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(Received for publication, April 24, 1996, and in revised form, June 27, 1996)
§,
¶,
,
§ and
From the
Division of Cell, Molecular and Oncology
Research, Charing Cross and Westminster Medical School, University of
London and '' Cancer Research Campaign Laboratories, Department of
Medical Oncology and
Department of Biochemistry, Charing Cross
Hospital, Fulham Palace Road, London W6 8RF, United Kingdom
The c-erbB-2 receptor tyrosine kinase is often overexpressed in human tumors, but the functional implications of this phenotype remain unclear. We previously used phosphorylation-specific antibodies to define major differences in c-erbB-2 tyrosine kinase activity between overexpressing human tumor cell lines (Epstein, R. J., Druker, B. J., Roberts, T. M., and Stiles, C. D. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 10435-10439). Here we extend this approach to define the relationship between c-erbB-2 tyrosine phosphorylation and protein kinase C (PKC)-dependent transmodulation. Phosphorylation-specific antibodies to the juxtamembrane PKC site Thr686 recognize tyrosine-dephosphorylated wild-type c-erbB-2 following G8/DHFR 3T3 cell treatment with PKC agonists. B104-1-1 cells transformed by activated c-erbB-2 express a subset of tyrosine-phosphorylated receptors that are homologously phosphorylated on Thr686, indicating that Thr686 phosphorylation alone is insufficient to abrogate receptor tyrosine phosphorylation. Similarly, the c-erbB-2-overexpressing human cancer cell lines SK-Ov-3 and BT-474 express constitutively Thr686-phosphorylated receptors. SK-Ov-3 cells express predominantly kinase-inactive c-erbB-2 that is heavily Thr686-phosphorylated, indicating that Thr686 phosphorylation in this line is heterologous in origin. In contrast, BT-474 cells express constitutively autophosphorylated c-erbB-2 despite Thr686 phosphorylation. These results indicate that Thr686 phosphorylation does not directly abolish c-erbB-2 activity and suggest that such phosphorylation reflects constitutive PKC activity induced by either receptor-activating mutations or heterologous growth factors. The latter possibility suggests in turn that c-erbB-2 interacts in an as yet undefined way with heterologous growth factor receptors in human tumor cells.
The c-erbB-2 (neu, HER-2) receptor is a type I receptor tyrosine kinase commonly overexpressed in human tumors such as breast cancer (1). A plausible explanation for this phenotype is that functional kinase activity predisposes to the clonal outgrowth of a neoplastic subpopulation. This hypothesis has been supported by transgenic studies of mutant neu overexpression, which have confirmed in vivo tumorigenesis (2, 3). Extrapolating from such studies to the clinic has proven problematic, however, for various reasons. First, tyrosine phosphorylation of the wild-type receptor in vitro has been associated with growth arrest (4) and differentiation (5) as well as with mitogenesis. Second, transforming c-erbB-2 mutations are readily inducible by point mutation (6), yet few if any such mutations have been documented in sporadic tumors (7). Third, correlation of functional c-erbB-2 activity with clinical outcome is still awaited, whereas certain c-erbB-2 antibodies used in such studies preferentially detect catalytically inactive receptors (8). Finally, overexpression of c-erbB-2 occurs more commonly in subtypes of preinvasive (in situ) disease than in established breast cancer (9), casting doubt on the evolutionary role of this molecule in tumor growth.
How c-erbB-2 is functionally regulated in human tumor cells therefore remains an open question with potentially important therapeutic implications. Unlike its homologue, the epidermal growth factor receptor (EGFR), c-erbB-2 has no known homodimerizing ligand and remains largely uncharacterized with respect to autoregulatory homeostatic mechanisms. Catalytic activation of c-erbB-2 appears inducible by either mutation-driven homodimerization (10) or perhaps by membrane overexpression alone. However, the latter mechanism does not explain the variability in net receptor autophosphorylation between cell lines with similar c-erbB-2 expression (11), suggesting that other variables, such as heterodimer formation, paracrine ligand expression, or signaling crosstalk, are likely to influence in vivo receptor function.
