(Received for publication, May 16, 1994; and in revised form, October 20, 1994)
From the
Several receptor tyrosine kinases generate soluble ligand
binding domains either by differential splicing resulting in a
truncated RNA transcript, or by proteolytic cleavage. Although the
exact role in vivo of these soluble extracellular domains is
unclear, proteolysis may function to down-regulate the receptor, and
soluble extracellular domains (ECD) may compete with the intact
receptor binding to ligand. Axl is a member of a new class of receptor
tyrosine kinases characterized by an ECD resembling cell adhesion
molecules and unique sequences in the kinase domain. In addition, Axl
is transforming in both fibroblast and hematopoietic cells, and appears
to be involved in mesenchymal development. We now find that Axl is
post-translationally processed by cleavage in a 14 amino acid region
immediately NH-terminal to the transmembrane domain
resulting in a soluble ECD and a membrane bound kinase domain. The
sequence of this putative cleavage site shares no homology with
recognition sites of known proteases. Characterization of this
proteolytic processing shows that it does not require protein synthesis
or transport but is augmented by phorbol ester treatment. Since the
cleavage of Axl enhances turnover of the kinase on the cell surface, we
suggest that proteolytic processing down-regulates Axl kinase activity.
The ECD ()of RTKs plays a regulatory role in
controlling receptor kinase activity. Studies of several retrovirally
transduced oncogenes including v-kit, v-ros, and
v-erbB suggest that truncation of the ECD of RTKs renders
these proteins
oncogenic(1, 2, 3, 4) . Indeed, in vitro deletion of the ECD of a number of RTKs, including
the EGFR, the insulin receptor, and neu/erbB2 renders these
receptors transforming in the absence of
ligand(5, 6, 7) . Studies of the sevenless (sev) RTK of Drosophila provide
compelling evidence for the importance of the ECD in regulation of the
activity of RTKs. sev is required for the proper development
of the R7 photoreceptor of the compound eye(8) .
Loss-of-function sev alleles result in the absence of R7
photoreceptor cells. Conversely, mutations which activate the sev pathway, such as removal of the ECD of sev, result in
supernumerary R7 cells(9) . Flies that contain this truncated sev gene do not require the presence of the sev ligand encoded by the bride-of-sevenless gene (boss). These results are evidence that deletion of the
extracellular ligand binding domain relieves negative constraints that
regulate the kinase activity of the receptor at the cell surface.
In addition to these cis regulatory functions, processed and soluble ECDs of various receptors also appear to modulate receptor function. The most notable examples of biologically functional secreted receptors are the forms of the interleukin-6 and ciliary neurotrophic factor receptors which are capable of interacting with their respective ligands and gp130 to trigger transmembrane signaling(10, 11) . In RTKs, a secreted form of the EGFR ECD is produced as the result of alternative splicing of the message(12, 13, 14) . The function of this soluble ECD is unclear but may serve to regulate the activity of the full-length receptor(14, 15) . Additionally, soluble forms of CSF-1R, MET, and HER-2/neu have also been detected in the CM of cells expressing the receptor(16, 17, 18, 19) . In contrast to the soluble EGFR, the soluble CSF-1R and MET ECDs arise through proteolytic cleavage of the intact receptor by an, as yet, unknown protease. This proteolytic process is regulated in part by protein kinase C and is thought to down-regulate the receptor. The mechanism which generates the soluble neu/erbB2 protein is not clear but may also involve proteolytic processing(18) . Thus, proteolytic cleavage may represent a general mechanism to modulate RTK function.
In this study, we describe the processing of the Axl RTK
at the cell surface. This receptor was first identified in the DNA of
affected cells from leukemia patients employing
transfection/tumorigenicity assays and is capable of transforming
murine fibroblast and a myeloid cell line through
overexpression(20, 21, 22) . We now
demonstrate that cells expressing Axl produce a soluble protein
corresponding to the ECD of Axl (Axl) which arises in
vivo through post-translational proteolytic processing of the
full-length receptor and may play a role in Axl-dependent signaling.
The Axl-expressing cell lines AF6295,
TF14B12, and TF17B were generated as described previously(20) .
