Focal Adhesion Kinase–Mediated Activation of Glycogen Synthase Kinase 3β Regulates IL-33 Receptor Internalization and IL-33 Signaling

Jing Zhao, Jianxin Wei, Rachel K. Bowser, Russell S. Traister, Ming-Hui Fan and Yutong Zhao
J Immunol January 15, 2015, 194 (2) 795-802; DOI: https://doi.org/10.4049/jimmunol.1401414

Abstract

IL-33, a relatively new member of the IL-1 cytokine family, plays a crucial role in allergic inflammation and acute lung injury. Long form ST2 (ST2L), the receptor for IL-33, is expressed on immune effector cells and lung epithelia and plays a critical role in triggering inflammation. We have previously shown that ST2L stability is regulated by the ubiquitin-proteasome system; however, its upstream internalization has not been studied. In this study, we demonstrate that glycogen synthase kinase 3β (GSK3β) regulates ST2L internalization and IL-33 signaling. IL-33 treatment induced ST2L internalization, and an effect was attenuated by inhibition or downregulation of GSK3β. GSK3β was found to interact with ST2L on serine residue 446 in response to IL-33 treatment. GSK3β binding site mutant (ST2LS446A) and phosphorylation site mutant (ST2LS442A) are resistant to IL-33–induced ST2L internalization. We also found that IL-33 activated focal adhesion kinase (FAK). Inhibition of FAK impaired IL-33–induced GSK3β activation and ST2L internalization. Furthermore, inhibition of ST2L internalization enhanced IL-33–induced cytokine release in lung epithelial cells. These results suggest that modulation of the ST2L internalization by FAK/GSK3β might serve as a unique strategy to lessen pulmonary inflammation.

Introduction

Interleukin-33 is highly expressed in endothelial and epithelial cells, both of which frequently encounter threats from the surrounding environment (1, 2). During infection or injury, IL-33 acts as an alarmin and is released from injured or dying host cells (3, 4). IL-33 plays a crucial role in allergic inflammation and sepsis-induced injury. Our laboratory and others showed that increases in immunoreactive IL-33 are detected in bronchoalveolar lavage fluid from LPS- or Pseudomonas aeruginosa–treated mice (5, 6). IL-33 induces IL-6 and IL-8 release in lung cells and increases lung endothelial permeability (7–9). IL-33–deficient mice exhibit reduced mortality and cytokine release in a LPS sepsis model (10). However, a recent study demonstrated a critical role for IL-33 in bacterial sepsis as administration of IL-33 enhanced neutrophil influx and bacterial killing (11). Inhibition of IL-33 by administration of neutralizing IL-33 Ab or IL-33 decoy receptor attenuates lung inflammation in murine models of asthma (12, 13). In addition, administration of exogenous IL-33 to mice lacking an adaptive immune system induces cytokine release and goblet cell hyperplasia (14).

ST2 is a member of the IL-1R family, consisting of two major isoforms: a soluble, secreted form (sST2) and a transmembrane, long form (ST2L) (15, 16). ST2L is the receptor for IL-33 and is expressed on immune effector cells and lung epithelia and plays a critical role in triggering inflammation (7, 17). ST2L is a classic type I membrane receptor, containing three extracellular IgG-like domains, a transmembrane domain, and an intracellular Toll/IL-1R domain (18). We have demonstrated that lysophosphatidic acid regulates sST2 gene expression in human lung epithelia (19). Recently, we also showed that ST2L is ubiquitinated and degraded in response to IL-33 (5).

Glycogen synthase kinase 3β (GSK3β) is a key signaling Ser/Thr kinase that has diverse biological effects. Some of these are proapoptotic while others are antiapoptotic (20–23), and GSK3β also influences the stability of several signaling proteins (e.g., β-catenin and smad3) (24, 25). GSK3β activity is known to be enhanced by tyrosine 216 phosphorylation (26). IL-33 induces phosphorylation of tyrosine 216 within GSK3β, suggesting that IL-33 increases GSK3β activity (5). Overexpression of GSK3β attenuates TNF-α– or IL-1β–induced cytokine expression and plays an anti-inflammatory role in endotoxin-induced septic inflammation (27). We previously showed that GSK3β mediates ST2L phosphorylation at serine residue 442, thereby promoting its ubiquitination and degradation (5); however, the role of GSK3β in IL-33–induced cytokine release has not been examined.

Membrane receptor internalization is often triggered in response to agonist binding. It is important in controlling agonist-induced cellular responses by regulating the receptor level on the cell surface. Internalized receptors can subsequently be degraded in the lysosome or proteasome (5, 28) or return back to cell surface through an early endosome recycling pathway (29, 30). GSK3β has been shown to regulate cell surface protein internalization (31). GSK3β interacts with the 5-hydroxytryptamine (5-HT) receptor and stabilizes the 5-HT receptor on the cell surface. To our knowledge, in this study, we show for the first time that ST2L internalization and signaling are regulated by focal adhesion kinase (FAK)–activated GSK3β. These results might serve as a basis for new approaches to lessen the severity of inflammation by regulating ST2L internalization through activation of FAK/GSK3β pathway.

Materials and Methods

Cells and reagents

Murine lung epithelial (MLE12) cells (American Type Culture Collection, Manassas, VA) were cultured with HITES medium containing 10% FBS. RAW264 cells were cultured with DMEM containing 10% FBS. The cells were cultured at 37°C in a 5% CO2 incubator. V5 Ab, mammalian expressional plasmid pcDNA3.1 TOPO /His-V5, Escherichia coli Top 10 competent cells, and phospho-serine Ab were from Invitrogen (Carlsbad, CA). FAK, pY576/577-FAK, Flag tag (9A3), GSK3β, and pY216GSK3β Abs were from Cell Signaling Technology (Danvers, MA). Recombinant mouse IL-33 protein, KC, and mouse IL-6 ELISA kits were from R&D Systems (Minneapolis, MN). ST2L Ab was from Abcam (Cambridge, MA). FITC-conjugated ST2L (FITC-ST2L) Ab (DJ8) and a rat FITC–conjugated IgG1 isotope control Ab were from MD Bioproducts (St. Paul, MN). GSK3β short hairpin RNA (shRNA) and scrambled shRNA were from Sigma-Aldrich (St. Louis, MO). FAK inhibitor and TWS119 were from Cayman Chemical (Ann Arbor, MI). Immunobilized protein A/G beads were from Santa Cruz Biotechnology (Santa Cruz, CA). All materials used in these experiments were of the highest grade commercially available.

Construction of wild-type ST2L-Flag and mutant plasmids

The mouse ST2L cDNA with a Flag tag on the C terminus was inserted into a pcDNA3.1 TOPO/His-V5 vector. Site directed mutagenesis was performed to generate ST2L serine mutants according to the manufacturer’s instructions (Agilent Technologies, Santa Clara, CA). All the sequences of ST2L wild-type and mutants were confirmed by DNA sequencing.

