, and 20 mM Tris, pH 7.5, using enhanced
chemiluminescence (ECL) detection.
DNA Constructs--
The C842S, D811A, and Y676F/D811A mutations
in PTPH1 cDNA were made by site-directed mutagenesis using the
Muta-Gene kit (Bio-Rad) and confirmed by double strand DNA sequencing.
The catalytic domain (residues 634 to 913) of PTPH1 was fused to
glutathione S-transferase (GST) in a pGEX vector. These
GST-PTPH1 fusion proteins were produced in three forms: wild type,
D811A, and C842S. For transfection of mammalian cells, wild type or
mutant PTPH1 was tagged at the C terminus with the HA epitope
(SYPYDVPDYAS). After confirmation by sequencing, these constructs were
cloned into vector pCDNA3 (Invitrogen) and retroviral vector pBSTR1
(S. Reeves, Massachusetts General Hospital), Myc-tagged murine VCP
constructs, wild type (VCPmyc), and mutant with a Y796F/Y805F double
mutation (VCPmyc-FF) were generous gifts of Dr. L. Samelson (National
Institutes of Health).
Cell Culture, Stable Cell Lines, and Cell
Treatments--
Mammalian cell lines, including 293, A431, COS-7,
HepG2, MDCK, NIH3T3, REF-52, Saos-2 and Vero, were all maintained in
Dulbecco's modified Eagle's medium supplemented with 10% fetal
bovine serum (Life Technologies, Inc.). Stable NIH3T3 cell lines
expressing full-length PTPH1 under the control of a
tetracycline-repressible promoter were made using a retroviral gene
delivery system (15, 16). Briefly, viral packaging line LinX (G. Hannon, Cold Spring Harbor Laboratory) was transfected by
calcium-phosphate precipitation with 15 µg of PTPH1 retroviral
constructs. All the following steps of establishing and maintaining the
stable cell lines were performed in the presence of 2 µg/ml
tetracycline to suppress the expression of PTPH1. Retroviruses were
produced at 30 °C and filtered to remove packaging cells (0.45-µm
filter; Millipore). NIH3T3 cells were then infected overnight at
30 °C with the viral supernatants supplemented with 4 µg/ml
Polybrene (Sigma). Two days after infection, cells were selected with 2 µg/ml puromycin. Individual colonies were isolated and maintained in
puromycin and tetracycline. To induce PTPH1 expression, cells were
washed and reseeded in new dishes in the presence of puromycin but the
absence of tetracycline.
For cell synchronization, cells were induced to express PTPH1 for
24 h and then cultured in the presence of 1 mM
hydroxyurea for 18 h to arrest cells at the G1/S
boundary. The block was released by washing the cells with fresh medium
3 times. At different times after the release, cells were lysed in
Nonidet P-40 buffer for immunoblot analysis. Serum starvation of cells
was achieved by culturing in Dulbecco's modified Eagle's medium with
0.5% fetal bovine serum for 16 h. Stimulation of these starved
cells was performed with 10 µg/ml insulin (Roche Molecular Biochemicals).
Substrate Trapping in Vitro--
The GST fusion proteins were
expressed in Escherichia coli and purified on
glutathione-Sepharose beads according to the manufacturer's protocol
(Pharmacia). Mammalian cells were treated with 50 µM pervanadate for 30 min, washed with phosphate-buffered saline, and
lysed in substrate-trapping buffer (50 mM HEPES, pH 7.5, 5 mM EDTA, 1% Triton X-100, 150 mM NaCl, 10 mM sodium phosphate, 50 mM sodium fluoride, 5 mM iodoacetic acid, and 1 × protease inhibitor
mixture). Before clarifying the lysates, 10 mM
dithiothreitol was added to inactivate any unreacted iodoacetic acid.
These lysates were incubated for 2 h at 4 °C with GST or
GST-PTPH1 catalytic domain fusion proteins bound on beads, then the
beads were washed 4 times with trapping buffer. Bound proteins were
resolved by SDS-PAGE and blotted onto Imobilon-P membranes. Blots were
then incubated with anti-phosphotyrosine antibodies and developed by ECL. Tyrosine-phosphorylated proteins identified by this assay were
subjected to scale-up purification and sequenced by Edman degradation
of K-endopeptidase-digested peptides (17).
Ectopic Expression of PTPH1 Inhibited Cell Growth--
In order to
gain insight into the biological function of PTPH1, we made stable
NIH3T3 cell lines expressing the phosphatase under the control of a
tetracycline-repressible promoter. As shown in Fig.
