(Received for publication, October 20, 1995; and in revised form, December 12, 1995)
From the
The RET proto-oncogene encodes a member of the receptor tyrosine kinase family. Multiple endocrine neoplasia type 2B (MEN 2B) is caused by the mutation of a conserved methionine to a threonine in the catalytic domain of the RET kinase. When the MEN 2B point mutation was introduced into the epidermal growth factor (EGF) receptor (M857T EGFR), the intrinsic tyrosine kinase activity of the mutant receptor was similar to that of wild-type EGF receptor and remained ligand-dependent. However, the mutant receptor showed an enhanced transforming capacity compared to the wild-type receptor as judged by its ability to mediate the growth of NIH 3T3 cells in soft agar. Using the oriented peptide library approach to examine substrate specificity, the M857T mutation was found to be associated with a decrease in the selectivity of the receptor for Phe and an increase in the selectivity for acidic residues at the P + 1 position as compared to wild-type EGF receptor. Short-term responses to EGF were similar in cells expressing wild-type and M857T EGF receptors. However, significant differences in receptor down-regulation were observed between the two receptors. These data demonstrate that the MEN 2B point mutation alters the substrate specificity of receptor tyrosine kinases and suggest that the enhanced oncogenesis associated with the MEN 2B mutation may be due in part to alterations in receptor regulation.
RET was first identified as a transforming gene by
transfection experiments using DNA isolated from a human T-cell
lymphoma(1) . Subsequently, physical and genetic mapping data
have implicated the RET gene in several dominantly inherited
human neoplasias, including multiple endocrine neoplasia type 2A (MEN
2A), ()multiple endocrine neoplasia type 2B (MEN 2B), and
familial medullary thyroid carcinoma
(FMTC)(2, 3, 4, 5, 6) . MEN
2A and FMTC are both characterized by the development of medullary
thyroid carcinoma between the ages of 20 and 50. Approximately
one-third to one-half of MEN 2A patients also develop pheochromocytomas
and parathyroid hyperplasia. MEN 2B is a more severe form of the
disease with onset of symptoms during the first decade of life.
Patients exhibit ganglioneuromas of the intestinal tract, mucosal
neuromas, and opthalmalogic and skeletal abnormalities, in addition to
a particularly aggressive form of medullary thyroid carcinoma.
The RET proto-oncogene encodes a protein that is a member of the
family of transmembrane tyrosine kinase growth factor receptors. The
RET protein is comprised of a 635-amino acid extracellular domain, a
single transmembrane spanning region, and an intracellular tyrosine
kinase domain that contains a short, 26-amino acid kinase insert
domain. Residues 515 to 634 of the extracellular domain constitute a
region of high cysteine content similar to those found in the EGF
receptor and the insulin receptor(7, 8) . Immediately
amino-terminal to this high cysteine region is a domain of 110
amino acids that is 20-30% homologous to cadherins (9) The presence of this cadherin-like domain is unique among
receptor tyrosine kinases and its function within the RET protein is
not known. A ligand for the RET receptor tyrosine kinase has not been
identified.
Mutations in several conserved cysteine residues in the high cysteine region of the ret extracellular domain account for the majority of cases of MEN 2A and FMTC(2, 3) . However, mutations in the tyrosine kinase domain of RET have also been associated with FMTC(10) . Biochemical characterization of MEN 2A RET proteins in which Cys-634 is altered to tyrosine, arginine, or tryptophan suggests that the mutation causes constitutive dimerization and activation of the RET tyrosine kinase(11, 12) . The MEN 2B phenotype is associated with a single missense mutation in the tyrosine kinase domain of RET that converts Met-918 to threonine(4, 5) . To date, this is the only mutation that has been detected in patients with MEN 2B. Interestingly, Met-918 is conserved as a methionine among the receptor tyrosine kinases but is present as a conserved threonine residue in the cytosolic, non-receptor tyrosine kinases such as src(13) .
Comparison of the sequence of the RET tyrosine kinase domain with the structure of the cyclic AMP-dependent protein kinase with bound inhibitor peptide suggests that Met-918 forms part of the binding site for the substrate residue immediately COOH-terminal to the site of phosphorylation(14, 15) . Based on this information, Carlson et al.(4) predicted that the MEN 2B mutation would alter the substrate specificity of the RET tyrosine kinase rather than constitutively activate the enzyme. The properties of the MEN 2B RET protein appear to be consistent with this hypothesis. Only a modest increase in basal autophosphorylation has been demonstrated but there are differences between the two-dimensional gel electrophoresis patterns of tyrosine phosphorylated proteins in cells expressing MEN 2A RET and MEN 2B RET(11, 12) . More recently, Songyang et al.(16) have shown that wild-type RET and MEN 2B RET exhibit different activity toward three synthetic tyrosine-containing peptides.
