©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Transforming Receptor Tyrosine Kinase, Axl, Is Post-translationally Regulated by Proteolytic Cleavage (*)

(Received for publication, May 16, 1994; and in revised form, October 20, 1994)

John P. O'Bryan (1) (2)(§) Yih-Woei Fridell (2) Ray Koski (3) Brian Varnum (3) Edison T. Liu (1) (2)(¶)

From the  (1)Curriculum in Genetics, (2)Department of Medicine, Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599 and the (3)Amgen Corporation, Thousand Oaks, California 91320

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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(2)-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.


INTRODUCTION

The ECD (^1)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.


EXPERIMENTAL PROCEDURES

Materials

Tissue culture plasticware was purchased from Corning. Cell culture media, FBS, PBS, and antibiotics were purchased from the Lineberger Comprehensive Cancer Center Tissue Culture Facility. N-Glycanase was obtained from Genzyme. Immobilon-P membranes were purchased from Millipore. Amicon was the source of Centricon ultrafiltration devices. The Tissue-Tearor homogenizer was obtained from Fisher Scientific. Horseradish peroxidase conjugates of anti-rabbit IgG and anti-mouse IgG, ECL chemiluminescence reagent kits and Hyperfilm-ECL were purchased from Amersham Corp. Isotopically labeled compounds and EN^3HANCE were obtained fron DuPont NEN. X-Omat AR x-ray film was purchased from Eastman Kodak Co. Protein A-Sepharose, anti-phosphotyrosine monoclonal antibody (PT-66), and other reagents were purchased from Sigma.

Cell Culture and Immunoprecipitation

All cells were grown at 37 °C in the presence of 5% CO(2), 95% air. A549, a human lung carcinoma cell line, T24, a human bladder carcinoma cell line which expresses a mutated H-ras allele, NIH/3T3 cells, and axl-transfected NIH/3T3 cells were grown in Dulbecco's modified Eagle's medium containing high glucose (4500 mg/liter) and supplemented with 10% FBS. The human proerythroblastic leukemia cell line K562 was grown in RPMI 1640 media supplemented with 10% FBS. K562 cells were differentiated along the megakaryocytic pathway by the addition of TPA as described previously(23) .

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) ).

Production of Axl-specific Antibodies

A fragment of Axl corresponding to amino acids 33-136 was generated by polymerase chain reaction amplification. This fragment was cloned into the bacterial expression vector pCFM1656(24) . Recombinant protein was expressed as described previously(25) . Inclusion bodies were solubilized in 5 M urea, 50 mM Tris-HCl, pH 8.0, 10 mM DTT and protease inhibitors. The soluble protein was fractionated on a Q-Sepharose column. Axl protein was pooled, and the urea was removed by sequential dialysis. The dialyzed pool was reloaded on a Q-Sepharose column and step eluted with 0.25 M NaCl. The eluted protein was then fractionated by gel filtration in the presence of PBS. This material was injected into rabbits for the generation of polyclonal antibodies by standard methods.

Analysis of Cell Lysates and CM for Axl Proteins

Adherent cells were grown in 10-cm plates to approximately 75% confluence, washed with cold PBS, and lysed in 1 ml of cold Nonidet P-40 lysis buffer (1% Nonidet P-40, 20 mM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA, 10% glycerol, 0.1 mg/ml PMSF, 100 µM Na(3)VO(4)). All immunoprecipitations were performed at 4 °C. Samples were gently rotated for 30 min and centrifuged to pellet insoluble material. Affinity-purified anti-IgL (5 µg/ml lysate) was added to a fraction of the samples and then incubated with gentle rotation for 1 h. Protein A-Sepharose beads (50 µl/ml lysate; Sigma) were added to samples and incubation continued for an additional h. Immune complexes were recovered by centrifugation for 3 min followed by a brief wash with cold lysis buffer. Samples were resuspended in 1 times Laemmli gel sample loading buffer (10 mM Tris-HCl, pH 7.8, 3% SDS, 5% glycerol, 2% 2-mercaptoethanol, and 0.05% bromphenol blue), boiled, resolved by SDS-PAGE, transferred to Immobilon-P filters, and analyzed by Western blotting techniques as described previously(20) . Filters were blocked for 1 h in TBST (50 mM NaCl, 10 mM Tris-HCl, pH 7.5, 0.05% Tween-20) containing 1% bovine serum albumin. Filters were then incubated with antibodies as described previously (20) . After incubation with secondary antibody, filters were extensively washed with TBST, incubated with ECL reagents and then placed on Hyperfilm-ECL.