Cell surface protein isolation

Proteins expressed on the cell surface were isolated using a cell surface protein isolation kit (Pierce Biotechnology, Rockford, IL) with biotin labeling following the manufacturer’s instructions. Briefly, cell surface proteins were labeled with a cell-impermeable, cleavable biotinylation reagent, Sulfo-NHS-SS-Biotin (0.25 mg/ml), for 30 min at 4°C with constant rocking. Cells were collected and cell lysates were incubated with Immobilized NeutrAvidin gel for 60 min at room temperature with an end-over mixing, followed by column purification. The isolated cell surface proteins were analyzed for ST2L or Flag tag by immunoblotting.

Immunoblotting and immunoprecipitation

After treatment, cells were washed with cold PBS and collected in cell lysis buffer containing 20 mM Tris HCl (pH 7.4), 150 mM NaCl, 2 mM EGTA, 5 mM β-glycerophosphate, 1 mM MgCl2, 1% Triton X-100, 1 mM sodium orthovanadate, 10 μg/ml protease inhibitors, 1 μg/ml leupeptin, and 1 μg/ml pepstatin. An equal amount of cell lysates (20 μg) was subjected to SDS-PAGE gel, electrotransferred to membranes and immunoblotted. For immunoprecipitation, equal amounts of cell lysates (1 mg) were incubated with specific primary Abs overnight at 4°C, followed by the addition of 40 μl protein A/G–agarose beads and incubation for an additional 2 h at 4°C. The immunoprecipitated complex was washed three times with 1% Triton X-100 in ice-cold PBS and analyzed by immunoblotting with indicated Abs.

Immunostaining

MLE12 cells were plated on 35-mm glass-bottom culture dishes. For ST2L-Flag staining, after treatments, cells were fixed in 3.7% formaldehyde for 20 min, followed by permeabilization with 0.1% Triton X-100 for 2 min. Cells were incubated with 1:200 dilution of Flag tag Ab, followed by a 1:200 dilution of fluorescence-conjugated secondary Ab sequentially for immunostaining. For endogenous cell surface ST2L staining, cells were incubated with FITC-labeled ST2L Ab for 1 h and then were treated with IL-33. Cells were then fixed with 3.7% formaldehyde for 20 min, followed by washes with PBS. Immunofluorescent cell imaging was performed on a Nikon confocal microscope.

Flow cytometry

MLE12 or RAW264 cells were harvested by mild trypsinization. Cell surface endogenous ST2L or overexpressed ST2L-Flag expression were determined with FITC-labeled ST2L Ab using an Accuri C6 Flow Cytometer.

Plasmid transfection

MLE12 cells were nucleofected with plasmids in the Nucleofection II System (Lonza, Gaithersburg, MD), and the cells were cultured in complete HITES medium for 48 h for overexpression and 72 h for knockdown experiments.

Cytokine ELISA measurement

Cell culture medium was replaced with fresh medium before IL-33 treatment. After 6 h of IL-33 treatment, cell supernatants were collected, centrifuged at 1000 × g for 5 min at 4°C, and frozen at −80°C for later analysis of KC and mouse IL-6 by ELISA, according to the manufacturer’s instructions.

Statistics

All results were subjected to statistical analysis using two-way ANOVA and, wherever appropriate, Student t test. Data are expressed as mean ± SD of triplicate samples from at least three independent experiments and p values <0.05 were considered statistically significant.

Results

IL-33 induces ST2L internalization

To investigate the effect of IL-33 on ST2L internalization, MLE12 cells were labeled with fluorescent-conjugated ST2L Ab and then treated with IL-33 for 30 min. In untreated cells, ST2L was localized on the cell surface, whereas IL-33 treatment induced ST2L internalization to the cytoplasm (Fig. 1A). IL-33 treatment-decreased ST2L cell surface expression was confirmed by FACS analysis in macrophage cell line, RAW264 cells (Fig. 1B). Furthermore, MLE12 cells were transfected with a plasmid containing ST2L cDNA with a Flag tag in the C terminus, and then were stimulated with IL-33 for 5, 15, and 30 min. Immunostaining with an anti-Flag Ab showed that in unstimulated cells ST2L was primarily localized on the cell surface, with a small amount of ST2L detectable in the cytoplasm. IL-33 treatment induced ST2L trafficking from the cell surface to the cytoplasm at both 15 and 30 min (Fig. 1C). IL-33–induced ST2L-Flag internalization in MLE12 cells was also confirmed by FACS analysis (Fig. 1D). These data suggest that IL-33 induces ST2L internalization.

FIGURE 1.
FIGURE 1.

IL-33 induces ST2L internalization. (A) MLE12 cells grown on glass-bottom dishes were incubated with FITC-conjugated ST2L Ab for 1 h and then were treated with mouse rIL-33 (10 ng/ml, 30 min). Cells were fixed with 3.7% formaldehyde. ST2L: green; nuclei: red (DAPI). Representative images from >40 cells with >80% positive in three independent experiments are shown. (B) RAW264 cells were treated with IL-33 (10 ng/ml) for 0, 5, 15, and 30 min, and expression of ST2L on the cell surface was determined by flow cytometry. IL-33 treatment reduces ST2L cell surface expression and produces a left shift of the curve. (C) MLE12 cells grown on glass-bottom dishes were transfected with ST2L-Flag plasmid for 2 d. Cells were treated with IL-33 (10 ng/ml; 0, 5, 15, and 30 min). Immunostaining was performed with a Flag Ab. ST2L-Flag: green; nuclei: red (DAPI). Representative images from >40 ST2L-Flag overexpressing cells with >80% positive in three independent experiments are shown. (D) MLE12 cells were transfected with ST2L-Flag plasmid for 2 d prior to treatment with IL-33 (10 ng/ml; 0, 15, and 30 min). Expression of ST2L-Flag on the cell surface was determined by flow cytometry. IL-33 treatment reduces ST2L-Flag cell surface expression and produces a left shift of the curve. Original magnification ×600.

GSK3β interacts with ST2L on serine residue 446

We have shown that GSK3β phosphorylates ST2L (5). To investigate whether GSK3β regulates ST2L internalization, we first examined whether IL-33 induces serine phosphorylation of ST2L-Flag and whether ST2L interacts with GSK3β. ST2L-Flag overexpressing MLE12 cells were treated with IL-33 for 30 min, and then, the phosphorylation of ST2L-Flag was examined by immunoprecipitation with a phospho–serine Ab, followed by Flag immunoblotting. As shown in Fig. 2A, IL-33 triggered ST2L-Flag serine phosphorylation in a dose-dependent manner. Furthermore, we found that GSK3β binds to ST2L in response to IL-33 treatment in RAW264 (Fig. 2B) and MLE12 cells (Fig. 2C). Double immunostaining with ST2L and phospho–Y216-GSK3β Abs revealed that IL-33 treatment increased tyrosine phosphorylation of GSK3β and colocalization of ST2L and activated GSK3β in the cytoplasm (Fig. 2D). ST2L contains a GSK3β conserved phosphorylation motif: 442-SxxxS-446 (Fig. 3A). Usually, the first serine residue is the GSK3β phosphorylation site and a phosphorylated serine residue on the C terminus of the motif is the GSK3β binding site. We have shown that serine 442 within ST2L is phosphorylated by GSK3β (5). To examine whether serine 446 is the GSK3β binding site, serine 446 (S446) within ST2L-Flag was substituted with an alanine residue (A). We found that GSK3β binds to ST2L-Flag wild-type and ST2LS442A-Flag, but not ST2LS446A-Flag (Fig. 3B, 3C). Furthermore, ST2LS446A-Flag impaired IL-33–induced serine phosphorylation (Fig. 3D). These results demonstrate that serine 446 within ST2L is in fact a binding site for GSK3β.