Because the ligand for RET has not yet been identified, previous studies of the MEN 2B RET protein have been unable to determine the effect of this point mutation on either the ligand dependence of RET tyrosine kinase activity, the activation of downstream signaling pathways, or receptor regulation. To address these issues, we introduced the MEN 2B point mutation into the kinase domain of the EGF receptor and stably expressed this construct in NIH 3T3 cells. Met-857 of the EGF receptor is equivalent to Met-918 in RET. Thus, we refer to this mutant as the M857T EGF receptor. Substrate specificity studies using the oriented peptide library approach demonstrated that the wild-type and M857T EGF receptors preferentially phosphorylated different tyrosine-containing peptides confirming the hypothesis that the MEN 2B mutation changes the substrate specificity of RET. The mutation was found to enhance the transforming capacity of the EGF receptor but had little effect on the ability of EGF to stimulate downstream signaling pathways. However, M857T EGF receptors exhibited differences in down-regulation, resensitization, and trafficking compared to wild-type receptors. The data suggest that the MEN 2B mutation is generally transforming for growth factor receptors and that the enhanced oncogenesis may result from a change in receptor kinase specificity that leads to alterations in long-term receptor regulation. These data provide insight into the mechanism by which the MEN 2B mutation induces malignant activation of RET.
The
synthetic peptide combinatorial library with the sequence
EVPEYXXXSPLLL, where X stands for any amino acid
except cysteine and tryptophan, was prepared using a t-butyloxycarbonyl strategy. The amino acids (Bachem,
Torrance, CA) were coupled as the 1-hydroxybenzotriazole esters on an
Applied Biosystems Model 431A (Perkin Elmer, Norwalk, CT). The fixed
amino acids were double coupled with a 5-fold excess of protected amino
acid to ensure high coupling efficiencies.
(4-Oxymethyl)phenylacetamidomethyl resin preloaded with leucine (0.2
mmol, Perkin Elmer) had a loading of 0.75 mmol/g. The synthesis was
interrupted when the first degenerate site was reached. The resin was
washed with dichloromethane and dried in vacuo overnight. The
dried resin was weighed and divided into four parts. Parts 1, 2, and 3
each contained 5/18 of the total resin. Part 4 contained 3/18 of the
total resin. These individual parts were coupled with four separate
groupings of protected amino acids. Group I included alanine, glycine,
methionine, aspartic acid (OcHex), and tyrosine (Br-Z). Group II
contained glutamic acid (OcHex), lysine (Cl-Z), phenylalanine,
histidine (Bom), and proline. Group III contained asparagine,
glutamine, arginine (Tos), valine, and leucine. Group IV included
serine (Bzl), threonine (Bzl), and isoleucine. Note that the first
three groups each contained five amino acids and were therefore coupled
with 5/18 of the resin while group IV had only three amino acids and
was coupled with 3/18 of the resin. For each site, resin divisions 1
through 3 were coupled with groups I through III of amino acids and
resin division 4 was always coupled with group IV. The amino acids in
each group were loaded into a single cartridge for the synthesizer. In
the cartridge for group I, 0.1 mmol of each amino acid was present. For
group II proline was present at 0.15 mmol while the others were present
at 0.1 mmol. In group III valine was present at 0.15 mmol while the
others were present at 0.1 mmol. In group IV, 0.15 mmol of each amino
acid was present. Each of the four resin divisions were placed back
onto the synthesizer and single coupled to the appropriate amino acid
grouping. The coupling time on the instrument was increased from 35 to
56 min. To assess the extent of coupling efficiency, the amount of free
NH terminus was determined using the colorimetric
quantitative ninhydrin assay(18) . After coupling of each
group, no detectable color was seen indicating that the couplings had
gone to completion. After all four divisions had been coupled with the
appropriate amino acid grouping, the resins were recombined and mixed.
The resin was then dried overnight and separated by weight into four
divisions identical to those described above. These division were
coupled to the four amino acid groupings as before. The procedure was
repeated to synthesize the third degenerate position. At the end of the
synthesis of the degenerate sites, the resins were pooled and the final
five fixed amino acids were added following the same protocol used for
the first fixed residues. The peptide mixture was cleaved from the
resin using anhydrous HF at 0 °C for 120 min in the presence of the
scavengers dimethyl sulfide and anisol. The cleaved peptide was washed
exhaustively with ether, dissolved in acetic acid, and lyophilized. The
peptide mixture eluted as a single (slightly broadened peak) on
reversed phase high pressure liquid chromatography. MALDI-TOF-MS
revealed an envelop of masses centered about the expected average
molecular weight for the peptide series. Sequence analysis of the
peptide library indicated the following ratios of amino acids at the
degenerate positions: Ala-1, Asp-1.6, Glu-1.4, Phe-0.85, Gly-2.5,
His-1.4, Ile-0.32, Lys-1.0, Leu-1.3, Met-0.62, Asn-1.6, Pro-1.8,
Gln-1.6, Arg-1.1, Ser-0.67, Thr-0.59, Val-0.96, and Tyr-1.4.