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 times 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.

[S]Methionine Labeling of AF6295 Cells

AF6295 cells (5 times 10^5) were seeded in a 3.5-cm plate. On the following day, cells were washed once with PBS and placed in methionine free-Dulbecco's modified Eagle's medium, 10% FBS. After growth for 1 h at 37 °C, the medium was supplemented with 50 µCi L-[S]methionine and cells were grown for an additional 1 (Fig. 2) or 4 h (Fig. 5B). Cultures were then washed once in PBS, placed in unlabeled complete medium (time 0), and lysed in Nonidet P-40 lysis buffer at the designated time points. For phorbol ester treatment, TPA was added following the 4 h labeling and 1-h incubation in unlabeled medium (time 0). Cells were then lysed after the designated time points following TPA addition. Axl proteins were immunoprecipitated from half of the lysate with anti-IgL antibody as described above. Half of the immunoprecipitate was fractionated by SDS-PAGE with a 7.5% polyacrylamide gel. Gels were fixed for 1 h in methanol/acetic acid/water (1:3:6) and then placed in EN^3HANCE reagent for 30 min followed by a 30-min incubation in cold water. Gels were dried and exposed to Kodak XAR film for 3 days at -70 °C with an intensifying screen.


Figure 2: [S]Methionine pulse labeling of AF6295 cells reveal that Axl is the fully processed form of the holoenzyme. Cells (5 times 10^5) 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^3HANCE 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.



N-Glycanase Treatment of Secreted Axl Proteins

Samples of 24-h CM from AF6295 cells (50 µl from a total of 7 ml) and A549 cells (400 µl from a total of 7 ml) were lyophilized and then resuspended in 10 ml of a solution containing 0.4 M NaP(i), pH 8.6, 0.5% SDS, 53.4 mM 2-mercaptoethanol. Samples were boiled for 5 min followed by the addition of 5 µl of 7.5% Nonidet P-40, 13 µl of H(2)O, 1 µl of PMSF (10 mg/ml), and 1 unit of N-glycanase and overnight incubation at 37 °C. Reactions were stopped by the addition of 15 µl of 4 times Laemmli gel-loading buffer. Samples were boiled, fractionated by SDS-PAGE with a 10% polyacrylamide gel, transferred to Immobilon-P filters, and analyzed by Western blotting with anti-IgL as described above.

Cycloheximide Treatment of Axl-expressing Cells

10-cm plates containing equivalent numbers of AF6295 cells were grown in complete medium in the presence or absence of cycloheximide (10 µg/ml) for 30 min. Cells were then washed once with PBS and placed in serum-free medium in the presence or absence of both TPA and cycloheximide. Samples were analyzed by Western blot as described above.

Membrane Fractionation

Ten plates (10 cm) of AF6295 cells were grown to approximately 90% confluence. Cells were washed once with PBS and then placed in media supplemented with 100 nM TPA but lacking FBS and antibiotics. Cells were grown for an additional hour at 37 °C, washed once with cold PBS, and then scraped into PBS and transferred to a 50-ml conical tube. Plates were washed a final time with cold PBS, and this wash was combined with the resuspended cells. Cells were pelleted by gentle centrifugation, resuspended in 2 ml of ice-cold TSA (2 mM Tris, pH 8.0, 0.14 M NaCl), and stirred gently on ice for 1 h. Cells were homogenized with a Tissue Tearor two to three times on ice and brought to a final volume of 2.5 ml of TSA containing 0.25 M sucrose, 1 mM EDTA, 0.1 mg/ml PMSF, 25 µg/ml leupeptin, 100 µM Na(3)VO(4) and then centrifuged 10 min at 125,000 times g at 4 °C to remove nuclei. The supernatant was saved, and the pellet was resuspended in 2.5 ml of the TSA/sucrose solution and centrifuged again. The supernatants were combined and centrifuged at 125,000 times g for 2 h at 4 °C to pellet the membrane fraction. The supernatant was removed and saved. The pellet was resuspended in 1 ml of TSA containing 0.5% Triton X-100 and 0.5% sodium deoxycholate and mixed for 30 min at 4 °C to solubilize membranes. Equivalent amounts of total lysate, cytosol and membrane fractions were fractionated by SDS-PAGE on 7.5% polyacrylamide gels and analyzed by Western blot as described above.