AF6295 cells express genomic sequences, whereas TF14B12 and TF17B are
independent clones derived from NIH-3T3 cells transfected with axl cDNAs. TF14B12 overexpresses axl cDNA1-4 which
encodes the full-length 894 amino acid Axl protein
(Axl, (20) ). TF17B overexpresses axl cDNA6-2 which encodes an Axl isoform lacking 9 amino acids
amino-terminal to the transmembrane domain (Axl
, (20) ).
For
analysis of secreted Axl proteins, cells were grown in 10-cm plates to
approximately 75% confluence and then placed in 7 ml of serum-free
media. After 24 h, CM from cell lines were removed and a portion either
concentrated on Centricon-10 ultrafilters as per the
manufacture's instructions or lyophilized and resuspended in 1
Laemmli gel sample loading buffer. Samples were boiled,
resolved by SDS-PAGE, and then transferred to Immobilon-P filters and
analyzed by Western blotting as above.
Figure 2:
[S]Methionine pulse
labeling of AF6295 cells reveal that Axl
is the fully
processed form of the holoenzyme. Cells (5
10
) were
grown in a 3.5-cm plate in the presence of
[
S]methionine followed by a 1-h chase with cold
methionine for the designated number of hours (0-4 h). Cells were
then lysed and Axl proteins immunoprecipitated as described under
``Experimental Procedures.'' The immunoprecipitate was
fractionated by SDS-PAGE on a 7.5% polyacrylamide gel. Thereafter, the
gel was fixed for 1 h in methanol/acetic acid/water (1:3:6). Signal was
amplified by the addition of EN
HANCE reagent for 30 min
followed by a 30-min incubation in cold water. The gel was then dried
and placed on XAR film for 3 days at -70 °C with an
intensifying screen. The positions of the fully processed Axl receptor
(Axl
) and the partially processed forms (Axl
and Axl
) are indicated with arrows. The
positions of molecular mass markers are indicated with dashes.
Figure 5:
Production of Axl is
augmented by phorbol esters. A, AF6295 cells were grown in 7
ml of complete medium in the presence or absence of 100 nM TPA. At 0, 1, 2, 4, 8, 12, and 24 h, 1-ml samples were removed,
filtered, and frozen. The equivalent of 70 µl of CM from each
sample was fractionated by SDS-PAGE in a 10% polyacrylamide gel,
transferred to Immobilon-P filters, and the Western blot incubated with
either the anti-IgL or anti-Axl-box antibody (indicated above each gel)
as described under ``Experimental Procedures.'' Dashes indicate the position of molecular mass markers of 97, 68, and 46
kDa. -, TPA not added; +, TPA added. B, AF6296
cells were grown in media containing
[
S]methionine for 4 h, washed with PBS, and then
placed in fresh medium to chase label. TPA (100 nM) was added
to fresh medium after 1 h (time 0). Axl proteins were
immunoprecipitated from cell lysates (B) and fractionated by
SDS-PAGE in a 7.5% polyacrylamide gel and processed as described under
``Experimental Procedures.'' Exposure time was 28 h at
-70 °C with an intensifying screen. -, TPA not added;
+, TPA added.
Figure 1:
An antibody directed against the
amino-terminal immunoglobulin domain of Axl recognizes Axl-specific
proteins in the lysates and conditioned media (CM) from Axl-expressing
cells. For analysis of CM, 4 ml of CM from AF6295 or NIH/3T3 cells were
concentrated to 1 ml. Five µg of affinity-purified anti-Igl were
added to 1 ml of concentrated CM and 1 ml of cell lysate.
Immunoprecipitations were performed as described under
``Experimental Procedures.'' Immune complexes were
resuspended in 50 µl of 4 SDS-sample buffer and boiled to
disrupt complexes. Half of each sample was fractionated by SDS-PAGE on
a 10% polyacrylamide gel and transferred to Immobilon-P filters.
Filters were hybridized with affinity-purified anti-Axl IgL (1
µg/ml). Immunoreactive bands were visualized by ECL kit reagents.
Axl-specific proteins are designated on the right of the
figure. The position of molecular mass markers is shown to the right. CM, conditioned media; LYS, cell
lysate. The dark band at the bottom of the figure corresponds
to heavy chain IgG.