Phosphopeptides were
purified as described by Songyang et al.(24) with
some modifications. The trichloroacetic acid supernatants from 10
reactions were pooled and applied in three aliquots to an 18-ml G-15
Sephadex column equilibrated with 30% acetic acid. The column was
eluted with the same buffer and void volume fractions were collected.
Following three runs, the fractions containing phosphopeptides were
pooled and applied to a 2-ml DEAE-Sephacel column. The column was
developed with 30% acetic acid. Fractions containing P
were pooled and applied to a 1-ml Fe
-iminodiacetic
acid-agarose column. The column was washed sequentially with 5 column
volumes of 50 mM glycine, 100 mM NaCl, pH 3.0, 3
column volumes of 50 mM MES, 1 M NaCl, pH 5.5, and 5
column volumes of 50 mM MES, 1 M NaCl, pH 7.0. The
phosphorylated peptides were eluted with 500 mM ammonium
bicarbonate, pH 8.0. Fractions containing
P were pooled,
lyophilized, and resuspended in 400 µl of H
O. Excess
ammonium bicarbonate was removed by desalting over a 6-ml G-15 Sephadex
column. Contamination of the purified product by non-phosphorylated
peptide was estimated to be <5% by comparing protein concentration
as determined by BCA assay with phosphopeptide present as determined by
P content. The purified phosphopeptides were sequenced and
data analyzed as described(24) .
Cytoskeletons were prepared by
incubating cells in 100-mm dishes for 30 min at 4 °C with constant
agitation in 10 ml of buffer containing 40 mM HEPES, pH 7.2, 2
mM MgCl, 2 mM MnCl
, 1 mM CaCl
, 250 mM NaCl, 0.5% Triton X-100 plus
protease and phosphatase inhibitors as described above. The buffer was
removed and the monolayers washed twice with the same Triton
X-100-containing buffer and twice with phosphate-buffered saline. The
Triton-insoluble material was scraped into 1 ml of RIPA buffer and
homogenized by passage through a 25-gauge needle. The homogenate was
then clarified by centrifugation at 12,000
g for 10
min. The protein present in lysates or cytoskeletal preparations was
determined by BCA assay and aliquots containing 100 µg of protein
were analyzed by SDS-polyacrylamide gel electrophoresis.
Two clones expressing wild-type EGF receptor and two clones expressing M857T EGF receptor were assayed for tyrosine kinase activity. Kinase activity was assessed by quantitating the ability of membranes derived from the different cell lines to catalyze the phosphorylation of the Arg-Arg-Src synthetic peptide. Parental NIH 3T3 cells exhibited no EGF stimulated peptide kinase activity (not shown). However, as shown in Fig. 1A, all transfected cell lines exhibited an EGF-stimulated increase in tyrosine kinase activity indicating that both the wild-type and the M857T EGF receptors were functional with respect to kinase activity. In addition, the concentration at which half-maximal activation of the receptor kinase was attained was similar in lines expressing wild-type and M857T EGF receptors. These data demonstrate that introduction of the M857T point mutation into the EGF receptor does not generate a constitutively active kinase but rather yields a receptor that responds to EGF over the same dose range as does the wild-type receptor.
Figure 1:
Phosphorylation of Arg-Arg-Src peptide
by wild-type and M857T EGF receptors. Panel A, dose response
to EGF. Membranes were prepared from cells expressing the wild-type EGF
receptor (WT) or the M857T EGF receptor (Mut) and
assayed for the ability of increasing concentrations of EGF to
stimulate the phosphorylation of Arg-Arg-Src as described under
``Experimental Procedures.'' Data shown represent the average
of duplicate determinations. Panel B, Lineweaver-Burk analysis
of Arg-Arg-Src phosphorylation by WT9 and Mut6 cells. Membranes were
prepared from WT9 or Mut6 cells and equal amounts of membrane protein
were assayed for their ability to catalyze the phosphorylation of
increasing concentrations of Arg-Arg-Src in the presence of 3
10
M EGF. Data shown represent the average
of triplicate determinations.
A clone expressing 96,000 ± 8,000 wild-type EGF receptors/cell (WT9) and a clone expressing 112,000 ± 12,000 M857T EGF receptors/cell (Mut6) were selected for further study. Scatchard analysis of the parental NIH 3T3 cells demonstrated that they contained fewer than 2000 EGF receptors/cell. Thus, the human EGF receptor is expressed at approximately 50-fold higher levels in WT9 and Mut6 cells than is the endogenous mouse EGF receptor.
