From the Human prostatic acid phosphatase (PAcP) is a
prostate epithelium-specific differentiation antigen. In prostate
carcinomas, the cellular PAcP is decreased. We investigated its
functional role in these cells. Several lines of evidence support the
hypothesis that cellular PAcP functions as a neutral protein-tyrosine
phosphatase and is involved in regulating prostate cell growth. In this
study, we identify its in vivo substrate. Our results
demonstrated that, in different human prostate cancer cell lines, the
phosphotyrosine (Tyr(P)) level of a 185-kDa phosphoprotein (pp185)
inversely correlates with the cellular activity of PAcP. On SDS-PAGE,
this pp185 co-migrates with the c-ErbB-2 oncoprotein. Immunodepletion
experiments revealed that c-ErbB-2 protein is the major pp185 in cells.
Results from subclones of LNCaP cells indicated the lower the cellular
PAcP activity, the higher the Tyr(P) levels of c-ErbB-2. This inverse correlation was further observed in PAcP cDNA-transfected cells. In
clone 33 LNCaP cells, L-(+)-tartrate suppresses the
cellular PAcP activity and causes an elevated Tyr(P) level of c-ErbB-2 protein. Epidermal growth factor stimulates the proliferation of LNCaP
cells, which concurs with a decreased cellular PAcP activity as well as
an increased Tyr(P) level of c-ErbB-2. Biochemically, PAcP
dephosphorylates c-ErbB-2 at pH 7.0. The results thus suggest that
cellular PAcP down-regulates prostate cell growth by dephosphorylating Tyr(P) on c-ErbB-2 oncoprotein in those cells.
Protein tyrosine phosphorylation and dephosphorylation play a key
role in regulating the proliferation and differentiation of normal and
tumor cells (1, 2). In cells, the tyrosine phosphorylation status is
apparently regulated by a dynamic equilibrium of protein-tyrosine
kinases and PTPases1 (3, 4). In
human cancers, an increased level of
tyrosine phosphorylation is implicated in a high cellular proliferation rate and an eventual development of tumors (5). This effect of
increased tyrosine phosphorylation could be due to activation of
protein-tyrosine kinases, inactivation of PTPases, or both.
The biological function of PTPases in antagonizing protein-tyrosine
kinase activity and cellular transformation has been demonstrated in
cell culture models (for a review, see Ref. 6). For example, protein-tyrosine phosphatase 1B and LAR PTPase dephosphorylated the IR
on tyrosine residues, resulting in a decreased tyrosine kinase specific
activity of IR (7, 8). Additionally, SHP-1, an Src homology 2 domain-containing PTPase, could be recruited by an erythropoietin
receptor and subsequently down-regulated JAK2 through a
dephosphorylation mechanism (9, 10). Furthermore, by cDNA
transfection, the expression of an exogenous protein-tyrosine phosphatase 1B suppressed the malignant transformation of NIH3T3 cells
induced by the c-erbB-2 oncogene (11). Similarly, an induced expression of DEP-1, a PTPase, inhibited cell growth by 5-10-fold in
human breast cancer cell line MCF-7, which is
c-erbB-2-amplified (12). In a xenograft animal model,
c-erbB-2 gene-transformed 18-Hn1 human breast carcinoma
cells developed tumors in athymic nude mice. Following LAR
transfection, the tumor growth was significantly inhibited (13). Thus,
PTPases could counteract the growth-stimulating and oncogenic activity
of protein-tyrosine kinases.
Human PAcP is a prostate epithelium-specific differentiation antigen.
Two forms of PAcP have been identified: one stays intracellular, while
the other is secreted (14). Its cellular enzyme activity and mRNA
level is decreased in prostate carcinomas, compared with normal or
benign prostate hypertrophy cells (15, 16). Results from many studies
have demonstrated that the cellular form of PAcP could function as a
PTPase in cells. Briefly, PAcP exhibits endogenous PTPase activity and
dephosphorylates tyrosine-phosphorylated proteins (17, 18).
