From the Lombardi Cancer Center, Georgetown
University, Washington, D.C. 20007, ¶ AMGEN, Thousand Oaks,
California 91320, and
Ciphergen Biosystems, 490 San Antonio
Road, Palo Alto, California 94306
Received for publication, November 27, 2000, and in revised form, February 6, 2001
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ABSTRACT |
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Pleiotrophin (PTN) is a secreted growth factor
that induces neurite outgrowth and is mitogenic for fibroblasts,
epithelial, and endothelial cells. During tumor growth PTN can serve as
an angiogenic factor and drive tumor invasion and metastasis. To identify a receptor for PTN, we panned a phage display human cDNA library against immobilized PTN protein as a bait. From this we isolated a phage insert that was homologous to an amino acid sequence stretch in the extracellular domain (ECD) of the orphan receptor tyrosine kinase anaplastic lymphoma kinase (ALK). In parallel with PTN,
ALK is highly expressed during perinatal development of the nervous
system and down-modulated in the adult. Here we show in cell-free
assays as well as in radioligand receptor binding studies in intact
cells that PTN binds to the ALK ECD with an apparent
Kd of 32 ± 9 pM. This receptor
binding is inhibited by an excess of PTN, by the ALK ECD, and by
anti-PTN and anti-ECD antibodies. PTN added to ALK-expressing cells
induces phosphorylation of both ALK and of the downstream effector
molecules IRS-1, Shc, phospholipase C- Polypeptide growth factors induce their effects by interacting
with cell surface receptors. Frequently these receptors are composed of
an extracellular ligand-binding domain
(ECD),1 a single
transmembrane region and may contain an intracellular kinase
domain. Upon binding, ligands induce protein kinase activity and signal
via intracellular adaptor or effector molecules that are recruited to
the intracellular domain and modulate phosphorylation of downstream
targets (1, 2). Here we identify a receptor for pleiotrophin (PTN), a
growth factor initially described a decade ago and shown to induce
mitogenesis in cultured cells of epithelial, endothelial, and
mesenchymal origin as well as neurite outgrowth in neuronal cells
(3-8). Animal studies demonstrated that PTN can serve as a
rate-limiting angiogenic factor during tumor growth, invasion, and
metastasis (8-12). Clinical studies showed elevated serum levels and
tumor expression of PTN in samples from patients with colon, stomach,
pancreatic, and breast cancer (5, 13). Furthermore, PTN has been
implicated in neonatal brain development as well as in
neurodegenerative disorders (reviewed in Ref. 14). Obviously,
understanding of PTN-mediated signal transduction as well as
identification of a receptor for PTN would enhance studies on the
biology and pathology of this growth factor family.
Our previous studies have shown that the activation of
mitogen-activated protein kinase and PI 3-kinase pathways is required for mitogenic activity of PTN, and we had found that the adaptor molecule Shc participated in signal transduction (15). Based on the
work of different laboratories in various cell types, it was
hypothesized that proteins of 170-220 kDa that are
tyrosine-phosphorylated in response to PTN could be part of the
receptor complex (15-17). More recently, several cell membrane-located
proteins were shown to bind PTN at low affinity and serve as potential
coreceptors or modulators of signal transduction (18-21), but none of
these molecules carried the hallmarks of a signal transducing receptor predicted from the earlier work.
To identify a receptor for PTN, we rationalized that panning of a phage
display cDNA library against immobilized PTN as a bait would allow
us to isolate phage containing a ligand binding fragment of the
receptor on their surface. Because of the high levels of expression of
PTN during the perinatal development of the nervous system, we
hypothesized that fetal brain would most likely also express a PTN
receptor. We therefore panned a human fetal brain cDNA phage
display library over several rounds against purified PTN that had been
tested for biological activity (15). From this we isolated a phage
insert homologous to an amino acid sequence stretch in the ECD of the
receptor tyrosine kinase anaplastic lymphoma kinase (ALK), a recently
described orphan receptor with an apparent molecular mass of 200-220
kDa (22, 23). Similar to PTN, ALK is highly expressed during perinatal
development of the nervous system and down-modulated in the adult (3,
22, 23).
The ALK tyrosine kinase domain was originally discovered because of its
oncogenic activity resulting from a t(2,5) translocation and fusion of
the ALK intracellular domain with nucleophosmin (24) (see Fig.
1a). More recently, the full-length ALK protooncogene was
cloned as a transmembrane receptor (22, 23) of an apparent molecular
mass of ~220 kDa. The closest homologue of ALK is leukocyte-tyrosine kinase (LTK) (25), a smaller (100 kDa) transmembrane protein that lacks
60% of the N terminus of the ECD of ALK. We now describe PTN as an
activating ligand for the ALK orphan tyrosine kinase and show in
receptor binding studies in intact cells as well as in cell-free
binding studies with PTN and the ALK ECD that ALK is a receptor for
PTN.
Phage Display Cloning--
An M13 phage display library of human
fetal brain cDNA (EasyMATCH Phage Display) was obtained from
CLONTECH. The human cDNA fragments are located
downstream of the phage gene III leader sequence to generate gene III
fusion proteins that are exposed on the phage surface. Phages
containing candidate PTN receptor cDNA fragments as inserts were
selected by repeated panning of the library with purified PTN (~1
µg/well) (6, 15) that had been immobilized in the wells of a 96-well
plate. Panning of selected clones against fibroblast growth
factor-2 was used as a negative control. Several rounds of panning
against purified, biologically active PTN resulted in the isolation of
two distinct phages that bound to PTN. One of the phage inserts was
homologous to a cDNA of unknown function that was identified during
screening of metastatic melanoma cells (GenBankTM D50525;
see Ref. 26) and contains only a very short open reading frame. The
other phage contained a cDNA insert that encoded for a peptide
sequence homologous to a region in the ECD of the orphan tyrosine
kinase receptor ALK (22, 23), and we report on this below.
