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• W2135492013 abstract "Article24 November 2005free access A promiscuous liaison between IL-15 receptor and Axl receptor tyrosine kinase in cell death control Vadim Budagian Vadim Budagian Research Center Borstel, Borstel, Germany Search for more papers by this author Elena Bulanova Elena Bulanova Research Center Borstel, Borstel, Germany Search for more papers by this author Zane Orinska Zane Orinska Research Center Borstel, Borstel, Germany Search for more papers by this author Lutz Thon Lutz Thon Institute of Immunology, University of Kiel, Kiel, Germany Search for more papers by this author Uwe Mamat Uwe Mamat Research Center Borstel, Borstel, Germany Search for more papers by this author Paola Bellosta Paola Bellosta Department of Medical Sciences, Novara, Italy Search for more papers by this author Claudio Basilico Claudio Basilico Department of Microbiology, School of Medicine, New York, NY, USA Search for more papers by this author Dieter Adam Dieter Adam Institute of Immunology, University of Kiel, Kiel, Germany Search for more papers by this author Ralf Paus Ralf Paus Department of Dermatology, University of Lübeck, Lübeck, Germany Search for more papers by this author Silvia Bulfone-Paus Corresponding Author Silvia Bulfone-Paus Research Center Borstel, Borstel, Germany Search for more papers by this author Vadim Budagian Vadim Budagian Research Center Borstel, Borstel, Germany Search for more papers by this author Elena Bulanova Elena Bulanova Research Center Borstel, Borstel, Germany Search for more papers by this author Zane Orinska Zane Orinska Research Center Borstel, Borstel, Germany Search for more papers by this author Lutz Thon Lutz Thon Institute of Immunology, University of Kiel, Kiel, Germany Search for more papers by this author Uwe Mamat Uwe Mamat Research Center Borstel, Borstel, Germany Search for more papers by this author Paola Bellosta Paola Bellosta Department of Medical Sciences, Novara, Italy Search for more papers by this author Claudio Basilico Claudio Basilico Department of Microbiology, School of Medicine, New York, NY, USA Search for more papers by this author Dieter Adam Dieter Adam Institute of Immunology, University of Kiel, Kiel, Germany Search for more papers by this author Ralf Paus Ralf Paus Department of Dermatology, University of Lübeck, Lübeck, Germany Search for more papers by this author Silvia Bulfone-Paus Corresponding Author Silvia Bulfone-Paus Research Center Borstel, Borstel, Germany Search for more papers by this author Author Information Vadim Budagian1, Elena Bulanova1, Zane Orinska1, Lutz Thon2, Uwe Mamat1, Paola Bellosta3, Claudio Basilico4, Dieter Adam2, Ralf Paus5 and Silvia Bulfone-Paus 1 1Research Center Borstel, Borstel, Germany 2Institute of Immunology, University of Kiel, Kiel, Germany 3Department of Medical Sciences, Novara, Italy 4Department of Microbiology, School of Medicine, New York, NY, USA 5Department of Dermatology, University of Lübeck, Lübeck, Germany *Corresponding author. Department of Immunology & Cell Biology, Research Center Borstel, Parkallee 22, 23845 Borstel, Germany. Tel.: +49 4537 188200; Fax: +49 4537 188403; E-mail: [email protected] The EMBO Journal (2005)24:4260-4270https://doi.org/10.1038/sj.emboj.7600874 Retraction(s) for this article A promiscuous liaison between IL-15 receptor and Axl receptor tyrosine kinase in cell death control02 February 2011 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Discrimination between cytokine receptor and receptor tyrosine kinase (RTK) signaling pathways is a central paradigm in signal transduction research. Here, we report a ‘promiscuous liaison’ between both receptors that enables interleukin (IL)-15 to transactivate the signaling pathway of a tyrosine kinase. IL-15 protects murine L929 fibroblasts from tumor necrosis factor α (TNFα)-induced cell death, but fails to rescue them upon targeted depletion of the RTK, Axl; however, Axl-overexpressing fibroblasts are TNFα-resistant. IL-15Rα and Axl colocalize on the cell membrane and co-immunoprecipitate even in the absence of IL-15, whereby the extracellular part of Axl proved to be essential for Axl/IL-15Rα interaction. Most strikingly, IL-15 treatment mimics stimulation by