From the Department of Biochemistry and Molecular
Biology, § Section of Urologic Surgery, Department of
Surgery, and ¶ Eppley Cancer Institute, University of Nebraska
Medical Center, Omaha, Nebraska 68198
Received for publication, July 26, 2000, and in revised form, October 18, 2000
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ABSTRACT |
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The cellular form of human prostatic acid
phosphatase (PAcP) is a neutral protein-tyrosine phosphatase (PTP) and
may play a key role in regulating the growth and androgen
responsiveness of prostate cancer cells. The functional role of the
enzyme is at least due in part to its dephosphorylation of c-ErbB-2, an in vivo substrate of the enzyme. In this study, we
investigated the molecular mechanism of phosphotyrosine
dephosphorylation by cellular PAcP. We mutated several amino acid
residues including one cysteine residue that was proposed to be
involved in the PTP activity of the enzyme by serving as the phosphate
acceptor. The cDNA constructs of mutant enzymes were transiently
transfected into C-81 LNCaP and PC-3 human prostate cancer cells that
lack the endogenous PAcP expression. The phosphotyrosine level of
ErbB-2 in these transfected cells was subsequently analyzed. Our
results demonstrated that the phosphotyrosine level of ErbB-2 in cells expressing H12A or D258A mutant PAcP is similar to that in control cells without PAcP expression, suggesting that these mutants are incapable of dephosphorylating ErbB-2. In contrast, cells expressing C183A, C281A, or wild-type PAcP had a decreased phosphotyrosine level
of ErbB-2, compared with the control cells. Similar results were
obtained from in vitro dephosphorylation of
immunoprecipitated ErbB-2 by these mutant enzymes. Furthermore,
transient expression of C183A, C281A, or the wild-type enzyme, but not
H12A or D258A, decreased the growth rate of C-81 LNCaP cells. The data
collectively indicate that His-12 and Asp-258, but not Cys-183 or
Cys-281, are required for the PTP activity of PAcP.
Protein tyrosine phosphorylation plays a pivotal role in
controlling multiple eukaryotic cellular processes including
metabolism, proliferation, differentiation, migration, and survival
(1-3). Therefore, aberrant tyrosine phosphorylation can lead to
abnormal outcomes such as neoplasm or cancer. Many oncogenes or
protooncogenes encode protein-tyrosine kinases
(PTKs)1 (4-6). Uncontrolled
activation of these PTKs could be due to mutation, amplification, or
loss of regulatory factors, which contributes to enhanced tyrosine
phosphorylation. For receptor PTKs, mutation could lead to a
ligand-independent activation by maintaining the receptor in a constant
activated state. Amplification of these receptors can also lead to
transformation through an increase in overall tyrosine kinase activity.
For example, amplification of receptor PTKs has been found in several
types of human cancers including breast, ovarian, lung, and colon
cancers (7-9). In the case of breast cancer, overexpression of ErbB-2,
due to amplification of the gene, is one of the consistent genetic
alterations (7, 8). In addition, overexpression of ErbB-2 correlates
with a poor patient prognosis in breast cancer as well as other cancers including ovarian (8) and gastric (10) cancers.
The homeostasis of protein tyrosine phosphorylation in the cell is
maintained by PTKs and PTPs (2, 11). PTPs can directly interact with
PTKs, resulting in dephosphorylation of the kinases. Alternatively,
PTPs may act on the upstream or downstream signaling of PTKs. Most of
known PTPs serve as negative regulators of critical signal transduction
pathways (12-16) and are able to suppress transformation phenotype
induced by PTKs in cultured cells (17, 18). For example, expression of
PTP1B in NIH-3T3 cells leads to a reduced transformation by human
NEU oncogene (19). Expression of cytoplasmic PTP-TD14 in
NIH-3T3 cells is able to inhibit Ha-ras-mediated
transformation (20). The wild-type PTP1B, but not the inactive mutant,
inhibits transformation of rat 3Y1 fibroblasts by v-crk,
v-src, and v-ras (21). Similarly, expression of a
low molecular weight bovine liver PTP in v-erbB,
v-src, and v-raf-transformed NIH-3T3 fibroblast cells can inhibit anchorage-independent growth in soft agar (22). In a
tumorigenic human breast carcinoma cell line that overexpresses p185neu, expression of leukocyte common antigen-related PTP
correlates with a decreased proliferation rate in vitro and
a reduced tumor growth rate in athymic nude mice (23). Zander et
al. (24) have demonstrated that T cell PTP can suppress
transformation by murine v-fms in rat 2 cells. In the cell,
the targets of PTPs include receptor PTKs (25-27) and other key
signaling molecules such as adapter proteins (25), docking proteins
(28, 29), Src family kinases (18, 30, 31), ERKs (32), and other PTKs
(33).