FIGURE 2.
FIGURE 2.

GSK3β interacts with ST2L. (A) MLE12 cells were transfected with ST2L-Flag plasmid (2 μg) for 2 d and then treated with IL-33 (0, 5, 10, and 20 ng/ml) for 30 min. Cell lysates were subjected to immunoprecipitation with an Ab against IgG or phospho (p)-serine, followed by immunoblotting with a Flag Ab. Input lysates were analyzed by immunoblotting with Flag and β-actin Abs. (B) RAW264 cells were treated with IL-33 (10 ng/ml; 30 min). Cell lysates were subjected to immunoprecipitation with an Ab against GSK3β, followed by immunoblotting with a ST2L Ab. Input lysates were immunoblotted with ST2L and β-actin Abs. (C) MLE12 cells were transfected with ST2L-Flag plasmid (2 μg) for 2 d and then treated with IL-33 for 0, 5, 15, and 30 min. Cell lysates were subjected to immunoprecipitation with an Ab against IgG or Flag, followed by immunoblotting with a GSK3β Ab. Input lysates were analyzed by immunoblotting with GSK3β, Flag, and β-actin Abs. (D) MLE12 cells grown on glass-bottom dishes were treated with IL-33 (10 ng/ml; 30 min). Immunostaining was performed to examine the localization of ST2L (green) and phospho–Y216-GSK3β (p-GSK3β, red). Representative images from >40 cells, with >80% of these staining positive, in three independent experiments are shown. Original magnification ×600.

FIGURE 3.
FIGURE 3.

Serine residue 446 is a GSK3β binding site within ST2L. (A) ST2L has a GSK3β consensus phosphorylation sequence. (B) MLE12 cells were transfected with wild-type ST2L-Flag or ST2LS446A-Flag plasmid for 2 d. Cell lysates were subjected to immunoprecipitation with an Ab against Flag tag, followed by immunoblotting with GSK3β and Flag Abs. Input lysates were immunoblotted with a GSK3β Ab. (C) MLE12 cells were transfected with wild-type ST2L-Flag, ST2LS442A-Flag, or ST2LS446A-Flag plasmid for 2 d. Cell lysates were subjected to immunoprecipitation with an Ab against GSK3β, followed by immunoblotting with a Flag Ab. Input lysates were immunoblotted with Flag tag and GSK3β Abs. (D) MLE12 cells were transfected with wild-type ST2L-Flag or ST2LS446A-Flag plasmid for 2 d prior to IL-33 treatment (10 ng/ml; 30 min). Cell lysates were subjected to immunoprecipitation with an Ab against phospho (p)-serine, followed by immunoblotting with a Flag Ab, or subjected to immunoprecipitation with an Ab against Flag tag, followed by immunoblotting with a p-serine Ab. Input lysates were immunoblotted with Flag tag and β-actin Abs.

GSK3β modulates ST2L internalization

ST2L-Flag overexpressing MLE12 cells were pretreated with a GSK3β inhibitor, TWS119, prior to IL-33 stimulation. IL-33 induced ST2L-Flag internalization, an effect was inhibited in TWS119-pretreated cells (Fig. 4A). Next, we examined ST2L expression on the cell surface. IL-33 reduced ST2L levels on the cell surface (Fig. 4B–E), which is consistent with our previous findings (5). Inhibition of GSK3β by TWS119 (Fig. 4B, 4C) or downregulation of GSK3β by GSK3β shRNA transfection (Fig. 4D, 4E) reduced the effect of IL-33, suggesting that GSK3β plays a critical role in the regulation of ST2L internalization. Furthermore, we examined whether the GSK3β phosphorylation site mutant ST2LS442A-Flag and the GSK3β binding site mutant ST2LS446A-Flag were resistant to agonist-modulated internalization. ST2LS442A-Flag or wild-type ST2L-Flag overexpressing MLE12 cells were treated with IL-33 for 30 min. IL-33 induced wild-type ST2L-Flag internalization without altering ST2LS442A-Flag localization (Fig. 5A) or its expression on the cell surface (Fig. 5B, 5C). In addition, IL-33 had no effect on ST2LS446A-Flag localization (Fig. 5D). Internalized ST2L-Flag colocalized with early endosome marker (Rab5a-V5), but not late endosome marker (Rab7a-V5). TWS119 was able to block the ST2L-Flag internalization to the early endosome (Supplemental Data). These data support our hypothesis that GSK3β modulates ST2L internalization through interaction with and phosphorylation of ST2L.

FIGURE 4.
FIGURE 4.

TWS119 or GSK3β shRNA attenuates IL-33–induced ST2L internalization. (A) ST2L-Flag overexpressing MLE12 cells grown on glass-bottom dishes were treated with TWS119 (10 μM; 1 h) prior to IL-33 treatment (10 ng/ml, 30 min). Immunostaining was performed with a Flag Ab. ST2L-Flag: green; nuclei: red (DAPI). Representative images from >40 ST2L-Flag overexpressing cells with >80% positive in three independent experiments are shown. Original magnification ×600. (B) MLE12 cells grown on D-100 dishes were treated with TWS119 (10 μM; 1 h) prior to IL-33 treatment (10 ng/ml; 30 min). Cell surface proteins were isolated as described in Materials and Methods. Cell surface lysates were analyzed by immunoblotting with a ST2L Ab. Whole-cell lysates were analyzed by immunoblotting with ST2L and β-actin Abs. (C) MLE12 cells were treated with TWS119 (10 μM; 1 h) prior to IL-33 treatment (10 ng/ml; 30 min). Expression of ST2L on the cell surface was determined by flow cytometry. (D) MLE12 cells were transfected with scrambled (Scram) shRNA or GSK3β shRNA plasmid for 3 d prior to IL-33 treatment (10 ng/ml, 30 min). Cell surface proteins were isolated as described in Materials and Methods. Cell surface lysates were analyzed by immunoblotting with a ST2L Ab. Whole-cell lysates were analyzed by immunoblotting with ST2L, GSK3β, and β-actin Abs. (E) MLE12 cells were transfected with scrambled (Scram) shRNA or GSK3β shRNA plasmid for 3 d prior to IL-33 treatment (10 ng/ml; 30 min). Expression of ST2L on the cell surface was determined by flow cytometry.