Membranes were prepared from WT9 cells and Mut6 cells and
the kinetic parameters for phosphorylation of Arg-Arg-Src were
determined by Lineweaver-Burk analysis. As shown in Fig. 1B, the K and V
for EGF-stimulated tyrosine kinase activity
were essentially identical in the two cell lines. Values for the K
and V
in WT9 cells were
1.5 mM and 630 pmol/min/mg protein, respectively. In Mut6
cells, these values were 1.7 mM and 590 pmol/min/mg protein.
The observation that the WT9 and Mut6 cells exhibit similar kinetic
constants for the utilization of this substrate suggests that the
intrinsic tyrosine kinase activities of the two forms of the receptor
are similar.
Although the binding and kinase activities of the wild-type and M857T EGF receptors were similar, Mut6 cells exhibited a markedly enhanced transforming potential in soft agar assays. When WT9 cells were plated in soft agar, EGF induced a dose-dependent increase in the ability of the cells to form colonies (Fig. 2A). Likewise, EGF induced an increase in the growth of Mut6 cells in soft agar. However, at optimal concentrations of EGF, approximately 4-fold more colonies were formed by the Mut6 cells than by the WT9 cells. Furthermore, the colonies formed by the Mut6 cells were significantly larger than those formed by WT9 cells and exhibited a less compact morphology (Fig. 2B). Parental NIH 3T3 cells did not produce colonies in soft agar at any concentration of EGF (not shown).
Figure 2:
Growth of WT9 and Mut6 cells in soft agar. Panel A, dose response to EGF. WT9 cells and Mut6 cells were
plated in soft agar in the presence of the indicated concentrations of
EGF as described under ``Experimental Procedures.'' After 14
days for WT9 or 8 days for Mut6 cells, the plates were examined
microscopically and 10 random fields were scored for colonies
containing 20 cells. Panel B, photographs of soft agar
colonies formed by WT9 and Mut6 cells.
The wild-type EGF receptor selected primarily hydrophobic residues at the P + 1 position, including those with both aliphatic and aromatic side chains. A slight preference for glutamic acid was also observed. The M857T EGF receptor exhibited a different set of preferences. While amino acids with aliphatic side chains, such as Val, Ile, and Leu, were selected at this position, the M857T receptor showed no selectivity for the aromatic side chain of Phe (ratio = 1.1 in M857T EGFR). In addition, the M857T EGF receptor showed increased selectivity for Glu relative to wild-type receptor and a preference for Asp at this site appeared. These data suggest that the M857T mutation decreases the phosphorylation of substrates with aromatic side chains at the P + 1 position and increases the phosphorylation of substrates with acidic residues at this position.
Subtle changes in substrate specificity were also observed at the P + 2 position. Both the wild-type and M857T EGF receptors showed strong selectivity for acidic residues at this position but also showed some preference for amino acids with hydrophobic side chains. However, the wild-type receptor selected the large aromatic Phe residue whereas the M857T EGF receptor selected the much smaller Val residue. Preferences at the P + 3 position were similar for the two receptors.
One noteworthy finding not reported in Table 1was that both wild-type and M857T EGF receptors strongly selected against Arg in either of the first two degenerate positions. The ratio of Arg present at the P + 1 and P + 2 positions in the phosphorylated peptides to Arg present in these positions in the total peptide library was 0.3 to 0.4 for both receptors. In addition, both receptors selected against Asn at the P + 1 position (ratio = 0.5). Wild-type EGF receptors also selected against Arg at the P + 3 position (ratio = 0.5) whereas the M857T EGF receptor did not (ratio = 0.8).
To determine whether the observed
changes in selectivity in vitro lead to changes in the
phosphorylation of substrates in vivo, the EGF-stimulated
phosphorylation of endogenous proteins in the two cell lines was
compared (Fig. 3). In total cell lysates the phosphorylation of
60- and 90-kDa proteins was stimulated more strongly by EGF in WT9
cells than in Mut6 cells. Conversely, in cytoskeletal preparations, the
phosphorylation of several proteins of 95 kDa was stimulated by
EGF more strongly in Mut6 cells than in WT9 cells. These data suggest
that the differences in substrate specificity between wild-type and
M857T EGF receptors observed in vitro translate into
differences in substrate phosphorylation in vivo.
Figure 3: EGF-stimulated phosphorylation of endogenous proteins in WT9 and Mut6 cells. WT9 and Mut6 cells were stimulated with or without 50 nM EGF for 2 min at 37 °C. Cells were then lysed in RIPA buffer (left panel) or cytoskeletons (right panel) were prepared as described under ``Experimental Procedures.'' An aliquot of each extract containing 100 µg of protein was analyzed by SDS-polyacrylamide gel electrophoresis followed by Western blotting with an anti-phosphotyrosine antibody.