the Axl ligand, Gas6, resulting in a rapid tyrosine phosphorylation of both Axl and IL-15Rα, and activation of the phosphatidylinositol 3-kinase/Akt pathway. This is also seen in mouse embryonic fibroblasts from wild-type but not Axl−/− or IL-15Rα−/− mice. Thus, IL-15-induced protection from TNFα-mediated cell death involves a hitherto unknown IL-15 receptor complex, consisting of IL-15Rα and Axl RTK, and requires their reciprocal activation initiated by ligand-induced IL-15Rα. Introduction The cytokine interleukin (IL)-15 is a potent apoptosis inhibitor (Bulfone-Paus et al, 1997b, 1999) with many immunomodulatory activities that overlap with those of IL-2 (Bulfone-Paus et al, 1997a; Waldmann and Tagaya, 1999). IL-15 binds to a heterotrimeric receptor complex that shares the IL-2 receptor β (IL-2Rβ) and IL-2 receptor γ (IL-2Rγ/γc) chains, but has a unique IL-15Rα chain that binds IL-15 with high affinity even in the absence of the other two receptor subunits (Giri et al, 1995; Fehninger and Caligiuri, 2001). Receptor tyrosine kinases (RTKs) are cell-surface receptors that transduce specific signals from the extracellular environment, posses an intrinsic tyrosine kinase activity, and control growth, differentiation, and survival of cells (Bennasroune et al, 2004). The mammalian Axl RTK family includes Axl, Tyro3, and Mer (Graham et al, 1994; Mark et al, 1994), and is widely expressed (Lai et al, 1994; Lu et al, 1999; Healy et al, 2001; Lu and Lemke, 2001). Axl, Tyro3, and Mer display an extracellular region (two immunoglobulin-related domains linked to two fibronectin type III repeats) and an intracellular region that contains an intrinsic RTK domain. The only known bona fide ligand for the Axl RTK family members is Gas6, the product of growth-arrest-specific gene 6 (Nagata et al, 1996), a soluble member of the vitamin K-dependent protein family (Manfioletti et al, 1993). Gas6/Axl signaling modulates cell growth and inhibits apoptosis (Bellosta et al, 1997; Melaragno et al, 2004). Axl promotes survival of pulmonary endothelial cells (Healy et al, 2001) and neuronal cells (Allen et al, 1999), and protects murine fibroblasts and human endothelial cells from apoptosis induced by TNFα or other stimuli (Bellosta et al, 1997; Goruppi et al, 1997). Axl is overexpressed in a variety of tumor cells (Chen et al, 1999; Goruppi et al, 2001), and overexpression of Axl can transform fibroblasts even in the absence of ligand (O'Brian et al, 1991; Burchert et al, 1998). Among signaling molecules activated by Axl in various cell types are phosphatidylinositol 3-kinase (PI3K), Akt, Src, extracellular signal-regulated kinase (ERK), and nuclear factor kappaB (NF-κB) (Goruppi et al, 1997; Lee et al, 2002). For example, in NIH 3T3 mouse fibroblasts, antiapoptotic Gas6/Axl signaling induces NF-κB activity and increases protein level of Bcl-xL (Demarchi et al, 2001). IL-15 protects mouse L929 fibroblasts from TNFα-induced apoptosis through recruitment of TRAF2 to the IL-15Rα chain and NF-κB activation (Bulfone-Paus et al, 1999). In human Raji cells, IL-15 stimulation reduces ceramide-induced apoptosis, accompanied by the recruitment of Syk, a non-receptor protein-tyrosine kinase, to the IL-15Rα chain, which phosphorylates a single tyrosine in the cytoplasmic tail of IL-15Rα (Bulanova et al, 2001). L929 cells lack Syk, yet IL-15 induces IL-15Rα phosphorylation in these cells. Therefore, the goal of this study was to investigate whether the IL-15-mediated antiapoptotic signaling in these model cells involves the activity of other, yet unknown kinase(s). Here, we unravel an unexpected alliance between IL-15Rα and Axl RTK, which is indispensable for the IL-15-mediated survival of L929 cells upon TNFα stimulation. Our results point to a so far unprecedented cooperation in signal transduction between a cytokine receptor and an RTK, and demonstrate how direct interactions between heterologous receptor systems can regulate qualitatively distinct cellular responses to death-inducing and death-opposing stimuli. Results Axl RTK is essential for the IL-15-mediated protection of murine fibroblasts from TNFα cytotoxicity IL-15 protects mouse L929 fibroblasts from TNFα-mediated cell death via recruitment of TRAF2 to its high-affinity receptor