The ability of protein kinase C (PKC)1-dependent signaling pathways to induce homologous desensitisation and/or heterologous transmodulation of receptor tyrosine kinases has been recognized for over a decade (12, 13, 14, 15, 16, 17, 18). In recent years, much of this work has focused on PKC-dependent phosphorylation of threonine-654 (Thr654) in the juxtamembrane domain of EGFR (19, 20, 21, 22). The importance of this Thr654 site for negative EGFR regulation has now been established by site-directed mutagenesis (23, 24, 25, 26). This regulatory domain is approximately 70% homologous to the Thr686-containing region of the c-erbB-2 receptor (27, 28). We and others have previously used in vivo labeling and phosphoaminoacid analysis to show that threonine phosphorylation of c-erbB-2 occurs in response to PKC agonists such as phorbol esters (11, 29, 30) and heterologous growth factors (4, 29), and have confirmed that such exposures are tightly linked to c-erbB-2 tyrosine dephosphorylation (4, 31). Because oncogene-inhibitory signaling pathways are potentially attractive targets for anticancer drug development, clarification of PKC-dependent c-erbB-2 transmodulation could prove useful in developing novel therapies.
In an earlier report, we demonstrated the utility of phosphorylation-specific c-erbB-2 antibodies for identifying intrinsic differences in receptor catalytic activity between human tumor cell lines (8, 11). Here we use phosphothreonine-specific c-erbB-2 antibodies to analyze regulatory patterns of PKC-inducible receptor desensitisation in human and rodent cell lines expressing different forms of the receptor. The results support the paradigm that juxtamembrane threonine phosphorylation is linked to negative c-erbB-2 regulation in some cell systems and confirm differences in phosphorylation patterns between wild-type and mutant receptors and between malignant and nonmalignant cell lines. They also indicate, however, that the neoplastic phenotype may be affected by cell signaling alterations distinct from (though not necessarily independent of) c-erbB-2 overexpression.
G8/DHFR cells (NIH 3T3-derived murine fibroblasts) expressing a transfected and methotrexate-amplified dicistronic rat c-neu/dihydrofolate reductase clone (32) were a gift of Dr. Robert Weinberg (Whitehead Institute, Cambridge, MA). The cell lines BT-474, SK-Ov-3, and B104-1-1 were obtained from the American Type Culture Collection. G8/DHFR cells were maintained in a humidified 5% CO2 incubator at 37 °C in Dulbecco's minimal essential medium supplemented with 10% bovine calf serum, glutamine, and antibiotics; for G8/DHFR stock cultures, 0.3 µM methotrexate was added to the medium. SK-Ov-3 cells were maintained in RPMI plus 10% fetal calf serum, antibiotics, and glutamine. Experimental cell samples were seeded into tissue culture dishes and treated by adding either phorbol dibutyrate (PdBU, Sigma; 100 ng/ml) or sodium orthovanadate (Sigma; 1 mM) directly to the medium.
Immunological ReagentsThe pAb-1 rabbit polyclonal antibody to the tyrosine 1248-containing C-terminal peptide sequence of c-erbB-2 (anti-Tyr1248; Triton Biosciences, Alameda, CA) was reconstituted in water and diluted 1:1000 in TBST buffer (10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.05% Tween 20) for immunoblotting. The Ab-3 mouse monoclonal antibody raised against the same peptide sequence (Oncogene Science) was used at 1:100 for immunoprecipitations. Monoclonal 4G10 antiphosphotyrosine antibody (a kind gift of B. Druker and T. Roberts) was purified over a Staph aureus protein A affinity column, diluted in TBST buffer (10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.05% Tween 20) and used at 1:1000 for immunoblotting.
Phosphorylation-specific c-erbB-2 AntibodiesThese were
developed as described previously (11). Briefly,
phosphothreonine-specific c-erbB-2 antibodies were developed
by immunizing rabbits with a threonine-phosphorylated peptide
KIRKY
MRRLL (Calbiochem-Novabiochem) corresponding to the
juxtamembrane Thr686-containing erbB-2 sequence
(28). The resulting antisera were screened by immunoblotting (1:10,000)
using control and PdBU-treated G8/DHFR cell lysates.
Immunoprecipitation and immunocytochemical studies were performed at
1:5000 dilution. Adsorption of crude antisera to the unphosphorylated
peptide to eliminate contaminating non-phosphorylation-specific
antibodies was carried out where described.