1, when expression of wild type PTPH1 was
induced by removal of tetracycline from the media, cell growth was
strongly inhibited. The same stable cell line, containing the wild type
PTPH1 expression construct, accumulated 7-fold fewer cells upon
induction of PTPH1 (Fig. 1B). Approximately 10% of the
cells gradually detached and floated off the dish during the induction
of wild type PTPH1. These floating cells were stained by trypan blue,
indicating that they were no longer viable. In contrast, overexpression
of the catalytically impaired D811A mutant of PTPH1 had no effect on either cell growth or viability, suggesting that the catalytic activity
of PTPH1 is required for growth inhibition. Similar results were
obtained in 3 individual cell lines for each PTPH1 construct, indicating that the results presented do not arise from clonal variation.
In light of the fact that ~10% of cells induced to express wild type
PTPH1 died, we considered that one explanation for the growth
inhibition imposed by PTPH1 could be the induction of apoptosis. However, using a DNA fragmentation assay, we did not detect any apoptotic traits in cells induced to express PTPH1 (data not shown). Since there was no apparent effect on apoptosis, we investigated whether the growth inhibition may arise from interference with the cell
cycle machinery. Using flow cytometric assays of DNA content, we
observed that upon induction of PTPH1 expression the distribution of
cells throughout the cell cycle was not altered compared with control
cells, indicating that arrest did not occur in a particular phase of
the cell cycle (data not shown). In addition, we investigated the
effects of PTPH1 expression on reentry into the cell cycle during the
recovery of cells from G1/S arrest by hydroxyurea. As shown
in Fig. 2, the elevation of cyclin D1
during cell cycle reinitiation and progression was totally abolished by
the induction of PTPH1. These data indicate that expression of PTPH1
disrupts cell cycle progression, thus slowing down or arresting cell
growth.
Identification of VCP as a Substrate of PTPH1 in Vitro--
In
order to understand the molecular mechanism of PTPH1-induced growth
inhibition, we generated a substrate-trapping mutant form of the PTP
(PTPH1-D811A) with which to identify critical substrates.
Pervanadate-treated cell lysates were incubated with GST-PTPH1
catalytic domain fusion proteins bound on beads. Bound proteins were
eluted with sample buffer, fractionated by SDS-PAGE, and immunoblotted
with anti-phosphotyrosine antibodies. We observed that a prominent
tyrosine-phosphorylated protein of 97 kDa (pp97) was isolated
specifically by the PTPH1-D811A mutant from 293 cell lysates, but not
by either the wild type or the PTPH1-C842S mutant (Fig.
3). Furthermore, pp97 was also
consistently recovered as the major tyrosine-phosphorylated protein
from other cell lines tested including A431, COS-7, HepG2, MDCK,
REF-52, Saos-2, and Vero (data not shown). Other proteins were
occasionally isolated at lesser intensities, but due to their variable
recovery they were not considered further. However, it is important to
note that in any starting lysate, which contains hundreds of
tyrosine-phosphorylated proteins, pp97 is not a major component, thus
indicating the selectivity of the interaction between the isolated
catalytic domain of PTPH1 and this substrate.
We subjected pp97 to a large scale purification and obtained protein
sequence by Edman degradation of K-endopeptidase-digested peptides.
Sequences of 7 individual peptides were obtained and were all found to
match sequences in VCP, a membrane-associated ATPase (18). VCP, also
named p97, and its yeast ortholog CDC48, has been demonstrated to play
an essential role in regulating many membrane activities and is a well
established cell cycle regulator (14). This function of VCP as a
regulator of cell cycle progression is interesting in light of the
suppression of cell proliferation induced by ectopic expression of PTPH1.