chain, followed by NF-κB activation (Bulfone-Paus et al, 1999). To further dissect the antiapoptotic effects of IL-15, we have established an IL-15-dependent L929 subline that is resistant to TNFα-induced cell death, referred to hereafter as L929R (see Materials and methods). The L929R cells are indistinguishable from the parental cell line, both morphologically and with respect to the expression levels of TNFα receptors, IL-15Rα and IL-2Rγ (Supplementary Figure 1) and the absence of IL-2Rβ (data not shown). However, cDNA microarray revealed a significant upregulation of an RTK, Axl, in L929R cells compared to parental L929 cells (data not shown). High expression of Axl was confirmed by RT–PCR, Western blotting (WB), and flow cytometry analysis (FACS) (Supplementary Figure 1A and B). Given the high expression of Axl in L929R cells, we tested how IL-15 treatment affects Axl expression in L929 cells. In fact, IL-15 stimulation for 24 h induced a significant upregulation of Axl RTK mRNA and protein to a level comparable to that of L929R cells (Supplementary Figure 2A and B). In L929R cells, IL-15 failed to further elevate Axl expression. Gas6 did not modulate the expression of its own receptor (neither at the transcriptional nor at the protein level) within 24 h (Supplementary Figure 2A and B), 48, or 72 h (data not shown). Instead, Gas6 significantly upregulated IL-15Rα expression (Supplementary Figure 2A). As previously reported (Kumaki et al, 1996), IL-15 downregulated the expression of its own high-affinity α chain (Supplementary Figure 2A). The fact that Gas6/Axl signaling protects murine fibroblasts from apoptosis induced by serum withdrawal or treatment with TNFα (Bellosta et al, 1997) raised the possibility that the observed overexpression of Axl in L929R cells was the sole cause for the acquired TNFα resistance. In this case, targeted deletion of Axl was expected to restore TNFα sensitivity in L929R cells. Thus, L929R cells, as well as the parental cells, were transfected with small interfering RNA oligonucleotides (Axl siRNA) to disrupt Axl transcripts and were analyzed by RT–PCR and WB for the knockdown of Axl 48 h post-transfection. Transfection with Axl siRNA resulted in a significant decrease of Axl at the mRNA (Figure 1A) and protein (Figure 1B) levels in both cell lines. As expected, the targeted depletion of Axl indeed restored TNFα sensitivity in L292R cells, resulting in a dramatic increase in the number of apoptotic cells in the presence of TNFα, as assessed by propidium iodide (PI) and annexin V staining and FACS analysis. About 70–80% of Axl-targeted L929R cells died, whereas cells transfected with scrambled control siRNA oligonucleotides remained viable (Figure 1C). Moreover, IL-15 failed to prevent the TNFα-induced apoptosis in parental L929 cells deficient in Axl expression, but effectively rescued non-transfected fibroblasts (Figure 1C). The protective properties of Axl were corroborated by the fact that L929 cells stably overexpressing a vector coding for Axl were resistant to TNFα (Supplementary Figure 2C). Transfection of L929 cells with siRNA oligonucleotides targeting expression of IL-2Rγ had no influence on the IL-15-mediated protection of these cells from TNFα-induced apoptosis (data not shown). This indicates that IL-2Rγ is dispensable for the antiapoptotic function of IL-15 in L929 cells. Figure 1.Axl is required for resistance to TNFα cytotoxicity. L929 and L929R cells were transfected with Axl siRNA. (A) Axl mRNA expression was analyzed by RT–PCR (upper panel). β-Actin is used as an internal control (lower panel). (B) WB analysis of Axl protein expression (upper panel). IL-15Rα expression served as a control (lower panel). (C) Cells were treated for 18 h with TNFα (5 ng/ml), IL-15 (100 ng/ml), or both, and cell viability was assessed by PI and annexin V staining and FACS analysis. (D) L929, L929R, and Axl-overexpressing L929 cells were treated or not with TNFα (100 ng/ml) alone or together with 20 mM zVAD-fmk and ceramide generation was determined. All ceramide determinations were performed in duplicate. Download figure Download PowerPoint Since TNFα induces rapid generation of ceramide in L929 cells, which induces apoptosis in these fibroblasts (Strelow et al, 2000), we asked