Human PAcP is a prostate epithelium-specific enzyme with intracellular
and secreted forms (34, 35). Although the activity of the enzyme in
circulation has been studied extensively as a marker for tumor
progression, the functional role of the cellular form of the enzyme
remains to be explored further. It is known that the cellular form of
this enzyme has a high level of expression in normal, well
differentiated prostate epithelial cells but is diminished in prostate
carcinoma cells. In human prostate cancer cells, the cellular level of
the enzyme is inversely correlated with the cell growth rate. For
instance, LNCaP cells that express cellular PAcP have a slow growth
rate, compared with PC-3 and DU-145 cells that lack the endogenous PAcP
expression (36, 37). Introduction of cellular PAcP into prostate cancer
cells results in a decreased growth rate (36-38). It has been shown
that cellular PAcP is also involved in regulating the
androgen-stimulated growth of prostate cells (39). C-33 LNCaP cells
that express PAcP and androgen receptor are responsive to androgen
stimulation, whereas C-81 LNCaP and PC-3 prostate carcinoma cells that
express functional androgen receptor but lack PAcP expression are
androgen-unresponsive. Moreover, androgen responsiveness of these cell
lines can be restored by reintroducing cellular PAcP expression (39).
The collective data clearly indicate that cellular PAcP is critical for
regulating proliferation and androgen responsiveness of human prostate
cancer cells.
It has been known for nearly 2 decades that human PAcP possesses
intrinsic PTP activity. First, the enzyme is copurified with a PTP
activity and exhibits a high specificity toward phosphotyrosyl proteins
(40, 41). Subsequently, it is shown that PAcP dephosphorylates epidermal growth factor receptor preferentially at neutral pH, resulting in a decreased PTK-specific activity of the receptor protein
(42). Recent studies clearly show that the phosphotyrosine level of a
185-kDa protein (pp185) is inversely correlated with the cellular PAcP
activity in prostate cancer cells (39, 43, 44). The pp185 is found to
be c-ErbB-2/Neu/HER-2. Several lines of evidence together suggest that
c-ErbB-2 is indeed an in vivo substrate of the cellular form
of PAcP (43).
Little is known so far about the molecular mechanism by which PAcP
dephosphorylates phosphotyrosyl proteins. In the present study, we
performed experiments to determine the specific residues that are
important for the PTP catalytic activity of human PAcP. We constructed
several PAcP cDNA mutants by site-directed mutagenesis and
transfected them into human prostate cancer cells. Subsequently, we
analyzed the AcP activity of these mutants and the effect of their
expression on the tyrosine phosphorylation of cellular proteins including c-ErbB-2. The results clearly demonstrated that replacement of His-12 or Asp-258 by Ala abolishes the PTP activity of PAcP toward
ErbB-2, whereas mutation of Cys-183 or Cys-281 has no significant effect on the activity.
Reagents and Antibodies--
Gentamicin,
L-glutamine, fetal bovine serum, RPMI medium 1640, OPTI-MEM
I reduced-serum medium, LipofectAMINE PLUS transfection reagent, and
BamHI and XbaI restriction enzymes were purchased from Life Technologies, Inc. SDS, glycine, acrylamide/bisacrylamide solution, prestained and unstained standard protein markers, and the
Coomassie-based protein assay reagent were obtained from Bio-Rad. Pfu DNA polymerase and the QuickChange site-directed
mutagenesis kit were from Stratagene (La Jolla, CA). T4 DNA ligase and
the Site-directed Mutagenesis--
A 1.2-kilobase pair human PAcP
cDNA (36) was subcloned into the vector pBluescript II SK
(Stratagene) and used for mutagenesis. Replacement of His-12 or Cys-281
by Ala was carried out using the QuickChange mutagenesis kit. Mutation
of Cys-183 and Asp-258 to Ala was generated as described previously
(46) using two adjacent, nonoverlapping primers. The oligonucleotide
primer sets used were as follows: H12A,
5'GTGACTTTGGTGTTTCGCGCTGGAGACCGAAGTCCC3' and 5'GGGACTTCGGTCTCCAGCGCGAAACACCAAAGTCAC3';
C183A, 5'GCGGAGAGTGTTCACAATTTCACTTTACC3' and
5'GTATAAAGGGTCGTAGACTTTACTCCAAATTCC3'; D258A,
5'GCGACTACTGTGAGTGGCCTACAGATGG3' and
5'GTGCGCAGAATACATGATAAGTTTTTTGTAGCTTGG3'; C281A,
5'CTCCTTCCTCCCTATGCTTCCGCGCACTTGACGGAATTGTAC3'
and 5'GTACAATTCCGTCAAGTGCGCGGAAGCATAGGGAGGAAGGAG3'.