Cell Lines, Transfections, and Plasmid Constructs--
Stable
transfection of SW-13 human adrenal carcinoma cells (6) and of 32D
murine interleukin-3-dependent myeloid cells (27, 28) with
an ALK expression vector (22) was performed by electroporation. Cells
were allowed to recover for 1 day (32D cells) or 2 days (SW-13 cells)
before selection of stable transfectants with 0.750 mg/ml of G-418 (9).
SW-13 cells have been used by our laboratory in a number of studies
with PTN (5, 6, 13). The 32D cells are a standard model for receptor
studies (28) and do not express ALK mRNA as assessed by RT-PCR (see
Table I). Furthermore, 32D cells grow in suspension culture and are
thus easily amenable to radioligand ligand binding studies.
Protein/Protein Interaction Studies on Nitrocellulose
Membranes--
PTN purified from the culture supernatant of cells
transfected with the human PTN cDNA (~5 ng of biologically active
protein in 5 µl) (15) or the ECD of ALK produced as an Fc fusion
protein in Chinese hamster ovary cells as described (22, 29) (~7.5 ng
in 10 µl), or fetal bovine serum as a negative control (2 µl) were
immobilized by spotting onto a nitrocellulose membrane. Nonspecific binding sites were blocked by incubation of the membranes with 5%
(w/v) skim milk in Tris-buffered saline containing 0.1% (v/v) Tween 20 (TBST). The membranes were then incubated for 2 h at room
temperature with PTN (in TBST), ALK ECD protein (2 µg/ml in TBST), or
TBST alone (negative control) as appropriate, washed in TBST, and
exposed overnight to rabbit antibodies to the ALK ECD (22) or mouse
monoclonal antibodies to PTN (4B7 (13)). Antibodies were at a 1:20
dilution in PBS, 0.1% Tween 20, 1% bovine serum albumin. After
washing, bound antibody was visualized using commercially available
reagents (see Refs. 13 and 15).
Protein/Protein Interaction Studies on Protein Arrays ("Chip
Assay")--
The analysis was performed with a SELDI (30) Protein
Biology System I (Ciphergen, Palo Alto, CA). The different
PTN-containing ligand preparations (1 µl of a 20 µg/ml solution)
(15) were placed on a normal phase protein array, which was then
washed, and 1 µl of Receptor Binding Studies in Intact Cells--
32D cells
(ALK-transfected or vector controls; 15 million in 2 ml of growth
medium with 10% fetal calf serum) were incubated with radioligand or
the respective additions for 1 h at 37 °C. Cells were then
pelleted, the supernatant was aspirated, and after two additional
washes in 10 ml of growth medium, bound radioactivity in the final cell
pellet was detected by scintillation counting. The radiolabeled PTN was
purified from supernatants of PTN-overexpressing SW-13 cells (clone 8;
see Ref. 5) that had been metabolically labeled overnight with 0.25 mCi
of [35S]cysteine in cysteine-free Dulbecco's modified
Eagle's medium with 2% fetal calf serum (further details in Refs. 5
and 15). The concentration of PTN protein in the ligand preparations
was measured by Silverstain and Western blot using commercially
available PTN (Sigma) as a standard and an affinity-purified Cell Growth, Immunoprecipitation, and
Immunoblotting--
For metabolic labeling experiments cells were
plated at ~15% confluence in T-162 flasks and starved in serum-free
medium for 2 days with one intermittent media change. Cell were then
incubated for 2 h in phosphate-free Iscove's modified
Eagle's medium (Biofluids) and subsequently incubated in 7 ml of
phosphate-free Iscove's modified Eagle's medium with 0.5 mCi of
[32P]orthophosphate (64014L; ICN Biomedical, Irvine, CA)
for 4 h and stimulated for the indicated times with PTN (10 ng/ml)
(15). Cell lysates were then prepared, and a total of 3 mg of cellular proteins were subjected to immunoprecipitation as described (15). For
uncoupled antibodies, Sepharose-bound protein G (Gammabind plus,
Amersham Pharmacia Biotech) was used to precipitate immunocomplexes. Antibodies were anti-phosphotyrosine (agarose-coupled 4G10, 30 µl;
Upstate Biotechnology Inc., Lake Placid NY), anti-ALK (a mixture of 1 µg/ml each of N-19, T-18, and C-19 from Santa Cruz Biotechnology, Santa Cruz, CA and p80 from Accurate Chemicals, Westburg, NY), anti-IRS-1 (3 µg of rabbit IgG, gift of Dr. L.M. Wang, NCI, National Institutes of Health). The resulting precipitates were analyzed by
SDS-polyacrylamide gel electrophoresis and autoradiography. Experimental procedures for immunoblots for phosphotyrosine, IRS-1 (anti-IRS-1, Transduction Laboratories, Lexington, KY), PLC- RT-PCR--
Total RNA was prepared from cell lines as described
(9). Random-primed cDNAs were generated using avian moloney
virus reverse transcriptase and specific fragments were
amplified using Taq polymerase (Life Sciences, Inc.,
St. Petersburg, FL) and primers from the region coding for the ECD of
human or murine ALK (human sense: 5'-CAA CGA GGC TGC AAG AGA GAT-3';
murine sense: 5'-CCA CAA CGA AGC TGG AAG AGA-3'; and antisense human
and murine 5'-GTC CCA TTC CAA CAA GTG AAG GA-3'). To amplify the ALK
DNA fragment, the sample was initially heated at 94 °C for 2 min
followed by 30 cycles consisting of heating at 94 °C for 30 s,
cooling at 56 °C for 45 s, and extension at 72 °C for
45 s. After separation in agarose gels, the PCR products were
blotted onto nitrocellulose, and specific products were visualized by
hybridization with nested, radiolabeled oligonucleotides (5'-ACT CCA
GGG AAG CAT GGT TGG-3' or 5'-GGA TGT TCC TTC ACT GCA GTT C-3').