PKC was purchased
from Promega. The sequence of the control
Ser1113-containing peptide was PLQRYSEDP. The assay was
performed by incubating 25 ng of PKC with or without 100 µmol of
peptide substrate at 30 °C for 20 min in PKC assay buffer (20 mM HEPES, pH 7.4, 1.5 mM CaCl2, 1 mM dithiothreitol, 10 mM MgCl2, 1 mM ATP, 100 µg/ml phosphatidylserine) containing 10 µCi
[
32P]ATP in a total volume of 25 µl. An identical
assay without PKC was used as a negative control. The reaction was
terminated by adding 25 µl of 1.5% H3PO4,
and the reaction mix was spotted onto a Whatman P-81 filter. After
being washed in 0.5% H3PO4 four times for 5 min each, the filter was air-dried and radioactivity determined by
scintillation.
Protein lysis was performed by washing monolayer cultures twice with ice-cold PBS, then adding lysis buffer (10 mM Na2HPO4·7H2O, 10 mM NaH2PO4·H2O, 150 mM NaCl, 1% Nonidet P-40 (v/v), 10% glycerol (v/v), 50 mM sodium fluoride, 10 mM sodium pyrophosphate) plus protease inhibitors (1 mM sodium orthovanadate, 40 µM leupeptin, 10 µg/ml aprotinin, 1 mM phenylmethylsulfonyl fluoride) for 15 min at 4 °C with gentle rocking. After being centrifuged to remove cell debris, lysates for immunoblotting studies were immediately boiled for 5 min in sample buffer (6.7% sodium dodecyl sulfate (w/v), 30% glycerol (v/v), 62.5 mM Tris base, pH 6.8, 0.01% bromphenol blue) then loaded onto a 7.5% polyacrylamide gel. For immunoprecipitations, 100 µl lysates were incubated with antibody overnight followed by a further 30-min incubation with 40 µl protein A-Sepharose CL4B beads (Pharmacia Biotech Inc.) prior to washing three times in ice-cold TBS, addition of sample buffer, and boiling. Samples were electrophoresed as above and then transblotted onto nitrocellulose as described (33). Membranes were blocked for 2 h at 37 °C, then incubated with primary antibody overnight at 4 °C with gentle shaking. Following three washes with TBS, membranes were incubated with alkaline-phosphatase-conjugated (Promega) polyclonal anti-rabbit/anti-mouse IgG for at least 2 h, washed, then developed immediately using colorimetric reagents.
In Vivo Phospholabeling and Phosphoaminoacid AnalysisCell monolayers were washed free of medium using PBS, rinsed once with phosphate-free Dulbecco's minimal essential medium, and incubated for 4 h with 3 ml of phosphate-free Dulbecco's minimal essential medium containing glutamine, 0.2% bovine calf serum, and 4mCi 32P-orthophosphate per 10-cm plate. Samples were subsequently washed twice with PBS and overlayered with 5 ml of medium plus or minus PdBU for 15 min at 37 °C. The cells were then lysed, immunoprecipitated using c-erbB-2 monoclonal antibody, washed, boiled, and loaded onto a denaturing 0.1% SDS 7.5% acrylamide gel. After exposure, the gel slices of interest were excised, cleaned, fixed in 30% methanol overnight, and then trypsinised for 24 h. The samples were dried, washed free of trypsin, resuspended in 100 µl of H2O, relyophilised, and then incubated in 90 µl of distilled 6 N HCl at 100 °C for 2 h. HCl was removed by four cycles of vacuum centrifugation with serial H2O washes. Dried samples were Cerenkov counted and then dissolved in buffer containing 0.3% each of xylene cyanol, orange G, and acid fuchsin dye stocks. After correction for total Cerenkov counts, approximately 0.5 µl of each sample was spotted onto a 20 × 20-cm plastic-backed cellulose thin layer chromatography plate. Electrophoresis was then performed at 800 V for 75 min, and the plate was exposed to x-ray film for 2 weeks.