FIGURE 5.
FIGURE 5.

Both ST2LS442A-Flag and ST2LS446A-Flag are resistant to IL-33–modulated internalization. (A) Wild-type ST2L-Flag or ST2LS442A-Flag overexpressing MLE12 cells grown on glass-bottom dishes were stimulated with IL-33 (10 ng/ml; 30 min). Immunostaining was performed with a Flag Ab. Flag: green; nuclei: red (DAPI). Representative images from >40 ST2L-Flag or ST2LS442A-Flag overexpressing cells with >80% positive in three independent experiments are shown. (B) Wild-type ST2L-Flag or ST2LS442A-Flag overexpressing MLE12 cells grown on D-100 dishes were treated with IL-33 (10 ng/ml; 30 min). Cell surface proteins were isolated as described in Materials and Methods. Cell surface lysates were analyzed by immunoblotting with a Flag tag Ab. Whole-cell lysates were analyzed by immunoblotting with Flag tag and β-actin Abs. (C) Wild-type ST2L-Flag or ST2LS442A-Flag overexpressing MLE12 cells treated with IL-33 (10 ng/ml; 30 min). Expression of ST2L-Flag or ST2LS442A-Flag on the cell surface was determined by flow cytometry. (D) Wild-type ST2L-Flag or ST2LS446A-Flag overexpressing MLE12 cells grown on glass-bottom dishes were stimulated with IL-33 (10 ng/ml; 30 min). Immunostaining was performed with a Flag Ab. Flag: green; nuclei: red (DAPI). Representative images from >40 ST2L-Flag or ST2LS446A-Flag overexpressing cells with >80% positive in three independent experiments are shown. Original magnification ×600.

FAK regulates GSK3β-modulated ST2L internalization

We have shown that IL-33 activates GSK3β by inducing phosphorylation of tyrosine 216 (5); however, the kinase responsible for the activation of GSK3β in response to IL-33 has not been studied. FAK is a tyrosine kinase that regulates a variety of cellular responses by targeting multiple substrates. In this study, we found that IL-33 induces FAK phosphorylation in a time-dependent manner (Fig. 6A). Inhibition of FAK by its small molecule inhibitor attenuated IL-33–induced tyrosine 216 phosphorylation of GSK3β in a both time- and dose-dependent manner (Fig. 6B, 6C). Furthermore, FAK inhibition blocked IL-33–mediated ST2L-Flag internalization and loss from the cell surface (Fig. 6D, 6E). These results indicate that FAK is a regulator of GSK3β-modulated ST2L internalization.

FIGURE 6.
FIGURE 6.

FAK regulates GSK3β-modulated ST2L internalization. (A) MLE12 cells were treated with IL-33 for the indicated lengths of time. Cell lysates were analyzed by immunoblotting with phospho (p)-FAK and FAK Abs. (B) MLE12 cells were pretreated with or without FAK inhibitor ([FAKi] 10 μM; 1 h) prior to IL-33 treatment (0, 5, 15, and 30 min). Cell lysates were analyzed by immunoblotting with pY216-GSK3β and GSK3β Abs. (C) MLE12 cells were pretreated with or without FAKi (1, 5, or 10 μM; 1 h) prior to IL-33 treatment (10 ng/ml; 30 min). Cell lysates were analyzed by immunoblotting with pY216-GSK3β and GSK3β Abs. (D) ST2L-Flag overexpressing MLE12 cells grown on D-100 dishes were treated with FAKi (10 μM; 1 h) prior to IL-33 stimulation (10 ng/ml; 30 min). Cell surface proteins were isolated as described in Materials and Methods. Cell surface lysates were analyzed by immunoblotting with a Flag tag Ab. Whole-cell lysates were analyzed by immunoblotting with Flag tag and β-actin Abs. (E) ST2L-Flag overexpressing MLE12 cells grown on glass-bottom dishes were treated with FAKi (10 μM; 1 h) prior to IL-33 stimulation (10 ng/ml; 30 min). Immunostaining was performed with a Flag Ab. Flag: green; nuclei: red (DAPI). Representative images from >40 ST2L-Flag overexpressed cells with >80% positive in three independent experiments are shown. Original magnification ×600.

GSK3β regulates IL-33/ST2L–induced cytokine release in lung epithelial cells

Because GSK3β modulates ST2L expression on the cell surface, we hypothesized that GSK3β may regulate IL-33/ST2L–modulated cellular responses. We and others have shown that IL-33 induces IL-8 and IL-6 release in human lung epithelial and endothelial cells (5, 7). In this study, we show that IL-33 increased KC release in a time-dependent manner in MLE12 cells (Fig. 7A). Downregulation of GSK3β by GSK3β shRNA transfection significantly increased IL-33–induced KC release (Fig. 7B). Furthermore, we compared the effects of ST2LS442A-Flag and ST2LS446A-Flag to wild-type ST2L-Flag on IL-33–induced KC release in MLE12 cells. Both mutants significantly increased KC release compared with wild-type ST2L-Flag. In addition, GSK3β shRNA transfection (Fig. 7D) and ST2LS442A-Flag (Fig. 7E) enhanced IL-33–induced IL-6 release in MLE12 cells. These data suggest that inhibition of GSK3β reduces ST2L internalization and leads to persistent cell surface receptor activation by IL-33, thereby promoting increased IL-33/ST2L–modulated cytokine release. GSK3β is therefore a key regulator of both ST2L internalization and IL-33/ST2L biological functions.

FIGURE 7.
FIGURE 7.

GSK3β regulates IL-33/ST2L–induced cytokine release. (A) MLE12 cells were treated with IL-33 (10 ng/ml) for 0, 3, 6, and 24 h. Supernatants were collected, and KC levels were measured by KC ELISA kit. (B) MLE12 cells were transfected with Scram shRNA or GSK3β shRNA for 3 d prior to IL-33 stimulation (10 ng/ml; 6 h). Supernatants were collected, and KC levels were measured by ELISA. (C) Wild-type ST2L-Flag, ST2LS442A-Flag, or ST2LS446A-Flag overexpressing MLE12 cells were treated with IL-33 (10 ng/ml; 6 h). Expression of ST2L-Flag and mutants before IL-33 stimulation were examined by Flag immunoblotting. Supernatants were collected, and KC levels were measured by ELISA. (D) MLE12 cells were transfected with Scram shRNA or GSK3β shRNA for 3 d prior to IL-33 stimulation (10 ng/ml; 6 h). Supernatants were collected, and IL-6 levels were measured by ELISA. Expression of GSK3β was examined by GSK3β and β-actin immunoblotting. (E) Wild-type ST2L-Flag or ST2LS442A-Flag overexpressing MLE12 cells were treated with IL-33 (10 ng/ml; 6 h). Expression of ST2L-Flag and ST2LS442A-Flag before IL-33 stimulation were examined by Flag immunoblotting. Supernatants were collected, and IL-6 levels were measured by ELISA.