The PTPH1-Y676F/D811A Double Mutant Trapped VCP in
Vivo--
Although the PTPH1-D811A mutant of PTPH1 trapped VCP in the
above assay in vitro, it was difficult to detect trapping
directly in mammalian cells. We noted that the PTPH1-D811A mutant
incorporated significant levels of phosphotyrosine when expressed in
mammalian cells (Fig. 4A),
whereas the GST-PTPH1-D811A fusion protein expressed in E. coli bore no phosphotyrosine (Fig. 3). We reasoned that the
presence of phosphate on the PTPH1-D811A mutant may impede access of
other tyrosine-phosphorylated substrates to its active site and thus
compromise its trapping ability. A conserved tyrosine residue is
located in the active site and provides a hydrophobic interaction with
the substrate phosphotyrosine, serving a role in orienting the
substrate (10). We thought it was possible that this residue may be a
receptor for a phosphate from the highly active thiophosphate
intermediate formed between the cysteine residue of the Asp PTPH1 Dephosphorylates the C-terminal Tyrosines of VCP--
The
tyrosines (Tyr796 and Tyr805) at the C terminus
of VCP have been reported to be the major sites of phosphorylation,
with Tyr805 accounting for more than 90% of the tyrosine
phosphorylation on the protein (19). Mutation of the C-terminal
tyrosine phosphorylation site in yeast CDC48, equivalent to
Tyr805 in VCP, abolished translocation of the protein to
the nucleus during mitosis and led to elongation of the cell cycle and
a growth defect (20). We tested whether PTPH1 recognized these residues in VCP in mammalian cells. Human 293 cells were co-transfected with
individual PTPH1 constructs and wild type VCP, or VCP containing a
Y796F/Y805F double mutation. Immunoprecipitates of PTPH1 from lysates
of these co-transfected 293 cells were monitored for the presence of
VCP (Fig. 5). We found that only wild
type VCP was trapped by the PTPH1-Y676F/D811A mutant and that the wild
type or the D811A single mutant of PTPH1 were ineffective substrate traps. The Y796F/Y805F VCP mutant was not associated with any of the
PTPH1 constructs. It is important to note that under the conditions of
these experiments, the wild type and mutant PTPH1 were expressed to
similar levels, as were both forms of VCP (data not shown). Therefore,
these results indicate that phosphorylation of the C-terminal tyrosines
of VCP is required for its recognition as substrate by PTPH1.
PTPH1 Specifically Dephosphorylated VCP while Retaining the Overall
Phosphotyrosine Profile in Cells--
Our trapping studies have
identified the cell cycle regulator VCP as a substrate of PTPH1.
Therefore, it was important to study the phosphorylation status of VCP
in stable cell lines expressing wild type PTPH1. However, tyrosine
phosphorylation of VCP has been found to be very transient in
vivo and difficult to measure (19, 21). We addressed this issue in
two ways. First, we examined the level of phosphotyrosine in VCP
immunoprecipitated from cells that were pretreated with 1 mM vanadate before lysis. Vanadate is a PTP inhibitor which
preserves phosphotyrosine residues in cellular proteins and will
minimize nonspecific dephosphorylation events during the experimental
manipulations. We observed a 3-5-fold decrease in the phosphotyrosine
level of VCP following ectopic expression of PTPH1 (Fig.
6a). Second, we determined the
distribution of VCP in the total population of
phosphotyrosine-containing proteins immunoprecipitated from randomly
growing cells. In pools containing similar amounts of
tyrosine-phosphorylated proteins, the population of
tyrosine-phosphorylated VCP was dramatically reduced following the
induction of PTPH1 (Fig. 6B). In contrast, the tyrosine
phosphorylation of FAK, a well known tyrosine-phosphorylated protein in
NIH3T3 cells, was unchanged when analyzed under the same conditions. Furthermore, expression of PTPH1 did not alter the global pattern of
tyrosine phosphorylation in starved, randomly growing or
insulin-stimulated cells (Fig. 6C). This indicates that the
overexpression of PTPH1 results in selective, rather than random,
dephosphorylation of tyrosine-phosphorylated proteins and highlights
the importance of VCP as a target in the cell growth arrest induced by
PTPH1 expression.