whether high Axl expression interferes with ceramide generation. Parental, TNFα-resistant, and Axl-overexpressing L929 cells were treated with TNFα for 4 h or were left untreated. In parallel, cells were cultured in the presence of TNFα in combination with the broad-spectrum caspase inhibitor zVAD-fmk, because such treatment induced a more pronounced increase in intracellular ceramide than TNFα alone (Thon et al, 2005). TNFα induced a rapid accumulation of ceramide in L929 cells, whereas ceramide generation was clearly impaired in L929R fibroblasts (Figure 1D). Importantly, transfection with Axl construct also reduced ceramide generation. TNFα combined with zVAD-fmk induced a dramatic increase in the intracellular ceramide level, yet only in TNFα-sensitive fibroblasts (Figure 1D). Therefore, Axl RTK is required for the survival of mouse fibroblasts in the presence of TNFα, and Axl affects the TNFα-induced generation of intracellular ceramide. Since IL-15 treatment no longer protected Axl-deficient L929 cells from TNFα (Figure 1C), Axl must be an essential player in the IL-15-mediated protection of these fibroblasts from TNFα-induced cell death. Axl RTK and IL-15Rα associate and colocalize with each other To study whether and how Axl RTK participates in IL-15 signaling, immunoprecipitation (IP) experiments were performed. Parental L929 cells were stimulated with IL-15 or left untreated, and IL-15Rα or Axl was precipitated from the cell lysates using specific antibodies (Abs). Surprisingly, anti-Axl Abs co-immunoprecipitated IL-15Rα, and vice versa, in L929 fibroblasts even in the absence of IL-15 stimulation (Figure 2A). This unexpected physical association between IL-15Rα and Axl was confirmed by a two-site ELISA, where plates were coated with monoclonal Abs directed against Axl and incubated with cell lysates to bind target protein. Next, polyclonal Abs directed against IL-15Rα were added to detect Axl-associated IL-15Rα (Figure 2B). This also allowed quantitative analyses, by comparing results from two-site ELISA with specific ELISA for Axl or IL-15Rα (not shown). Arbitrarily defining the amount of Axl or IL-15Rα detected by specific ELISA as 100%, 63.1% of Axl and 87.4% of IL-15Rα molecules in L929 cells were in complex, whereas in L929R cells characterized by high Axl expression, 60.5% of Axl and 98% of IL-15Rα molecules were in complex. Thus, only a fraction of Axl molecules is associated with IL-15Rα in living cells, which may also depend on the cell type and may be relevant for the IL-15-mediated biological responses. To test whether the observed association occurs only in viable cells, we performed crosslinking experiments. However, no difference in the amount of IL-15Rα associated with Axl was observed after crosslinking, as detected by IP followed by WB, and two-site ELISA (data not shown). Figure 2.IL-15 induces phosphorylation of IL-15Rα and Axl, which constitutively associate. (A) L929 cells were stimulated with IL-15 (100 ng/ml). Cell lysates were precipitated with specific Abs against Axl, IL-15Rα, or IL-2Rγ and analyzed by WB. (B) Axl/IL-15Rα coassociation was detected in a two-site ELISA. Plates were coated with anti-Axl Abs. Axl (white bars) or Axl/IL-15Rα complexes (gray bars) were detected in cell lysates using biotinylated anti-Axl or anti-IL-15Rα Abs, respectively. COS-7 cells served as a negative control. (C) L929 cells were treated for 15 min with IL-15, TNFα, or both. Axl was precipitated from lysates and its phosphorylation was assessed with anti-pTyr Abs (upper panel). Axl detection served as a loading control (lower panel). (D) Axl and IL-15Rα colocalization analysis by confocal microscopy. Cells were stained with anti-IL-15Rα Abs (red), anti-Axl Abs (green), and TOTO-3 for nuclei (blue). Yellow color indicates IL-15Rα and Axl colocalization (right panels). Download figure Download PowerPoint Moreover, both anti-IL-15Rα and anti-Axl Abs precipitated IL-2Rγ, which constitutes (together with IL-15Rα) the high-affinity IL-15R complex in these L929 cells (Figure 2A). Remarkably, co-IP of IL-2Rγ with either receptor was much more prominent in the presence of IL-15 (Figure 2A). To further validate these results, we tested whether anti-γc Abs precipitate Axl or IL-15Rα upon IL-15 