Biochemically, the tyrosine phosphorylation level and the tyrosine
kinase-specific activity of EGFR could be down-regulated by PAcP at a
neutral pH (19). Expression of an exogenous PAcP via either cDNA
transfection (20, 21) or protein incorporation (22, 23) was associated
with decreased Tyr(P) levels of cellular proteins. Additionally,
crystallographic analyses and titration experiments revealed that PAcP
could indeed function as a "cysteine"-mediated PTPase (24, 25).
Taken collectively, these data indicate that the cellular form of PAcP
is involved in regulating tyrosine phosphorylation in prostate
epithelia.
Although previous investigations suggested that PAcP acts as a negative
regulator of tyrosine phosphorylation signals, the in vivo
substrate of PAcP was not identified yet. In this study, we analyzed
protein tyrosine phosphorylation in several prostate cancer cells that
express different levels of cellular PAcP. Our results demonstrated
that the Tyr(P) level of pp185, a 185-kDa phosphoprotein, negatively
correlates with the cellular activity of PAcP. We clarified the
identity of pp185 to be the c-ErbB-2 oncoprotein. Furthermore, using
PAcP cDNA transfection, we elucidated the relationship between PAcP
expression and the Tyr(P) level of c-ErbB-2 in prostate cancer cells.
Thus, our data suggest that c-ErbB-2 protein is an in vivo
substrate of cellular PAcP.
Materials--
FBS, RPMI 1640 culture medium, gentamicin, EGF,
and horseradish peroxidase-conjugated anti-mouse and anti-rabbit IgGs
were purchased from Life Technologies, Inc. Charcoal/dextran-treated, certified FBS (lot AGD6463, testosterone Cell Culture--
Human prostate carcinoma cell lines LNCaP, DU
145, and PC-3 were obtained from the American Type Culture Collection
(Rockville, MD) and routinely maintained in RPMI 1640 medium
supplemented with 5% FBS, 1% glutamine, and 0.5% gentamicin. Cells
were split once per week, defined as one passage. LNCaP cells that had
passage numbers less than 33 were designated as clone 33, greater than 80 as clone 81, and between 34 and 80 as clone 51 (28). Since cellular
PAcP activity is regulated by androgen in prostate cells (20, 29), a
steroid-reduced medium, i.e. RPMI 1640 medium containing 5%
charcoal-treated, heat-inactivated FBS, was used in this study (26,
28). For doubling time determination, after 72 h of culture, one
set of attached cells was harvested and counted, defined as cell number
on day 0. The remaining cells were fed with fresh medium and then
counted on days 3, 5, and 7. Fresh medium (3 ml/well in six-well
plates) was added to the remaining cultures on days 3 and 5. To
quantify cell growth, attached cells were trypsinized and combined with
the suspended cells, and the total cell number was counted using a
Coulter Counter Z1 model.
PAcP cDNA Transfectants--
PC-411, -412, and -416 cells
were subclones of PC-3 parental cells transfected with a full-length
human PAcP cDNA driven by a pCMV-neo expression vector, as
described previously (20). Clone 81 LNCaP cells were also transfected
with the same expression vector containing human PAcP cDNA. Two
subclones designated as LNCaP-28 and -40 were obtained as the stable
transfectants after neomycin selection. LNCaP-CMV-13 is a subline of
clone 81 LNCaP cells transfected with the pCMV-neo vector alone.
Acid Phosphatase Activity Assay--
p-Nitrophenol
phosphate was utilized as the substrate to quantify the acid
phosphatase activity at pH 5.5 by measuring the absorbency of released
PNP at 410 nm (30). Since L-(+)-tartrate is a classic
inhibitor of PAcP and since greater than 90% of
L-(+)-tartrate-sensitive acid phosphatase in LNCaP cells
can be precipitated by anti-PAcP antiserum, the
L-(+)-tartrate-sensitive acid phosphatase activity was used
to represent the PAcP activity (30).