Discussion

IL-33 is a cytokine belonging to the IL-1 family and plays a critical role in the pathogenesis of pulmonary inflammatory diseases (7, 32). IL-33 induces IL-6 and IL-8 release in lung epithelial cells and macrophages, and increases lung endothelial permeability (7, 9). The biological effects of IL-33 are through interactions with its receptor ST2L. Therefore, understanding the regulation of cell surface expression of ST2L is important for limiting lung inflammation and injury. Ligand-induced receptor internalization and downregulation are crucial steps to control the magnitude and duration of extracellular signals in eukaryotes. Our previous studies demonstrated that ST2L is ubiquitinated and degraded in response to IL-33 in a GSK3β activation–dependent manner (5). In this study, we extend our understanding of ST2L downregulation by demonstrating a role for GSK3β in the regulation of ST2L receptor internalization. The current study indicates that FAK-activated GSK3β modulates ST2L internalization and signaling.

Receptor internalization is mediated by ligand-induced intracellular signaling including activation of intracellular protein kinases. For example, JAK kinase regulates IL-5R internalization (33). We have shown that GSK3β, a proline-directed serine/threonine kinase, regulates IL-33–induced ST2L phosphorylation; however, the role of GSK3β in the regulation of ST2L internalization has not been studied. GSK3β is a multifunctional kinase which is involved in a wide range of cellular processes including cell proliferation, cytokine release, and cell death (21, 22, 34, 35). In addition, the effect of GSK3β on receptor expression on the cell surface has been reported previously (31). Mutation of the GSK3β phosphorylation site of 5-HT type 1B serotonin receptor reduced the receptor expression on the cell surface. GSK3β also contributes to receptor endocytosis by interaction with the endosome (36, 37). The phosphorylation of substrates by GSK3β is directed in part by the recognition motif Ser/Thr-X-X-X-Ser/Thr (38). IL-33 treatment induces ST2L phosphorylation on serine 442, which is identified as a GSK3β phosphorylation site. In this study, combined immunocytochemical and biochemical evidence indicates that activation of GSK3β reduces ST2L expression on the cell surface, therefore attenuating IL-33–induced cytokine release. In this study, we show phosphorylated serine 446 within ST2L is the GSK3β docking site. Loss of interaction between the ST2LS446A mutant and GSK3β is not due to mislocalization of the modified receptor, as we show that this mutant protein is expressed on the cell surface at levels comparable to wild-type and still functions as an IL-33R. Overexpression of this GSK3β docking site mutant reduces ST2L internalization, therefore enhancing IL-33–induced cytokine release. These data suggest that interruption of the interaction between ST2L and GSK3β may abrogate the negative feedback loop for controlling ST2L-modulated pathways. The protein kinase for phosphorylation of serine 446 within ST2L is still unclear. IL-33 treatment induces ST2L trafficking into the early endosome, not late endosome. Our previous data show that ST2L is degraded in the proteasome but not the lysosome (5), which fuses with the late endosome. This conclusion is consistent with the findings of Hemar et al. that IL-2Rα-chain internalization is mediated by the early endosome, but not the late endosome (39). However, the mechanism by which ST2L is transported from early endosome to proteasome remains unclear. Gorbea et al. (40) have demonstrated a protein interaction network linking the early endosome and 26S proteasome ST2L is polyubiquitinated (5), which may also play a key role in the regulation of the receptor trafficking to endosome or proteasome.

The enzymatic activity of GSK3β is inhibited by serine 9 phosphorylation and enhanced by tyrosine 216 (Y216) phosphorylation (26, 41–43). Several studies have demonstrated that AKT inhibits GSK3β by serine 9 phosphorylation (41, 42). However, the regulation of GSK3β Y216 phosphorylation has not been well studied. IL-33 increases GSK3β activation by stimulating phosphorylation of GSK3β on Y216. This study provides the first evidence that IL-33 activates FAK, a tyrosine kinase, with FAK subsequently regulating GSK3β Y216 activation and thereby regulating ST2L internalization. John et al. (44) and Bianchi et al. (45) have shown that inhibition of GSK3β decreased phosphorylation of FAK during cell spreading and migration. FAK has been known to regulate the recycling of receptor tyrosine kinases back to the plasma membrane (46). To our knowledge, this is the first report regarding the role of FAK in the regulation of receptor internalization. FAK negatively regulates endothelial barrier integrity as knockdown of FAK increases basal myosin II L chain phosphorylation and VE–cadherin accumulation on the cell membrane. We have shown that IL-33 reduces lung endothelial barrier integrity (5). This effect may be due to increased FAK activation. Finally, IL-1R accessory protein is a coreceptor for IL-33. The effect of the FAK/GSK3β pathway on regulation of IL-1R accessory protein internalization and stability has not been determined yet; this will be one focus of our future research studies.

The IL-33R, ST2, exists in two forms: the membrane-bound form ST2L and the secreted form sST2 (15, 16). sST2 functions as an IL-33 decoy receptor, serving to block ST2L activation (13, 47). ST2-deficient mice lack both forms of ST2, leading to confusion regarding the role of ST2 in inflammatory lung diseases. For example, ST2-deficient mice are protected from sepsis-induced lung injury (48), whereas blockade of the IL-33 pathway by sST2 reduces airway inflammation in allergic asthma (49). We have shown that downregulation of ST2L by the ubiquitin–proteasome system reduces endotoxin-mediated lung injury (5). In this study, we reveal a new pathway by which FAK/GSK3β regulates ST2L internalization and expression on the cell surface, therefore modulating IL-33 inflammatory effects. FAK and GSK3β are widely considered as potential targets for the treatment of cancer (50, 51). In this paper, we provide evidence to support a novel approach to treating inflammatory lung diseases: activating FAK/GSK3β reduces ST2L expression on the cell surface and therefore limits the inflammatory response to IL-33.

Disclosures

The authors have no financial conflicts of interest.

Footnotes

  • This work was supported by National Institutes of Health Grants RO1 HL01916 and HL112791 (to Y.Z.) and American Heart Association Award 12SDG9050005 (to J.Z.).

  • The online version of this article contains supplemental material.

  • Abbreviations used in this article:

    FAK
    focal adhesion kinase
    GSK3β
    glycogen synthase kinase 3β
    5-HT
    5-hydroxytryptamine
    MLE
    murine lung epithelial
    shRNA
    short hairpin RNA
    sST2
    secreted form ST2
    ST2L
    long form ST2.

  • Received June 2, 2014.
  • Accepted November 5, 2014.