An important step in understanding the function of PTPs is to
identify their physiological substrates. Use of catalytically impaired,
substrate-trapping mutants, in which the invariant general acid
aspartate residue is changed to alanine, has proved to be a potent
strategy for identifying substrates of a number of PTPs (11-13). In
our pursuit of the substrate(s) of PTPH1, we have refined the approach
by combining the Asp A Refinement of the Method for Producing Substrate-trapping Mutant
PTPs--
In the initial substrate trapping experiments performed
in vitro, we utilized a GST-PTPH1-D811A mutant fusion
protein, which was produced in bacteria, to identify VCP as a potential
substrate of PTPH1. However, no tyrosine-phosphorylated proteins
co-immunoprecipitated with the PTPH1-D811A mutant following expression
in mammalian cells. We found that, unlike the bacterially expressed
D811A mutant, the PTPH1-D811A expressed in mammalian cells was itself
recovered in a tyrosine-phosphorylated form and could not trap
substrates. We have also observed this problem with several other
members of the PTP family.3
Interestingly, the inactive Cys
Mutation of Tyr676 to phenylalanine should preserve the
hydrophobic interaction with the substrate and maintain the integrity of the active site cleft, while removing the hydroxyl group that may
act as a phosphate acceptor. We expect that this mutant would be
structurally similar to the wild type enzyme but would not contain
phosphotyrosine. As shown, indeed the PTPH1-Y676F/D811A double mutant
does not accumulate a significant amount of phosphotyrosine and,
importantly, traps VCP as a substrate in mammalian cells. Interestingly, in the Asp Expression of PTPH1 Leads to Suppression of Cell Growth and
Specific Dephosphorylation of VCP--
VCP/p97/CDC48 and other members
of the AAA family of ATPases are involved in cell cycle regulation,
protein degradation, organelle biogenesis, and vesicle-mediated protein
transport (14). The importance of VCP for normal cellular function can
also be implied by the fact that it is highly conserved in protein
sequence from yeast, Caenorhabditis elegans, plants to
animals. Mutants of CDC48, the yeast ortholog of mammalian VCP, are
arrested late in mitosis and accumulate elongated nuclei that appear
unable to undergo fission, a process that involves membrane fusion
(22). A homozygous insertion mutation in smallmind, a
Drosophila gene encoding a protein in the subfamily of
VCP/p97/CDC48, arrests development at the larval stage with neuronal
defects (23). VCP/p97/CDC48 was found to be a critically required
component in organelle membrane fusions, such as the fusion of
endoplasmic reticulum membrane and the rebuilding of Golgi cisternae
from mitotic Golgi fragments, that occur at the end of mitosis in both
yeast and animal cells (24-26). VCP/CDC48 is mainly attached to the
endoplasmic reticulum in quiescent cells and relocalizes in a cell
cycle-dependent manner: CDC48 enters the nucleus during
late G1 in yeast and VCP aggregates at the centrosomes
during mitosis in mammalian cells (20).
Tyrosine phosphorylation of VCP was stimulated upon activation of T
cell antigen receptors and tyrosine 805 of VCP accounted for more than
~90% of the total phosphorylation (18, 19, 21). Rather than directly
effecting the ATPase activity of VCP (19), tyrosine phosphorylation of
VCP and CDC48 appears to play a role in regulating subcellular
distribution (20). Failure of CDC48/VCP to migrate into the nucleus in
yeast, or to centrosomes in mammalian cells, would be expected to
result in disturbance of cell division. Mutation of tyrosine 834 of
CDC48, which is equivalent to the major phosphorylation site
Tyr805 in VCP, to phenylalanine abolishes this
translocation and causes an elongated cell cycle and growth
retardation, whereas mutation of the same residue to glutamate, to
mimic phosphorylation, allows translocation to the nucleus and results
in normal cell growth (20). The growth inhibition mediated by ectopic
expression of PTPH1 is reminiscent of the yeast CDC48 mutant that
harbors this tyrosine to phenylalanine mutation. We demonstrated that
ectopic expression of wild type PTPH1 inhibited cell growth in the
stable NIH3T3 cell lines and that the phosphatase activity of PTPH1 is required for this growth arrest. In cells in which PTPH1 expression had
been induced, tyrosine phosphorylation of VCP was dramatically and
selectively reduced, with no change detected in the phosphotyrosine levels on FAK or the total profile of tyrosine-phosphorylated proteins
in the cell. These results suggest that a selective interaction between
VCP and PTPH1 was not compromised by overexpression of the phosphatase
and that the growth inhibition by PTPH1 may be due to the specific
dephosphorylation of VCP. Moreover, the PTPH1-Y676F/D811A double mutant
recognizes tyrosine-phosphorylated VCP specifically and engagement of
VCP in a complex with this trapping mutant, presumably interfering with
the interaction between VCP and its appropriate targets in the
membrane, yields the same phenotype as expression of the wild type
enzyme. These results argue that PTPH1 specifically dephosphorylates
the C-terminal tyrosines of VCP, thus interfering with the
phosphotyrosine-dependent regulation of VCP in the cell
cycle, resulting in growth inhibition.