In addition to changing the desired residue to Ala, the mutations
(underlined) also create an MvnI restriction site (bold) to
facilitate the initial mutant screening. Generation of this restriction
site does not result in a change in amino acid. All mutant cDNAs
were completely sequenced. The sequence-confirmed cDNA was
subcloned into mammalian expression vectors pcDNA 3.1 and pCEP4 for
transfection and expression in prostate cancer cells.
Cell Culture and Transfection--
Human prostate cancer cells
LNCaP and PC-3 were obtained originally from the American Type Culture
Collection (Manassas, VA) and maintained in RPMI 1640 medium
supplemented with 5% fetal bovine serum, 1% glutamine, and 0.5%
gentamicin as described (36, 47). Cells were passaged once a week.
LNCaP cells with passage numbers less than 33 and greater than 81 are
defined as C-33 and C-81, respectively (39). The liposome-mediated
transfection was performed using LipofectAMINE PLUS reagent. C-81 LNCaP
or PC-3 cells were plated and grown in 6-well plates or 100-mm culture dishes for 40 h. About 1 and 6 µg of plasmids carrying PAcP
cDNAs were used for transfection of the cells in each well and
dish, respectively. To monitor the transfection efficiency, the
pSV- PAcP Activity and Protein Concentration Determination--
AcP
activity was assayed at pH 5.5 using pNPP as the substrate (48), and
the tartrate-sensitive AcP activity was used to represent PAcP activity
(43, 48). Protein concentration was determined using the Bio-Rad
protein assay reagent. Bovine serum albumin was used as the protein reference.
Immunoprecipitation--
Cells were washed twice with ice-cold
Hepes-buffered saline, harvested by scraping, and lysed in a lysis
buffer containing 20 mM Hepes, pH 7.0, 0.5% deoxycholic
acid, 0.1% SDS, 1% Nonidet P-40, 150 mM NaCl, 2 mM EDTA, 10 mM NaF, 0.1 mM
ZnCl2, 2 mM sodium orthovanadate, and a mixture
of protease inhibitors as described previously (43). Lysates were
centrifuged at 12,000 × g for 10 min. The supernatant
proteins (1-2 mg in ~1 ml) were preabsorbed by incubating with 20 µl of washed protein A-Sepharose beads for 30 min at 4 °C. The
supernatant fractions were further incubated with 5-8 µg of
monoclonal mouse anti-ErbB-2 antibody preconjugated with protein
A-Sepharose for 3 h at 4 °C. The immunoprecipitated ErbB-2
complexes were washed four times with the lysis buffer and dissolved in
SDS sample buffer.
SDS-PAGE and Western Blotting--
Proteins in total cell
lysates or from immunoprecipitation were separated by SDS-PAGE and then
electrophoretically transferred to nitrocellulose membranes (Micron
Separation, MA). To increase the transfer efficiency of ErbB-2, 0.1%
SDS was included in the blotting buffer (39, 43). The nitrocellulose
membranes were probed with appropriate primary and horseradish
peroxidase-conjugated secondary antibodies. Specific proteins on the
membrane were visualized using the SuperSignal West Pico
chemiluminescent reagents from Pierce.
In Vitro Dephosphorylation of ErbB-2--
ErbB-2 protein was
immunoprecipitated from C-81 LNCaP cells as described above. After
washed with the lysis buffer, the immunocomplexes were further washed
three times with dephosphorylation buffer (20 mM Hepes, pH
7.0, 150 mM NaCl, 2 mM 2-mercaptoethanol) and incubated at room temperature with secreted PAcP in the conditioned medium. The ErbB-2 immunocomplexes were then washed with the lysis buffer and suspended in SDS-sample buffer for further analysis by
SDS-PAGE and Western blotting.
Exogenous Expression of PAcP in Prostate Cancer Cells--
To
analyze the functional role of cellular PAcP as a PTP, a human PAcP
cDNA was cloned into two types of mammalian expression vectors,
pcDNA3.1 and pCEP4. The pCEP4 vector, but not the pcDNA3.1, can
replicate episomally in human cells. Both constructs were individually
transfected into C-81 LNCaP cells that lack the expression of
endogenous PAcP (39). Control cells were transfected with the vector
alone. To quantify the transfection efficiency, a reporter plasmid
containing the Effect of Mutation on PAcP Activity--
To investigate the amino
acid residues that are potentially involved in the PTP activity of
PAcP, we performed site-directed mutagenesis of the PAcP molecule.
Mutation of individual amino acid residues was carried out as described
under "Experimental Procedures." Based on the results from previous
studies (50-53), four amino acid residues, i.e. His-12,
Cys-183, Asp-258 and Cys-281, were chosen for mutation by substituting
the corresponding residue with Ala. The mutated cDNA was sequenced,
subcloned into the expression vectors, and subsequently transiently
transfected into human prostate cancer cells for functional analysis.