As mentioned earlier, the juxtamembrane Thr654-containing peptide sequence of EGFR is the main protein kinase C site of the receptor (20, 23, 34) and exhibits major homology to the Thr686-containing sequence in c-erbB-2, which has been characterized as a PKC consensus site (27, 28). To confirm that this region is indeed a PKC substrate, we compared the PKC-inducible phosphorylation of this peptide sequence in vitro with that of a second c-erbB-2 peptide sequence homologous to a second EGFR desensitization domain believed to represent a CaM kinase II site (35). The results of these experiments, presented in Table I, confirm that the Thr686-containing peptide is phosphorylated by PKC almost 100-fold more efficiently than the Ser1113-containing peptide, or a peptide sequence from the extracellular domain of c-erbB-2 (data not shown). The explanation for the phosphorylation detected on the peptide incubated with isotope but without PKC is not clear (see Table I), though we note that this accounts for less than 2% of the signal seen with PKC. We therefore conclude that the Thr686-containing peptide is an excellent PKC substrate in vitro.
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Given the foregoing, the
Thr686-containing peptide sequence of c-erbB-2
seemed an appropriate immunogen for raising antibodies against the
PKC-modified receptor, with the resulting antisera capable of being
screened using protein lysates obtained from cells treated with known
PKC agonists and antagonists. The former include PdBU (36) as well as
the tyrosine phosphatase inhibitor sodium orthovanadate (because
tyrosine phosphorylation directly activates phospholipase
C-
37, an activator of PKC), whereas the latter include
calphostin C38. Fig. 1A
illustrates the screening of crude antisera to the
Thr(P)686 immunogen and shows the inverse pattern of
immunoreactivity when compared with an antiphosphotyrosine antibody.
The anti-Thr(P)686 antiserum also selectively recognizes
the native form of PKC-modified c-erbB-2 as demonstrated by
immunoprecipitation (Fig. 1B).
) and PdBU-treated G8/DHFR cells (aPT-686,
+). aY-1248,
and + were
immunoprecipitated using the Ab-3 c-erbB-2 antibody
(Oncogene; 1:100). After gel electrophoresis, immunoblotting was
carried out using the pAb-1 c-erbB-2 antibody (Triton;
1:1000).
Binding of anti-Thr(P)686 to c-erbB-2 Is Phosphorylation-specific, Receptor-specific, and PKC-dependent
The phosphorylation status of synthetic
phosphopeptides used for immunization was confirmed using mass
spectrometry (data not shown). These phosphopeptides were then used in
conjunction with unphosphorylated peptides of identical sequence to
establish the phosphorylation specificity of anti-Thr(P)686
binding. Immunoreactivity of control and PdBU-treated G8/DHFR lysates
is abolished by preincubation of anti-Thr(P)686 for 30 min
with the immunizing phosphopeptide KIRKY
MRRLL (+Thr(P), 10 µg/ml; Fig. 2A, lanes 3 and
4). In contrast, incubation with the unphosphorylated
peptide (+Thr, 10 µg/ml) has only a minor effect on immunoreactivity,
abolishing non-phosphorylation-specific ``background'' antiserum
binding from control cells (Fig. 2A, lane 5) but
sparing anti-Thr(P)686 immunorecognition of the
PdBU-treated sample (Fig. 2A, lane 6). This
indicates that the crude anti-Thr(P)686 antiserum binds
preferentially to the threonine-phosphorylated peptide and that a minor
degree of non-phosphorylation-specific background binding of the
polyclonal can be eliminated by competing with the unphosphorylated
peptide, as demonstrated in a previous report (8). A 10-fold molar
excess (w/v) of soluble phosphothreonine fails to abrogate
anti-Thr(P)686 immunoreactivity when compared with the
phosphopeptide (data not shown), indicating that the antiserum does not
recognize phosphothreonine alone but specifically detects the
threonine-phosphorylated c-erbB-2 juxtamembrane sequence.
Similarly, immunoreactivity of anti-Thr(P)686 for
PKC-modified c-erbB-2 is not attenuated by preincubating the
antibody with tyrosine- or serine-phosphorylated peptides (data not
shown). Phosphoaminoacid analysis confirms phorbol-inducible
enhancement of c-erbB-2 threonine (and serine)
phosphorylation associated with c-erbB-2 tyrosine
dephosphorylation (Fig. 2B).
and
+) following 30-min antibody preincubation with either the
phosphorylated peptide immunogen (+Thr(P), 10 µg/ml; +PThr,
and +) or an unphosphorylated peptide of identical
amino acid sequence (+Thr, 10 µg/ml; +Thr,
and
+). B, phosphoaminoacid analysis. Cell samples
were stimulated with PdBU (100 ng/ml, 10 min) then lysed together with
control samples. Immunoprecipitation of c-erbB-2 was carried
out using an extracellular domain antibody (see ``Experimental
Procedures'').