Immunoprecipitation and Immunoblotting--
Subconfluent cells
were harvested by scraping, pelleted, and rinsed with ice-cold 20 mM Hepes-buffered saline, pH 7.0, and then lysed in
ice-cold cell lysis buffer containing protease and phosphatase
inhibitors (i.e. 20 mM Hepes, pH 7.0, 0.5%
deoxycholic acid, 0.15 M NaCl, 0.1% SDS, 1% Nonidet P-40,
4 mM EDTA, 10 mM NaF, 0.1 mM
ZnCl2, 10 mM
Na4P2O7, 2 mM sodium
orthovanadate, 2 mM phenylmethylsulfonyl flouride, 1 trypsin-inhibitory unit of aprotinin, 4 µM leupeptin, and
1 µM pepstatin A). The lysates were spun at 1,600 × g for 10 min at 4 °C. The supernatants were transferred,
and protein concentration was determined using a Bio-Rad protein assay
kit. For immunoprecipitation, 1 mg of supernatant protein was incubated
with 9G6 anti-ErbB-2 Ab-conjugated protein A-Sepharose complex for
2 h at 4 °C. The immunocomplexes were spun at 700 × g for 5 min, washed four times with ice-cold lysis buffer,
and suspended in SDS-PAGE sample buffer. For immunoblotting, an aliquot
of total lysate protein (50 µg) or the supernatant of
immunoprecipitated complexes in SDS-PAGE sample buffer was electrophoresed and transferred to a nitrocellulose filter (Micron Separation Inc., MA). Filters were incubated with appropriate antibodies, and the protein bands were visualized by the ECL detection system (28). For rehybridization, the filters were stripped by a
stripping buffer, i.e. 62.5 mM Tris-HCl, pH 6.7, containing 100 µM 2-mercaptoethanol and 2% SDS, for 30 min at 50 °C. After two washes with TBST buffer (i.e. 20 mM Tris-buffered saline, pH 7.5, containing 0.1% Tween
20), the filters were reacted with specific Abs, and the signal was
detected by the ECL method.
PAcP Expression and Tyrosine Phosphorylation of pp185 in Different
Prostate Cancer Cells--
Previous studies demonstrated that cellular
PAcP could function as a neutral PTPase in prostate cancer cells (19,
22, 23). To understand its biological role, we investigated its in vivo substrate. We initially examined PAcP expression in
different subclones of LNCaP cells as well as PC-3 and DU 145 cells. As shown in Fig. 1A, PAcP protein
level in clone 33 LNCaP cells was higher than that in clone 51 and 81 cells, while PC-3 and DU 145 cells did not express a detectable PAcP
protein. To examine the relationship between PAcP expression and
protein tyrosine phosphorylation, the same membrane was rehybridized
with anti-Tyr(P) Ab. As shown in Fig. 1B, among different
subclones of LNCaP cells, the tyrosine phosphorylation level of a
protein with a molecular mass of approximately 185 kDa (pp185)
inversely correlates with the cellular level of PAcP. Significantly,
levels of pp185 tyrosine phosphorylation were high in PC-3 and DU 145 cells (Fig. 1B), which lack PAcP expression (Fig.
1A).
Department of Biochemistry and Molecular
Biology, ¶ Section of Urologic Surgery,
Eppley Cancer Institute, University of Nebraska Medical
Center, Omaha, Nebraska 68198
ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
3.0 ng/dl) was from Hyclone (Logan, UT). Protein molecular weight standard marker, acrylamide, and a protein assay kit were obtained from Bio-Rad. Polyclonal anti-ErbB-2 Abs (C-18 and K-15) for immunoblotting, monoclonal anti-ErbB-2 Ab (9G6) for immunoprecipitation, and normal mouse IgG were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Rabbit anti-PAcP antiserum was obtained as described previously (26). Monoclonal anti-Tyr(P) antibody (4G10) was from
Upstate Biotechnology Inc. (Lake Placid, NY). Purified PAcP was
isolated from human seminal fluid as described (27). An enhanced
chemiluminescence (ECL) detection system was purchased from Amersham
Pharmacia Biotech. All other reagents were from Sigma.
RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References
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Fig. 1.
Endogenous PAcP expression and tyrosine
phosphorylation of pp185. Subconfluent PC-3, DU 145, and different
subclones of LNCaP cells were harvested, lysed, and aliquoted for
immunoblotting experiments. Total cell lysates (50 µg) were
electrophoresed on a 7.5% SDS-PAGE and transferred to a nitrocellulose
filter. A, immunoblotting with rabbit anti-PAcP antiserum.
B, tyrosine phosphorylation level of total cell proteins.
The filter shown in A was stripped and then rehybridized
with anti-Tyr(P) ( -p-tyr) Ab. The arrow
indicates the pp185. PC, PC-3 cells; DU, DU 145 cells; LNCaP C-33, C-51, and C-81,
LNCaP clone 33, clone 51, and clone 81 cells, respectively.
IB, immunoblotting.
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Immunodepletion of pp185 by Anti-ErbB-2 Ab-- To examine if c-ErbB-2 is the major phosphoprotein at 185 kDa, c-ErbB-2 was immunodepleted from total lysates of clone 81 LNCaP cells. As shown in Fig. 3A, the immunoprecipitated amounts of c-ErbB-2 protein increased following the dosage of Ab. The immunodepleted cell lysates were then immunoblotted with anti-Tyr(P) Ab. In these depleted lysates, the tyrosine phosphorylation level of pp185 decreased, proportionally to the increase of Ab dosage (Fig. 3B). Greater than 80% of the Tyr(P) level of the pp185 was depleted from 1 mg of cell lysate by 10 µg of Ab (Fig. 3B). However, normal mouse IgG had no effect on immunoprecipitating c-ErbB-2 protein (Fig. 3A) and did not affect the Tyr(P) level of pp185 (Fig. 3B). The same membrane was subsequently reacted with anti-ErbB-2 Ab. A diminished amount of c-ErbB-2 protein in immunodepleted lysates corresponded to the decreased tyrosine phosphorylation level of pp185 (Fig. 3, C versus B). Thus, c-ErbB-2 protein is the major pp185 whose Tyr(P) level inversely correlates with the cellular PAcP activity in prostate cancer cells.
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Tyrosine Phosphorylation of c-ErbB-2 in Different Prostate Cancer Cells-- To investigate whether c-ErbB-2 could be a substrate of cellular PAcP, we first examined the Tyr(P) level of c-ErbB-2 in subclones of LNCaP cells. The c-ErbB-2 protein was immunoprecipitated, and its tyrosine phosphorylation level was analyzed by immunoblotting with anti-Tyr(P) Ab. The results demonstrated the lower the cellular PAcP activity, the higher the Tyr(P) level of c-ErbB-2 protein in LNCaP cells (top of Fig. 4A versus Fig. 1A). The same membrane was reacted with anti-ErbB-2 Ab after stripping. As shown in the bottom of Fig. 4A, there was no significant change in c-ErbB-2 protein levels among clone 33, 51, and 81 LNCaP cells. The relative Tyr(P) level of c-ErbB-2 protein was semiquantified by normalizing to its protein level after densitometric analyses. In clone 81 cells, the tyrosine phosphorylation of c-ErbB-2 was approximately 5-fold higher than that in clone 33 cells, while the PAcP activity was 5-fold lower (Table I).