As shown in Fig. 2A, PAcP
proteins including the wild-type and mutants were expressed in C-81
LNCaP cells as demonstrated by Western blotting analyses. Using pNPP as
the substrate, the AcP activity of the wild-type and mutant enzymes was
determined. As shown in Fig. 2B, the endogenous PAcP
activity in the control cells that were transfected with the vector
alone was negligible as reported previously (39). The AcP-specific
activity of the H12A and D258A mutants decreased about 95% compared
with that of the wild-type enzyme. In contrast, the specific activity
for the C183A or C281A mutant was very similar to that of the wild-type PAcP (Fig. 2B). These results indicate that His-12 and
Asp-258, but not Cys-183 and Cys-281, are required for the AcP activity toward pNPP hydrolysis.
Effect of Point Mutation on the in Vivo PTP Activity of
PAcP--
To examine the PTP activity of PAcP mutants, we initially
analyzed the pattern of tyrosine phosphorylation of total cellular proteins in cells transfected with the wild-type or mutant PAcP cDNA (Fig. 3). As shown in Fig.
3A, immunoblotting analyses with anti-phosphotyrosine
antibodies showed that expression of the wild-type PAcP in C-81 LNCaP
cells correlated with a decreased tyrosine phosphorylation of a pp185.
Results from immunodepletion studies revealed that ErbB-2 is the major
pp185 and contributes more than 80% of the phosphotyrosine at the
position (43). Immunoblotting with anti-phosphotyrosine antibodies
(Fig. 3A, top panel) demonstrated that expression of the
H12A or D258A PAcP mutant did not have a significant effect on the
tyrosine phosphorylation of ErbB-2, indicating a lack of PTP activity
for these mutants. The loss of activity is not due to a global
structure change of the enzyme since both mutants and the wild-type
PAcP have a very similar overall structure (50). Expression of the
C183A or C281A mutant decreased the tyrosine phosphorylation level of
ErbB-2, suggesting that these mutant enzymes retain their PTP activity.
A similar level of ErbB-2 protein (Fig. 3A, middle panel)
was detected in all cell lysates by stripping and reprobing the same
membrane with anti-ErbB-2 antibodies. Exogenous expression of the
wild-type PAcP and its mutants was also clearly shown by Western
blotting (Fig. 3A, bottom panel). To analyze further the
specificity of dephosphorylation by PAcP, we semi-quantified the
phosphorylation level of two other unknown phosphotyrosyl proteins,
pp160 and pp50, in LNCaP cell lysates by densitometric analyses (Fig.
3A). Although the phosphotyrosine level of pp160 inversely
correlated with the PTP activity of PAcP, the phosphotyrosine level of
pp50 was not significantly affected by PAcP expression. The pp160 might be directly or indirectly regulated by ErbB-2 as a downstream or
interacting molecule. Further studies are required to determine the
actual relationship. Thus, these results suggest the substrate specificity of PAcP.
The effect of PAcP expression on protein tyrosine phosphorylation was
also examined in PC-3 cells, another PAcP-deficient human prostate
cancer cell line. Similarly, both H12A and D258A mutants lost their PTP
activity (Fig. 3B). Although the overall tyrosine
phosphorylation pattern of PC-3 cells was somewhat different from that
of LNCaP cells, the tyrosine phosphorylation level of the pp185
inversely correlated with the functional PAcP activity as in LNCaP
cells (Fig. 3). To exclude the possible interfering effect by pCEP4
vector, both C-81 LNCaP and PC-3 cells were transfected with PAcP
cDNA constructs cloned into another expression vector, pcDNA3.1. The same results were obtained as described above,
i.e. the H12A and D258A PAcP mutants lost their PTP
activity, whereas the C183A and C281A mutants retained the activity
(data not shown). Again, the phosphotyrosine level of ErbB-2 inversely
correlated with the cellular PAcP activity as shown in Fig. 3.
Dephosphorylation of ErbB-2 by PAcP Mutants--
The PTP activity
of PAcP mutants toward ErbB-2 was further analyzed. ErbB-2 protein was
immunoprecipitated from C-81 LNCaP cells transiently transfected with
the mutant and wild-type PAcP cDNAs. The phosphotyrosine level of
the immunoprecipitated ErbB-2 was detected by Western blotting using
the anti-phosphotyrosine antibody. As shown in Fig.
4A, the tyrosine
phosphorylation level of ErbB-2 in C-81 LNCaP cells which expressed the
H12A or D258A PAcP was very similar to that in control cells without
PAcP expression. In contrast, the phosphotyrosine level of ErbB-2 in
the cells expressing the C183A or C281A PAcP was decreased by ~40%
compared with that in the control cells, while comparable to that in
the cells expressing the wild-type PAcP. These data further indicated that the C183A and C281A PAcP mutant proteins retain the PTP activity in prostate cancer cells. In contrast, the H12A and D258A PAcP mutants
lost their PTP activities.