Because the phosphopeptide immunogen used to raise
anti-Thr(P)686 exhibits significant amino acid homology to
the corresponding EGFR sequence, we next sought to exclude
crossreactivity of the antiserum with protein lysates from ligand- and
phorbol-treated EGFR-expressing cells. Fig. 3 shows that
neither high concentrations of EGF (100 ng/ml) nor high concentrations
of PdBU (100 ng/ml) induce anti-Thr(P)686 immunorecognition
in EGFR-expressing chimeric 293 cell transfectants, thus confirming the
receptor specificity of this reagent.
The PKC dependence of anti-Thr(P)686 immunoreactivity was
tested by pretreating G8/DHFR cells with the fluorescence-activated
protein kinase C antagonist calphostin C38 prior to PdBU or
vanadate exposure. This inhibitor reduces anti-Thr(P)686
immunoreactivity induced by these agonists (Fig.
4A), consistent with the PKC dependence of
these effects. In contrast, treatment of unstimulated G8/DHFR cells
with the serine-threonine phosphatase inhibitor okadaic acid does not
affect anti-Thr(P)686 immunoreactivity (Fig.
4B), consistent with the findings of other groups studying
EGFR39. However, pretreatment of cells with PdBU is
associated with enhancement of anti-Thr(P)686 binding by
okadaic acid exposure (Fig. 4B), indicating that a
phosphatase acting at this site is itself dependent upon PKC for
activation.
Thr686 Phosphorylation of Wild-type c-erbB-2 Is Heterologously Inducible in G8/DHFR Cells, Whereas Mutant Tyrosine-phosphorylated Receptors in B104-1-1 Cells Are Homologously Thr686-phosphorylated
Experimental use of phorbol
ester provides an artificial means of manipulating PKC such that enzyme
in all cell sites is nonselectively activated; in other words, this
treatment represents a way of effecting heterologous receptor
transmodulation. In contrast, ligand-inducible receptor activation
might be expected to induce homologous receptor desensitization via
phosphorylation events localized to activated receptor subsets
(e.g. within focal adhesions). In the case of vanadate,
however, it is unclear whether anti-Thr(P)686 binding (and,
by inference, PKC activity) is induced heterologously or homologously.
We therefore carried out a series of phosphorylation-specific
immunoprecipitations and immunoblots to permit assessment of the
relationship between phosphorylation states of c-erbB-2
subsets (e.g. tyrosine-phosphorylated receptors), their
``cleared'' supernatants (containing tyrosine-dephosphorylated
receptors) and other phosphorylated receptor subsets within the cell
(e.g. threonine-phosphorylated receptors). The efficiency of
the immunoprecipitation procedure to clear the supernatant of the
relevant receptor phosphotype was confirmed in control experiments (not
shown). The anti-Tyr1248 monoclonal recognizes
c-erbB-2 in control G8/DHFR cells somewhat more efficiently
than in vanadate-treated cells (Fig. 5), consistent with
the specificity of this antibody for its tyrosine-dephosphorylated
epitope (8). PdBU-treated G8/DHFR cells express highly
anti-Thr(P)686-reactive c-erbB-2, which is
tyrosine-dephosphorylated, indicating heterologous induction of
threonine phosphorylation; the same is true of vanadate-treated
samples, with the pattern of anti-Thr(P)686 binding closely
matching that of anti-Tyr1248 irrespective of the receptor
tyrosine phosphorylation state (Fig. 5). Induction of PKC activity by
vanadate thus appears to be a nonselective process occurring in
trans, rather than one that is restricted to
tyrosine-phosphorylated receptor subsets. Hence, this is consistent
with vanadate-inducible phospholipase C-
activation (37).
To simulate the effects of ligand stimulation, analysis of
transmembrane mutant receptors from B104-1-1 cells was undertaken.