Figure 4: Autophosphorylation of the EGF receptor in WT9 and Mut6 cells. WT9 and Mut6 cells were stimulated with or without 50 nM EGF at 37 °C for the time indicated and RIPA lysates were prepared. Samples containing 100 µg of protein were analyzed by SDS-polyacrylamide gel electrophoresis followed by Western blotting using an anti-EGF receptor antibody or an anti-phosphotyrosine antibody.
Figure 5: Stimulation of MAP kinase (panel A) or PI 3-kinase (panel B) by EGF in WT9 and Mut6 cells. Panel A, WT9 and Mut6 cells were treated with 50 nM EGF at 37 °C for the times indicated. RIPA lysates were then prepared and a 100-µg sample of each extract was analyzed by SDS-polyacrylamide gel electrophoresis. Proteins were transferred to nitrocellulose and subjected to Western blotting using an anti-MAP kinase antibody. Panel B, WT9 and Mut6 cells were treated with the indicated concentrations of EGF for 5 min at 37 °C. Cells were lysed in PI 3-kinase solubilization buffer and the extracts immunoprecipitated with anti-phosphotyrosine antibodies coupled to agarose. The immunoprecipitates were then assayed for PI 3-kinase activity as described under ``Experimental Procedures.'' The data shown represent the mean ± S.D. of duplicate determinations. Under the same conditions, parental NIH 3T3 cells exhibited a basal activity of 378 cpm and a stimulated activity of 504 cpm at 50 nM EGF.
The tyrosine phosphorylation of several
downstream signaling molecules was also examined (Fig. 6). EGF
stimulated the phosphorylation of the focal adhesion kinase, FAK, to
the same extent in WT9 and Mut6 cells. In addition, the EGF-stimulated
phosphorylation of Ras-GAP and the associated p190 and p62 proteins was
similar in WT9 and Mut6 cells. EGF also induced the phosphorylation of
the p46, p52, and p66 forms of SHC in WT9 and Mut6 cells. A tyrosine
phosphorylated protein at 170 kDa, which most likely represents
the EGF receptor, was also present in anti-SHC immunoprecipitates from
both cell lines indicating that in both cell types phosphorylated SHC
was also associated with the EGF receptor. The slightly lower level of
phosphorylated EGF receptor present in immunoprecipitates from Mut6
cell probably reflects the lower level of receptor autophosphorylation
observed in these cells (Fig. 4). The band at approximately 145
kDa that was present in anti-SHC immunoprecipitates from both cell
lines may be the p145 protein observed by Kavanaugh et al.(27) in anti-SHC immunoprecipitates.
Figure 6: EGF-stimulated phosphorylation of FAK, Ras-GAP, and SHC in WT9 and Mut6 cells. WT9 and Mut6 cells were treated with or without 50 nM EGF for 2 min at 37 °C. Nonidet P-40 lysates were then prepared and aliquots of each extract containing 300 µg of protein were immunoprecipitated with anti-focal adhesion kinase antibody, anti-Ras-GAP antibody, or anti-SHC antibody. Immunoprecipitates were analyzed by SDS-polyacrylamide gel electrophoresis followed by Western blotting with a horseradish peroxidase-coupled anti-phosphotyrosine antibody.
Figure 7:
Down-regulation and internalization of EGF
receptors in WT9 and Mut6 cells. Panel A, WT9 and Mut6 cells
were incubated with 2 nM EGF at 37 °C in DMEM containing
0.1% bovine serum albumin for the times indicated. Cultures were then
washed twice for 2 min with 50 mM glycine, 100 mM NaCl, pH 3.0, to remove surface-bound EGF and twice with
Hanks' balanced buffer solution. Cell surface binding was then
assessed using 1 nMI-EGF as described under
``Experimental Procedures.'' Data shown represent the mean
± S.D. of triplicate determinations. Panel B, WT9 and
Mut6 cells were washed twice in Hanks' balanced buffer solution
and incubated with 1 nM
I-EGF at 37 °C in
DMEM containing 40 mM HEPES and 0.1% bovine serum albumin for
the times indicated. At each time point, half of the cultures were
washed with Hanks' balanced salt solution and processed as usual
to determine total cell associated
I-EGF binding. The
second half of the cultures were washed twice for 2 min with 50 mM glycine, 100 mM NaCl, pH 3.0, to remove surface-bound EGF
and then washed twice with Hanks' balanced salt solution. The
cultures were then processed for counting of
I to
determine internalized
I-EGF. Surface
I-EGF
binding was calculated as the difference between total cell association
and internalized
I-EGF. The results are presented as the
ratio of internalized/surface
I-EGF (36) and
represent the mean ± S.D. of triplicate
determinations.