RESULTS

Processing of the Axl Protein

In order to examine the processing of the intact Axl receptor, we analyzed cell lysates of a secondary nude mouse tumor cell line expressing the genomic axl (AF6295, 20). Cell lysates were immunoprecipitated with a polyclonal antibody directed against a bacterially expressed first immunoglobulin loop of Axl (see ``Experimental Procedures'') and then analyzed by Western blot. A 140-kDa protein corresponding to the full-length Axl receptor as well as several smaller immunoreactive species of 120 and 104 kDa were detected (Fig. 1). In cells pulse labeled with [S]methionine, the 104-kDa Axl immunoreactive protein disappears first followed by loss of the 120-kDa protein and finally by the disappearance of the 140-kDa band (Fig. 2). N-Glycanase treatment of Axl protein immunoprecitated with anti-IgL reduces the apparent molecular mass of these species to 104 kDa (data not shown). These results suggest that the smaller 120- and 104-kDa proteins, respectively, are partially glycosylated and unglycosylated precursors for the full-length protein which is consistent with the predicted molecular mass of the unprocessed Axl protein(20) . In addition, the disappearance of the S-labeled Axl proteins seen in Fig. 2suggests that the half-life of the receptor in AF6295 cells is on the order of 2 h.


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 times 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.



A Soluble ECD of the Axl Receptor Results from Proteolytic Cleavage of the Holoenzyme

As mentioned previously, a number of isoforms of RTKs exist, including that of a soluble ECD without the catalytic kinase domain. To test for the presence of a soluble form of Axl, we analyzed CM from AF6295 cells. Using anti-IgL antibodies, we detect an immunoreactive band of 80 kDa in CM (Fig. 1). Inclusion of the Axl immunoglobulin peptide antigen along with the primary antibody blocks immunoreactivity (data not shown). Furthermore, this 80-kDa soluble protein does not react with an antibody directed against the carboxyl-tail of the receptor. These results demonstrate that the soluble Axl-related protein, which we will hereafter refer to as Axl, corresponds to the ECD of the full-length receptor.

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 times 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.



Production of Axl Is Regulated by Protein Kinase C

Proteolytic cleavage of both the CSF-1R and MET receptors is augmented by protein kinase C(16, 19) . To test for the involvement of protein kinase C in the production of Axl, we treated both Axl genomic and cDNA transfected NIH/3T3 cell lines with TPA and analyzed the CM. Treatment of AF6295 cells with 100 nM TPA dramatically increases the level of Axl released into the culture medium as compared with untreated cells (Fig. 5A). This induction is seen as early as 15 min of treatment (data not shown) and is still evident after 12 h. By 24 h, however, treated and untreated cells produce equivalent amounts of Axl, suggesting that the effects of TPA are transient. Similar results are seen with an axl cDNA-transfected cell line, TF14B12 (data not shown). Furthermore, production of both forms of Axl (the Axl and the Axl forms) is induced by addition of TPA suggesting that there is no difference in the TPA-induced processing of either Axl forms (Fig. 5A). Analysis of the cell lysates of TPA-treated cells indicates there is a concomitant decrease in the amount of Axl as Axl is released into the media (data not shown). Extending our methionine labeling experiments in AF6295 cells, we found that treatment of [S]methionine-labeled cells with TPA results in a rapid decrease in the amount of Axl (Fig. 5B). Concomitant with the loss of Axl in cell lysates is the appearance of radiolabeled Axl in the CM of AF6295 cells (data not shown). These data support the premise that Axl is generated by the proteolytic processing of the full-length Axl receptor and that TPA regulates this protease activity.