stimulation. The γc chain indeed associates with Axl and IL-15Rα upon stimulation with IL-15, as demonstrated by the ability of anti-γc Abs to co-precipitate these molecules (Figure 2A). Isotype-matched control Abs did not precipitate these proteins (data not shown). IL-15 treatment induced phosphorylation of both Axl and the IL-15Rα chain (Figure 2A). The pattern of IL-15-induced Axl phosphorylation was rather similar in the presence or absence of TNFα (Figure 2C). The constitutive association of Axl and IL-15Rα was further supported by confocal microscopy, which suggested that IL-15Rα and Axl colocalize on the cell membrane in the absence of IL-15 (Figure 2D). To analyze the physical association of IL-15Rα and Axl on the cell membrane in more detail, we next tested by density gradient centrifugation and confocal microscopy whether IL-15Rα and Axl associate in low-density lipid microdomains of the cell membrane (lipid rafts). These are enriched in many signaling molecules and segregate proteins to regulate signaling and membrane trafficking (Marmor and Julius, 2001). Under our experimental conditions, however, we did not detect IL-15Rα or Axl in membrane microdomains by either method (data not shown). The extracellular portion of Axl contains features that are reminiscent of cell adhesion molecules, and Axl mediates cell aggregation and adhesion by homotypic/homophilic association of its extracellular domains (Bellosta et al, 1995). Moreover, Axl-Fc chimeric protein, which consists of the extracellular domain of Axl (Faust et al, 1992) fused to the carboxy-terminal Fc region of human IgG1, promotes the migration of L929 cells upon immobilization on plastic (Budagian et al, 2005). Given the ability of IL-15Rα to interact with Axl, we asked whether IL-15 stimulation might provide a costimulatory signal for cell adhesion and migration through heterophilic interactions of IL-15Rα with Axl. To test this hypothesis, plates coated with Axl-Fc were seeded with equal numbers of cells. After 18 h, the cells were cleared within a defined area by scratching, photographed in phase-contrast, and allowed to migrate into the cleared area in the presence of IL-15. Unstimulated cells served as a control. However, the migratory properties of IL-15-stimulated and unstimulated cells did not differ (data not shown). These limited, initial data do not support a costimulatory role of IL-15 in the Axl-dependent mechanism(s) that mediate cell migration. Since the other two members of the Axl RTK family, Tyro3 and Mer, share the same ligand with Axl, we also examined their potential interaction with IL-15Rα. Remarkably, the expression of Tyro3 and Mer at mRNA (Supplementary Figure 3A) and protein (Supplementary Figure 3B) levels was rather low in both cell lines, compared to Axl. Also, unlike Axl, surface staining of Tyro3 and Mer was more intense in parental L929 fibroblasts than in the TNFα-resistant subline (Supplementary Figure 3C). Importantly, in line with the two-site ELISA results, no associations between IL-15Rα and either of these two RTKs were observed in IP experiments (data not shown). Thus, IL-15Rα specifically interacts with Axl and not other members of this RTK family. Contrary to current views on the trimeric structure of IL-15R (Fehninger and Caligiuri, 2001), Axl RTK may serve as an integral component of the functional IL-15R complex due to a constitutive physical association of the IL-15Rα chain with Axl even in the absence of ligand stimulation. The extracellular portion of Axl is essential for its association with IL-15Rα To better understand the molecular basis for this physical interaction, we co-transfected COS-7 cells with plasmids encoding the full-length IL-15Rα and one of two Axl mutant constructs, followed by IP with anti-IL-15Rα Abs (Figure 3). A chimeric protein (Bek-Axl) in which the extracellular domain of Axl was substituted with that of fibroblast growth factor receptor (BEK) (Bellosta et al, 1995) exhibited a dramatic decrease in the ability to associate with IL-15Rα in co-IP studies (Figure 3A). Conversely, Axl-DN, a dominant-negative mutant of Axl that lacks the kinase domain (Bellosta et al, 1995), still preserved the capacity to co-precipitate with IL-15Rα (Figure 3B). Next, we performed in vitro