The PTP activity of the wild-type and mutant PAcP enzymes toward ErbB-2
was also studied by in vitro dephosphorylation. ErbB-2 protein was immunoprecipitated from C-81 LNCaP cell lysates and subsequently dephosphorylated by PAcP in the conditioned media from
cells expressing the wild-type or mutant PAcP. As shown in Fig.
4B, the wild-type, C183A, and C281A mutants dephosphorylated ErbB-2 at pH 7. The extent of dephosphorylation by these enzymes was
~40% in comparison with the control and did not change significantly by extending the incubation time from 20-60 min or by increasing the
amount of PAcP (data not shown). This observation suggests that PAcP
may specifically target one or some of the multiple phosphotyrosyl
residues on the ErbB-2 protein. The phosphotyrosine level of ErbB-2
protein was not significantly changed after incubation with the H12A or
D258A mutant enzyme, indicating a lack of PTP activity for these
mutants. Taken together, the data indicated that His-12 and Asp-258 of
PAcP are involved in the phosphotyrosine dephosphorylation of
ErbB-2.
Effect of PAcP Expression on ERK Phosphorylation and Prostate
Cancer Cell Growth--
To address whether dephosphorylation of ErbB-2
by PAcP has any impact on its signaling capacity, we examined the
phosphorylation status of the downstream ERK/MAP kinases. PC-3 cells
were transiently transfected with the wild-type PAcP cDNA or vector
alone since these cells exhibit a higher transfection efficiency and
basal phosphorylation level of ERK/MAP kinase than C-81 LNCaP cells. The phosphorylation of ERK was then detected by Western blotting using
the antibodies specific to the activated MAP kinase protein that is
phosphorylated at both Thr-202 and Tyr-204. The phosphorylation of
ErbB-2 was also examined by using the antibody against an activated ErbB-2 protein that is phosphorylated at Tyr-1248. The data in Fig.
5A showed that the expression
of exogenous cellular PAcP correlates not only with a decreased ErbB-2
phosphorylation at Tyr-1248 but also with a reduced phosphorylation
level of the p42 ERK/MAP kinase in the cells, suggesting a
down-regulation of the MAP kinase activity. The phosphorylation level
of p44 ERK/MAP kinase is below the detection limit. These results thus
indicate that dephosphorylation of ErbB-2 by PAcP can suppress its
mitogenic signaling abilities.
We further analyzed the effect of PAcP expression on the proliferation
of prostate cancer cells. C-81 LNCaP cells were transiently transfected
with various PAcP cDNAs or the vector alone. After 3 days, the cell
number was counted. As shown in Fig. 5B, the growth rate of
the cells transfected with the wild-type, C183A, or C281A mutant PAcP
decreased significantly, ~20% lower than the control cells
transfected with the vector alone. In contrast, the H12A or D258A
mutant PAcP had no effect on the growth of LNCaP cells. To clarify
further the significance of PAcP effect on cell growth, stable subclone
cell lines were established from LNCaP and PC-3 cells transfected with
the wild-type PAcP. As shown in Fig. 5, C and D,
the expression of exogenous PAcP correlated with a decreased cellular
proliferation. The data together indicated that dephosphorylation of
ErbB-2 by PAcP results in a reduced mitogenic signaling, leading to a
diminished growth rate of prostate cancer cells.
PAcP has been extensively studied as a differentiation antigen of
prostate epithelia and as a marker for prostate cancer (54). Several
lines of evidence indicate that cellular PAcP functions as a neutral
PTP (40-42) and is involved in the regulation of proliferation and
androgen responsiveness of human prostate cancer cells (36, 37, 39).
The intrinsic PTP activity of the enzyme is apparently critical for its
functional role in the cancer cells (43, 44, 55).
PTPs are a large family of enzymes with diverse structure and
complexity (3, 12, 15, 56). Most of them contain an ~240-residue
conserved PTP domain with the exception of some members such as the low
molecular weight PTPs (13, 16, 57). They all utilize an invariant Cys
residue located in the signature motif (CXXXXXR(S/T)) as the
phosphate mediator. Based on the structural studies, enzymes in the PTP
family have a very similar active site and catalytic mechanism (3, 16,
57-59).