Hybridization was overnight at 42 °C in 5× SSC, 5× Denhardt's,
0.5% SDS, 0.1 mg/ml denatured salmon sperm DNA. After washes at
42 °C twice in 2× SSC, 0.1% SDS and once in 0.1× SSC, 0.1% SDS
membranes were exposed to Hyperfilm MP (Amersham Pharmacia Biotech).
Data Analysis--
The Prism-Graphpad software was used for data
analysis by chi square, linear, or nonlinear regression analysis. For
saturation binding studies the equation B = Bmax × L/(L + Kd) + Identification of ALK as a Candidate Receptor for PTN--
To
identify a receptor for PTN, we used immobilized human PTN protein as a
bait to screen a phage display human fetal brain cDNA library for
phage expressing a ligand binding fragment of the putative receptor on
its surface. Fibroblast growth factor-2 was used as a negative control.
Several rounds of panning against purified, biologically active PTN
resulted in the isolation of a phage cDNA insert that coded for a
peptide sequence homologous to a region in the ECD of the orphan
tyrosine kinase receptor ALK (amino acids 396-406 in
GenBankTM accession number U66559; Ref. 22). This putative
binding site for PTN is contained within a signature sequence patterns typical of the ECDs of various transmembrane proteins (MAM
domains) (Fig. 1a). The
closest homologue of the ~220-kDa ALK is LTK (25), a 100-kDa
transmembrane protein with a short ECD that lacks the N-terminal 60%
of the ALK ECD and thus does not include the putative PTN binding
domain. In contrast, the Drosophila ALK protein contains this region (including the MAM domains) and shows an overall 42% similarity with the human homologue.
PTN Binding to ALK in Cell-free Assays--
Initially, we
investigated the interaction between PTN and a recombinant ALK ECD
protein that was generated as a fusion protein with the Fc region of
immunoglobulin G (29). In an experimental design that mimics the phage
display library panning, we immobilized PTN on nitrocellulose
membranes, and binding of the ALK ECD protein was tested.
Immunodetection revealed that immobilized PTN binds the ALK ECD (Fig.
1b).
Next, we used the converse approach and immobilized the recombinant ALK
ECD protein (or fetal calf serum) on nitrocellulose membranes and
incubated that with various concentrations of PTN present in a
partially purified preparation. Bound PTN was detected using a
monoclonal antibody (13). PTN showed specific and saturable binding to
the immobilized ALK ECD protein (Fig. 1c), whereas only
background signals were detected in the negative control (Fig.
1c, inset).
Obviously, the immunoblots will show whether or not PTN binds to the
ALK ECD, but they will not reveal as to whether other unidentified
proteins present in the PTN preparation also bind. To address this
question, we used protein chip technology coupled to mass spectrometry
(30) (Fig. 1d). The ALK ECD was immobilized on a protein
chip and incubated with a partially purified PTN preparation. An excess
of a control receptor protein (transforming growth factor- Receptor Binding in Intact Cells--
To assess receptor binding
of PTN in intact cells, we used 32D murine myeloid cells. These cells
are dependent on interleukin-3 for their growth in suspension culture
and have been used extensively for the study of tyrosine kinase
receptors (e.g. Refs. 27, 28, and 31). The 32D cells do not
express express ALK mRNA as assessed by RT-PCR (see below), and we
hence stably expressed the ALK cDNA to compare radioligand binding
in 32D/control and 32D/ALK-transfected cells. PTN radioligand was
purified from supernatants of PTN-overexpressing cells that were
metabolically labeled with [35S]cysteine (5). As expected
for a protein, the PTN radioligand showed background binding to
32D/control cells that increased linearily with increasing radioligand
concentrations and was not saturated within the concentration range
used (Fig. 2a, open
symbols, 32D/ctrl). This binding to the 32D/control
cells was also not competed by excess cold PTN or by anti-PTN
antibodies (not shown) and is hence considered nonspecific binding. In
contrast, ALK-expressing 32D cells showed significantly increased
binding of the PTN radioligand (Fig. 2a, filled
symbols, 32D/ALK) in comparison with the 32D/control cells. The ALK-dependent binding was satured within the
concentration range of PTN used and an equilibrium dissociation
constant (Kd) for PTN of 32 ± 9 pM
(0.5 ng/ml) was calculated from nonlinear regression analysis of these
data. At PTN concentrations equivalent to this high affinity
Kd, the nonspecific binding to the 32D/control cells
was ~25% of the binding to 32D/ALK cells (Fig. 2a; 0.5 ng/ml of PTN). In addition to the evaluation by nonlinear regression,
the binding data were also subjected to the more traditional Scatchard
analysis (Fig. 2b). For this, nonspecific binding of the PTN
radioligand to 32D/control cells was subtracted from the total binding
to 32D/ALK cells to obtain the amount of PTN bound to ALK at different
concentrations of PTN. These data were plotted against the respective
ratio of bound/free PTN (Fig. 2b). A linear regression
analysis of the Scatchard plot showed that all of the data points were
within the 95% confidence interval (dotted lines in Fig.