co-IP of IL-15Rα-Fc with different recombinant Axl proteins that contained either one of two IgG domains of Axl (Axl-Ig(1) or Axl-Ig(2), or both the domains (Axl-Ig(1+2)). However, these proteins failed to precipitate with IL-15Rα-Fc (Supplementary Figure 4). These data suggest that the Axl and IL-15Rα association requires the intact extracellular domain of Axl RTK. Figure 3.Mapping analysis of IL-15Rα/Axl interaction. COS-7 cells were transfected with IL-15Rα, Axl, Axl-Bek, and Axl-DN expression constructs in different combinations. IL-15Rα (A) or Axl (B) was precipitated from the cell lysates using specific Abs and analyzed by WB using anti-IL-15Rα or Axl Abs (upper two panels). Transfection efficiency was proved by protein expression analysis in lysates (lower two panels). (C) Cells were stimulated with IL-15 or Gas6 for 15 min. IL-15Rα or Axl was precipitated from the cell lysates and its phosphorylation was analyzed by WB. IL-15Rα or Axl was detected as a control for transfection and loading. Download figure Download PowerPoint Next, we determined whether the IL-15-induced phosphorylation of IL-15Rα is mediated by Axl. COS-7 cells were co-transfected with IL-15Rα and Axl constructs, followed by cell stimulation with IL-15 and assessment of IL-15Rα phosphorylation. IL-15Rα phosphorylation was seen only in cells expressing both constructs, whereas transfection of IL-15Rα with an empty vector or Axl-DN had no effect (Figure 3C). This strongly suggests that the IL-15-induced phosphorylation of IL-15Rα is mediated by Axl RTK. IL-15 transactivates the Axl signaling pathway Axl stimulation by Gas6 suppresses apoptosis in NIH 3T3 fibroblasts by upregulating Bcl-xL through activation of the PI3K/Akt pathway and NF-κB (Goruppi et al, 1997; Demarchi et al, 2001). Because IL-15 induced Axl phosphorylation, we compared the effects of IL-15 and Gas6 on L929 cells. Intriguingly, both IL-15 and Gas6 induced similar patterns of Axl phosphorylation within the first minutes of action (Figure 4A). IL-15 stimulation essentially mirrored Gas6-mediated signaling events, also resulting in the phosphorylation of PI3K and Akt. A modest phosphorylation of ERK1/2 was detected (Figure 4A), whereas the phosphorylation state of JNK and p38 kinase was unaffected (data not shown). Figure 4.IL-15 mimics Gas6-mediated signaling. (A) L929 cells were activated with IL-15 or Gas6. Axl (first panel) and PI3K (third panel) were precipitated from the cell lysates and analyzed by WB using anti-pTyr Abs. Phosphorylation of Akt (fifth panel) and ERK (seventh panel) was analyzed using phospho-specific Abs. For loading control, Axl, PI3K, Akt, or ERK was detected after stripping (even panels). (B) Bcl-xL and Bcl-2 were detected in cells stimulated for 24 h. Download figure Download PowerPoint On analyzing Bcl-xL and Bcl-2 protein expression in L929 cells treated with IL-15 or Gas6 for 24 h, we found that IL-15 and Gas6 upregulated these proteins in L929 cells (Figure 4B). L929R fibroblasts exhibited high basal expression of these two proteins, which could not be upregulated further by IL-15 (data not shown). To explore whether the similarity in downstream signaling events in response to Gas6 or IL-15 stimulation reflected transactivation of the Axl signaling cascade by IL-15, we repeated our experiments in Axl-depleted cells. Transfection with Axl siRNA revealed that IL-15 failed to activate PI3K, Akt, or ERK1/2 in Axl-deficient cells (Figure 5). This suggests that IL-15 transactivates the Axl signaling pathway in L929 fibroblasts. Figure 5.Axl-mediated signaling is impaired in L929 cells upon Axl depletion. L929 cells were transfected with Axl siRNA. After 48 h, cells were harvested, serum-starved for 4 h, and activated with IL-15 or Gas6. Axl (first panel) and PI3K (third panel) were precipitated from the cell lysates and analyzed by WB using anti-pTyr Abs. Phosphorylation of Akt (fifth panel) and ERK (seventh panel) was analyzed by phospho-specific Abs. For loading control, Axl, PI3K, Akt, or ERK was detected on the same blots after stripping (even panels). Download figure Download PowerPoint Gas6 is not involved in the IL-15-mediated Axl transactivation Next, we assessed the role of Gas6 in the IL-15-mediated transactivation of Axl under