EGF receptor down-regulation leads to a decrease in the responsiveness of cells to EGF. Because a major difference in the biological response of WT9 cells and Mut6 cells to EGF occurred in an assay (soft agar) in which the cells were incubated with EGF for many days, we wondered whether the observed differences in down-regulation led to differences in the long-term responsiveness of these cells to EGF. To address this question, duplicate cultures of WT9 and Mut6 cells were incubated with increasing concentrations of EGF for 24 h to down-regulate the EGF receptors. One monolayer from each set was then re-challenged with 50 nM EGF for 2 min. Receptor levels and autophosphorylation were then assessed by Western blotting to evaluate the ability of the cells to respond to stimulation by EGF. As shown in Fig. 8, control WT9 cells showed a strong increase in receptor autophosphorylation in response to acute stimulation with EGF. Pretreatment of WT9 cells with 0.5 nM EGF for 24 h did not significantly alter the ability of these cells to respond to a subsequent challenge with 50 nM EGF. However, down-regulation for 24 h with concentrations of EGF of 2 nM or greater completely blunted the ability of the receptors to autophosphorylate in response to an acute challenge with EGF. Anti-EGF receptor blots (Fig. 8) demonstrated a similar dose response to EGF for the decline in total cellular EGF receptor levels. Mut6 cells appeared to be significantly less sensitive to down-regulation by EGF than WT9 cells. Even following down-regulation with 2 nM EGF for 24 h, Mut6 cells showed little loss of total cellular EGF receptors and exhibited a nearly normal autophosphorylation response to EGF. Only at doses of EGF of 10 nM or greater were the M857T EGF receptors significantly down-regulated with respect to either receptor number or receptor autophosphorylation.
Figure 8: Dose response to EGF for the long-term down-regulation of the EGF receptor. WT9 and Mut6 cells were incubated with the indicated concentrations of EGF in normal growth media for 24 h. Subsequently, half of the cultures were stimulated with 50 nM EGF for 2 min at 37 °C. RIPA lysates were then prepared and 100-µg samples of each extract were analyzed by SDS-polyacrylamide gel electrophoresis. Proteins were transferred to nitrocellulose and subjected to Western blot analysis using anti-EGF receptor antibodies, anti-phosphotyrosine antibodies, or anti-MAP kinase antibodies.
These differences in receptor down-regulation were also apparent in the ability of the wild-type and M857T EGF receptors to mediate activation of MAP kinase. As shown in Fig. 8, activation of MAP kinase following treatment of WT9 cells with 50 nM EGF for 2 min was only apparent in control cells and cells down-regulated for 24 h with 0.5 nM EGF. As was true for receptor autophosphorylation, the ability of the wild-type receptor to mediate activation of MAP kinase was completely ablated following down-regulation with 2 nM or higher concentrations of EGF for 24 h. By contrast, a significant decrease in the ability of M857T EGF receptors to stimulate MAP kinase did not occur until long-term down-regulation was carried out with doses of 10 nM or greater EGF.
Further analysis of the long-term
down-regulation of the EGF receptor in WT9 and Mut6 cells was carried
out. WT9 cells and Mut6 cells were incubated with 2 nM EGF for
periods of time ranging from 1 to 24 h. At the end of the incubation,
cell surface I-EGF binding was assessed as well as total
cellular receptor levels and receptor autophosphorylation. The data in Fig. 9A demonstrate that when cells were incubated with
2 nM EGF for up to 24 h, cell surface
I-EGF
declined in both cell types. However, after 24 h, Mut6 cells exhibited
approximately twice as much
I-EGF binding. Total cellular
levels of EGF receptors also declined rapidly in WT9 cells and remained
low throughout the incubation (Fig. 9B). By contrast,
treatment of Mut6 cells with EGF led to a modest decrease in total
cellular receptor levels over the first 6 h but the levels of EGF
receptor increased thereafter. Receptor autophosphorylation was
markedly different over this time course in the two cell lines (Fig. 9B). Whereas the wild-type EGF receptor was
phosphorylated to some extent throughout the entire 24-h time course,
the M857T EGF receptor appeared to be completely dephosphorylated
within 1 h after treatment with EGF and remained unphosphorylated
throughout the incubation.
Figure 9:
Time course of the long-term
down-regulation of the EGF receptor. WT9 and Mut6 cells were incubated
with 2 nM EGF in normal growth media for the times indicated.