Since AF6295 cells were transformed by the
human Axl genomic DNA, it is possible that Axl is
translated from a differentially spliced transcript encoding a
truncated Axl protein. However, we have not detected expression of axl transcripts encoding a soluble Axl ECD in AF6295 cells
making this mechanism unlikely. More importantly, using the anti-IgL
antibody, we also detect Axl
in the CM of TF14B12, an
NIH/3T3 derived cell line which overexpresses a full-length axl cDNA (Fig. 4B)(20) . However, CM from
untransfected NIH/3T3 cells shows no detectable Axl indicating that
Axl
arises through proteolytic processing of the
full-length receptor and not by alternative splicing of the RNA ( Fig. 1and Fig. 4B).
Figure 4:
Axl protein expression in various cell
lines. A, cell lysates were analyzed as in Fig. 1using
the anti-IgL antibody except one-fifth of the immunoprecipitate from
each lysate was separated by SDS-PAGE on a 10% polyacrylamide gel. B, for analysis of CM, 500 µl of medium from each cell
line was concentrated on a Centricon-30 ultrafilter, resuspended in a
final volume of 80 µl 1 SDS sample buffer, and one-tenth of
each sample analyzed by Western blot using the anti-IgL antibody as in Fig. 1.
Axl is not
restricted to expression in mouse fibroblasts. A similar Axl
protein is seen in the CM from T24, a human bladder carcinoma
cell line possessing an activated H-ras oncogene (Fig. 4B). A slightly smaller soluble protein is
detected in the CM from A549, a human lung carcinoma cell line. This
difference in size in the soluble ECD is due to differential
glycosylation of the protein between mouse and human cells as treatment
of CM from A549 and AF6295 cells with N-glycanase reduces the
apparent molecular mass of the two secreted proteins to identical sizes
(58 kDa) consistent with the predicted molecular mass of the
unprocessed Axl ECD (Fig. 3). Both T24 and A549 express
significant amounts of the Axl holoenzyme as assessed by Western
blotting (Fig. 4A).
Figure 3:
N-Glycanase treatment of
Axl. Samples of 24 h CM from AF6295 cells and A549 cells
were lyophilized and then digested overnight with N-glycanase
as described under ``Experimental Procedures.'' Half of each
sample was fractionated by SDS-PAGE on a 10% polyacrylamide gel,
transferred to Immobilon-P, and the Western blot incubated with
anti-IgL (1 µg/ml). -, samples without the glycanase; +,
samples with the glycanase. The secreted and glycosylated Axl proteins
are designated Axl
, whereas the deglycosylated Axl
proteins are designated Axl.
We reported previously the
isolation of two alternatively spliced forms of the Axl message. These
transcripts encode proteins that either contain or lack 9 amino acids
carboxyl-terminal to the fibronectin III domains in the ECD of Axl (Fig. 6)(20) . These two forms are designated
Axl and Axl
to refer to the presence
or absence, respectively, of these 9 amino acids. Although these two
forms of the receptor are variably expressed in a number of cell lines,
both are equivalent in their transforming potential(20) . Using
an affinity purified, polyclonal antibody directed against the 9 amino
acid stretch (hereafter referred to as the Axl-box), we analyzed the CM
from various Axl expressing cell lines. As shown in Fig. 5A,
Axl
in AF6295 is recognized by the anti-IgL and
anti-Axl-box antibodies. Similar results were obtained with Axl
from TF14B12, an NIH/3T3 cell line expressing the axl
cDNA(20) . In TF17B, an NIH/3T3
cell line expressing the axl
cDNA, we detect
a soluble Axl
protein in the CM immunoreactive only with
the anti-IgL antibody (data not shown). These results map the
proteolytic cleavage site to a 14-amino acid region between the Axl-box
and the transmembrane domain harboring the amino acid sequence
VKEPSTPAFSWPWW (between amino acids 438 and 451, Fig. 6).
Comparison of this region of Axl with known proteins does not reveal
homology to any known protease cleavage sites.
Figure 6: Schematic representation of the predicted Axl protein structure. The immunoglobulin (IgL) and fibronectin III (FNIII) domains are indicated with arrows. The amino acid sequence of Axl between the final FNIII domain and the transmembrane domain is shown to the right. The boxed 9 amino acids correspond to the differentially spliced Axl-box region(20) . The remaining 14 amino acids represent the putative protease cleavage site.