This mutation, originally induced by the carcinogen ethylnitrosourea,
causes constitutive c-erbB-2 homodimerization (10) and cell
transformation (40). Fig. 6 shows that most
anti-Thr(P)686 immunoreactivity is found in the
tyrosine-phosphorylated c-erbB-2 receptor subset, even
though this subset comprises only a small proportion of total cellular
receptor expression. The coexistence of this relatively heavy threonine
phosphorylation within the tyrosine-phosphorylated receptor subset
suggests either that receptor autophosphorylation is not efficiently
abolished by PKC-dependent c-erbB-2 modification
or else that tyrosine-phosphorylated mutant receptors are not
efficiently down-regulated by Thr686 phosphorylation.
Thr686 Is Constitutively Phosphorylated in SK-Ov-3 Human Ovarian Cancer Cells and BT-474 Human Breast Cancer Cells
We previously showed that G8/DHFR cells overexpress
c-erbB-2, which is constitutively tyrosine-phosphorylated
(i.e. independent of medium conditioning or cell contact),
but that receptor tyrosine dephosphorylation occurs rapidly following
exposure to PKC agonists, such as calf serum, platelet-derived growth
factor, or phorbol ester (4, 31). In addition, we showed that a human
ovarian cancer cell line (SK-Ov-3) that overexpresses
c-erbB-2 to a similar extent, on the other hand, expresses
c-erbB-2 receptors that are minimally kinase-active by
comparison with some other cell lines, whereas BT-474 cells express
receptors that are both strongly tyrosine-phosphorylated and
kinase-active (11). To determine the relationship between
c-erbB-2 threonine and tyrosine phosphorylation events in
these cell lines, patterns of receptor tyrosine and threonine
phosphorylation were analyzed in control, vanadate-treated, and
phorbol-treated samples. Exposure to the tyrosine phosphatase inhibitor
orthovanadate increases tyrosine phosphorylation in G8/DHFR and SK-Ov-3
cells but not in BT-474 cells; the latter cells express strong tyrosine
phosphorylation at 185 kDa without treatment (Fig. 7),
raising the possibility that a defect in phosphatase action may
contribute to the constitutive kinase activity of this receptor
(11).
Fig. 8 shows further that both tumor cell
lines exhibit anti-Thr(P)686 immunoreactivity (though more
pronounced in SK-Ov-3 than in BT-474 cells) whereas control G8/DHFR
cells express little detectable threonine-phosphorylated
c-erbB-2. Moreover, unlike G8/DHFR cells, minimal
enhancement of anti-Thr(P)686 binding in SK-Ov-3 and BT-474
cells is induced by either vanadate or PdBU, indicative of constitutive
receptor threonine phosphorylation; the intensity of this
phosphorylation varies with serum batches (data not shown), consistent
with mediation in part by heterologous growth factors. We note also
that the greater Thr686 phosphorylation of SK-Ov-3 relative
to BT-474 cells correlates with the respective sensitivity of
c-erbB-2 in these cell lines to vanadate.
As in B104-1-1 cells, the coexistence of Thr686 phosphorylation and c-erbB-2 autophosphorylation in BT-474 cells indicates that the former is not sufficient to eliminate the latter. The apparent resistance of BT-474 cells to vanadate (Fig. 7) may be consistent with a receptor-activating mutation; however, we cannot exclude the possibility that primary or secondary reductions in tyrosine phosphatase action may contribute to both the constitutive autophosphorylation and Thr686 phosphorylation in these cells. A similar pattern of coexisting c-erbB-2 tyrosine and Thr686 phosphorylation is seen in SK-Br-3 human breast cancer cells (data not shown).
Receptor tyrosine kinases are among the most potent transforming molecules known, making them prime targets for antineoplastic medical therapies. An obstacle to rational drug development of this kind has been the limited understanding of mechanisms controlling the kinase activity of these molecules. PKC-dependent transmodulation of the EGF receptor is a case in point: although many studies have suggested a critical role for Thr654 phosphorylation in this process (23, 24, 25, 41), others have cast doubt upon this conclusion (42). Loss of high affinity ligand binding (17, 25, 43) has been associated with phorbol-inducible receptor transmodulation, though less specifically with Thr654 phosphorylation per se (24, 41). Such PKC-dependent reductions in ligand binding may in turn reflect induction of receptor internalization (44), either with (45) or without receptor degradation (18, 23, 26). The complexity and interdependence of the signaling pathways involved in these phenomena is suggested here by the PKC-dependent enhancements of Thr686 phosphorylation induced by inhibitors of tyrosine and serine-threonine phosphatases (Fig. 4).