In panel A, cultures were washed with acid wash buffer and
cell surface I-EGF binding determined as described in the
legend to Fig. 7. Results represent the mean ± S.D. of
triplicate determinations. In panel B, RIPA lysates were
prepared and analyzed by SDS-polyacrylamide gel electrophoresis
followed by Western blotting with anti-EGF receptor antibodies or
anti-phosphotyrosine antibodies.
Multiple endocrine neoplasia type 2B is the result of a Met
Thr mutation in the catalytic domain of the RET tyrosine kinase.
Because the RET protein has not been extensively characterized and no
ligand has been identified for this receptor, we investigated the
effects of this Met
Thr mutation within the context of the well
studied EGF receptor. Our findings demonstrate that this mutation is
capable of enhancing the transforming capacity of the EGF receptor as
judged by the ability of cells to grow in soft agar. This suggests that
transformation elicited by the MEN 2B mutation is not specific for the
RET tyrosine kinase and implies that signaling or regulatory pathways
utilized by all receptor tyrosine kinases are likely to be the basis of
MEN 2B-induced oncogenesis.
The MEN 2B mutation lies at position 857
in the cytoplasmic domain of the EGF receptor. Consistent with the
intracellular location of this mutation, we found no evidence for a
change in the ability of EGF to interact with its receptor.
Lineweaver-Burk analysis of the phosphorylation of the synthetic
Arg-Arg-Src peptide by cells that expressed similar numbers of
wild-type or M857T EGF receptors yielded similar K and V
values for the phosphorylation of
this peptide by both receptors. This demonstrates that the intrinsic
tyrosine kinase activity of the wild-type and mutant EGF receptors is
the same.
It is noteworthy that the M857T EGF receptor was not constitutively activated. EGF was still required to induce a biological response through this mutant receptor. These findings suggest that the oncogenicity of MEN 2B RET is not due to the constitutive activation of the kinase. This conclusion contrasts with the findings from studies of the activity of the MEN 2A RET protein. MEN 2A mutations are found primarily in the high cysteine region of the extracellular domain(2, 3) . Characterization of MEN 2A RET suggests that it is constitutively dimerized and active(11, 12) . The fact that the MEN 2A and MEN 2B phenotypes are clinically distinguishable is consistent with the apparent differences between a constitutively activated MEN 2A RET and a ligand-dependent but structurally altered MEN 2B RET.
The Met
Thr mutation in MEN 2B RET was hypothesized to change the
substrate specificity of the RET receptor kinase, in particular at the
P + 1 position(4) . Using the oriented peptide library
approach(24) , we found that the corresponding Met
Thr
mutation in the EGF receptor decreased its selectivity for aromatic
residues and increases its selectivity for acidic residues at the P
+ 1 position. Minor changes relating to the size of the preferred
hydrophobic side chains also occurred at the P + 2 position but no
significant differences were observed at the P + 3 position. These
findings directly demonstrate that the MEN 2B mutation alters the
substrate specificity of a receptor tyrosine kinase. The preference of
the M857T EGF receptor for Glu and Asp residues at the P + 1
position is similar to that observed for the src family
kinases(16) . Since the src family kinases all contain a
threonine residue at the position equivalent to that of Met-857 in the
EGF receptor, a hydrophilic residue at this position in the kinase
sequence appears to be a strong determinant for the selection of acidic
residues at the P + 1 position of the kinase substrate.
Our experiments with the wild-type and M857T EGF receptor utilized a degenerate peptide library that differed from that used by Songyang et al.(16) . In addition, our studies were performed with cell membranes as the source of tyrosine kinase activity rather than with purified recombinant proteins. Nonetheless, our findings with respect to the selectivity of the wild-type EGF receptor are essentially identical to the results previously reported by Songyang et al.(16) . Thus, the oriented peptide library method appears to be a consistent and reliable approach to characterize differences in protein kinase specificity. However, primary sequence selectivity probably represents only one component of substrate specificity, as none of the known sites of autophosphorylation in the EGF receptor (28, 29, 30) conform to the amino acid preferences identified in the oriented peptide library experiment. Hence, substrate localization due to intramolecular interactions or SH2-dependent protein binding probably plays a major role in substrate selection by receptor tyrosine kinases.
The observation that there are differences in the phosphorylation of endogenous proteins in response to EGF in cells expressing wild-type or M857T EGF receptors suggests that the changes in substrate specificity identified using the in vitro oriented peptide library approach reflect significant changes in substrate specificity in vivo. Together with the demonstration that the wild-type and M857T EGF receptors possess similar intrinsic kinase activities, these findings support the hypothesis that MEN 2B-induced oncogenesis is the result of a change in kinase substrate specificity rather than an increase in total enzyme activity.