Since PAcP shares little sequence homology with other PTPs, it is not
known about the catalytic mechanism whereby this enzyme uses to
dephosphorylate phosphotyrosyl proteins. Results from titration
experiments indicate that each of the two subunits contains three
disulfide bonds (51). Nevertheless, analyses on the crystal structure
of rat PAcP reveal that only four of the six Cys residues participate
in the formation of disulfide bridges for each monomer (52). It has
also been shown that there is no covalent linkage between the two
subunits. Given the high homology in sequence and similarity in
three-dimensional structure between rat and human PAcP (52, 53, 61), it
is conceivable that each subunit of human PAcP also possesses two Cys
residues with free sulfhydryls. The existence of two free sulfhydryl
groups in human PAcP is further demonstrated by biochemical studies
(50). Thus, it is proposed that one of the two Cys residues, Cys-183
and Cys-281, is indeed involved in the phosphotyrosine
dephosphorylation (50, 52). In fact, based on the three-dimensional
structure of rat PAcP, Schneider et al. (52) have proposed
that Cys-183 is located in the cleft near the active site and could
participate in the substrate binding and/or catalysis. They further
propose that the conformational change triggered by substrate binding
can bring residues including Cys-183 closer to the catalytic center.
The notion that the free "Cys" can function as the phosphate
acceptor is further indicated by titration experiments (50). Thus, PAcP may belong to the family of "cysteine" protein-tyrosine phosphatases.
In this report, our data clearly show that mutation of either Cys-183
or Cys-281 to Ala has little effect on the AcP activity toward pNPP or
the PTP activity as indicated by the in vivo phosphotyrosine level of c-ErbB-2 (Figs. 2B, 3, and 4). Furthermore, over a
period of 72 h, C-81 LNCaP cells that were transiently transfected
with the C183A or C281A mutant PAcP cDNA had a growth rate about
20% lower than the cells transfected with the vector alone (Fig.
5B). The extent of the down-regulation on cell growth by
these mutants is similar to that caused by transient expression of the
wild-type PAcP. Compared with LNCaP or PC-3 subclone cells stably
expressing wild-type PAcP, cells transiently transfected with the
wild-type, C183A, or C281A cDNA exhibited a higher growth rate
(Fig. 5). It should be noted that the relatively low level of growth
suppression is in part due to a low transfection efficiency of those
cells (49). In addition, those cells have a slow growth rate (39). It
appears that the suppression of prostate cancer cell growth by PAcP is
due to down-regulation of the mitogenic MAP kinase pathway since the
phosphorylation of ERK2/MAP kinase is down-regulated in PAcP-expressing
cells (Fig. 5). Furthermore, our results show that phospho-Tyr-1248 of
ErbB-2, one of the major autophosphorylation sites, is targeted by PAcP
(Fig. 5A). Preliminary data indicate that the tyrosine
phosphorylation level of p52Shc is also decreased in the
cells expressing PAcP.2
Therefore, the data collectively indicate that a Cys residue is not
required for the PTP catalytic activity of PAcP. To the best of our
knowledge, this is the first study that clearly demonstrates a novel
mechanism of dephosphorylation by a PTP in the cell.
In vitro studies indicated that a group of residues,
including His-12 and Asp-258, are essential for the catalytic activity of human PAcP to hydrolyze pNPP or phenyl phosphate (50). His-12 is
involved in the formation of the phosphate-binding site and is
phosphorylated during catalysis as an acceptor of the phosphate group.
Asp-258 may function as a general acid to donate a proton for the
substrate leaving group during phosphoester hydrolysis (50, 52).
Similarly, other PTPs also require an Asp residue (equivalent to
Asp-181 in PTP1B) to protonize the phenolic oxygen of the tyrosyl
leaving group during catalysis (59). Consistent with previous
observations (50), the H12A and D258A mutants are inactive to pNPP
(Fig. 2B). More importantly, our results demonstrate that
mutation of His-12 or Asp-258 to Ala has resulted in a loss of the
in vivo PTP activity toward ErbB-2 in human prostate cancer
cells (Figs. 3 and 4A). The lack of PTP activity for these PAcP mutants is further confirmed by in vitro
dephosphorylation of ErbB-2 (Fig. 4B) and the cell growth
study (Fig. 5). It should be noted that mutation of His-12 or Asp-258
to Ala does not cause a global structural change of PAcP (50). Thus,
our results from in vivo and in vitro studies
indicate that both His-12 and Asp-258 residues are essential for the
PTP activity of PAcP in human prostate cancer cells.
Human PAcP may represent a distinct subgroup of PTPs. Although sequence
analyses reveal some homologous segments (data not shown, see Refs. 51
and 60), there is no overall sequence similarity between PAcP and other
members of the PTP family. In addition, PAcP protein does not contain
the signature motif (-CXXXXXR(S/T)-) despite the existence
of two free sulfhydryls in each subunit. Previous studies (50, 52)
suggest that one Cys residue could be involved in dephosphorylating
phosphotyrosyl proteins, raising the possibility that the enzyme might
use different molecular mechanisms to dephosphorylate pNPP in
vitro and phosphoproteins in vivo. Studies in this
report clearly demonstrate that neither of the two Cys residues,
Cys-183 and Cys-281, is required for the PTP activity of human PAcP.