As previously reported, G8/DHFR cells exhibit a minor degree of constitutive erbB-2 tyrosine autophosphorylation that is rapidly abolished by a variety of PKC agonists (4, 31). In contrast, SK-Ov-3 ovarian cancer cells express c-erbB-2 receptors that virtually lack autocatalytic activity in vitro (8, 11). The present study provides a potential explanation for the differential tyrosine phosphorylation of these two cell lines by revealing reciprocal differences in anti-Thr(P)686 immunoreactivity. Absence of the latter in G8/DHFR cells is associated with moderate c-erbB-2 catalytic activity (4), whereas selective induction of threonine phosphorylation by phorbol ester is tightly associated with tyrosine dephosphorylation; conversely, constitutive receptor threonine phosphorylation is associated with absent receptor autocatalytic activity in SK-Ov-3 cells (11), with receptor tyrosine autophosphorylation exclusively demonstrable in these cells by inhibiting tyrosine phosphatases (Figs. 7 and 8). BT-474 cells, on the other hand, appear constitutively phosphorylated on both tyrosine and threonine residues, with this lack of inducibility suggesting a mutational basis for receptor activation. Our own sequencing studies, however, indicate that no mutation is present in either the transmembrane or juxtamembrane c-erbB-2 region in these cells (data not shown).
Our data thus far do not explain the well-documented relationship between PKC activity and tyrosine phosphatase function. A coherent hypothesis is that inducible c-erbB-2 threonine phosphorylation is linked to inducible receptor interaction with phosphatases in G8/DHFR cells. Similarly, constitutive threonine phosphorylation may be linked to constitutive c-erbB-2 tyrosine dephosphorylation in SK-Ov-3 cells expressing the wild-type receptor (again consistent with a preferential interaction of tyrosine phosphatases with the Thr686-phosphorylated receptor subset), whereas the less intense Thr686 phosphorylation seen in the BT-474 cell line may be related to the vanadate-resistant phenotype of these cells. Interestingly, sequencing of the c-erbB-2-associated PTP1B tyrosine phosphatase (46, 47) has revealed numerous N-terminal mutations in this and other tumor cell lines (data not shown). Mutagenesis studies are planned to clarify the mechanism(s) by which juxtamembrane threonine phosphorylation may facilitate c-erbB-2 interaction with phosphatases.
To our knowledge, this is one of the first published studies to use a high specificity antiphosphothreonine antibody raised against synthetic phosphopeptides. The immunogenicity of phosphotyrosine is long established (48), consistent with the major structural consequences now known to be induced by this critical posttranslational modification (49). However, the structural changes induced by serine/threonine phosphorylation remain unclear, and high quality antibodies selectively detecting such modifications have proven difficult to obtain. The findings of this study confirm that sensitive detection of threonine phosphorylation events is indeed possible, and we are currently extending our use of this approach to the analysis of c-erbB-2-overexpressing tumour specimens.
In conclusion, our findings suggest that c-erbB-2 Thr686 phosphorylation may be constitutively induced in human tumor cell lines, and we speculate that the etiology of this constitutive event may be either heterologous or mutational. A third possibility is that primary dysregulation of cellular PKC activity could play a role in modulating the neoplastic phenotype in vivo, a possibility consistent with studies suggesting a role for PKC in cell transformation (50), tumorigenesis (51), and metastasis (52). Perhaps the most intriguing of these possibilities, however, is that c-erbB-2 acts as a heterologous integration point for growth factor signaling, because this possibility has additional implications for the physiological role of c-erbB-2. We predict that more extensive use of phosphothreonine-specific antibodies will permit functional clarification of these signaling interactions both in vitro and in vivo.
To whom correspondence should be addressed. R. E. is supported
by the Cancer Research Campaign.
We thank R. Weinberg for providing the G8/DHFR cell line, B. Druker and T. Roberts for a gift of 4G10 monoclonal antibody, A. Chantry for supplying reagents and constructs for control experiments, and C. Coombes for support.
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