References

  1. ↵
    1. Moussion C.,
    2. N. Ortega,
    3. J. P. Girard
    . 2008. The IL-1-like cytokine IL-33 is constitutively expressed in the nucleus of endothelial cells and epithelial cells in vivo: a novel “alarmin?” PLoS One 3: e3331.
    OpenUrlCrossRefPubMed
  2. ↵
    1. Kouzaki H.,
    2. K. Iijima,
    3. T. Kobayashi,
    4. S. M. O’Grady,
    5. H. Kita
    . 2011. The danger signal, extracellular ATP, is a sensor for an airborne allergen and triggers IL-33 release and innate Th2-type responses. J. Immunol. 186: 4375–4387.
    OpenUrlAbstract/FREE Full Text
  3. ↵
    1. Cayrol C.,
    2. J. P. Girard
    . 2009. The IL-1-like cytokine IL-33 is inactivated after maturation by caspase-1. Proc. Natl. Acad. Sci. USA 106: 9021–9026.
    OpenUrlAbstract/FREE Full Text
  4. ↵
    1. Lüthi A. U.,
    2. S. P. Cullen,
    3. E. A. McNeela,
    4. P. J. Duriez,
    5. I. S. Afonina,
    6. C. Sheridan,
    7. G. Brumatti,
    8. R. C. Taylor,
    9. K. Kersse,
    10. P. Vandenabeele,
    11. et al
    . 2009. Suppression of interleukin-33 bioactivity through proteolysis by apoptotic caspases. Immunity 31: 84–98.
    OpenUrlCrossRefPubMed
  5. ↵
    1. Zhao J.,
    2. J. Wei,
    3. R. K. Mialki,
    4. D. F. Mallampalli,
    5. B. B. Chen,
    6. T. Coon,
    7. C. Zou,
    8. R. K. Mallampalli,
    9. Y. Zhao
    . 2012. F-box protein FBXL19-mediated ubiquitination and degradation of the receptor for IL-33 limits pulmonary inflammation. Nat. Immunol. 13: 651–658.
    OpenUrlCrossRefPubMed
  6. ↵
    1. Talabot-Ayer D.,
    2. C. Lamacchia,
    3. C. Gabay,
    4. G. Palmer
    . 2009. Interleukin-33 is biologically active independently of caspase-1 cleavage. J. Biol. Chem. 284: 19420–19426.
    OpenUrlAbstract/FREE Full Text
  7. ↵
    1. Yagami A.,
    2. K. Orihara,
    3. H. Morita,
    4. K. Futamura,
    5. N. Hashimoto,
    6. K. Matsumoto,
    7. H. Saito,
    8. A. Matsuda
    . 2010. IL-33 mediates inflammatory responses in human lung tissue cells. J. Immunol. 185: 5743–5750.
    OpenUrlAbstract/FREE Full Text
    1. Hong Y. J.,
    2. M. H. Jeong,
    3. Y. H. Choi,
    4. J. S. Ko,
    5. M. G. Lee,
    6. W. Y. Kang,
    7. S. E. Lee,
    8. S. H. Kim,
    9. K. H. Park,
    10. D. S. Sim,
    11. et al
    . 2009. Positive remodeling is associated with more plaque vulnerability and higher frequency of plaque prolapse accompanied with post-procedural cardiac enzyme elevation compared with intermediate/negative remodeling in patients with acute myocardial infarction. J. Cardiol. 53: 278–287.
    OpenUrlCrossRefPubMed
  9. ↵
    1. Choi Y. S.,
    2. H. J. Choi,
    3. J. K. Min,
    4. B. J. Pyun,
    5. Y. S. Maeng,
    6. H. Park,
    7. J. Kim,
    8. Y. M. Kim,
    9. Y. G. Kwon
    . 2009. Interleukin-33 induces angiogenesis and vascular permeability through ST2/TRAF6-mediated endothelial nitric oxide production. Blood 114: 3117–3126.
    OpenUrlAbstract/FREE Full Text
  10. ↵
    1. Oboki K.,
    2. S. Nakae,
    3. K. Matsumoto,
    4. H. Saito
    . 2011. IL-33 and airway inflammation. Allergy Asthma Immunol. Res. 3: 81–88.
    OpenUrlCrossRefPubMed
  11. ↵
    1. Alves-Filho J. C.,
    2. F. Sônego,
    3. F. O. Souto,
    4. A. Freitas,
    5. W. A. Verri Jr..,
    6. M. Auxiliadora-Martins,
    7. A. Basile-Filho,
    8. A. N. McKenzie,
    9. D. Xu,
    10. F. Q. Cunha,
    11. F. Y. Liew
    . 2010. Interleukin-33 attenuates sepsis by enhancing neutrophil influx to the site of infection. Nat. Med. 16: 708–712.
    OpenUrlCrossRefPubMed
  12. ↵
    1. Liu X.,
    2. M. Li,
    3. Y. Wu,
    4. Y. Zhou,
    5. L. Zeng,
    6. T. Huang
    . 2009. Anti‑IL-33 antibody treatment inhibits airway inflammation in a murine model of allergic asthma. Biochem. Biophys. Res. Commun. 386: 181–185.
    OpenUrlCrossRefPubMed
  13. ↵
    1. Yin H.,
    2. X. Y. Li,
    3. B. H. Yuan,
    4. B. B. Zhang,
    5. S. L. Hu,
    6. H. B. Gu,
    7. X. B. Jin,
    8. J. Y. Zhu
    . 2011. Adenovirus-mediated overexpression of soluble ST2 provides a protective effect on lipopolysaccharide-induced acute lung injury in mice. Clin. Exp. Immunol. 164: 248–255.
    OpenUrlCrossRefPubMed
  14. ↵
    1. Kondo Y.,
    2. T. Yoshimoto,
    3. K. Yasuda,
    4. S. Futatsugi-Yumikura,
    5. M. Morimoto,
    6. N. Hayashi,
    7. T. Hoshino,
    8. J. Fujimoto,
    9. K. Nakanishi
    . 2008. Administration of IL-33 induces airway hyperresponsiveness and goblet cell hyperplasia in the lungs in the absence of adaptive immune system. Int. Immunol. 20: 791–800.
    OpenUrlAbstract/FREE Full Text
  15. ↵
    1. Li H.,
    2. K. Tago,
    3. K. Io,
    4. K. Kuroiwa,
    5. T. Arai,
    6. H. Iwahana,
    7. S. Tominaga,
    8. K. Yanagisawa
    . 2000. The cloning and nucleotide sequence of human ST2L cDNA. Genomics 67: 284–290.
    OpenUrlCrossRefPubMed
  16. ↵
    1. Trajkovic V.,
    2. M. J. Sweet,
    3. D. Xu
    . 2004. T1/ST2—an IL-1 receptor-like modulator of immune responses. Cytokine Growth Factor Rev. 15: 87–95.
    OpenUrlCrossRefPubMed
  17. ↵
    1. Löhning M.,
    2. A. Stroehmann,
    3. A. J. Coyle,
    4. J. L. Grogan,
    5. S. Lin,
    6. J. C. Gutierrez-Ramos,
    7. D. Levinson,