Figure 7:
The proteolytic production of Axl is independent of protein synthesis. 10-cm plates containing
equivalent numbers of AF6295 cells were grown in the presence (+)
or absence(-) of cycloheximide (10 µg/ml) for 30 min. Cells
were then washed with PBS and placed in serum-free medium ± TPA
and ± cycloheximide for 1 or 2 h. Equivalent amounts of CM (70
µl) from each sample were analyzed by SDS-PAGE in a 7.5%
polyacrylamide gel, transferred to Immobilon-P, and the Western blot
incubated with anti-IgL as described under ``Experimental
Procedures.'' The position of the secreted Axl extracellular
domain (Axl
) is indicated by an arrow.
Figure 8:
Proteolytic cleavage of Axl results in the
production of a COOH-terminal phosphorylated protein. AF6295 cells were
grown as in Fig. 1. Medium was removed and cells washed once
with PBS and then placed in serum-free medium ± TPA (100
nM) for 0, 1, 2, or 4 h. Cells were then lysed and equivalent
amounts of protein from each sample fractionated by SDS-PAGE in a 7.5%
polyacrylamide gel. Western blots of gels were probed with antibodies
directed against phosphotyrosine (-PTYR) (A) or
the carboxyl-tail of Axl (
-COOH) (B). The fully
and partially processed Axl proteins are designated Axl
and Axl
, respectively. The COOH-terminal Axl
protein is marked Axl
. The positions of molecular mass
markers are designated with dashes.
Figure 9:
The COOH-terminal Axl cleavage product,
Axl, partitions with the membrane fraction of cells.
AF6295 cells were grown for 1 h in the presence of TPA (100
nM), lysed, and fractionated into membrane (M) and
cytosol (C) fractions. Equivalent amounts of protein from each
sample, including total lysate (T), were fractionated by
SDS-PAGE on a 7.5% polyacrylamide gel and analyzed by Western blot
using antibodies directed against the carboxyl-tail of Axl (a-COOH) and anti-phosphotyrosine (a-PTYR). The
carboxyl-terminal cleavage product corresponding to the kinase domain
of Axl is designated Axl
. The positions of molecular mass
markers are designated with dashes.
Figure 10: Proteolytic cleavage of Axl occurs at the plasma membrane. AF6295 cells were placed on ice for 15 min. Growth medium was supplemented with 20 mM HEPES, pH 7.3, to prevent media from becoming basic during the course of the experiment. Medium was removed and cells were washed once with ice-cold PBS then placed in 7 ml of cold serum-free medium with (+) or without(-) TPA (100 nM) at 4 °C. CM from each sample was analyzed by Western blot using anti-IgL as described under ``Experimental Procedures.'' The secreted Axl ECD is designated with an arrow.
Axl is a novel RTK isolated from patients with myeloid
leukemic disorders(20, 21) . The structure of Axl,
with its ECD resembling cell adhesion molecules and the intracellular
domain bearing the unique consensus kinase sequence KW(I/L)A(I/L)E,
defines a new class of receptor tyrosine kinases which include
v-ryk, sky/tif/rse, eyk, and the neural
specific kinases tyro3/brt, and
tyro12(28, 29, 30, 31, 32, 33) .
We have demonstrated previously that Axl is transforming due to
overexpression as opposed to genetic alteration (20) and have
shown (foster) ligand-dependent transforming activity in the murine
interleukin-3-dependent 32D myeloid cell line(22) . During
mouse development, axl is expressed in the developing
mesenchyme and may be involved in organogenesis(34) . In the
adult, axl message is detected in a variety of tissues and at
especially high levels in the heart, skeletal muscle, and ovarian
follicles, as well as in myeloid precursors in the bone
marrow(27, 34) . In this study, we have examined the
biochemical regulation of the receptor at the cell surface. Using
antibodies directed against different epitopes in the ECD, we
demonstrate that a truncated form of the receptor, Axl,
is released into the CM of Axl expressing cells. Although Axl
may arise through two mechanisms, alternative splicing of the
message or proteolysis of the full-length receptor, our data support
the latter scenario. Axl
is present in cells transfected
with the genomic Axl as well as full-length axl cDNA clones
but not in untransfected NIH/3T3 cells. Thus, Axl
is
derived from the full-length Axl protein and not an alternative
transcript. Although alternatively spliced transcripts encode truncated
forms of the trkB and EGF receptor
kinases(3, 12, 13, 35, 36) ,
we have not, as yet, found evidence for axl transcripts
encoding a truncated protein.