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L-(+)-Tartrate Effect on Tyrosine Phosphorylation of c-ErbB-2-- Since results from PAcP cDNA transfection studies revealed that an additional PAcP expression correlates with a decreased Tyr(P) level on c-ErbB-2 protein in prostate cancer cells, we investigated this relationship further by treating clone 33 LNCaP cells with L-(+)-tartrate, a classic inhibitor of PAcP (23, 30). Cells were exposed to different doses of L-(+)-tartrate, and c-ErbB-2 protein was immunoprecipitated and followed by immunoblotted for analyzing tyrosine phosphorylation status. As shown in Fig. 5A, the tyrosine phosphorylation level of c-ErbB-2 increased following an increase of inhibitor concentrations. However, the c-ErbB-2 protein expression levels were very similar among the cells exposed to different doses of L-(+)-tartrate (Fig. 5B). The relative Tyr(P) level of c-ErbB-2 in cells exposed to 5 mM L-(+)-tartrate was approximately 4.5-fold of that in control cells (Fig. 5B). Conversely, the cellular PAcP activity was decreased by around 20% (data not shown), as we reported previously (22, 23). Thus, an inhibition of PAcP activity concurs with an increase of tyrosine phosphorylation of c-ErbB-2 in prostate cancer cells.
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EGF Effect on Cell Growth, PAcP Activity, and Tyrosine Phosphorylation of c-ErbB-2-- EGF stimulates the growth of LNCaP cells (20) and prostate primary cells (32). Since cellular PAcP is possibly involved in down-regulating the growth of prostate cells, we also analyzed whether a stimulation of cell growth by EGF correlates with a decrease of PAcP activity in clone 33 LNCaP cells. EGF caused an increase in cell growth indicated by total cellular protein amounts with the optimal dose at 10 ng/ml (Fig. 6A). In contrast, the enzyme activity of cellular PAcP diminished following the dosage of EGF (Fig. 6A). The EGF effect in regulating tyrosine phosphorylation status of c-ErbB-2 in these cells was further examined. Results shown in Fig. 6B (top) demonstrated that the Tyr(P) level of c-ErbB-2 increased, in proportion to the EGF concentrations with the optimal induction at 10 ng/ml. The c-ErbB-2 protein level was also inspected and shown in Fig. 6B (bottom). Densitometric analyses demonstrated an over 3-fold induction of tyrosine phosphorylation of c-ErbB-2 by 10 ng/ml of EGF, in comparison with that in the control group (Fig. 6B). These data therefore indicate that EGF down-regulates cellular PAcP activity, thus resulting in the elevation of Tyr(P) level on c-ErbB-2 protein, which leads to an increased cell proliferation rate.
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Dephosphorylation of c-ErbB-2 by PAcP in Vitro-- To elucidate a direct interaction between these two proteins, we analyzed the dephosphorylation of c-ErbB-2 by PAcP at a neutral pH in vitro. c-ErbB-2 protein was immunoprecipitated from clone 81 LNCaP cells. The immunocomplexes were resuspended in Hepes-buffered saline, and purified PAcP was added. The remaining tyrosine phosphorylation level of c-ErbB-2 was examined by immunoblotting with anti-Tyr(P) Ab. As shown in Fig. 7A, PAcP dephosphorylated c-ErbB-2 protein on tyrosine residues following a time course. c-ErbB-2 protein amounts in each sample were analyzed by immunoblotting with anti-ErbB-2 Ab (Fig. 7B). The relative tyrosine phosphorylation level of c-ErbB-2 was semiquantified. The results indicated that PAcP dephosphorylated c-ErbB-2 protein in a time-dependent fashion (Fig. 7C). However, in the presence of a PTPase inhibitor, sodium orthovanadate, no dephosphorylation of c-ErbB-2 by PAcP was observed (Fig. 7C). As a negative control, there was no dephosphorylation in the absence of PAcP protein (Fig. 7C).