In summary, using a modification of the substrate-trapping strategy
developed in the laboratory, we have identified the cell cycle
regulator VCP as a major cellular substrate of PTPH1. Ectopic expression of PTPH1 led to selective dephosphorylation of VCP in stable
NIH3T3 cell lines and inhibited cell cycle progression and growth. In
light of reports that disruption of VCP/CDC48 function, and in
particular the disruption of tyrosine phosphorylation site in
VCP/CDC48, also inhibits cell proliferation, our data are consistent with the interpretation that the effects of ectopic expression of PTPH1
are manifested primarily through its dephosphorylation of VCP. These
results further demonstrate the importance of tyrosine phosphorylation
in regulating the function of VCP in cell cycle progression.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Ala mutant enzyme. By applying this strategy in this study, we have been able to identify the first substrate for the human protein-tyrosine phosphatase, PTPH1.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Growth inhibition of stable NIH3T3 cell lines
overexpressing PTPH1. Cells were plated at a density of 4 × 105 per 10-cm dish and PTPH1 was either induced (+) or left
uninduced ( ). At the indicated time (days) after plating, cells were
either photographed with a CCD camera or counted after removal from the
dish by trypsin treatment. A, photographs of the cells at
days 1 and 4 after replating. B, total cell numbers (the
mean from triplicate plating).
,
wild type;
, +wild type;
,
PTPH1 D811A mutant;
, +PTPH1 D811A mutant.
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Fig. 2.
Overexpression of PTPH1 inhibits cell cycle
progression. Stable NIH3T3 cells were synchronized by treatment
with hydroxyurea, with (+) or without ( ) induction of PTPH1. Cells
were collected at different times (h) after release from the
hydroxyurea block and subjected to immunoblot analysis with antibodies
against the HA epitope (for PTPH1) and cyclin D1.
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Fig. 3.
Identification of pp97, VCP, as a major
substrate of PTPH1 in vitro using the D811A
mutant. Pervanadate-treated 293 cell lysates were incubated with
GST or GST fusion proteins of the catalytic domain of PTPH1 immobilized
on beads. Bound materials were resolved by SDS-PAGE and immunoblotted
by anti-phosphotyrosine antibodies (4G10). Lane 1 is cell
lysate alone. Other lanes are bound materials from GST (lane
2), GST-PTPH1-WT (lane 3), GST-PTPH1-C842S (lane
4), GST-PTPH1-D811A (lane 5). Upper panel,
immunoblot (IB) with anti-phosphotyrosine antibodies showing
the isolation of pp97. Lower panel, immunoblot with anti-GST
antibodies showing that similar amounts of each fusion protein were
used in the assays (GST alone ran off gel). Peptide sequencing of pp97
revealed it was VCP.
Ala
mutant and the phosphate group of its substrates. When we mutated this
tyrosine residue (Tyr676) in PTPH1-D811A to phenylalanine
to make a Y676F/D811A double mutant, we observed that the level of
phosphotyrosine was dramatically reduced compared with the PTPH1-D811A
single mutant (Fig. 4B). More importantly, the
PTPH1-Y676F/D811A double mutant specifically trapped VCP in
vivo. These results indicate that mutation of this conserved
tyrosine residue may improve the efficiency of substrate trapping,
particularly for those PTPs in which the Asp
Ala mutants are
susceptible to modification of the tyrosine residue that defines the
active site.
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Fig. 4.
Identification of VCP as a substrate of PTPH1
in vivo. Immunoprecipitates using antibodies
against the HA epitope were prepared from lysates of 293 cells
transfected with HA-tagged PTPH1 (WT, wild type; CS, C842S; DA, D811A;
or YFDA, Y676F/D811A). The immunoprecipitates were then analyzed by
immunoblotting with antibodies against VCP, phosphotyrosine (G98) or
the HA epitope. A, accumulation of phosphotyrosine on the
D811A mutant inhibits its trapping ability. Upper panel,
immunoblot with an anti-phosphotyrosine antibody showing the presence
of phosphotyrosine in the PTPH1-D811A mutant. Lower panel,
immunoblot with an anti-HA epitope antibody showing equal loading of
the HA-tagged PTPH1 proteins. B, the PTPH1-Y676F/D811A
double mutant displayed greatly reduced levels of phosphotyrosine and
trapped VCP in vivo. Upper panel, immunoblot with anti-VCP
antibodies showing that VCP was only isolated in a complex with the
PTPH1-Y676F/D811A double mutant. Middle panel, immunoblot
with an anti-phosphotyrosine antibody showing the levels of
phosphotyrosine in the PTPH1 mutants. Lower panel,
immunoblot with an anti-HA epitope antibody showing equal loading of
the HA-tagged PTPH1 mutants.