There are several mechanisms through which alterations in substrate specificity could enhance the transforming potential of the EGF receptor in vivo. First, changes in substrate specificity could lead to the phosphorylation of novel downstream targets resulting in the activation of new signaling pathways. Alternatively, changes in substrate specificity could affect the normal pattern of EGF receptor regulation leading to an alteration in receptor down-regulation or desensitization. Our findings with respect to the M857T EGF receptor support the latter hypothesis.
Although the M857T EGF receptor was more transforming than the wild-type EGF receptor and exhibited differences in substrate specificity, many of the downstream signaling pathways activated in response to EGF were unchanged in cells expressing the M857T EGF receptor. This included activation of MAP kinase and PI 3-kinase as well as EGF-stimulated phosphorylation of SHC, focal adhesion kinase, Ras-GAP and its associated p62 and p190 proteins. Thus the enhanced transforming capacity of the M857T EGF receptor apparently is not due to changes in the acute activation of these common signaling pathways.
By contrast, regulation of receptor function appeared to be
significantly altered by the Met to Thr mutation. Treatment with EGF
for up to 60 min led to the loss of cell surface I-EGF
binding activity in cells expressing both forms of the receptors.
However, the loss of cell surface receptors was more rapid and more
extensive in cells expressing the M857T EGF receptor than in cells
expressing the wild-type EGF receptor. The enhanced clearance of M857T
receptors from the cell surface was not due to an increased rate of
receptor internalization (Fig. 7). As down-regulation of cell
surface receptors at short times represents a balance between the rate
of receptor internalization and the rate of receptor recycling back to
the surface, these findings suggest that there is less recycling of the
M857T EGF receptor than of the wild-type receptor. Thus, receptor
trafficking appears to have been affected by the MEN 2B mutation. EGF
receptor kinase activity has been shown to be important in receptor
trafficking. Kinase-negative EGF receptors undergo ligand-induced
internalization but do not become down-regulated because the receptors
are recycled back to the cell surface rather than targeted to the
lysosomes for degradation(31, 32) . It is possible
that the change in substrate specificity observed in the M857T EGF
receptor leads to alterations in the phosphorylation of a protein
involved in sorting such that more M857T EGF receptors are targeted to
the lysosomes and fewer are shunted into the recycling pathway.
In
addition to differences in short-term down-regulation, WT9 and Mut6
cells also exhibited alterations in their long-term responsiveness to
EGF. When incubated with EGF for 24 h to down-regulate the receptors,
Mut6 cells required a 5-fold higher concentration of growth factor to
ablate their responsiveness to EGF than did WT9 cells. That this
difference in regulation can lead to changes in the growth properties
of cells is apparent from the soft agar growth curves shown in Fig. 2. In that experiment, the response of WT9 cells peaked at
0.7 nM EGF, a concentration at which Mut6 cells showed
approximately half-maximal response. By 2 nM EGF, Mut6 cells
showed maximal response to EGF but the response of WT9 cells was
beginning to decline. Only at a concentration of 20 nM EGF did
the response decline in Mut6 cells. Since the affinity of EGF was the
same in both cell lines (K = 0.6
nM), it is clear that a much larger proportion of the EGF
receptors could be occupied and activated in Mut6 cells prior to the
engagement of the down-regulatory response. The activation of more
receptors and the consequent generation of a stronger response could be
one mechanism that contributes to the enhanced growth in soft agar of
the cells expressing the M857T EGF receptor.
Analysis of the time course of long-term down-regulation of wild-type and M857T EGF receptors suggested that although EGF receptor levels initially declined in both cell lines, recovery of receptor expression was more rapid in Mut6. The process through which this recovery occurs is at present unknown. Interestingly, tyrosine phosphorylation of the wild-type EGF receptor was readily detectable throughout the 24-h incubation with EGF whereas phosphorylation of the M857T EGF receptor was negligible over this time course. The increased dephosphorylation of the M857T EGF receptor could be linked to the more rapid recovery of receptor levels and may be the result of elevated phosphotyrosine phosphatase activity. As phosphotyrosine phosphatases have been shown to provide a positive growth signal in many systems(33, 34, 35) , an increase in this activity could contribute to the enhanced cellular proliferation seen in the Mut6 cells.
In these studies we have examined the consequences of putting the MEN 2B mutation into the EGF receptor. Our data indicate that there was little change in a variety of short-term responses to EGF. However, in assays examining the response of cells to long-term treatment with EGF, we observed marked differences in the behavior of the wild-type and M857T EGF receptors. These results suggest that the enhanced oncogenicity associated with the MEN 2B mutation correlates with changes in substrate specificity and may be due to alterations in the long-term response to growth factor rather than changes in the acute response to EGF. We have identified significant alterations in the down-regulation of the M857T EGF receptor. These changes in the long-term regulation of EGF receptor function may be responsible, at least in part, for the enhanced transforming potential of the M857T EGF receptor.