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The Relationship among the Growth Rate, PAcP Expression, and Phosphotyrosine Level of c-ErbB-2 in Prostate Cancer Cells-- Our data indicate that tyrosine phosphorylation levels of c-ErbB-2 can be regulated by cellular PAcP in human prostate cancer cells. Since c-ErbB-2 is one of the major protein-tyrosine kinases involved in growth factor-stimulated proliferation of mammalian cells including prostate cells (32, 33), we investigated the biological significance of decreased tyrosine phosphorylation of c-ErbB-2 protein by analyzing the cell proliferation rate. We first examined the growth rate of different LNCaP subcloned cells. As shown in Table I, the doubling time of clone 33, 51, and 81 cells in a steroid-reduced medium was approximately 112, 57, and 49 h, respectively. Similar studies indicated that growth rates of PAcP cDNA-transfected LNCaP subcloned cells (LNCaP-28 and -40) and cDNA-transfected PC-3 subcloned cells (PC-412 and -416) were lower than that of their parental control cells (Table I). The cellular PAcP activity in each cell type was further measured. Data taken collectively show that in all three independent prostate cancer cell systems, the higher the activity of cellular PAcP is, the lower the tyrosine phosphorylation levels of c-ErbB-2 and the slower the cell growth rates (Table I).
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DISCUSSION |
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The protein-tyrosine phosphatase activity of PAcP has been demonstrated in many biochemical studies over the last decade (17-19, 22-25, 28). In normal canine prostate glands, well differentiated epithelia expressed a high level of PAcP and a low level of tyrosine-phosphorylated proteins (34). In contrast, poorly differentiated canine prostate basal cells did not express a detectable PAcP, while the Tyr(P) level was high (34). Treatment of canine differentiated prostate tissue with orthovanadate resulted in an increase in cellular Tyr(P) level and a decrease in PAcP activity (35). Additionally, in human prostate cancer cell cultures, the tyrosine phosphorylation of cellular proteins inversely correlated with cellular PAcP activity (20, 23). These studies imply that PAcP is involved in regulating protein tyrosine phosphorylation in differentiated prostate epithelia. Nevertheless, the in vivo substrate(s) of PAcP has not yet been identified.
In the current study, our results clearly indicate that the tyrosine phosphorylation status of c-ErbB-2 protein is regulated by PAcP in prostate cancer cells. For example, our initial experiments using clone 81 LNCaP and PC-3 cells lacking PAcP expression demonstrated a poor immunoprecipitation of c-ErbB-2 by an Ab (C-18) against its C terminus, but not by an Ab (9G6) recognizing its N terminus (data not shown). Perhaps the inaccessibility of C-18 Ab is due to the high extent of tyrosine phosphorylation on C terminus of c-ErbB-2 protein in these two cell lines. Among different subclones of LNCaP cells, loss of PAcP expression correlated with an elevation of Tyr(P) level on c-ErbB-2 (Figs. 1 and 4). PAcP cDNA transfection in clone-81 LNCaP and PC-3 cells concurred with a diminished tyrosine phosphorylation of c-ErbB-2 (Figs. 2 and 4). When clone 33 LNCaP cells were exposed to a PAcP inhibitor, L-(+)-tartrate, a significant induction of tyrosine phosphorylation on c-ErbB-2 protein was observed (Fig. 5). Furthermore, EGF treatment on the same cells caused a decrease in cellular PAcP activity, with a parallel increase in Tyr(P) level of c-ErbB-2 (Fig. 6). Biochemically, PAcP dephosphorylated c-ErbB-2 on tyrosine residues in a time-dependent fashion (Fig. 7). To the best of our knowledge, this is the first demonstration of a putative in vivo substrate of PAcP, a differentiation-associated PTPase.