2b) and resulted in a Kd value (36 ± 8 pM) indistinguishable from the value derived by
nonlinear regression analysis of the direct binding data (see above).
This evaluation of PTN radioligand binding using isogenic cells
(32D/ALK and 32D/control cells) demonstrates that ALK can serve as a
high affinity receptor for PTN in intact cells.
In addition to the direct radioligand binding studies, we used distinct
competitors to provide independent evidence that the high affinity PTN
binding to 32D/ALK cells is due to PTN binding to the ALK receptor
expressed in these cells. First, an excess of added cold PTN as well as
anti-PTN antibodies was able to compete for radioligand binding (Fig.
2c), whereas fibroblast growth factor-2 did not compete (not
shown). This supports ligand specificity of the receptor binding.
Second, the comparison between 32D/ALK and 32D/control cells already
showed that high affinity binding of PTN is only observed after the
expression of ALK (see above). In support of this receptor specificity,
added ALK ECD protein was also able to inhibit the PTN binding (Fig.
2c). This finding further corroborates the results of the
in vitro protein/protein binding studies that had suggested
a high affinity interaction between the ALK ECD protein and the PTN
ligand (Fig. 1, b-d). Third, from the phage display screen
we had derived a putative ligand binding domain in the ALK ECD (Fig.
1a, diamond). We raised antibodies against a
fusion protein containing this domain (anti-ECD; see "Materials and
Methods") and found that these IgGs inhibit high affinity binding of
PTN to the 32D/ALK cells (Fig. 2c). Unrelated IgGs did not
compete for binding (not shown). This suggests that the putative
binding domain in the ALK ECD participates in the PTN receptor binding.
We conclude from this series of experiments that PTN specifically binds
to the ALK orphan receptor as a high affinity ligand at least in part
via the putative ligand binding domain described above. Of note,
biologically effective concentrations of PTN are within the range of
the Kd values derived from these receptor binding
studies supporting a role of ALK for PTN-induced effects (see below and
Refs. 5 and 6).
PTN-induced Signal Transduction through ALK--
To assess how ALK
participates in PTN signal transduction and affects the growth response
to PTN, we used SW-13 cells. These cells form colonies in soft agar in
response to exogenously added PTN protein (5) and were used as
indicator cells to purify and N-terminally sequence biologically active
PTN from the supernatants of human breast cancer cells (6).
Furthermore, after expression of PTN SW-13 cells become clonogenic
in vitro (6, 9) and tumorigenic in mice (6). From these
previous findings we reasoned that SW-13 cells contain the complete
machinery required for PTN effects and could thus be used in signal
transduction as well as in growth studies of a candidate PTN receptor.
Expression studies showed that SW-13 cells express only low levels of
endogenous ALK mRNA that is detectable by RT-PCR (Table
I) and below detection by Northern
analysis (not shown). We thus generated ALK-overexpressing SW-13 cells
(SW-13/ALK cells) for further analysis of signal transduction pathways
(Fig. 3) as well as comparative growth
response to PTN (Fig. 4). Immunoblot
analysis showed that the endogenous ALK is below detection in control
SW-13 cells and readily found in cell extracts from SW-13/ALK cells
(Fig. 4, inset).
To assess whether the endogenous ALK is utilized for the signal
transduction by PTN, SW-13 cells were stimulated with PTN, and cell
lysates were subjected to immunoprecipitation with anti-ALK antibodies
and subsequent Western blotting with antiphosphotyrosine antibodies.
This approach showed that ALK is indeed phosphorylated in response to
PTN (Fig. 3a, arrow), although the endogenous
receptor level in SW-13 cells is low (see above), and the resulting
phosphorylation signal hence is only small. Overexpression of ALK
enhances protein phosphorylation of SW-13 cells in response to PTN
(Fig. 3, b-d), and we utilized the SW-13/ALK cells for a
more detailled analysis of signal transduction.
In one experimental approach, tyrosine-phosphorylated proteins were
immunoprecipitated from PTN-stimulated SW-13/ALK cell extracts and
further analyzed by immunoblotting (Fig. 3, b and c). As an alternative approach, the SW-13/ALK cells as well
as the respective control cells were metabolically labeled with
[32P]orthophosphate, and protein phosphorylation in
response to PTN stimulation was analyzed after immunoprecipitation with
different antibodies (
To evaluate the specificity of the ligand/receptor interaction in the
SW-13/ALK cells, we included an
Immunoprecipitation of SW-13/ALK cell extracts with antibodies directed
against ALK revealed that a number of phosphoproteins are associated
with the receptor after PTN stimulation of the [32P]orthophosphate-labeled cells (Fig. 3d;
Effect of ALK Overexpression on the Growth of Cells--
As
described in the previous section, PTN induces ALK phosphorylation
in SW-13 cells, although the relatively low levels of ALK protein
expression in SW-13 cells is reflected in only a very small induction
of overall protein phosphorylation in response to PTN. This PTN-induced
protein phosphorylation is strongly enhanced in the SW-13/ALK cells
(Fig. 3). In parallel with this increase in phosphorylation,
PTN-induced soft agar growth of SW-13/ALK versus control
cells is also enhanced significantly (an 18-20-fold stimulation
versus a 3-6-fold stimulation, respectively). In addition, the sensitivity of the SW-13/ALK cells is shifted to ~3-fold lower PTN concentrations (Fig. 4). We conclude from this that overexpression of ALK enhances PTN-induced growth stimulation.