conditions that prevented endogenous Gas6 activity, using a chimeric Axl-Fc protein that captures Gas6 and inhibits its receptor binding (Costa et al, 1996). However, IL-15 still induced Axl phosphorylation in the presence of Axl-Fc (20–100 nM), whereas this chimeric protein abrogated Gas6-specific signaling in both L929 and L929R cells (Figure 6A). Importantly, IL-15 also stimulated the phosphorylation of Axl in L929 cells treated with warfarin (Figure 6B). Warfarin selectively inhibits the post-translational γ-carboxylation of Gas6, which is essential for its receptor binding and growth stimulation (Yanagita et al, 1999). These findings essentially rule out that endogenous Gas6 participates in the IL-15-mediated transactivation of Axl. To exclude a direct binding of IL-15 to Axl, we performed in vitro binding assays as described (Nagata et al, 1996), using purified recombinant proteins. However, no interactions other than the expected binding of IL-15 and Gas6 to their respective receptors were detected (Figure 6C). Figure 6.Gas6 does not affect IL-15-mediated Axl transactivation. (A) Cells were incubated for 10 min with 100 ng/ml Axl-Fc or control IL-3R-Fc proteins and then IL-15 or Gas6 (100 ng/ml) was added for another 5 or 15 min. Axl phosphorylation was detected by WB. (B) L929 cells were cultured in the presence of warfarin (100 μg/ml) for 24 h and stimulated with IL-15 or Gas6. Axl phosphorylation was detected by WB. (C) Recombinant Gas6 or IL-15 (10 nM) was incubated with 10 nM of Axl-Fc , IL-15Rα-Fc, or control IL-3R-Fc proteins. Complexes were captured on protein A-agarose and analyzed by WB using anti-Gas6 (upper panel) or anti-IL-15 (lower panel) Abs. Download figure Download PowerPoint IL-15 cross-modulates the Axl signaling pathway in mouse embryonic fibroblasts To check whether the ability of IL-15 to stimulate the IL-15Rα-mediated activation of Axl was restricted to L929 fibrosarcoma cells, mouse embryonic fibroblasts (MEFs) obtained from wild-type (WT), IL-15Rα-deficient (IL-15Rα−/−) (Lodolce et al, 1998), or Axl-deficient mice (Axl−/−) were also examined (Bellosta et al, 1997). MEFs from WT and Axl−/− mice almost equally expressed IL-15Rα, while MEFs from WT and IL-15Rα−/− animals had equal expression of Axl by WB (Figure 7A) and FACS analysis (Figure 7B). All three types of MEFs had comparable levels of IL-2Rγ expression (Figure 7A). To test whether IL-15 signaling in these cells depends on the expression of Axl, WT, Axl−/−, or IL-15Rα−/− MEFs were stimulated with IL-15 or Gas6. Subsequently, Axl was precipitated from the cell lysates using anti-Axl Abs, and protein precipitates were analyzed by WB. Figure 7.IL-15 and Gas6 signal in WT but not in Axl−/−, whereas Gas6 but not IL-15 induces signaling in IL-15Rα−/− MEFs. (A) WB analysis of Axl, IL-15Rα, and IL-2Rγ protein expression in WT, Axl−/−, and IL-15Rα−/− MEFs. (B) IL-15Rα expression was analyzed by FACS on cells labeled with IL-15Rα-IgG2b. (C) MEFs were stimulated with IL-15 or Gas6. Axl was precipitated from the lysates and phosphorylation was analyzed using anti-pTyr Abs (first panel). Same blots were analyzed for the association of Axl with IL-15Rα (third panel). Equal loading was confirmed by re-probing of blots with anti-Axl Abs (second panel). PI3K (fourth and fifth panels) and IL-15Rα (last two panels) were precipitated and their phosphorylation was detected using anti-pTyr Abs. Phosphorylation of Akt and ERK was detected using phospho-specific Abs. Blots were re-probed with PI3K, IL-15Rα, Akt, or ERK Abs for loading control. (D) Bcl-xL and Bcl-2 were detected in MEFs stimulated with IL-15 or Gas6 for 24 h. (E) Influence of IL-15 or Gas6 on serum deprivation-induced apoptosis in WT, Axl−/−, and IL-15Rα−/− MEFs. Apoptosis was induced by 48 h incubation in serum-free medium. IL-15 or Gas6 was added at a concentration of 100 ng/ml. Cells cultured in serum-free or serum-supplemented media were" @default.
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• W2135492013 date "2005-11-24" @default.
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• W2135492013 title "A promiscuous liaison between IL-15 receptor and Axl receptor tyrosine kinase in cell death control" @default.
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