The inverse correlation is exhibited between cellular PAcP expression and the Tyr(P) level of c-ErbB-2 as well as cell growth rate in different LNCaP subcloned cells and PAcP cDNA-transfected PC-3 cells (Table I). However, compared with LNCaP-40 subcloned cells, LNCaP-28 cells express a higher level of PAcP (Fig. 2A and Table I), while the tyrosine phosphorylation level of c-ErbB-2 as well as the cell growth rate in these two cell lines are very similar (Fig. 4B and Table I). To address this question, we analyzed the PAcP level in their conditioned media, since there are two forms of PAcP (14, 20). The results indicated that LNCaP-28 cells secreted a significantly higher amount of PAcP than LNCaP-40 cells (A410 = 0.209 of LNCaP-28 versus 0.002 of LNCaP-40). Thus, we proposed that a fraction of PAcP in LNCaP-28 cells is directed to the constitutively secretory pathways. Therefore, the high PAcP level in total cell lysates of LNCaP-28 cells includes the intermediate form of secretory PAcP, as described in our previous studies (20). Alternatively, the inhibition of the Tyr(P) level of c-ErbB-2 may reach to the optimum by the exogenous PAcP in LNCaP-40 cells. Although LNCaP-28 cells produce increased amounts of PAcP, the excess PAcP cannot function to further dephosphorylate c-ErbB-2 in vivo.
c-ErbB-2 is a transmembrane glycoprotein of Mr 185,000 that has an extensive sequence homology to EGFR (36, 37). Similar to EGFR, c-ErbB-2 is a putative growth factor receptor with an intrinsic tyrosine kinase activity, yet its ligand has not been identified (38). Amplification or overexpression of the c-erbB-2 gene has been found in approximately 20-30% of human breast cancers (39). Patients with breast tumors overexpressing the c-erbB-2 gene have a significantly lower survival rate and a shorter time to relapse than patients with tumors lacking overexpression of the c-erbB-2 gene (39). In prostate cancer, however, studies on the role of c-ErbB-2 have yielded conflicting results (40-43). Therefore, c-ErbB-2 has not been accepted as a marker for prostate tumor progression (44, 45). In the present study, we utilized a variety of prostate cancer subcloned cells as model systems to investigate not only the interaction between PAcP and c-ErbB-2, but also a possible functional role of c-ErbB-2 in regulating the growth of prostate cancer cells. Our results clearly demonstrate that the tyrosine phosphorylation level of c-ErbB-2 correlates with the proliferation rate of prostate cancer cells (for a summary, see Table I). Since cellular PAcP activity in prostate carcinomas is lower than that in noncancerous prostate cells (15, 16),2 we hypothesize that cells with a low level of PAcP have an unregulated tyrosine phosphorylation on c-ErbB-2, which eventually leads to the uncontrolled proliferation of cancers.
Several lines of evidence indicate that PTPases interact with
protein-tyrosine kinases intracellularly and regulate kinase signal
transduction pathways. For example, a physical association between LAR
and IR was shown by coimmunoprecipitation from Chinese hamster ovary
cells, which overexpressed the human IR gene (8). When rat fibroblast
cells with an overexpression of the human IR gene were transfected with
protein-tyrosine phosphatase 1B cDNA, insulin- and insulin-like
growth factor I-stimulated signalings were inhibited (9). Subsequently,
it was found that protein-tyrosine phosphatase 1B was coprecipitated
with the IR -subunit by specific Abs from insulin-stimulated cells
(46). SHP-1, an Src homology 2 domain-containing PTPase, bound
selectively to the tyrosine-phosphorylated erythropoietin receptor
through its Src homology 2 domain and henceforth inactivated JAK2 by
dephosphorylation (9, 10). Thus, a physical association indeed exists
between PTPases and their in vivo substrates. In previous
studies, we observed that the majority of cellular PAcP resides in the
cytosol and that a fraction of it could associate with the plasma
membrane using immunofluorescent staining with a confocal microscopy
(21).3 It indicated that
cellular PAcP and c-ErbB-2 are localized favorably for interaction.