Expression of ALK in Different Cell Lines and Their Response to
PTN--
The ALK protooncogene was described as an orphan receptor
that is highly expressed in the developing brain and down-regulated in
the adult (22, 23). This tissue expression pattern of ALK coincides
with the expression pattern of PTN (14), supporting the notion that PTN
and ALK could function as a ligand/receptor pair. In cultured cells of
different lineage we found ALK mRNA expressed in 7 of 10 cell
lines. The expression of ALK (and the lack thereof) correlated
significantly with the growth effect of PTN on the same cells (Table I;
p = 0.0016, chi square test). These data lend further
support to the notion of ALK as a receptor for PTN.
A number of laboratories have been studying the signal
transduction of the PTN growth factor family (reviewed in Ref. 14) and
have sought after a receptor for this growth factor. Our current studies provide evidence that PTN is a ligand for the orphan receptor ALK (anaplastic lymphoma kinase). The receptor binding and signal transduction studies reported here were initiated after a phage display
screen of a human fetal brain cDNA library with the PTN protein as
the bait. This screen resulted in the isolation of a phage containing
an insert homologous to a small fragment in the ECD of the
protooncogene ALK. The oncogenic ALK kinase was originally discovered
in a chromosomal (2, 5) translocation associated with anaplastic large
cell lymphomas. In this translocation the 3'-half of the ALK gene
(i.e. the intracellular portion of ALK with the kinase
domain) is fused to the 5'-portion of the nucleophosmin (NPM) gene
(Fig. 1a and Ref. 24). This fusion generates a
constitutively active ALK kinase because of the dimerization of NPM-ALK
via the NPM region (32, 33). Several more recent studies have suggested
that genes other than NPM can be fused to the ALK kinase and may serve
as dimerization and activation domains (34, 35). The activated ALK
kinase can act as an oncogene in different cell systems and induce
malignant transformation of fibroblasts (32, 36, 37) as well as Ba/F3
murine pro-B lymphoid cells (36). In addition, retroviral transduction
of murine bone marrow with an expression vector for NPM-ALK resulted in
B-cell lymphoma in animals transplanted with the transduced bone marrow
(38). More recently, the ALK kinase sequence from the NPM-ALK oncogene
was used by two different laboratories to clone the full-length ALK
receptor (22, 23). This work showed that this receptor contains a
transmembrane region and a large ECD that comprises over 60% of the
protein (Fig. 1a).
A number of independent studies showed that PTN is expressed in a
tightly regulated manner during perinatal organ development and in
selective populations of neurons and glia in the adult (reviewed in
Ref.14). Northern blot analysis of different tissues showed that the
ALK mRNA is mostly expressed in the brain and spinal cord. In
situ hybridization studies revealed that ALK expression in mice
initiates late during embryonic development (around embryonic day 11),
peaks during the neonatal period, but persists into adulthood in a few
select portions of the nervous system such as the thalamus, mid-brain,
and ganglionic cells of the gut. Immunoblotting with anti-ALK
antibodies corroborated the tissue expression and developmental regulation of ALK at the protein level (22, 23). Interestingly, more
recent immunohistochemistry studies with human tissues showed that the
ALK protein is expressed in the normal central nervous system, in
particular in some neurons as well as glial cells and is also found in
endothelial cells in these tissues (39). The expression profiles of ALK
and PTN suggest that this growth factor/receptor pair may play an
important role in the normal development and function of the nervous
system. However, the expression of ALK in endothelial cells in tissues
(39) as well as in cell culture (Table I) suggests a potentially
broader role of PTN/ALK.
A comparison of ALK mRNA expression and PTN response of 10 different cultured cell lines indicates that cells that lack ALK expression also fail to show a growth response to PTN and vice versa
(Table I). Based on the lack of a PTN response of ALK-negative cells,
it is tempting to speculate that there are only few distinct ALK-related receptors for PTN. To find potential ALK homologues in
addition to the known LTK (22, 23), we used the ALK ECD protein
sequence for a search of currently available data bases. From this
search only sequences coding for ALK (in different vertebrate and
invertebrate species) and LTK were identified. This search was done in
different modes against the protein data bases as well as with the
broader "tblastn" algorithm that searches the nucleotide data bases
(nr, EST, htgs, and Drosophila) and reveals any DNA
sequences that code for homologous protein fragments. Interestingly,
the Drosophila ALK contains the putative ligand binding
region in the human ALK ECD and also shows the same signature sequence
(MAM domains; Fig. 1a) in the vicinity of this region. In
contrast to ALK, the human and murine LTK proteins only comprise short
ECDs and lack this putative receptor binding portion present in the ALK
ECD, and we speculate that LTK might act as a coreceptor for ALK. The
Drosophila genome contains at least one gene homologous to
the PTN growth factor family ("miple"; GenBankTM
accession number AF149800), suggesting that a PTN/ALK interaction is
also possible in invertebrates.
In our studies we have mostly focused on the role of PTN and ALK in
human cancers. We and others demonstrated that PTN can be a
rate-limiting factor for tumor growth, invasion, angiogenesis, and
metastasis (8-12, 40). ALK could be involved in the activity of PTN as
an angiogenic factor, because ALK was found expressed in endothelial
cells in culture (Table I) as well as by immunohistochemistry in the
endothelium of human tissues (39). Furthermore, ALK might be a target
for the stimulation of stromal and mesenchymal responses to
tumor-derived PTN because of its expression in fibroblasts as well as
glial cells (Table I and Ref. 22, 23, and 39). Finally, coexpression of
PTN and of ALK in cancer cell lines (e.g. Colo357 pancreatic
cancer, Hs578T breast cancer, and U87 glioblastoma; Refs. 14 and
40)2 indicates that PTN and ALK could form an autocrine
loop of growth stimulation (41) of the tumor cells.