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Fig. 5.
PTPH1 specifically recognized the C-terminal
tyrosines of VCP. Immunoprecipitates of PTPH1, using antibodies to
the HA epitope, were prepared from 293 cells co-transfected with a
combination of VCPmyc (WT or FF) and PTPH1-HA (WT, DA, or YFDA). The
immunoprecipitates were then analyzed by immunoblotting with antibodies
against the Myc epitope to detect VCP (upper panel) or the
HA epitope to detect PTPH1 (lower panel).
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Fig. 6.
VCP was specifically dephosphorylated in
stable NIH3T3 cell lines expressing wild type PTPH1. A,
VCP was isolated by immunoprecipitation from cells treated with
vanadate. The immunoprecipitates were analyzed by immunoblotting with a
mixture of two horseradish peroxidase-coupled
anti-phosphotyrosine antibodies (PY20 and 4G10) (upper
panel) or anti-VCP (lower panel). B,
phosphotyrosine-containing proteins were isolated from cells by agarose
beads coupled with anti-phosphotyrosine antibody PT66. Then, the bound
materials were analyzed by immunoblotting with a mixture of two
horseradish peroxidase-coupled anti-phosphotyrosine antibodies (PY20
and 4G10). The same blot was also analyzed with anti-FAK antibodies.
C, the overall profile of tyrosine-phosphorylated proteins
was not altered by the overexpression of PTPH1. Stable NIH3T3 cells,
with (+) or without ( ) the induction of PTPH1, were treated as
indicated. Cell lysates were analyzed by immunoblotting with a mixture
of two horseradish peroxidase-coupled anti-phosphotyrosine antibodies
(PY20 and 4G10).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Ala mutation with a second mutation, which
changes a conserved tyrosine residue at the active site to
phenylalanine, to produce an effective substrate trap in a cellular
context. As a result, we have identified the cell cycle regulator
VCP/CDC48 as a substrate of PTPH1.
Ser mutants of these PTPs, in which
the essential nucleophilic cysteine was changed to serine, were not
tyrosine-phosphorylated, suggesting that the catalytic activity of
these PTPs was required for the accumulation of phosphotyrosine. Thus
the presence of phosphotyrosine may be a consequence of catalysis rather than these mutants being phosphorylated by protein-tyrosine kinases. These observations led us to speculate that the tyrosyl residue undergoing modification in PTPH1-D811A may be
Tyr676, the residue which helps to define the active site
cleft of the enzyme. The presence of phosphate on this residue, which
may come from transfer of the unstable phosphate in the
cysteine-phosphate intermediate, would be likely to impede access of
other tyrosine-phosphorylated proteins to the active site and thus
inhibit substrate trapping.
Ala mutants of PTP1B and TCPTP, which function as substrate traps in a cellular context, the incorporation of
phosphotyrosine has not been noted as a major problem (11, 13). The
reason for this difference between members of the PTP family is
currently unclear.
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ACKNOWLEDGEMENTS |
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We are grateful to the following colleagues for generously sharing reagents: Dr. Larry Samelson for the murine VCP constructs, Dr. Steve Reeves for retroviral vector pBSTR1, and Dr. Greg Hannon for packaging cell line LinX. We thank Dr. Mike Myers for stimulating discussion and critical reading of the manuscript, Kim Ivarson and Martha Daddario for technical support, Maria Coronesi for FACS analysis, and Nora Poppito for peptide sequencing.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grant CA53840 and the Mellam Family Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Recipient of a Glaxo-Wellcome Postdoctoral Fellowship. Current
address: Tanabe Research Laboratories, 4540 Towne Centre Court, San
Diego, CA 92121.
§ To whom correspondence should be addressed: Cold Spring Harbor Laboratory, 1 Bungtown Rd., Cold Spring Harbor, NY 11724. Tel.: 516-367-8846; Fax: 516-367-6812; E-mail: tonks{at}cshl.org.
2 S.-H. Zhang and N. K. Tonks, unpublished observations.
3 S.-H. Zhang, and N. K. Tonks, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: PTP, protein-tyrosine phosphatase; GST, glutathione S-transferase; VCP, valosin containing protein; PAGE, polyacrylamide gel electrophoresis.
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