Conversely, both His-12 and Asp-258 are required for the PTP and AcP
activities. These data therefore indicate that both AcP and PTP
activities of human PAcP share the same active site and apparently use
the same amino acid residues for catalysis. This notion is consistent
with the competitive inhibition phenomenon between pNPP and
phosphoangiotensin (41). However, the enzyme possesses the specificity
to different substrates. For example, kinetic studies indicate that the
Km value for PAcP toward phosphotyrosyl proteins is
more than 50-fold lower than toward phosphoseryl or phosphothreonyl
proteins (41). In this study, the in vivo preferential
dephosphorylation of ErbB-2 by PAcP in both C-81 LNCaP and PC-3 cells
also suggests that PAcP exhibits a substrate specificity toward
different phosphotyrosyl proteins (Fig. 3). Residues such as Tyr-123
and Arg-127 could be involved in determining the substrate specificity
(52). Interestingly, it is reported that a tartrate-resistant AcP from
osteoclasts and macrophages is also an active PTP (62). Based on the
competitive nature of the substrates, the enzyme may catalyze pNPP
hydrolysis and phosphotyrosyl protein dephosphorylation in the same
active site. However, further studies on this tartrate-resistant AcP are required to identify its active site and to delineate the catalytic mechanism.
To the best of our knowledge, this study represents the first effort to
determine the mechanism and residues used for the PTP catalytic
activity of human PAcP. Our collective data from both in
vivo and in vitro studies clearly demonstrate that
His-12 and Asp-258 are required for the PTP activity of the enzyme. In contrast, Cys-183 and Cys-281 are not essential for this activity. The
results thus indicate a novel PTP catalytic mechanism underlying the
role of PAcP in regulating the proliferation and androgen responsiveness of human prostate cancer cells.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-galactosidase assay system were from Promega (Madison, WI). Restriction enzyme MvnI was from Roche Molecular
Biochemicals. Mammalian expression vectors pcDNA 3.1 and pCEP4 were
obtained from Invitrogen (Carlsbad, CA). QIAprep Spin Miniprep and
QIAGen Maxi plasmid purification kits were obtained from Qiagen
(Valencia, CA). Polyclonal rabbit anti-human PAcP antibodies were
prepared as described previously (45). Polyclonal (C-18), monoclonal (9G6), anti-ErbB-2 and polyclonal anti-ERK protein antibodies were from
Santa Cruz Biotechnology (Santa Cruz, CA). Polyclonal anti-phospho-ErbB-2 and monoclonal anti-phosphotyrosine antibodies (4G10) were purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). Polyclonal anti-phospho-ERK MAP kinase (Thr-202/Tyr-204) antibody
was provided by New England Biolabs (Beverly, MA). Other reagents were
purchased from Sigma unless indicated otherwise.
-Galactosidase control vector (Promega) was used for
cotransfection. Cells were harvested 72 h after transfection.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-galactosidase gene was cotransfected into the same
cells (49). Activities of
-galactosidase and PAcP in cellular
lysates were quantified 72 h after transfection. The PAcP protein
level was analyzed by immunoblotting with a rabbit anti-PAcP antibody.
As shown in Fig. 1, PAcP had a higher
level of expression in the cells transfected with the pCEP4/PAcP
plasmid than in the cells transfected with the pcDNA 3.1/PAcP
although both plasmids had similar transfection efficiency (data not
shown). As a control,
-actin level was examined by probing the same
membrane with anti-
-actin antibodies. An approximately equal amount
of
-actin was detected in different lysates, indicating that a
similar amount of proteins was loaded for electrophoresis (Fig. 1).
Both constructs were subsequently used to analyze the effect of
exogenous PAcP expression on protein tyrosine phosphorylation.
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Fig. 1.
Expression of PAcP in C-81 LNCaP cells by
transient transfection. PAcP cDNA was subcloned into mammalian
expression vectors pcDNA 3.1 and pCEP4 and then transfected into
C-81 LNCaP human prostate carcinoma cells that lack endogenous PAcP
expression. About 72 h after transfection, cellular proteins were
extracted, separated by SDS-PAGE, and analyzed by Western blotting
using anti-PAcP antibodies. As an internal control, the -actin level
was also examined with the same membrane. IB,
immunoblotting.
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Fig. 2.
Expression and relative activity of the PAcP
mutants in prostate cancer cells. The expression vector pCEP4
alone or the constructs containing the wild-type (WT) or
mutant PAcP cDNAs was transfected into C-81 LNCaP cells.