Processing of Axl is regulated in part
by protein kinase C as demonstrated by the increase in cleavage of the
intact receptor upon phorbol ester treatment of Axl-expressing cells.
This proteolytic cleavage is independent of protein synthesis as
treatment of AF6295 cells with cycloheximide does not affect the
TPA-stimulated release of Axl. Moreover, TPA treatment
appeared to decrease the levels of the full-length receptor due to
cleavage of the holoenzyme and the subsequent release of soluble
Axl
.
Other RTKs have been reported to undergo similar
processing events as seen with Axl(16, 19) . Both MET
and CSF-1R are cleaved by an as yet unknown protease to release soluble
ligand binding domains. In addition, secreted isoforms of other RTKs,
such as EGFR, are produced as a result of alternative
splicing(13, 35) . In both cases, the presence of a
soluble ligand binding domain suggests that this truncated protein may
function in signal transduction as a possible inhibitor of the
membrane-bound receptor. Consistent with this possibility, Flickinger et al.(35) have shown that a secreted form of the
chicken EGFR is able to block the transforming growth
factor--dependent soft agar colony growth of chicken embryo
fibroblasts through binding of the growth factor. In addition, Basu et al.(15) have shown that a secreted human EGFR
protein is able to inhibit the activity of the full-length receptor
presumably through intermolecular interactions. In contrast to the
secreted chicken EGFR, the human form appears to inhibit the activity
of the intact membrane bound receptor through a mechanism other than
simple competition for ligand since excess EGF is not sufficient to
suppress the inhibitory effects of the truncated receptor(15) .
Although the possibility exists that Axl
may bind ligand,
preliminary data suggest that Axl
does not inhibit the
transforming ability of the intact Axl protein. AF6295 cells produce
large amounts of Axl
yet are still highly transformed
with high levels of phosphorylated Axl
and
Axl
. Furthermore, addition of a baculovirus-expressed
Axl ECD to the media of AF6295 cells does not appear to affect the
growth of cells or phosphorylation of intact Axl receptor. (
)These results suggest that Axl
is not a
dominant-negative inhibitor of the full-length receptor.
Using an
antibody directed against the intracellular portion of Axl, we detected
a membrane-bound, truncated kinase fragment, Axl. The
appearance of this fragment occurs with the same kinetics as the
release of Axl
into the CM of cells, indicating that
Axl
is derived from the cleavage of the full-length
protein. Although a carboxyl-terminal MET fragment was not detected
upon TPA-stimulated proteolysis of the full-length MET
receptor(19) , cleavage of the CSF-1R also generates a similar
carboxyl-terminal kinase fragment(16) . The exact role of
proteolytic cleavage in RTK function is unclear.
In some cases, removal of the ECD of RTKs has also been shown to activate the kinase domain. For example, removal of the ECD of the sevenless receptor, a protein involved in the development of the compound eye in Drosophila, results in supernumary photoreceptor cells, a phenotype characteristic of constitutive signaling through the sevenless pathway(9) . Furthermore, truncation of the EGFR ECD as a result of retroviral integration by avian leukosis virus or artificially by recombinant approaches constitutively activates the receptor(37, 7) .
In the case of Axl, Western blot
analysis of cell lysates from AF6295 cells treated with TPA revealed
tyrosine phosphorylation of the 55-kDa reminant COOH-terminal kinase
domain of Axl (Fig. 9). Since tyrosine phosphorylation of the
enzymatic portion of tyrosine kinases is a marker of enzymatic
activation, we suspect that the proteolytically generated Axl is at least a partially activated kinase. However, RTKs activated
by engineered ECD truncations are transforming in large part due to the
retention of the deregulated kinase on the cell surface. By contrast,
the considerably shortened half-life (<1 h) of the membrane bound,
truncated Axl kinase after TPA exposure suggests that proteolytic
cleavage results in receptor down-regulation. Further work is in
progress to confirm this possibility.
We have shown that Axl is proteolytically cleaved to release a soluble extracellular domain as well as a membrane-bound, truncated kinase domain. This finding adds to the growing list of RTKs that are naturally modulated by specific proteolysis. Although it is unclear whether this event is up-regulatory or down-regulatory, the specificity of the cleavage and its modulation by such agents as TPA suggest that this post-translational process is important in the functional regulation of RTKs such as CSF-1R, MET, and Axl and that proteases regulate these processes.