The intracellular domain of ALK is highly homologous to the LTK kinase
and to the protooncogene ros as well as the insulin receptor/insulin-like growth factor-1 receptor kinases (22). Furthermore, earlier studies have demonstrated that IRS-1 associates with NPM-ALK (32) as well as with LTK (42-44) and appears to be
required for growth simulatory or transforming activity. Finally, our
finding that IRS-1 is one of the signal transduction molecules that is
phosphorylated in response to PTN (Fig. 3d) also assigns the ALK
receptor more to the insulin receptor family. Although the phenotypic
significance of IRS-1 for PTN/ALK-mediated signal transduction remains
to be tested, it is likely that the IRS-1 docking protein will play a
role in PTN growth or survival signals. This would add the PTN/ALK
receptor to the insulin/insulin-like growth factor-1/interleukin-4
group of receptors that utilize IRS-1 as a major docking protein (for a
recent review see Ref. 2).
In addition to IRS-1, the docking protein Shc was shown to be utilized
by LTK for signaling through the Ras pathway (44). We demonstrate here
that Shc is phosphorylated by PTN signaling through ALK (Fig. 3c), and
we showed earlier that Shc is phosphorylated in response to PTN (15).
In contrast to Shc, IRS-1 not only mediates Ras signaling initiated by
LTK activation via a chimeric EGF-R/LTK receptor but also cell survival
through PI 3-kinase (44). This finding coincides with our earlier
studies on the signal transduction of PTN through PI 3-kinase (15) as
well as the PTN/ALK-mediated phosphorylation of the p85 subunit of PI
3-kinase (Fig. 3c). Finally, we demonstrate phosphorylation of PLC- In conclusion, the identification of ALK as a receptor for PTN will be
the basis for studies into the physiological as well as pathological
functions attributed to this growth factor. PTN can induce neurite
outgrowth and is thought to play a role during neonatal brain
development and maintenance of neuronal function (Ref. 4; reviewed in
Ref. 14). Furthermore, dysregulation of PTN has been described in
neurodegenerative disease processes (48), and it is conceivable that
studies of ALK in conjunction with PTN will generate novel insights
into the mechanisms underlying these diseases. Beyond a role in the
nervous system, PTN plays a significant role for tumor growth,
invasion, angiogenesis, and metastasis in some of the most aggressive
human cancer types, e.g. melanoma and pancreatic cancer (10,
11, 40). Also, ALK is expressed in a significant portion of human
cancer cell lines, and it is tempting to speculate that blockade of ALK
could be of therapeutic benefit in cancers or other diseases in which PTN plays a pathological role.
, and phosphatidylinositol
3-kinase. Furthermore, the growth stimulatory effect of PTN on
different cell lines in culture coincides with the endogenous
expression of ALK mRNA, and the effect of PTN is enhanced by ALK
overexpression. From this we conclude that ALK is a receptor that
transduces PTN-mediated signals and propose that the PTN-ALK axis can
play a significant role during development and during disease processes.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-cyano-4-hydroxy cinnamic acid (2 mg/ml) in
50% (v/v) acetonitrile and 0.5% (w/v) trifluoroacetic acid was added
to the spot. The retained proteins were then subjected to mass
spectrometry. For the analysis of the interaction of PTN with ALK, 3 µl of a 240 µg/ml solution of either the ALK ECD (see above) or the
human transforming growth factor-
receptor II (Sigma) in PBS
were applied to a preactivated protein array, which was then incubated
overnight in a humidified chamber at 4 °C. The protein solution was
removed, 3 µl of 1 M ethanolamine (pH 8.2) were added to
each spot, and the array was incubated for an additional 30 min at room
temperature. PTN (20 µg/ml), either alone or in the presence of the
ALK ECD or transforming growth factor-
receptor II each at a molar
ratio of 1:1.7 relative to PTN, was dissolved in PBS containing 0.1% Triton X-100, and 3 µl of each solution were added to the appropriate spots. After incubation of the chips for 1 h at room temperature in a humidified chamber, each spot was washed three times with 5 µl
of PBS containing 0.1% Triton X-100, and the entire array was then
washed once with 10 ml of 25% (v/v) ethylene glycol and twice with 10 ml of PBS. The proteins were then analyzed by mass spectrometry.
-PTN
antibody (R&D) as described (13, 15). Rabbit antibodies against the receptor binding domain in the ALK ECD (
-ECD) were raised using a
glutathione S-transferase fusion protein with a fragment of the human ALK ECD (amino acids 368-447; GenBankTM
NP_004295) that contains the putative ligand binding domain (human amino acids 396-406) as an insert. From this antiserum, IgGs were purified using protein G affinity chromatography (Pierce).
(anti-PLC-
1, Upstate Biotechnology Inc.), and PI 3-kinase (Upstate Biotechnology Inc.) using unlabeled cells were as described earlier (15).
× L was applied. In this
equation, B is the amount of ligand bound,
Bmax is the receptor binding capacity,
L is the ligand concentration, Kd is the
equilibrium dissociation constant, and
is the slope of linearily
increasing, nonspecific binding.
RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
View larger version (26K):
[in a new window]
Fig. 1.