A, the level of PAcP and -actin in total cell lysates was
detected by immunoblotting (IB) analyses with anti-PAcP and
-actin antisera, respectively. B, the AcP activity of
PAcP was assayed at 35 °C using pNPP as a substrate, as described
under "Experimental Procedures." The activity was normalized to the
PAcP protein level and shown as a percentage relative to that of the
wild-type enzyme. The standard deviations are indicated by error
bars. The data are the average of three sets of experiments.
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Fig. 3.
Effect of the wild-type and mutant PAcP
expression on tyrosine phosphorylation of cellular proteins in LNCaP
and PC-3 cells. C-81 LNCaP (A) or PC-3 (B)
human prostate cancer cells were transiently transfected with various
PAcP cDNA constructs or the vector alone. An equal amount of
cellular lysates was separated by SDS-PAGE and transferred to
nitrocellulose membranes. The phosphotyrosyl proteins (top),
ErbB-2 (middle), and PAcP (bottom) were analyzed
by probing the same membrane with anti-phosphotyrosine
(pTyr), ErbB-2, and PAcP antibodies, respectively. The
numbers on the left indicate the molecular weight
of the marker proteins. The results are representative of three
(A) or two (B) sets of independent experiments.
IB, immunoblotting.
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Fig. 4.
In vivo and in vitro
dephosphorylation of ErbB-2 by the wild-type and mutant
PAcPs. A, ErbB-2 protein was immunoprecipitated
(IP) from cellular lysates of C-81 LNCaP cells that were
transiently transfected with various PAcP cDNA constructs or the
vector alone. B, the ErbB-2 protein was immunoprecipitated
from C-81 LNCaP cells and then dephosphorylated in vitro for
30 min at room temperature by incubating with different PAcP proteins
in the conditioned medium, as described under "Experimental
Procedures." The tyrosine phosphorylation and protein levels of
ErbB-2 were detected by Western blotting analyses and semi-quantified
by densitometry. The level of PAcP expression is shown at the
bottom of each panel. The experiments were repeated twice or
more with similar results. IB, immunoblotting.
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Fig. 5.
Effect of PAcP expression on phosphorylation
of ERK/MAP kinases (MAPK) and growth of prostate
cancer cells. A, PC-3 cells were transiently
transfected with PAcP cDNA or the vector alone. The phosphorylation level of ErbB-2 and ERKs in the cells
was respectively analyzed with antibodies specific to the
phospho-ErbB-2 (Tyr-1248) and the phospho-ERK (Thr-202 and Tyr-204) by
Western blotting. The protein levels of ErbB-2, p44/42 ERKs and PAcP
were examined with the corresponding antibodies after the membranes
were stripped. B, C-81 LNCaP cells were transiently
transfected with the vector alone, wild-type, or mutant PAcP cDNAs.
After 3 days, the cell number was determined. The significance of
difference in cell number between control and experimental cells was
analyzed by Student's t test. * p < 0.05 (n = 3) and ** p < 0.01 (n = 3). C and D, PAcP-expressing
stable subclone cells, i.e. LNCaP-28 and LNCaP-40, or
PC-411, PC-412, and PC-416, were established from C-81 LNCaP
(C) or PC-3 (D) cells transfected with the
wild-type PAcP cDNA. The cell number was counted on the time points
as indicated in the figure. Similar results were obtained from two sets
of independent experiments in triplicates.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We thank Dr. Tzu-Ching Meng for helpful suggestions and discussions; Fen-Fen Lin for the cell growth curve determination, cell culture maintenance, and technical support; and Jitesh Pai and Xiaolin Shen for participating in part of the plasmid preparation and mutagenesis experiments. We thank the members of Dr. Richard MacDonald's laboratory, particularly Dr. James C. Byrd, for help and discussion.
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FOOTNOTES |
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* This work was supported in part by NCI Grants CA72274 and CA88184 from the National Institutes of Health and Nebraska Department of Health and Human Services Grant LB595.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..
To whom correspondence should be addressed: Dept. of
Biochemistry and Molecular Biology, University of Nebraska Medical
Center, 984525 Nebraska Medical Center, Omaha, NE 68198-4525. Tel.:
402-559-6658; Fax: 402-559-6650; E-mail: mlin@unmc.edu.
Published, JBC Papers in Press, November 6, 2000, DOI 10.1074/jbc.M006661200
2 M.-S. Lee, T. Igawa, X.-O. Zhang, F.-F. Lin, and M.-F. Lin, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are: PTK, protein-tyrosine kinase; AcP, acid phosphatase; ERK, extracellular signal-regulated kinase; PAcP, prostate acid phosphatase; pNPP, p-nitrophenyl phosphate; PTP, protein-tyrosine phosphatase; MAP, mitogen-activated protein; PAGE, polyacrylamide gel electrophoresis.
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