    8. A. Radbruch,
    9. T. Kamradt
    . 1998. T1/ST2 is preferentially expressed on murine Th2 cells, independent of interleukin 4, interleukin 5, and interleukin 10, and important for Th2 effector function. Proc. Natl. Acad. Sci. USA 95: 6930–6935.
    OpenUrlAbstract/FREE Full Text
  18. ↵
    1. Sims J. E.,
    2. C. J. March,
    3. D. Cosman,
    4. M. B. Widmer,
    5. H. R. MacDonald,
    6. C. J. McMahan,
    7. C. E. Grubin,
    8. J. M. Wignall,
    9. J. L. Jackson,
    10. S. M. Call,
    11. et al
    . 1988. cDNA expression cloning of the IL-1 receptor, a member of the immunoglobulin superfamily. Science 241: 585–589.
    OpenUrlAbstract/FREE Full Text
  19. ↵
    1. Zhao J.,
    2. Q. Chen,
    3. H. Li,
    4. M. Myerburg,
    5. E. W. Spannhake,
    6. V. Natarajan,
    7. Y. Zhao
    . 2012. Lysophosphatidic acid increases soluble ST2 expression in mouse lung and human bronchial epithelial cells. Cell. Signal. 24: 77–85.
    OpenUrlCrossRefPubMed
  20. ↵
    1. Loberg R. D.,
    2. E. Vesely,
    3. F. C. Brosius III.
    . 2002. Enhanced glycogen synthase kinase-3β activity mediates hypoxia-induced apoptosis of vascular smooth muscle cells and is prevented by glucose transport and metabolism. J. Biol. Chem. 277: 41667–41673.
    OpenUrlAbstract/FREE Full Text
  21. ↵
    1. Song L.,
    2. P. De Sarno,
    3. R. S. Jope
    . 2002. Central role of glycogen synthase kinase-3β in endoplasmic reticulum stress-induced caspase-3 activation. J. Biol. Chem. 277: 44701–44708.
    OpenUrlAbstract/FREE Full Text
  22. ↵
    1. Dickey A.,
    2. S. Schleicher,
    3. K. Leahy,
    4. R. Hu,
    5. D. Hallahan,
    6. D. K. Thotala
    . 2011. GSK-3β inhibition promotes cell death, apoptosis, and in vivo tumor growth delay in neuroblastoma Neuro-2A cell line. J. Neurooncol. 104: 145–153.
    OpenUrlCrossRefPubMed
  23. ↵
    1. Meares G. P.,
    2. R. S. Jope
    . 2007. Resolution of the nuclear localization mechanism of glycogen synthase kinase-3: functional effects in apoptosis. J. Biol. Chem. 282: 16989–17001.
    OpenUrlAbstract/FREE Full Text
  24. ↵
    1. Yost C.,
    2. M. Torres,
    3. J. R. Miller,
    4. E. Huang,
    5. D. Kimelman,
    6. R. T. Moon
    . 1996. The axis-inducing activity, stability, and subcellular distribution of β-catenin is regulated in Xenopus embryos by glycogen synthase kinase 3. Genes Dev. 10: 1443–1454.
    OpenUrlAbstract/FREE Full Text
  25. ↵
    1. Lim J. H.,
    2. H. Jono,
    3. K. Komatsu,
    4. C. H. Woo,
    5. J. Lee,
    6. M. Miyata,
    7. T. Matsuno,
    8. X. Xu,
    9. Y. Huang,
    10. W. Zhang,
    11. et al
    . 2012. CYLD negatively regulates transforming growth factor-β-signalling via deubiquitinating Akt. Nat. Commun. 3: 771.
    OpenUrlCrossRefPubMed
  26. ↵
    1. Hughes K.,
    2. E. Nikolakaki,
    3. S. E. Plyte,
    4. N. F. Totty,
    5. J. R. Woodgett
    . 1993. Modulation of the glycogen synthase kinase-3 family by tyrosine phosphorylation. EMBO J. 12: 803–808.
    OpenUrlPubMed
  27. ↵
    1. Vines A.,
    2. S. Cahoon,
    3. I. Goldberg,
    4. U. Saxena,
    5. S. Pillarisetti
    . 2006. Novel anti-inflammatory role for glycogen synthase kinase-3β in the inhibition of tumor necrosis factor-α‑ and interleukin-1β‑induced inflammatory gene expression. J. Biol. Chem. 281: 16985–16990.
    OpenUrlAbstract/FREE Full Text
  28. ↵
    1. Oksvold M. P.,
    2. C. B. Thien,
    3. J. Widerberg,
    4. A. Chantry,
    5. H. S. Huitfeldt,
    6. W. Y. Langdon
    . 2003. Serine mutations that abrogate ligand-induced ubiquitination and internalization of the EGF receptor do not affect c-Cbl association with the receptor. Oncogene 22: 8509–8518.
    OpenUrlCrossRefPubMed
  29. ↵
    1. Signoret N.,
    2. M. Marsh
    . 2000. Analysis of chemokine receptor endocytosis and recycling. Methods Mol. Biol. 138: 197–207.
    OpenUrlPubMed
  30. ↵
    1. Mueller A.,
    2. E. Kelly,
    3. P. G. Strange
    . 2002. Pathways for internalization and recycling of the chemokine receptor CCR5. Blood 99: 785–791.
    OpenUrlAbstract/FREE Full Text
  31. ↵
    1. Chen L.,
    2. G. D. Salinas,
    3. X. Li
    . 2009. Regulation of serotonin 1B receptor by glycogen synthase kinase-3. Mol. Pharmacol. 76: 1150–1161.
    OpenUrlAbstract/FREE Full Text
  32. ↵
    1. Kurowska-Stolarska M.,
    2. A. Hueber,
    3. B. Stolarski,
    4. I. B. McInnes
    . 2011. Interleukin-33: a novel mediator with a role in distinct disease pathologies. J. Intern. Med. 269: 29–35.
    OpenUrlCrossRefPubMed
  33. ↵
    1. Martinez-Moczygemba M.,
    2. D. P. Huston,
    3. J. T. Lei
    . 2007. JAK kinases control IL-5 receptor ubiquitination, degradation, and internalization. J. Leukoc. Biol. 81: 1137–1148.
    OpenUrlAbstract/FREE Full Text
  34. ↵
    1. Jacobs K. M.,
    2. S. R. Bhave,
    3. D. J. Ferraro,
    4. J. J. Jaboin,
    5. D. E. Hallahan,
    6. D. Thotala
    . 2012. GSK-3β: a bifunctional role in cell death pathways. Int. J. Cell Biol. 2012: 930710.
    OpenUrlCrossRefPubMed
  35. ↵
    1. Kerkela R.,
    2. L. Kockeritz,
    3. K. Macaulay,
    4. J. Zhou,
    5. B. W. Doble,
    6. C. Beahm,
    7. S. Greytak,
    8. K. Woulfe,
    9. C. M. Trivedi,