Cell-free ALK ECD/PTN binding studies.
a, organization of ALK (GenBankTM U66559). The
tentative ligand binding site identified by phage display (amino acids
391-401; diamond symbol) is contained in one of the domains
that are found in the ECD of a diverse family of transmembrane proteins
(Prosite data base PDOC 00604, MAM domain). The intracellular domain
(ICD) containing the tyrosine kinase (TK) and the
translocation site in the juxtamembrane region for fusion with
nucleophosmin (t(2,5) NPM-ALK; Ref. 24) are also depicted.
Consensus binding sites for insulin receptor substrate-1
(IRS-1), Shc, and PLC- are indicated by the
arrows. b, binding of the ALK ECD to PTN. The
purified PTN protein used as a bait for phage display was immobilized
on a nitrocellulose membrane, incubated without (
) or with (+) ECD-Fc
fusion protein, and bound ECD was visualized by immunodetection.
c, concentration-dependent binding of PTN to the
immobilized ALK ECD. The inset shows the experimental
setup as well as a representative immunoblot of the binding of the
highest concentration of PTN relative to control. The binding isotherm
from repeated measurements at different concentrations are shown. Data
represent the means ± S.E. of triplicate readings at each
concentration. The experiment was repeated twice. d, protein
chip analysis (SELDI (30)) of ligand binding to the ALK ECD. Mass
spectrometry analysis of the input ligand preparation in comparison to
proteins bound to the immobilized ALK ECD are shown. The
arrowheads indicate the peak corresponding to the PTN
protein. The ligand preparation was incubated with the transforming
growth factor-
RII protein as a control (middle panel) or
with the ALK ECD as a specific competitor (lower panel) and
then allowed to bind to the immobilized ALK ECD. Analysis of bound
proteins after SELDI shows that PTN binds to the immobilized ALK ECD
and that this binding is competed by preincubation of the ligand
solution with the ALK ECD.
RII) or
an equimolar concentration of the ALK ECD protein was included in the
binding reaction to assess specific and nonspecific binding. PTN (Fig.
1d, Input) specifically binds to the immobilized
ALK ECD and can be competed by added ALK ECD but not by the control
receptor protein. The immobilized ALK ECD protein also binds PTN in a
specific manner when the ligand is present at low abundance among a
complex mixture of proteins present in conditioned medium of
PTN-overexpressing cells (not shown). From independent experiments with
ligand preparations of different purity, the mass of the ligand bound
to the ALK ECD (15,882 ± 43 daltons; mean ± SD;
n = 4) was indistinguishable from the mass of purified
PTN (15,868 daltons; Fig. 1d, Input). We conclude from these immunodetection as well as the mass spectrometry studies that the ALK ECD specifically binds PTN.
View larger version (14K):
[in a new window]
Fig. 2.
ALK receptor binding studies of PTN in intact
cells. a, saturation binding of radiolabeled PTN to
32D/ALK (filled symbols) and 32D/control cells (open
symbols). Data were pooled from two independent experiments with
triplicate repeat measurements of data points. The curves
were obtained from nonlinear regression analysis for receptor binding
studies (see "Materials and Methods"). b, Scatchard
analysis of saturation binding of radiolabeled PTN. Binding of PTN to
32D/control cells was subtracted from the binding to 32D/ALK cells to
obtain the amount of PTN bound to ALK at different concentrations of
PTN (see panel a). This is plotted against the respective
ratio of bound/free PTN. The unit on the ordinate is
(pg/106 cells)/(ng/ml). The result from the linear
regression analysis (straight line) and the 95% confidence
intervals of the analysis (dotted lines) are shown.
c, competition for the binding of radiolabeled PTN (1 ng/ml)
to 32D/ALK-transfected cells by cold PTN (30X), ALK ECD protein (0.7 µg/ml), an affinity-purified anti-PTN antibody (2.5 µg/ml), or an
IgG raised against an ECD fragment containing the ligand-binding domain
(6 µg/ml; see "Materials and Methods").
Expression of ALK mRNA in different cell lines in comparison with
their growth in response to PTN
View larger version (66K):
[in a new window]
Fig. 3.
Signal transduction of PTN through ALK.
a, effect of PTN on ALK phosphorylation in SW-13 cells.
SW-13 cells were stimulated for 5 min with PTN (10 ng/ml). Cell lysates
were then prepared and subjected to immunoprecipitation with antibodies
to ALK (IP -ALK). The resulting precipitates
were analyzed by subsequent Western blot with an anti-phosphotyrosine
antibody (WB
-PY). The arrow
indicates the position of the ALK protein. b, effect of
addition of the ALK ECD-Fc fusion protein (0.7 µg/ml) or an
affinity-purified
-PTN antibody (2.5 µg/ml) on PTN-induced
tyrosine phosphorylation in SW-13/ALK cells. SW-13/ALK cells were
stimulated for 5 min with PTN that had been preincubated with
-PTN
or with the ALK ECD. An
-phosphotyrosine Western blot (WB
-PY) of immunoprecipitates with an
-PY antibody
(IP
-PY) is shown. c,
identification of phosphoproteins after PTN-stimulation of SW-13/ALK
cells. Cells were stimulated with PTN for different times, and extracts
were subjected to immunoprecipitation with an
-PY antibody and
subsequent Western blots with the antibodies indicated. PI
3-K refers to the p85 subunit of PI 3-kinase. Details are under
"Materials and Methods." d, SW-13 control or
SW-13/ALK-transfected cells were metabolically labeled with
[32P]orthophosphate and stimulated for the indicated
times with PTN (10 ng/ml). Cell lysates were then prepared and
subjected to immunoprecipitation with antibodies to phosphotyrosine
(
-PY), ALK, or IRS-1. The resulting
precipitates were analyzed by SDS-polyacrylamide gel electrophoresis
and autoradiography. Changes in phosphoproteins that were analyzed
further are indicated by arrows.