Production of Axl Is Independent of Protein Synthesis

The finding that TPA dramatically induces the amount of Axl suggests that production of Axl involves a protein kinase C-regulated protease. To rule out the possibility that TPA is inducing transcription of Axl message in 3T3 cells, an effect seen in other cell lines of myeloid origin(26, 27) , we tested the effect of cycloheximide on production of Axl in Axl-transfected 3T3 cells. Cycloheximide treatment does not affect the basal production of Axl (Fig. 7). In addition, there does not appear to be any effect of cycloheximide on TPA-stimulated proteolysis of the intact receptor (data not shown). These data suggest that the effects of TPA on the production of Axl are independent of protein synthesis and further support the notion that Axl is derived from proteolytic cleavage and not translation of an alternatively spliced transcript. In addition, the level of Axl decreases over time with cycloheximide treatment in comparison with Axl (data not shown). This finding is in agreement with the previous pulse-chase experiments that point to Axl as a precursor for Axl.


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.



Cleavage of Full-length Axl Results in the Production of a Membrane-bound Phosphorylated Carboxyl-terminal Peptide

Since Axl derives from proteolytic cleavage of the full-length receptor at or near the transmembrane domain, a predicted 55-kDa carboxyl-terminal cleavage product containing the kinase domain should also be produced. Using an affinity-purified polyclonal antibody directed against the carboxyl terminus of Axl (see ``Experimental Procedures''), we are able to detect a 55-kDa tyrosine-phosphorylated protein, Axl, suggesting that Axl corresponds to the kinase domain of Axl (Fig. 8, A and B). Separation of the membrane and cytosol fractions of AF6295 cells treated for 1 h with TPA demonstrated that this carboxyl-terminal fragment of Axl partitioned with the membrane fraction of cells further supporting the model that cleavage of the holoenzyme occurs between the Axl-box region and the transmembrane spanning region (Fig. 9). Furthermore, secretion of the soluble Axl ECD occurs when these experiments were carried out under conditions which block protein trafficking (Fig. 10). These data suggest that cleavage of the receptor occurs on the surface of the plasma membrane as opposed to within intracellular vesicles. Whereas the half-life of the holoenzyme is in the range of 2 h, the half-life of the truncated kinase domain is considerably less in that no Axl is detectable after 2 h of TPA exposure.


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 (alpha-PTYR) (A) or the carboxyl-tail of Axl (alpha-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.




DISCUSSION

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-alpha-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. (^2)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.


FOOTNOTES

*
This work was supported by National Institutes of Health Grant NCI RO1-CA49240 (to E. T. L.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Supported by a Howard Hughes Medical Institute Predoctoral Fellowship. Present address: Dept. of Molecular and Developmental Biology, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, 600 University Ave., Toronto, Ontario, M5G 1X5 Canada.

Scholar of the Leukemia Society of America. To whom correspondence should be addressed: CB#7295 Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7295. Tel.: 919-966-1352; Fax: 919-966-3015; medetl{at}med.unc.edu.

(^1)
The abbreviations used are: ECD, extracellular domain; RTK, receptor tyrosine kinase; EGF(R), epidermal growth factor (receptor); CSF-1R, colony-stimulating factor 1 receptor; MET, hepatocyte growth factor receptor; TPA, 12-O-tetradecanoylphorbol-13-acetate; PMSF, phenylmethylsulfonyl fluoride; FBS, fetal bovine serum; PBS, phosphate-buffered saline; CM, conditioned medium; PAGE, polyacrylamide gel electrophoresis; anti-IgL, antibody against the first immunoglobulin-like domain of Axl.

(^2)
Y.-W. Fridell, et al., unpublished observations.


ACKNOWLEDGEMENTS

We wish to thank Edward W. Baptist for his technical assistance.


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