However, we have had limited success in demonstrating a physical
interaction between PAcP and c-ErbB-2 by coimmunoprecipitation techniques. Several plausible reasons could explain this phenomenon. For example, since there is no significant sequence homology between PAcP and other members of the PTPase family (47, 48), the interaction
mechanism of PAcP with its in vivo substrate(s) could be
different from other PTPases. Alternatively, it is possible that the
association between PAcP and c-ErbB-2 is very labile and that the
binding is highly sensitive to the experimental conditions. Thus, the
dissociation between these two proteins occurs during experimental
procedures. Crystallographic results clearly demonstrated that PAcP has
two critical free sulfhydryl groups at cysteine residues that could
serve as phosphate acceptors (24). Subsequently, titration experiments
revealed a "S-32P" intermediate product of PAcP
occurring during the dephosphorylation reaction (25). Thus,
substrate-trapping mutants generated by site-directed mutagenesis of
these cysteine residues should provide us with new tools for studying
in vivo association between PAcP and c-ErbB-2.
The treatment of LNCaP cells with L-(+)-tartrate and EGF resulted in an increased tyrosine phosphorylation level of c-ErbB-2 protein. Since L-(+)-tartrate is a classic inhibitor of PAcP and also since its inhibition effect on cellular PAcP in LNCaP cells has been illustrated (22, 23), we propose that the induction of the Tyr(P) level of c-ErbB-2 by L-(+)-tartrate is directly due to the suppression of cellular PAcP activity. However, the molecular mechanism of EGF effect on tyrosine phosphorylation of c-ErbB-2 is not completely understood. It is possible that the suppression of cellular PAcP activity by EGF leads to a hyperphosphorylated c-ErbB-2. Other possibilities also exist. For example, we observed a coimmunoprecipitation of c-ErbB-2 protein by anti-EGFR Ab in clone 33 LNCaP cells (data not shown), indicating that a heterodimerization occurs between these two receptors. Since EGF treatment stimulates the tyrosine kinase-specific activity of EGFR, the increase of Tyr(P) level of c-ErbB-2 is possible via a cross-phosphorylation mechanism (49). Furthermore, the PAcP activity could be suppressed by direct phosphorylation through the active EGFR because of favorable subcellular localization, or by shutdown of its in vivo synthesis machinery. Detailed investigations are needed to delineate the molecular mechanism of interaction between cellular PAcP and c-ErbB-2 in LNCaP cells.
In conclusion, our data clearly demonstrated that cellular PAcP can dephosphorylate c-ErbB-2 oncoprotein on tyrosine residues in prostate cancer cells. This dephosphorylation results in a diminished proliferation rate of these cells. Further studies will help to elucidate their in vivo interactions and molecular sites of dephosphorylation of c-ErbB-2 by PAcP. These results will aid in understanding the mechanism of one step in multistage prostate carcinogenesis.
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ACKNOWLEDGEMENTS |
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We thank Dr. Prathibha S. Rao and Paul Beum for critical reading of this manuscript.
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FOOTNOTES |
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* This work was supported in part by National Cancer Institute, NIH, Grant CA 72274 and Nebraska Cancer and Smoking Disease Research Program Grant LB 506, 98-29.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.
§ A recipient of the Graduate Student Fellowship Award from the Graduate Studies Program, University of Nebraska Medical Center.
** To whom all correspondence and reprint requests should be addressed: Dept. of Biochemistry and Molecular Biology, College of Medicine, University of Nebraska Medical Center, 600 S. 42nd St., Omaha, NE 68198-4525. Tel.: 402-559-6658; Fax: 402-559-6650; E-mail: mlin{at}mail.unmc.edu.
The abbreviations used are: PTPase, protein tyrosine phosphatase; PAcP, prostatic acid phosphatase; IR, insulin receptor; LAR, leukocyte common antigen-related; EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; FBS, fetal bovine serum; Ab, antibody; Tyr(P), phosphotyrosine; PAGE, polyacrylamide gel electrophoresis; CMV, cytomegalovirus; pp185, a phosphoprotein with a molecular mass of 185 kDa.
2 J. Andresson, P. S. Rao, and M. F. Lin, unpublished data.
3 M. F. Lin, M. L. Chen, and J. K. Christman, unpublished data.
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REFERENCES |
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