View larger version (29K):
[in a new window]
Fig. 4.
Effect of ALK overexpression on
PTN-stimulated growth. Dose-response relations for the effect of
PTN on colony formation in soft agar by SW-13/control and SW-13/ALK
cells. One representative of three independent experiments with
mean ± S.E. of triplicate dishes is shown. Inset,
Western blot analysis for ALK protein in lysates of ALK-transfected or
control SW-13 cells.
-PY,
-ALK, or
-IRS-1; Fig.
3d). Analysis of the time course of the PTN effect shows
that induction of phosphorylation is obvious at the earliest time
point, i.e. 1 min after addition of PTN (Fig. 3d,
right panels). The anti-phosphotyrosine antibody (15)
precipitated several distinct phosphoproteins from SW-13/ALK cells
after [32P]orthophosphate labeling (arrowheads
in Fig. 3d;
-PY), and the induction of protein
phosphorylation was maintained at all three time points tested (1, 2, and 10 min). Furthermore, detection of tyrosine-phosphorylated proteins
by
-PY immunprecipitation followed by
-PY immunoblot detected a
similar pattern of phosphoproteins after ligand stimulation (Fig.
3b, two leftmost lanes). In contrast with this
dramatic induction of protein phosphorylation in SW-13/ALK cells by
PTN, control SW-13 cells showed only very small increases in overall
protein phosphorylation (Fig. 3d, left
panel).2
-PTN antibody and an excess of the
ALK-ECD protein in the phosphorylation studies. As shown in Fig.
3b, the PTN-stimulated tyrosine phosphorylation of proteins
in SW-13/ALK cells was inhibited significantly by preincubation of the
ligand with the recombinant ALK-ECD protein or with the
-PTN
antibody. We conclude from this set of data that the ligand-induced
protein phosphorylation in intact cells occurs rapidly and is specific
to the PTN ligand as well as the ALK receptor.
-ALK). Thus, we analyzed signaling proteins for which a consensus
binding site was found on the intracellular domain of ALK (Fig.
1a); i.e. Shc, IRS-1, and PLC-
. Furthermore,
our previous studies had shown that PI 3-kinase was a target for
PTN-induced phosphorylation (15), and we included this in our analysis.
Immunoprecipitation of 32P-labeled proteins with an
-IRS-1 antibody showed that the adaptor molecule IRS-1 is
phosphorylated upon PTN stimulation of SW-13/ALK cells (Fig.
3d, rightmost panel). Furthermore,
immunoprecipitation with an
-PY antibody and subsequent Western
blots revealed that PLC-
, PI 3-kinase, and Shc are also
phosphorylated after PTN stimulation of SW-13/ALK cells (Fig.
3c). These findings in conjunction with our earlier studies
(15), suggest that PTN induces the phosphorylation of ALK and
subsequent signal propagation via the adaptor molecules IRS-1 and Shc
as well as the enzymes PLC-
, ERK, and PI 3-kinase.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
in response to PTN stimulation of SW-13/ALK cells (Fig. 3c). This
finding corroborates PLC-
binding to the activated NPM-ALK kinase in
Ba/F3 and Rat-1 cells (36). Furthermore, PLC-
activation was
suggested as a crucial step for mitogenic activity of the ALK and
potentially the LTK kinase (36, 45). Typically PLC-
is activated by
fibroblast growth factor, hepatocyte growth factor, or
platelet-derived growth factor in a variety of cell lines (46).
Although the sequence homology of the kinase domain and IRS-1 signaling
assigns the ALK/LTK kinases to the insulin receptor family, a
role of PLC-
suggests an overlap with other growth factor
receptors and would be in line with an effect of PTN on cell lines of
distinct germ layer origin (Table I; reviewed in Refs. 2 and 47).
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ACKNOWLEDGEMENTS |
---|
We thank S. D. Lyman (Immunex, Seattle, WA) for review and suggestions and A. Schulte and E. Bowden (Georgetown University) for discussion and help with the experiments.
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FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grant CA58185 (Special Program of Research Excellence) and by a United States Army Medical Research Materiel Command Breast Cancer Program grant (to A. W.) as well as funds from the Studienstiftung des Deutschen Volkes (to A. A.).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.
The nucleotide sequences reported in this paper have been submitted to the GenBankTM/EBI Data Bank with accession numbers U66559, M57399, AF236106, and AF149800.
§ Both authors contributed equally to this work.
** To whom Correspondence should be addressed: Lombardi Cancer Center, Research Bldg. E311, Georgetown University, 3970 Reservoir Rd. NW, Washington D.C. 20007. Tel.: 202-687-3672; Fax: 202-687-4821; E-mail: wellstea@georgetown.edu.
Published, JBC Papers in Press, February 8, 2001, DOI 10.1074/jbc.M010660200
2 A. Wellstein, unpublished data.
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ABBREVIATIONS |
---|
The abbreviations used are:
ECD, extracellular
domain;
ALK, anaplastic lymphoma kinase;
IRS, insulin receptor
substrate;
LTK, leukocyte-tyrosine kinase;
PI, phosphatidylinositol;
PLC-, phospholipase C-
;
PTN, pleiotrophin;
SELDI, surface-enhanced laser desorption/ionization;
PCR, polymerase
chain reaction;
RT, reverse transcriptase;
PBS, phosphate-buffered
saline;
NPM, nucleophosmin.
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