    10. J. R. Woodgett,
    11. et al
    . 2008. Deletion of GSK-3β in mice leads to hypertrophic cardiomyopathy secondary to cardiomyoblast hyperproliferation. J. Clin. Invest. 118: 3609–3618.
    OpenUrlCrossRefPubMed
  36. ↵
    1. Taelman V. F.,
    2. R. Dobrowolski,
    3. J. L. Plouhinec,
    4. L. C. Fuentealba,
    5. P. P. Vorwald,
    6. I. Gumper,
    7. D. D. Sabatini,
    8. E. M. De Robertis
    . 2010. Wnt signaling requires sequestration of glycogen synthase kinase 3 inside multivesicular endosomes. Cell 143: 1136–1148.
    OpenUrlCrossRefPubMed
  37. ↵
    1. Pálfy M.,
    2. A. Reményi,
    3. T. Korcsmáros
    . 2012. Endosomal crosstalk: meeting points for signaling pathways. Trends Cell Biol. 22: 447–456.
    OpenUrlCrossRefPubMed
  38. ↵
    1. ter Haar E.,
    2. J. T. Coll,
    3. D. A. Austen,
    4. H. M. Hsiao,
    5. L. Swenson,
    6. J. Jain
    . 2001. Structure of GSK3β reveals a primed phosphorylation mechanism. Nat. Struct. Biol. 8: 593–596.
    OpenUrlCrossRefPubMed
  39. ↵
    1. Hémar A.,
    2. A. Subtil,
    3. M. Lieb,
    4. E. Morelon,
    5. R. Hellio,
    6. A. Dautry-Varsat
    . 1995. Endocytosis of interleukin 2 receptors in human T lymphocytes: distinct intracellular localization and fate of the receptor α, β, and γ chains. J. Cell Biol. 129: 55–64.
    OpenUrlAbstract/FREE Full Text
  40. ↵
    1. Gorbea C.,
    2. G. Pratt,
    3. V. Ustrell,
    4. R. Bell,
    5. S. Sahasrabudhe,
    6. R. E. Hughes,
    7. M. Rechsteiner
    . 2010. A protein interaction network for Ecm29 links the 26 S proteasome to molecular motors and endosomal components. J. Biol. Chem. 285: 31616–31633.
    OpenUrlAbstract/FREE Full Text
  41. ↵
    1. Cross D. A.,
    2. D. R. Alessi,
    3. P. Cohen,
    4. M. Andjelkovich,
    5. B. A. Hemmings
    . 1995. Inhibition of glycogen synthase kinase-3 by insulin mediated by protein kinase B. Nature 378: 785–789.
    OpenUrlCrossRefPubMed
  42. ↵
    1. Dudek H.,
    2. S. R. Datta,
    3. T. F. Franke,
    4. M. J. Birnbaum,
    5. R. Yao,
    6. G. M. Cooper,
    7. R. A. Segal,
    8. D. R. Kaplan,
    9. M. E. Greenberg
    . 1997. Regulation of neuronal survival by the serine-threonine protein kinase Akt. Science 275: 661–665.
    OpenUrlAbstract/FREE Full Text
  43. ↵
    1. Bhat R. V.,
    2. J. Shanley,
    3. M. P. Correll,
    4. W. E. Fieles,
    5. R. A. Keith,
    6. C. W. Scott,
    7. C. M. Lee
    . 2000. Regulation and localization of tyrosine216 phosphorylation of glycogen synthase kinase-3β in cellular and animal models of neuronal degeneration. Proc. Natl. Acad. Sci. USA 97: 11074–11079.
    OpenUrlAbstract/FREE Full Text
  44. ↵
    1. John J. K.,
    2. K. H. Paraiso,
    3. V. W. Rebecca,
    4. L. P. Cantini,
    5. E. V. Abel,
    6. N. Pagano,
    7. E. Meggers,
    8. R. Mathew,
    9. C. Krepler,
    10. V. Izumi,
    11. et al
    . 2012. GSK3β inhibition blocks melanoma cell/host interactions by downregulating N-cadherin expression and decreasing FAK phosphorylation. J. Invest. Dermatol. 132: 2818–2827.
    OpenUrlCrossRefPubMed
  45. ↵
    1. Bianchi M.,
    2. S. De Lucchini,
    3. O. Marin,
    4. D. L. Turner,
    5. S. K. Hanks,
    6. E. Villa-Moruzzi
    . 2005. Regulation of FAK Ser-722 phosphorylation and kinase activity by GSK3 and PP1 during cell spreading and migration. Biochem. J. 391: 359–370.
    OpenUrlCrossRefPubMed
  46. ↵
    1. Macagno J. P.,
    2. J. Diaz Vera,
    3. Y. Yu,
    4. I. MacPherson,
    5. E. Sandilands,
    6. R. Palmer,
    7. J. C. Norman,
    8. M. Frame,
    9. M. Vidal
    . 2014. FAK acts as a suppressor of RTK-MAP kinase signalling in Drosophila melanogaster epithelia and human cancer cells. PLoS Genet. 10: e1004262.
    OpenUrlCrossRefPubMed
  47. ↵
    1. Sweet M. J.,
    2. B. P. Leung,
    3. D. Kang,
    4. M. Sogaard,
    5. K. Schulz,
    6. V. Trajkovic,
    7. C. C. Campbell,
    8. D. Xu,
    9. F. Y. Liew
    . 2001. A novel pathway regulating lipopolysaccharide-induced shock by ST2/T1 via inhibition of Toll-like receptor 4 expression. J. Immunol. 166: 6633–6639.
    OpenUrlAbstract/FREE Full Text
  48. ↵
    1. Wieland C. W.,
    2. G. J. van der Windt,
    3. S. Florquin,
    4. A. N. McKenzie,
    5. T. van der Poll
    . 2009. ST2 deficient mice display a normal host defense against pulmonary infection with Mycobacterium tuberculosis. Microbes Infect. 11: 524–530.
    OpenUrlCrossRefPubMed
  49. ↵
    1. Hayakawa H.,
    2. M. Hayakawa,
    3. A. Kume,
    4. S. Tominaga
    . 2007. Soluble ST2 blocks interleukin-33 signaling in allergic airway inflammation. J. Biol. Chem. 282: 26369–26380.
    OpenUrlAbstract/FREE Full Text
  50. ↵
    1. Mitra S. K.,
    2. D. D. Schlaepfer
    . 2006. Integrin-regulated FAK-Src signaling in normal and cancer cells. Curr. Opin. Cell Biol. 18: 516–523.
    OpenUrlCrossRefPubMed
  51. ↵
    1. Garcea G.,
    2. M. M. Manson,
    3. C. P. Neal,
    4. C. J. Pattenden,
    5. C. D. Sutton,
    6. A. R. Dennison,
    7. D. P. Berry
    . 2007. Glycogen synthase kinase-3β: a new target in pancreatic cancer? Curr. Cancer Drug Targets 7: 209–215.
    OpenUrlCrossRefPubMed
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